CN1974765A

CN1974765A - Endogenous and non-endogenous versions of human G protein-coupled receptors

Info

Publication number: CN1974765A
Application number: CN 200610154066
Authority: CN
Inventors: 陈若平; 邓杭
Original assignee: Arena Pharmaceuticals Inc
Current assignee: Arena Pharmaceuticals Inc
Priority date: 1999-11-17
Filing date: 2000-11-16
Publication date: 2007-06-06
Also published as: CN1974766A; CN1847396A; CA2732120A1

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown (orphan GPCR receptors), and most particularly to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity.

Description

The endogenous form of human G protein-coupled receptor and non-endogenous form

The application for International Application PCT/US00/31509 on May 17th, 2002 enter the China national stage, application number is 00815869.X, denomination of invention is the dividing an application of " the endogenous form of human G protein-coupled receptor and non-endogenous form ".

Invention field

The disclosed invention of present patent application file relates to transmembrane receptor; Specifically, relate to and human g-protein coupled acceptor particularly endogenous people GPCR; Thereby what stress in particular is to be changed the non-endogenous form that has or strengthened the active GPCR of composition of acceptor.Preferably will this reformed GPCR be used for Direct Recognition as the candidate compound of receptor stimulant, inverse agonist or the partial agonist that might become therapeutical agent.

Background of invention

Although the acceptor of many types is arranged in human body, that up to the present the abundantest with the most relevant with treatment is g protein coupled receptor (GPCR).According to estimates, about 100,000 genes are arranged in human genome, the GPCR that encodes is estimated to be used in about 2% in them i.e. 2,000 genes.Comprise GPCR, the acceptor that its endogenous ligands has been familiar with is called as " known " acceptor, and the acceptor that endogenous ligands is not still known is called as " orphan " acceptor.GPCR is representing a key areas of medicament production exploitation: 60% prescription drug is developed about 20 in 100 known GPCR.

GPCR has an identical primitive (motif).All these acceptors have seven by 22 to 24 sequences that hydrophobic amino acid is formed, and they form seven α spirals, and each α spiral all strides across film (each span for example, is striden film-1 (TM-1), striden film-2 (TM-2) etc. all with numeral).Transbilayer helix connects by amino acid chain, and promptly " extracellular " amino acid chain is on one side being striden film-2 respectively and striden film-3, strides film-4 and stride film-5, stride film-6 and stride (these are called as " extracellular " district 1,2 and 3 (EC-1, EC-2 and EC-3) respectively) between the film-7 in the outside of cytolemma.At cytolemma inner i.e. " in the cell " on one side, transbilayer helix also connects by amino acid chain, and these amino acid chains are being striden film-1 and striden film-2, stride film-3 and stride film-4, stride film-5 and stride between the film-6 (these are called as " in the cell " district 1,2 and 3 (IC-1, IC-2 and IC-3) respectively) respectively." carboxyl " of acceptor (" C ") end is in intracellular zone, and " amino " of acceptor (" N ") is held in extracellular zone.

In general, (often be called as " activation " of acceptor) when endogenous ligands and receptors bind, the conformation in intracellular region territory changes, and carries out coupling with " G-albumen " in permissive cell inner compartment and the cell.It is reported that GPCR is " miscellaneous " for G albumen, that is to say, can with not only G protein-interacting.Referring to, Kenakin, T., 43, life science (LifeSciences) 1095 (1988).Although there is other G albumen, the current G albumen that has been identified is Gq, Gs, Gi, Gz and Go.The proteic coupling of endogenous ligands activatory GPCR and G-causes a signal cascade process (being called as " signal conduction ").Under common situation, the signal conduction finally causes cell activation or cell to suppress.It is believed that, the IC-3 of acceptor ring and carboxyl terminal all with the G protein-interacting.

Under physiological condition, GPCR is present on the cytolemma, and does not keep balance between the isomorphic map for these two kinds at " disactivation " state and " activation " state.Acceptor under inactive state can not with signal transduction path in the cell mutually coupling to produce biologically.Receptor conformation just makes it with pathway coupling mutually (by G-albumen) and produce biologically to the transformation of active condition.

Can acceptor be stabilized in its active condition by endogenous ligands or compound such as medicine.Recent discovery provide except that endogenous ligands or medicine, can promote and stable acceptor to the method for active condition conformation, this includes but not limited to the modification to the aminoacid sequence of acceptor.These methods are by be used for the effectively acceptor of stabilizing active state of imitation with the endogenous ligands of receptors bind.Stable being called as " composing type receptor activation " that forms by part dependent/non-dependent method so.

Summary of the invention

Disclosed herein is endogenous and non-endogenous form and the application thereof of people GPCR.

Brief description of the drawings

Fig. 1 has shown the second messenger IP that produces at the RUP12 (' RUP12 ') that is comparing following endogenous form with contrast (" CMV ") ₃

The second messenger that Fig. 2 is based on cell encircles the figure of AMP analytical results, shows the comparative result that the composing type signal of endogenous form RUP13 (" RUP13 ") and control vector (" CMV ") sends.

Fig. 3 is comparison CMV, endogenous form RUP13 (" RUP13wt ") and non-endogenous form composing type activatory RUP13 (" RUP13 (A268K) ") signal measurement result's a diagram, wherein uses the reporter plasmid with 8XCRE-Luc.

Fig. 4 be [ ³⁵S] synoptic diagram of GTP γ S analytical results, show the comparative result of the composing type signal transmission of RUP13:Gs fusion rotein (" RUP13-Gs ") and control vector (" CMV ").

Fig. 5 is comparison CMV, endogenous form RUP14 (" RUP14wt ") and non-endogenous form composing type activatory RUP13 (" RUP14 (L246K) ") signal measurement result's a diagram, wherein uses the 8XCRE-Luc reporter plasmid.

Fig. 6 is comparison CMV, endogenous form RUP15 (" RUP15wt ") and non-endogenous form composing type activatory RUP15 (" RUP15 (A398K) ") signal measurement result's a diagram, wherein uses the 8XCRE-Luc reporter plasmid.

The second messenger that Fig. 7 is based on cell encircles the figure of AMP analytical results, shows the comparative result that the composing type signal of endogenous form RUP15 (" RUP15wt "), non-endogenous form composing type activatory RUP15 (" RUP15 (A398K) ") and control vector (" CMV ") sends.

Fig. 8 be [ ³⁵S] synoptic diagram of GTP γ S analytical results, show the comparative result of the composing type signal transmission of RUP15:Gs fusion rotein (" RUP15-Gs ") and control vector (" CMV ").

Fig. 9 shows and contrasts (" CMV ") middle second messenger IP that produces of the RUP17 of endogenous form (" RUP17 ") by contrast ₃

Figure 10 shows and contrasts (" CMV ") middle second messenger IP that produces of the RUP21 of endogenous form (" RUP21 ") by contrast ₃

Figure 11 is comparison CMV, endogenous form RUP23 (" RUP23wt ") and non-endogenous form composing type activatory RUP23 (" RUP23 (W275K) ") signal measurement result's a diagram, wherein uses the 8XCRE-Luc reporter plasmid.

Figure 12 is the results of preliminary screening synoptic diagram of several candidate compounds at RUP13; The result of " compd A " is provided among the A2 of hole, the result of " compd B " is provided among the G9 of hole.

Describe in detail

This science document relates to acceptor and adopts some terms to describe acceptor is had the not part of same-action. For clear and self-consistentency, in document of the present invention, will use from the beginning to the end following definition. When these definition conflict with other definition of these words, select following definition:

Agonist means the material (for example, part, candidate compound) of activating cells internal reaction, and this moment, their bind receptors or promotion GTP combined with film.

At the amino acid abbreviations of this application A that is listed in the table below:

Table A

L-Ala	ALA	A
L-Ala	ALA	A	Arginine	ARG	R
L-asparagine	ASN	N	Arginine	ARG	R
L-asparagine	ASN	N	Aspartic acid	ASP	D
Halfcystine	CYS	C	Aspartic acid	ASP	D
Halfcystine	CYS	C	L-glutamic acid	GLU	E
Glutamine	GLN	Q	L-glutamic acid	GLU	E
Glutamine	GLN	Q	Glycine	GLY	G
Histidine	HIS	H	Glycine	GLY	G
Histidine	HIS	H	Isoleucine	ILE	I
Leucine	LEU	L	Isoleucine	ILE	I
Leucine	LEU	L	Methionin	LYS	K
Methionine(Met)	MET	M	Methionin	LYS	K
Methionine(Met)	MET	M	Phenylalanine	PHE	F
Proline(Pro)	PRO	P	Phenylalanine	PHE	F
Proline(Pro)	PRO	P	Serine	SER	S
Threonine	THR	T	Serine	SER	S
Threonine	THR	T	Tryptophane	TRP	W
Tyrosine	TYR	Y	Tryptophane	TRP	W
Tyrosine	TYR	Y	Xie Ansuan	VAL	V

Partial agonist means such material (for example, part, candidate compound), when they and receptors bind, and activating cells internal reaction or promote GTP and membrane-bound degree to be lower than agonist.

Antagonist (for example means such material, part, candidate compound), it and agonist combine with receptor competition ground in same site, but do not activate the cell internal reaction that the activity form by acceptor causes, and can so inhibition by agonist or the promoted cell internal reaction of partial agonist.Antagonist does not weaken the elementary cell internal reaction under the situation that does not have agonist or partial agonist.

Candidate compound means a molecule (such as but not limited to chemical compound) that will stand the triage techniques check.Preferably " candidate compound " do not comprise the known compound that is selected from inverse agonist, agonist or the antagonist of acceptor concerning the public, and they were determined (" compound of non-Direct Recognition ") by non-direct recognition methods in the past; More preferably do not comprise previous determined in a kind of Mammals, to have at least result of treatment by the compound of non-Direct Recognition; And, most preferably do not comprise previous determined in human body, have therepic use by the compound of non-Direct Recognition.

Composition is meant the material that comprises a kind of composition at least; Pharmaceutical composition promptly is an example of composition.

The compound effect means that compound suppresses or the measuring of the ability of costimulatory receptor function, and it is relative with receptors bind avidity.The typical method of measuring the compound effect is further open in the embodiment of present patent application part.

Codon is meant ternary Nucleotide (or speech suitable with Nucleotide), Nucleotide is made up of a nucleosides (adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)) phosphate group of coupling usually, amino acid of codon coding during translation.

Meaned the acceptor that is subject to the composing type receptor activation by the composing type activated receptors.By the composing type activated receptors can be endogenous also can right and wrong endogenous.

The composing type receptor activation means the method for the endogenous ligands of not utilizing it or its chemical equivalence thing and receptors bind and makes the acceptor under active condition stable.

Contact means puts two portions at least together, no matter is in vitro system or in vivo in the system.

Direct Recognition or by Direct Recognition, interrelate with term " candidate compound ", mean screening at composing type activated receptors, preferred pin to composing type activatory orphan receptor, most preferably at the candidate compound of the cell surface orphan receptor of composing type activatory and G albumen coupling.Term " non-directly identification " or " non-directly be identified " should do not explained or be understood that to be comprised or comprise in any case in this term.

The endogenous material that means by the natural generation of Mammals.These as illustration but be not the restriction.Compare with it, term " non-endogenous " means in this article and is not by Mammals (such as but not limited to the people) or viral natural generation.On the contrary, term " non-endogenous " means not to be material by Mammals (such as but not limited to the people) or viral natural generation herein.For example, be not the composing type activated receptors within it under the form of source, when making it the composing type activation when it is operated, this acceptor is most preferably censured is " non-endogenous by the composing type activated receptors ", this is as illustration rather than restriction.Two terms all can be used to describe " in the body " and " external " system.For example, in screening process, endogenous or non-endogenous acceptor can be used to the in-vitro screening system, and this is also only as illustration rather than restriction.As a further example rather than restriction, when the mammiferous genome of operation when comprising non-endogenous composing type activated receptor, can be by screening system candidate compound in the body.

In the context of this article, " g protein coupled receptor fusion rotein " and " gpcr fusion protein " are meant the non-intrinsic protein that is comprising endogenous composing type activatory GPCR, or the non-endogenous composing type activatory GPCR that merges with at least one G albumen, the preferred proteic α subunit of G (it is and GTP bonded subunit), the proteic type of G wherein preferably with under natural situation and the proteic type of endogenous orphan GPCR link coupled G be consistent.For example (but not limiting) at endogenous state, if G albumen " Gs α " is the G albumen main with the GPCR link coupled, is exactly the non-intrinsic protein that comprises the GPCR that merges with Gs α based on the gpcr fusion protein of this concrete GPCR.In some cases, as hereinafter will as described in, non-staple G albumen also can merge with GPCR.The C-terminal of G albumen and composing type activatory GPCR can directly merge, and also can have introns between the two at it.

Host cell means the cell that can insert plasmid and/or carrier therein.Under the prokaryotic host cell situation, plasmid is typically to duplicate (being separated to be introduced in the eukaryotic host cell after in the ordinary course of things, plasmid is duplicating) from the host molecule mode when host cell duplicates; Under the eukaryotic host cell situation, plasmid is integrated in the cell DNA of host cell, thereby, when eukaryotic cell duplicates, plasmid replication.Be purpose of the present invention disclosed herein, host cell is eukaryotic cell preferably, is more preferably mammalian cell, most preferably is the cell that chooses from 293,293T and the COS-7 cell.

Non-directly identification or non-directly be identified mean the traditional method of finding medicine, this method relate to the identification of the endogenous ligands of endogenous receptor-specific, screening at the candidate compound of acceptor, determine compound that those interference or competition ligand-receptor react to each other, measure the efficient that compound influences at least one second messenger's approach relevant with activated receptor.

Suppress, interrelate with term " reaction ", mean that when a compound exists a reaction is lowered or stops, this is opposite when just in time not existing with this compound.

Inverse agonist (for example means such material; part, candidate compound); they combine with the composing type activated form of endogenous recipient or acceptor; and it is below horizontal that the elementary cell internal reaction that will be caused by the activity form of acceptor is suppressed to arm's length basis; this activity level is to observe under the situation that does not have agonist or partial agonist, and perhaps they reduce combining of GTP and film.With do not having the primitive reaction under the inverse agonist situation to compare, the elementary cell internal reaction preferably is suppressed at least 30%, more preferably at least 50%, most preferably at least 75% in the presence of inverse agonist.。

Known receptor means the endogenous recipient that its special endogenous ligands has been identified.

Part means the molecule to the endogenous natural generation of the receptor-specific of endogenous natural generation.

Mean the specific transformation of these endogenous sequences about the Nucleotide of endogenous recipient and/or the sudden change of aminoacid sequence, thereby make the mutant of endogenous non-composing type activated receptor can cause the composing type of acceptor to activate.For the Equivalent of particular sequence, the follow-up mutant of people's acceptor is considered to the Equivalent of sudden change first of people's acceptor, if (a) indicated the same in itself of the composing type activation levels of follow-up mutant acceptor and suddenling change first of acceptor; (b) percentage ratio of the sequence homology between the sudden change first at follow-up mutant acceptor and acceptor is at least 80%, more preferably is at least 90%, most preferably is at least 95%.In ideal conditions, consider disclosed hereinly to be used to carry out the most preferred box of composing type activatory and to be included in single amino acid and/or the codon that changes between endogenous and the non-endogenous version GPCR that the percentage ratio of sequence homology should be at least 98%.

Non-orphan receptor is meant naturally occurring endogenous molecule, and naturally occurring endogenous ligands is shown specificity, and part makes intracellular signal by way of being activated with combining of acceptor.

Orphan receptor means such endogenous recipient, and its special endogenous ligands is still unrecognized or still unknown.

Pharmaceutical composition means and comprises at least a composition of active components, by this activeconstituents can study said composition can be in Mammals (such as but not limited to human body) specific effect.Those ordinarily skilled in the art can be understood and correct estimate those and be suitable for determining whether activeconstituents has the technology of the desired result that needs based on the technician.

Plasmid means the combination of carrier and cDNA.Generally, duplicate for cDNA and/or the purpose of marking protein is introduced host cell with plasmid.

The second messenger means in the cell that receptor activation produces and replys.For example, the second messenger comprises InsP3 (IP3), diacylglycerol (DAG), ring AMP (cAMP), and cyclo GMP (cGMP).The detection that the second messenger is replied can determine whether to exist receptor activation.In addition, the detection that the second messenger is replied can Direct Recognition candidate compound, for example comprises inverse agonist, agonist, partial agonist and antagonist.

Stimulate, interrelate, mean increased response when a kind of compound exists than when it does not exist with term " reaction ".

Carrier at cDNA means the annular DNA that at least one cDNA can be mixed wherein and can import in the host cell.

The sequence arrangement of lower part is for expression effect, and can not be interpreted as the restriction to following open or claim.

A. foreword

The tradition research of acceptor is based on so preposition supposition (based on history) always, and promptly endogenous ligands must at first be identified, and could find to act on antagonist and other molecules of acceptor then.Even under the situation that antagonist is at first found, the sight of search also extends to immediately searches endogenous ligands and gets on.Even after finding the composing type activated receptor, this thoughtcast also continues in acceptor research always.What be not realized before this is to be that the active condition of acceptor is the most useful to agonist, partial agonist and the inverse agonist of finding acceptor.The disease that causes because of the overactivity of acceptor and not enough activation for those wishes that the medicine that obtains is the compound that can be used for reducing the active condition of acceptor respectively or strengthen receptor active, and and need not be the antagonist of antagonism endogenous ligands.This is because a compound that reduces or strengthen the activated state receptor active does not need to be combined on the site the same with endogenous ligands.Thereby, as a method of the present invention is said, can begin by the compound of screening at part dependent/non-dependent activated state to any search of therapeutic compound.

B. the identification of people GPCR

The enforcement of the Human Genome Project causes being positioned at the identification of the bulk information of human genome related nucleic acid sequence, through this effort, in fact, we need not to understand or be familiar with the open reading-frame (ORF) information whether any special genes group sequence comprises interpreter's proteinoid, can obtain genetic sequence information, the method of several identification human genome amplifying nucleic acid sequences all is that those of ordinary skills are familiar with, such as (but not limiting), a large amount of human GPCR disclosed herein promptly is by looking back GenBank ^TMDatabase is found.Below table B, listed several by the endogenous GPCR that we find, and with disclosed other GPCR of GPCR homologous.

Table B

Disclosed human orphan GPCR	Go into to hide registration	Open reading-frame (ORF) (base pair)	With reference to homology GPCR	With indicated GPCR homology ratio
Disclosed human orphan GPCR	Go into to hide registration	Open reading-frame (ORF) (base pair)	With reference to homology GPCR	With indicated GPCR homology ratio	hRUP8	AL121755	1,152bp	NPY2R	27％
hRUP9	AC0113375	1,260bp	GAL2R	22％	hRUP8	AL121755	1,152bp	NPY2R	27％
hRUP9	AC0113375	1,260bp	GAL2R	22％	hRUP10	AC008745	1,014bp	C5aR	40％
hRUP11	AC013396	1,272bp	HM74	36％	hRUP10	AC008745	1,014bp	C5aR	40％
hRUP11	AC013396	1,272bp	HM74	36％	hRUP12	AP000808	966bp	Mas1	34％
hRUP13	AC011780	1,356bp	Fish GPRX-ORYLA	43％	hRUP12	AP000808	966bp	Mas1	34％
hRUP13	AC011780	1,356bp	Fish GPRX-ORYLA	43％	hRUP14	AL137118	1,041bp	CysLT1R	35％
hRUP15	AL016468	1,527bp	RE2	30％	hRUP14	AL137118	1,041bp	CysLT1R	35％
hRUP15	AL016468	1,527bp	RE2	30％	hRUP16	AL136106	1,068bp	GLR101	37％
hRUP17	AC023078	969bp	Mas1	37％	hRUP16	AL136106	1,068bp	GLR101	37％
hRUP17	AC023078	969bp	Mas1	37％	hRUP18	AC008547	1,305bp	Pitocin	31％
hRUP19	AC026331	1,041bp	HM74	52％	hRUP18	AC008547	1,305bp	Pitocin	31％
hRUP19	AC026331	1,041bp	HM74	52％	hRUP20	AL161458	1,011bp	GPR34	25％
hRUP21	AC026756	1,014bp	P2Y1R	37％	hRUP20	AL161458	1,011bp	GPR34	25％
hRUP21	AC026756	1,014bp	P2Y1R	37％	hRUP22	AC027026	993bp	RUP17 Mas1	67％ 37％
hRUP23	AC007104	1,092bp	Rat GPR26	31％	hRUP22	AC027026	993bp	RUP17 Mas1	67％ 37％
hRUP23	AC007104	1,092bp	Rat GPR26	31％	hRUP24	AL355388	1,125bp	SALPR	44％
hRUP25	AC026331	1,092bp	HM74	95％	hRUP24	AL355388	1,125bp	SALPR	44％
hRUP25	AC026331	1,092bp	HM74	95％	hRUP26	AC023040	1,044bp	Rabbit 5HT1D	27％
hRUP27	AC027643	158,700	MCH	38％	hRUP26	AC023040	1,044bp	Rabbit 5HT1D	27％

Effect in human body is useful to receptor homolog for further understanding acceptor, and at the present patent application file hereinafter, we will openly make these acceptors produce the technology of sudden change, so that set up the non-endogenous composing type activatory form of these acceptors.

These technology disclosed herein also have been applied to other orphan GPCR of people known in the art, and along with further describing of present patent application file, this technology can be more obvious.

C. acceptor screening

Filter out candidate compound corresponding to the non-endogenous composing type activated form of people GPCR disclosed herein, but the candidate compound that Direct Recognition works on this cell surface receptor, and do not need to use the endogenous ligands of acceptor.Utilize conventional, often be the technology that commerce can get, can determine that the zone of expressing is expressed and/or crossed to the endogenous form of people GPCR disclosed herein in vivo.Also might utilize these technology to determine and expression of receptor and/or the related relative disease/disorder of expression excessively that this method is disclosed in the present patent application file.

Manufacturing can prove the technology of people GPCR composing type activatory sudden change disclosed herein, be based on distance with proline residue, this residue is positioned at the TM6 inside of GPCR according to estimates, this algorithmic rule is disclosed in applied for the unsettled PCT of the examination of the common transfer of WO00/22129 announcement that among the PCT/US99/23938, it is for referencial use that this application and other patent documentation of enumerating are introduced this literary composition in the lump herein on April 20th, 2000.This algorithmic rule is not to predict according to traditional sequence alignment, but according to the specific range of above-mentioned TM6 proline residue (or also may be the endogenous composing type surrogate of this proline residue).Undergo mutation by making, preferably sport Methionin, can obtain this activation apart from the amino-acid residue at 16 amino-acid residue places of this residue (estimation is positioned at the IC3 district of acceptor).Other amino acid can be used to reach this purpose in this locational sudden change.

D. disease/disorder identification and/or selection

As what hereinafter will describe in detail, most preferably with inverse agonist and the agonist of method identification of the present invention at non-endogenous composing type activatory GPCR.So inverse agonist and agonist are the ideal candidates persons of lead compound during treatment is explored with the medicine of these receptor related diseases.Because but Direct Recognition is at the inverse agonist of these acceptors, therefore might develop and search at these receptor related diseases and disorderly pharmaceutical composition.For example, checking the existence of these GPCR in the ill and healthy tissues sample, is not only the problem of academic research now, the problem that also is on the research road of the endogenous ligands of the specific GPCR of identification to endeavour to solve.Can in the broad range of healthy and illing tissue, carry out tissue examination.So tissue examination provides the preferred first step that special acceptor and disease/disorder are interrelated.

The dna sequence dna of preferred people GPCR is used to make probe, is used to carry out (a) at the Dot blot of tissue mRNA and (b) the RT-PCR identification of expression of receptor described in the tissue sample.The existence of acceptor in tissue or diseased tissue, the concentration of perhaps comparing acceptor in diseased tissue with healthy tissues improves, and can preferably be used for discerning the related of methods of treatment (including but not limited to) and the sort of disease.In this way also can be well the zone of receptor mapping in organ.Be located in the known function of particular organization wherein based on acceptor, the imaginary functional role of acceptor can be derived.

E. the screening of candidate compound

1. general GPCR screening assay technology

When a kind of G protein receptor becomes composing type when activation, it and (for example, Gq, Gs, Gi, Gz, the Go) coupling of G albumen also stimulate GTP and the G protein binding.Then, by acceptor inactivation under normal circumstances, G albumen is hydrolyzed to GDP to GTP at leisure as the GTP enzyme, yet the composing type activated receptors continues GDP is converted into GTP.The analogue of GTP non-hydrolysable [ ³⁵S] GTP γ S, can be used to monitor and the combining of the film of expressing the composing type activated receptor.It is reported, [ ³⁵S] GTP γ S can be used to monitor part exist or non-existent situation under the coupling of G albumen and film.An illustration of this kind monitoring is arranged in other famous and feasible in the art illustrations, and it is reported in nineteen ninety-five by Traynor and Nahorski.A preferred application of this mensuration system is for the preliminary screening candidate compound, because native system is generally feasible to all albumen-coupled receptors, and does not consider and interactional that a kind of special G albumen of the cell intracellular domain of acceptor.

2. specific GPCR screening assay technology

Identify candidate compound in case use the receptor assays (i.e. screening is the method for the compound of agonist, partial agonist or inverse agonist) of " generally " G albumen coupling, preferred further screening is to confirm to act on the compound of acceptor site.For example, using compound that " generally " measuring method discerns can be not and receptors bind, but can be only from cell intracellular domain and G albumen " uncoupling " yet.

A.Gs, Gz and Gi

Gs stimulates adenylate cyclase.On the other hand, Gi (with Gz and Go) suppresses this enzyme.Adenylate cyclase enzyme catalysis ATP is to the conversion of cAMP; Therefore, be associated with cAMP level in the cell of the composing type activatory GPCR of Gs albumen coupling and rising.On the other hand, be associated with the interior cAMP level of the cell of reduction with the composing type activatory GPCR of Gi (with Gz or Go) albumen coupling.Generalized case is referring to " cynapse conduction non-direct mechanism (IndirectMechanisms of Synaptic Transmission) ", the 8th chapter, (From from the nerve to the brain Neuron To Brain) (third edition), Nichols, volumes such as J.G., Sinauer Associates, Inc. (1992).Therefore, the method that detects cAMP can be used to determine that whether an emulative compound is the inverse agonist (being the level that a such compound can reduce cAMP) of acceptor etc.The different methods of mensuration cAMP known in the art can be utilized; Most preferred method depends on the antibody of using anti--cAMP in based on the method for ELISA.The another kind of measuring method that can be employed is a kind of full cell second messenger reporter gene systems measurement method.Promoters driven on the gene is by the protein expression of a special coded by said gene.Ring AMP promotes expression of gene by following steps, i.e. its response promotes the combination of the conjugated protein or transcription factor (CREB) of the DNA of cAMP, and transcription factor then combines and drive genetic expression in the special site that is called as the cAMP response element with promotor.The reporter gene system can be built as has a promotor, and this promotor contains a plurality of cAMP response elements, for example beta-galactosidase enzymes or luciferase in the front of reporter gene.Thereby an acceptor by composing type activatory connection Gs causes the accumulation of cAMP, and cAMP then activates the gene and the expression of reporter protein matter.Reporter protein such as beta-galactosidase enzymes or luciferase matter available standards biochemical method detects (Chen etc., 1995).

B.Go and Gq

The activation of Go and Gq and Phospholipase C interrelates, and Phospholipid hydrolase is with posthydrolysis phosphoric acid ester PIP ₂, and discharge courier in two kinds of cells: diacylglycerol (DAG) and inositol-1,4,5-triphosphoric acid (IP ₃).The IP that accumulation increases ₃The acceptor related with Gq-and Go-is associated.Generalized case is referring to " cynapse conduction non-direct mechanism (Indirect Mechanisms of Synaptic Transmission) ", the 8th chapter, from the nerve to the brain ( From Neuron To Brain) (third edition), Nichols, volumes such as J.G., Sinauer Associates, Inc. (1992).Measure IP ₃The method of accumulation can be used to determine that whether a candidate compound is that for example (promptly so compound can reduce IP at the inverse agonist of Gq-or the related acceptor of Go-etc. ₃Level).The also available AP1 reporter-gene assays of the related acceptor of Gq method detects, because the Phospholipase C that Gq relies on causes the gene activation that contains the AP1 element; Thereby the related acceptor of activatory Gq will cause expression of gene like this to increase, and its inverse agonist will cause so reduction of expression, agonist will cause the rising of so expressing.The method that the commerce of so measuring can get can get.

3.GPCR fusion rotein

Endogenous composing type activatory orphan GPCR or non-endogenous composing type activatory orphan GPCR, be used to screen candidate compound, Direct Recognition inverse agonist, agonist and partial agonist, a significant screening difficult problem has been proposed, exactly, do not having under the endogenous ligands bonded situation, acceptor still has activity.Therefore, non-endogenous recipient when existing or not existing in order to distinguish candidate compound, the purpose of this differentiation is will understand inverse agonist that whether this compound is described acceptor, agonist, partial agonist or to this receptor not influence at all, best bet is strengthened this species diversity exactly, uses gpcr fusion protein a kind of method that comes to this.

In general, use above-mentioned analytical technology (technology that also has other), just may determine and endogenous GPCR link coupled advantage G albumen that the coupling of G albumen and GPCR provides can estimative signal pathway in case determine that non-endogenous orphan GPCR is the composing type activatory.Because preferably use mammalian expression system to screen, just wishing has endogenous G albumen to exist in this system, definite, and non-endogenous composing type activatory orphan GPCR continues to produce signal in this system.From this aspect, signal is strengthened, thereby when the inverse agonist of (for example) acceptor exists, distinguish the not isoacceptor that contacts with inverse agonist probably more easily, particularly in the whole process of screening.

The effect of gpcr fusion protein is to increase G albumen and non-endogenous GPCR link coupled effect, gpcr fusion protein is preferred for screening non-endogenous composing type activatory GPCR, because this method strengthens the signal very useful to such triage techniques, importantly help to produce very big " noise " ratio, this big signal to noise ratio is particularly preferred to screening candidate compound disclosed herein.

The constructing technology that is used for the construct of gpcr fusion protein expression is that those of ordinary skills are familiar with, commercial obtainable expression vector and system provide the various methods that can satisfy special requirement for the experimenter, the important criterion of this gpcr fusion protein construct, endogenous exactly GPCR sequence and G protein sequence all meet frame (preferably, the sequence of endogenous GPCR is positioned at G protein sequence upstream), and " termination " codon of necessary removal or alternative GPCR, thereby along with the expression of GPCR, G albumen also can be expressed.GPCR can directly link on the G albumen, or there is residue (preferably being no more than 12, though those of ordinary skill in the art can easily learn this numeral) at interval between.We like using introns (based on convenient), and the restriction site that is not used effectively in the expression has been formed introns.Before making the gpcr fusion protein construct, preferably at first confirm and non-endogenous GPCR link coupled G albumen, because have only G albumen seldom to be identified, can insert endogenous GPCR sequence therein so preferably comprise the construct of G protein sequence (as general G albumen construct), can screen on a large scale effectively like this and have not homotactic endogenous GPCR in a large number.

As mentioned above, expectation and Gi, Gz and Go link coupled composing type activation GPCR suppress the formation of cAMP, this just need people find analytical procedure based on these types GPCR (as, the cAMP signal reduces with activation, make that like this Direct Recognition (for example) inverse agonist (further weakening sort signal) is more interesting, as disclosed herein, we are verified, acceptor for these types, might make not based on the proteic gpcr fusion protein of endogenous G of endogenous GPCR, setting up feasible as possible is based analyzing method with the cyclase.For instance, endogenous Gi coupled receptor can be with the fusion of Gs albumen-we believe that such fusion constructs is when expressing, " driving " or " promotion " endogenous GPCR with as the Gi albumen coupling of Gs rather than " natural ", be based analyzing method thereby can set up with the cyclase.To with Gi, Gz, Go link coupled acceptor, be when being determined as the basis when using gpcr fusion protein and analysis with adenylate cyclase activity, we preferably use Gs (or the G albumen analogue that stimulates adenylate cyclase to form) to set up fusion constructs.

The G albumen fusion constructs that Gq albumen and Gs, Gi, Gz or Go albumen merge is effective too.The first six amino acid of Gq protein delation G protein alpha subunit (" G α q ") more preferably, and behind the C-terminal of G α q five amino acid by the fusion constructs that corresponding amino acid replacement obtained of purpose G protein alpha subunit.For example, fusion constructs may be Gq (lacking 6 amino acid) and the proteic fusion of Gi, produces " Gq/Gi fusion constructs ".We believe that this fusion constructs can promote the non-endogenous G albumen-Gq coupling with it of endogenous Gi coupled receptor, thus can be to second messenger's (for example, InsP3 or diacylglycerol) but not the generation of cAMP detect.

With target Gi link coupled GPCR and with signal enhanser Gs link coupled GPCR cotransfection (based on the analysis of cAMP)

Known Gi link coupled acceptor suppresses adenylate cyclase, and therefore reduces the output of cAMP, and this causes being difficult to assess the level of cAMP.The reduction that detects the cAMP output of indicating as the acceptor composing type activation of main coupling Gi when activating can be achieved by Gi and a signal enhanser (as the non-endogenous, constitutively activated acceptor of main coupling Gs when activating, as the TSHR-A623I of the following announcement) cotransfection of coupling GPCR.Obviously, the composing type of Gs coupled receptor activation can be judged based on the increase of cAMP output.The composing type activation of Gi coupled receptor causes the output of cAMP to reduce.Therefore, the method for this cotransfection is intended to effectively utilize the effect of these " opposed ".For example, non-endogenous composing type activated receptor of Gs link coupled (" signal enhanser ") and endogenous Gi coupled receptor (" target acceptor ") cotransfection provide basic cAMP signal (promptly, although Gi link coupled acceptor reduces the level of cAMP, the effect of this reduction is associated with the remarkable increase of composing type activatory Gs link coupled signal cAMP that enhanser causes).Thereby by signal enhanser and composing type activatory target acceptor cotransfection, the activity of Gi target increases (that is, reducing the cAMP level), estimates that the cAMP level will further reduce (with respect to basic horizontal).

Then, utilization is analyzed the candidate compound based on the analysis of cAMP will become possibility, but two restrictive conditions are arranged: at first with respect to Gi link coupled target acceptor, can produce the effect of " on the contrary ", promptly, the inverse agonist of Gi coupling target acceptor will strengthen determined cAMP signal, and the agonist of Gi coupling target acceptor will reduce this signal; The second, also can show following, utilize the candidate compound of this method Direct Recognition to assess individually, to guarantee it signal enhancing property acceptor there is not targeting (this can be before screening at the cotransfection acceptor or carries out later on).

F. pharmaceutical chemistry

General but be not under the frequent situation candidate compound Direct Recognition and the compound that produces by combinatorial chemistry technique to be united use, wherein prepare several thousand kinds of compounds at random and be used for this analysis.So results of screening generally will be the compound with unique division center; Thereafter, these compounds are preferably carried out extra chemically modified round a preferred division center, with its medicinal character of further reinforcement.Such technology is known in this field, need not describe in detail in this patent file.

G. pharmaceutical composition

The candidate compound of selecting for further exploitation can be used the known technology preparation in this area and become pharmaceutical composition.The appropriate drug acceptable carrier can get in the art; For example, referring to Remington ' s Pharmaceuctical Sciences, the 16th edition, 1980, MackPublishing Co. (volume such as Oslo).

H. other application

Although preferred application of disclosed non-endogenous people GPCR is for the candidate compound of Direct Recognition as inverse agonist, agonist or partial agonist (preferably as drug use), these forms of people GPCR also can be used to the usefulness of research.For example, have in the external or body of GPCR system and can be used to explain and understand the effect of these acceptors in normal and ill human body situation, also can understand composing type activatory role when it is applied to understand signal cascade and reacts.The value of these non-endogenous people GPCR is because its unique characteristics are them is reinforced as the purposes of research tool, and disclosed acceptor can be used to understand the effect of these acceptors in human body, even before the source part is identified within it.It will be significantly that other of disclosed acceptor are used for those skilled in the art, particularly after they have read present specification.

Embodiment

The embodiment that provides below, purpose is to illustrate rather than to limit the present invention.Special nucleotide sequence and aminoacid sequence are when this is open, and those of ordinary skill in the art can carry out less modification to these sequences, and obtain and the identical or similar substantially result who reports below.Use or understand to be based on the sequence correlation technique the traditional way of one and another sequence frame (as acceptor from the acceptor of mouse to the people or acceptor B) from people's acceptor A to the people, by series arrangement to recently determine the common zone as far as possible.Mutation method disclosed herein does not rely on this mode, and be based on algorithmic rule and be arranged in the position distance that the conservative proline residue in people GPCR TM6 district is separated by.Once this mode is reliable, believe that those of ordinary skill in the art can carry out less modification so that obtain and disclosed essentially identical result (as: composing type activation) herein.Think that these are modified all in the scope of the present disclosure.

Embodiment 1

Endogenous people GPCR

The identification of 1 people GPCR

Browsing GenBank ^TMOn the basis of database information, some disclosed endogenous people GPCR have been discerned.In searching database, following cDNA clone is also discerned, and is listed as follows (table C).

Table C

Disclosed people orphan GPCR	Go into to hide registration number	DNA complete sequence (base pair)	Open reading-frame (ORF) (base pair)	Nucleic acid sequence SEQ .ID.NO.:	Aminoacid sequence SEQ.ID.NO.
Disclosed people orphan GPCR	Go into to hide registration number	DNA complete sequence (base pair)	Open reading-frame (ORF) (base pair)	Nucleic acid sequence SEQ .ID.NO.:	Aminoacid sequence SEQ.ID.NO.	hRUP8	AL121755	147,566bp	1,152bp	1	2
hRUP9	AC0113375	143,181bp	1,260bp	3	4	hRUP8	AL121755	147,566bp	1,152bp	1	2
hRUP9	AC0113375	143,181bp	1,260bp	3	4	hRUP10	AC008745	94,194bp	1,014bp	5	6
hRUP11	AC013396	155,086bp	1,272bp	7	8	hRUP10	AC008745	94,194bp	1,014bp	5	6
hRUP11	AC013396	155,086bp	1,272bp	7	8	hRUP12	AP000808	177,764bp	966bp	9	10
hRUP13	AC011780	167,819bp	1,356bp	11	12	hRUP12	AP000808	177,764bp	966bp	9	10
hRUP13	AC011780	167,819bp	1,356bp	11	12	hRUP14	AL137118	168,297bp	1,041bp	13	14
hRUP15	AL016468	138,828bp	1,527bp	15	16	hRUP14	AL137118	168,297bp	1,041bp	13	14
hRUP15	AL016468	138,828bp	1,527bp	15	16	hRUP16	AL136106	208,042bp	1,068bp	17	18
hRUP17	AC023078	161,735bp	969bp	19	20	hRUP16	AL136106	208,042bp	1,068bp	17	18
hRUP17	AC023078	161,735bp	969bp	19	20	hRUP18	AC008547	117,304bp	1,305bp	21	22
hRUP19	AC026331	145,183bp	1,041bp	23	24	hRUP18	AC008547	117,304bp	1,305bp	21	22
hRUP19	AC026331	145,183bp	1,041bp	23	24	hRUP20	AL161458	163,511bp	1,011bp	25	26
hRUP21	AC026756	156,534bp	1,014bp	27	28	hRUP20	AL161458	163,511bp	1,011bp	25	26
hRUP21	AC026756	156,534bp	1,014bp	27	28	hRUP22	AC027026	151,811bp	993bp	29	30
hRUP23	AC007104	200,000bp	1,092bp	31	32	hRUP22	AC027026	151,811bp	993bp	29	30
hRUP23	AC007104	200,000bp	1,092bp	31	32	hRUP24	AL355388	190,538bp	1,125bp	33	34
hRUP25	AC026331	145,183bp	1,092bp	35	36	hRUP24	AL355388	190,538bp	1,125bp	33	34
hRUP25	AC026331	145,183bp	1,092bp	35	36	hRUP26	AC023040	178,508bp	1,044bp	37	38
hRUP27	AC027643	158,700bp	1,020bp	39	40	hRUP26	AC023040	178,508bp	1,044bp	37	38

2. full-length clone

a.hRUP8(Seq.Id.Nos.1&2)

The people RUP8 that announces is discerned by using est database information (dbEST).When search finds that is gone into to hide number to be the new GPCR of cDNA clones coding of AL121755 in dbEST.It is the RT-PCR clone of template that following PCR primer is used for people's testis Marathon-Ready cDNA (Clontech):

5 '-CTTGCAGACATCACCATGGCAGCC-3 ' (SEQ.ID.NO.:41; Just) and

5 '-GTGATGCTCTGAGTACTGGACTGG-3 ' (SEQ.ID.NO.:42; Antisense)

With Advantage cDNA polymerase (Clontech; Operate by businessman explanation) in 50 μ l reaction solutions, carry out PCR, program is: 94 ℃ 30 seconds; 94 ℃ 10 seconds; 65 ℃ 20 seconds, 72 ℃ 1.5 minutes, 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.

Isolate the PCR fragment of a 1.2kb, be cloned among the pCRII-TOPO (Invitrogen), and check order with ABI Big Dye Terminator test kit (P.E.Biosystem).See SEQ.ID.NO.:1, possible RUP8 aminoacid sequence shows in SEQ.ID.NO.:2.

b.hRUP9(Seq.Id.Nos.3&4)

The people RUP9 that announces is discerned based on the information of GenBank.Find in the search database that one is gone into to hide number cDNA clone for AC011375 from No. 5 chromosomal people's gene group sequences.This total length RUP9 carries out the PCR clone by following primer:

5 '-GAAGCTGTGAAGAGTGATGC-3 ' (SEQ.ID.NO.:43; Just) and

5 '-GTCAGCAATATTGATAAGCAGCAG-3 ' (SEQ.ID.NO.:44; Antisense),

And with human gene group DNA (Promega) as template.Use Taq Plus Precision polymerase (Stratagene) in containing the 100 μ l reaction solutions of 5%DMSO, to increase.Program is: 94 ℃ 1 minute; 94 ℃ 30 seconds; 56 ℃ 30 seconds; 72 ℃ 2 minutes; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, separate the PCR fragment of 1.3kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Terminator test kit (P.E.Biosystem).See SEQ.ID.NO.:3, possible RUP8 aminoacid sequence shows in SEQ.ID.NO.:4.Conform to the sequence that obtains from information storage from the isolating RUP9 clone's of human gene group DNA sequence.

c.hRUP10(Seq.Id.Nos.5&6)

The people RUP10 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC008754 for from No. 19 chromosomal people's gene group sequences.This total length RUP10 carries out the PCR clone by following primer:

5 '-CCATGGGGAACGATTCTGTCAGCTACG-3 ' (SEQ.ID.NO.:45; Just) and

5 '-GCTATGCCTGAAGCCAGTCTTGTG-3 ' (SEQ.ID.NO.:46; Antisense),

And be template with human leukocyte Marathon-Ready cDNA (Clontech).In 50 μ l reaction solutions, carry out PCR with Advantage cDNA polymerase (Clontech).Program is: 94 ℃ 30 seconds; 94 ℃ 10 seconds; 62 ℃ 20 seconds, 72 ℃ 1.5 minutes, 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.Isolate the PCR fragment of 1.0kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Terminator test kit (P.E.Biosystem).The nucleotide sequence of this new people's acceptor RUP10 is illustrated in SEQ.ID.NO.:5, and its possible aminoacid sequence is seen SEQ.ID.NO.:6.

d.hRUP11(Seq.Id.Nos.7&8)

The people RUP11 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC013396 for from No. 2 chromosomal people's gene group sequences.This total length RUP11 clone carries out the PCR clone by following primer:

5 '-CCAGGATGTTGTGTCACCGTGGTGGC-3 ' (SEQ.ID.NO.:47; Just) and

5 '-CACAGCGCTGCAGCCCTGCAGCTGGC-3 ' (SEQ.ID.NO.:48; Antisense),

And be template with human gene group DNA (Clontech).Use TaqPlus Precision DNA polymerase (Stratagene) in 50 μ l reaction solutions, to increase.Program is: 94 ℃ 3 minutes; 94 ℃ 20 seconds; 67 ℃ 20 seconds; 72 ℃ 1.5 minutes; 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.Isolate the PCR fragment of 1.3kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Terminator test kit (P.E.Biosystem).The nucleotide sequence of this new people's acceptor RUP11 is illustrated in SEQ.ID.NO.:7, and its possible aminoacid sequence is seen SEQ.ID.NO.:8.

e.hRUP12(Seq.Id.Nos.9&10)

The people RUP12 that announces is discerned based on the information of GenBank.Find in the search database to go into to hide for one number to be new GPCR of cDNA clones coding of AP000808, it has the remarkable homology with rat RTA and people mas1 oncogene GPCR.This total length RUP12 carries out the PCR clone by following primer:

5 '-CTTCCTCTCGTAGGGATGAACCAGAC-3 ' (SEQ.ID.NO.:49; Just) and

5 '-CTCGCACAGGTGGGAAGCACCTGTGG-3 ' (SEQ.ID.NO.:50; Antisense),

And be template with people's gene group cDNA (Clontech).Use TaqPlus PrecisionDNA polymerase (Stratagene) amplification.Program is: 94 ℃ 3 minutes; 94 ℃ 20 seconds; 65 ℃ 20 seconds; 72 ℃ 2 minutes; 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.Isolate the PCR fragment of 1.0kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and with ABI Big Dye Terminator test kit (P.E.Biosystem) check order fully (its nucleotides sequence is listed among the SEQ.ID.NO.:9 and illustrates, and its possible aminoacid sequence is seen SEQ.ID.NO.:10).

f.hRUP13(Seq.Id.Nos.11&12)

The people RUP13 that announces is discerned based on the information of GenBank.Find in the search database to go into to hide for one number to be new GPCR of cDNA clones coding of AC011780, it has the remarkable homology with GPCR fish GPRX-ORYLA.This total length RUP13 carries out the PCR clone by following primer:

5 '-GCCTGTGACAGGAGGTACCCTGG-3 ' (SEQ.ID.NO.:51; Just) and

5 '-CATATCCCTCCGAGTGTCCAGCGGC-3 ' (SEQ.ID.NO.:52; Antisense),

And be template with people's gene group cDNA (Clontech).Use TaqPlus PrecisionDNA polymerase (Stratagene) amplification.Program is: 94 ℃ 3 minutes; 94 ℃ 20 seconds; 65 ℃ 20 seconds; 72 ℃ 2 minutes; 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.Isolate the PCR fragment of 1.35kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and with ABI Big Dye Terminator test kit (P.E.Biosystem) check order fully (its nucleotide sequence is seen SEQ.ID.NO.:11, and possible aminoacid sequence is seen SEQ.ID.NO.:12).

g.hRUP14(Seq.Id.Nos.13&14)

The people RUP14 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AL137118 for from No. 13 chromosomal people's gene group sequence.This total length RUP14 carries out the PCR clone by following primer:

5 '-GCATGGAGAGAAAATTTATGTCCTTGCAACC-3 ' (SEQ.ID.NO.:53; Just) and

5 '-CAAGAACAGGTCTCATCTAAGAGCTCC-3 ' (SEQ.ID.NO.:54; Antisense),

And be template with human gene group DNA (Promega).Use TaqPlus Precision polymerase (Stratagene) and 5%DMSO amplification.Program is: 94 ℃ 3 minutes; 94 ℃ 20 seconds; 58 ℃ 2 minutes; 72 ℃ 10 minutes, go on foot the 3rd step circulation 35 times from second.

Isolate the PCR fragment of 1.1kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Terminator test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:13, and possible aminoacid sequence is seen SEQ.ID.NO.:14.) isolating RUP14 cloned sequence conforms to the sequence that obtains from information storage from the human gene group DNA.

h.hRUP15(Seq.Id.Nos.15&16)

The people RUP15 that announces is discerned based on the information of GenBank.Find in the search database that a cDNA clone who goes into to hide number for AC016468 is people's genome sequence.This total length RUP15 carries out the PCR clone by following primer:

5 '-GCTGTTGCCATGACGTCCACCTGCAC-3 ' (SEQ.ID.NO.:55; Just) and

5 '-GGACAGTTCAAGGTTTGCCTTAGAAC-3 ' (SEQ.ID.NO.:56; Antisense),

And be template with human gene group DNA (Promega).Use TaqPlus Precision polymerase (Stratagene) amplification.Program is: 94 ℃ 3 minutes; 94 ℃ 20 seconds; 65 ℃ 20 seconds; 72 ℃ 2 minutes; 72 ℃ 7 minutes, go on foot the 4th step circulation 35 times from second.

Isolate the PCR fragment of 1.5kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Terminator test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:15, and possible aminoacid sequence is seen SEQ.ID.NO.:16.) isolating RUP15 cloned sequence conforms to the sequence that obtains from information storage from the human gene group DNA.

i.hRUP16(Seq.Id.Nos.17&18)

The people RUP16 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AL136106 for from No. 13 chromosomal people's gene group sequence.This total length RUP16 carries out the PCR clone by following primer:

5 '-CTTTCGATACTGCTCCTATGCTC-3 ' (SEQ.ID.NO.:57; Justice, 5 ' end of atg start codon) and

5 '-GTAGTCCACTGAAAGTCCAGTGATCC-3 ' (SEQ.ID.NO.:58; Antisense, 3 ' end of terminator codon),

And be template with people's skeletal muscle Marathon-Ready cDNA (Clontech).In 50 μ l reaction solutions, carry out PCR (Clontech) with Advantage cDNA polymerase test kit.Program is: 94 ℃ 30 seconds; 94 ℃ 5 seconds; 69 ℃ 15 seconds; 72 ℃ 1 minute; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

Isolate the PCR fragment of 1.1kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with T7 Sequenase test kit (Amsham).(its nucleotide sequence is seen SEQ.ID.NO.:17, and possible aminoacid sequence is seen SEQ.ID.NO.:18).The RUP16 cloned sequence conforms to four non-order sections of AL136106, shows that the cDNA of RUP16 contains 4 exons.

j.hRUP17(Seq.Id.Nos.19&20)

The people RUP17 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC023078 for from the chromosomal people's gene group of o.11 sequence.This total length RUP17 carries out the PCR clone by following primer:

5 '-TTTCTGAGC ATGGATCCAACCATCTC-3 ' (SEQ.ID.NO.:59; Justice contains atg start codon) and

5 '-CTGTCTGACAGGGCAGAGGCTCTTC-3 ' (SEQ.ID.NO.:60; Antisense strand, 3 ' end of terminator codon),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, carry out PCR (Clontech) with Advantage cDNA polymerase mixture and 5%DMSO.Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 67 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds again; 72 ℃ 5 minutes, go on foot the 4th step circulation 30 times from second.

From 1% sepharose, isolate the PCR fragment of 970bp, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:19, and possible aminoacid sequence is seen SEQ.ID.NO.:20).

k.hRUP18(Seq.Id.Nos.21&22)

The people RUP18 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC008547 for from No. 5 chromosomal people's gene group sequence.This total length RUP18 carries out the PCR clone by following primer:

5 '-GGAACTCGTATAGACCCAGCGTCGCTCC-3 ' (SEQ.ID.NO.:61; Justice, 5 ' end of atg start codon) and

5 '-GGAGGTTGCGCCTTAGCGACAGATGACC-3 ' (SEQ.ID.NO.:62; Antisense, 3 ' end of terminator codon),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, carry out PCR (Clontech) with 5%DMSO with the accurate DNA polymerase of TaqPlus (Stratagene).Program is: 95 ℃ 5 minutes; 95 ℃ 30 seconds; 65 ℃ 30 seconds; 72 ℃ 2 minutes; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.3kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:21, and possible aminoacid sequence is seen SEQ.ID.NO.:22).

1.hRUP19(Seq.Id.Nos.23&24)

The people RUP19 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC026331 for from No. 12 chromosomal people's gene group sequence.This total length RUP19 carries out the PCR clone by following primer:

5 '-CTGCACCCGGACACTTGCTCTG-3 ' (SEQ.ID.NO.:63; Justice, 5 ' end of atg start codon) and

5 '-GTCTGCTTGT TCAGTGCCACTCAAC-3 ' (SEQ.ID.NO.:64; Antisense contains terminator codon),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, increase with 5%DMSO with TaqPlus Precision DNA polymerase (Stratagene).Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 70 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.1kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Termiantor test kit (P.E.Biosystem).Its nucleotide sequence is seen SEQ.ID.NO.:23, and possible aminoacid sequence is seen SEQ.ID.NO.:24).

m.hRUP20(Seq.Id.Nos.25&26)

The people RUP20 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AL161458 for from No. 1 chromosomal people's gene group sequence.This total length RUP20 carries out PCR by following primer:

5 '-TATCTGCAATTCTATTCTAGCTCCTG-3 ' (SEQ.ID.NO.:65; Justice, atg start codon 5 ' end) and

5 '-TGTCCCTAATAAAGTCACATGAATGC-3 ' (SEQ.ID.NO.:66; Antisense, terminator codon 3 ' end),

And be template with human gene group DNA (Promega).Increase with 5%DMSO with Advantage cDNA polymerase mixture (Clonetech).Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 60 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.0kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:25, and possible aminoacid sequence is seen SEQ.ID.NO.:26).

n.hRUP21(Seq.Id.Nos.27&28)

The people RUP21 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC026756 for from No. 13 chromosomal people's gene group sequence.This total length RUP21 carries out the PCR clone by following primer:

5 '-GGAGACAACCATGAATGAGCCAC-3 ' (SEQ.ID.NO.:67; Just) and

5 '-TATTTCAAGGGTTGTTTGAGTAAC-3 ' (SEQ.ID.NO.:68; Antisense strand),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, increase with 5%DMSO with TaqPlus Precision polymerase (Stratagene).Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 55 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 30 times from second.

From 1% sepharose, isolate the PCR fragment of 1014bp, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.:27, and possible aminoacid sequence is seen SEQ.ID.NO.:28).

o.hRUP22(Seq.Id.Nos.29&30)

The people RUP22 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC027026 for from the chromosomal people's gene group of o.11 sequence.This total length RUP22 carries out the PCR clone by following primer:

5 '-GGCACCAGTGGAGGTTTTCTGAGC ATG-3 ' (SEQ.ID.NO.:69; Justice contains atg start codon) and

5 '-CTGATGGAAGTAGAGGCTGTCCATCTC-3 ' (SEQ.ID.NO.:70; Antisense, terminator codon 3 ' end),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, increase with 5%DMSO with TaqPlus Precision DNA polymerase (Stratagene).Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 55 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 30 times from second.

From 1% sepharose, isolate the PCR fragment of 970bp, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.29, and possible aminoacid sequence is seen SEQ.ID.NO.30).

p.hRUP23(Seq.Id.Nos.31&32)

The people RUP23 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC007104 for from No. 4 chromosomal people's gene group sequence.This total length RUP23 carries out PCR by following primer:

5 '-CCTGGCGAGCCGCTAGCGCC ATG-3 ' (SEQ.ID.NO.71; Justice, ATG is an atg start codon) and

5 '-ATGAGCCCTGCCAGGCCC TCAGT-3 ' (SEQ.ID.NO.72; Antisense, TCA are terminator codon),

And be template with people's placenta Marathon-Ready cDNA (Clontech).Increase in 50 μ l reaction solutions with Advantage cDNA polymerase (Clontech).Program is: 95 ℃ 30 seconds; 95 ℃ 15 seconds; 66 ℃ 20 seconds; 72 ℃ 1 minute 20 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.0kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Terminator test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.31, and possible aminoacid sequence is seen SEQ.ID.NO.32).

q.HRUP24(Seq.Id.Nos.33&34)

The people RUP25 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC026331 for from No. 12 chromosomal people's gene group sequence.This total length RUP25 carries out the PCR clone by following primer:

5 '-GCTGGAGCATTCACTAGGCGAG-3 ' (SEQ.ID.NO.73; Justice, atg start codon 5 ' end) and

5 '-AGATCCTGGTTCTTGGTGACAATG-3 ' (SEQ.ID.NO.74; Antisense, terminator codon 3 ' end),

And be template with human gene group DNA (Promega).In 100 μ l reaction solutions, carry out PCR with 5%DMSO with Advantage cDNA polymerase mixture (Clontech).Program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 56 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.2kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Terminator test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.33, and possible aminoacid sequence is seen SEQ.ID.NO.34).

r.hRUP25(Seq.Id.Nos.35&36)

5 '-GCTGGAGCATTCACTAGGCGAG-3 ' (SEQ.ID.NO.75; Justice, atg start codon 5 ' end) and

5 '-AGATCCTGGTTCTTGGTGACAATG-3 ' (SEQ.ID.NO.76; Antisense, terminator codon 3 ' end),

And be template with human gene group DNA (Promega).Increase with Advantage cDNA polymerase mixture (Clontech) and 5%DMSO, program is: 94 ℃ 1 minute; 94 ℃ 15 seconds; 56 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.2kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Terminator test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.35, and possible aminoacid sequence is seen SEQ.ID.NO.36).

s.hRUP 26(Seq.Id.Nos.37&38)

The people RUP26 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC023040 for from No. 2 chromosomal people's gene group sequence.This total length RUP26 carries out the RT-PCR clone by the RUP26 Auele Specific Primer:

5 '-AGCCATCCCTGCCAGGAAGCA TGG-3 ' (SEQ.ID.NO.77; Justice contains atg start codon) and

5 '-CCAGACTGTGGACTCAAGAACT CTAGG-3 ' (SEQ.ID.NO.78; Antisense contains terminator codon),

And be template with people's pancreas Marathon-Ready cDNA (Clontech).In 100 μ l reaction solutions, carry out PCR (Clontech) with 5%DMSO with Advantage cDNA polymerase mixture (Clontech).Program is: 94 ℃ 5 minutes; 95 ℃ 30 seconds; 65 ℃ 30 seconds; 72 ℃ 2 minutes; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.1kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.37, and possible aminoacid sequence is seen SEQ.ID.NO.38).

t.hRUP27(Seq.Id.Nos.39&40)

The people RUP27 that announces is discerned based on the information of GenBank.Find in the search database one go into to hide number for the cDNA clone of AC027643 for from No. 12 chromosomal people's gene group sequence.This total length RUP27 carries out the PCR clone with the RUP27 Auele Specific Primer:

5 '-AGTCCACGAACA ATGAATCCATTTCATG-3 ' (SEQ.ID.NO.79; Justice contains atg start codon) and

5 '-ATCATGTCTAGACTCATGGTGATCC-3 ' (SEQ.ID.NO.80; Antisense, terminator codon 3 ' end),

And be template with Adult Human Brain Marathon-Ready cDNA (Clontech).In 50 μ l reaction solutions, carry out PCR with 5%DMSO with Advantage cDNA polymerase mixture (Clontech).Program is: 94 ℃ 1 minute; 94 ℃ 10 seconds; 58 ℃ 20 seconds; 72 ℃ 1 minute 30 seconds; 72 ℃ 5 minutes, go on foot the 4th step circulation 35 times from second.

From 1% sepharose, isolate the PCR fragment of 1.1kb, be cloned in the pCRII-TOPO carrier (Invitrogen), and check order fully with ABI Big Dye Termiantor test kit (P.E.Biosystem).(its nucleotide sequence is seen SEQ.ID.NO.35, and possible aminoacid sequence is seen SEQ.ID.NO.36).The cDNA cloned sequence of isolating RUP27 is determined with five non-order sections of AC027643 and conforms to from human brain, shows that the cDNA of RUP27 contains 5 exons.

Embodiment 2

Prepare non-endogenous composing type activatory GPCR

Believe that those of ordinary skill in the art has the ability to select to be used for the technology of nucleotide sequence sudden change, what provide below is exactly the method that is used to make the non-endogenous form of above disclosed several people GPCR.Following public sudden change is based on the algorithmic rule method, the 16th amino acids (being positioned at the IC3 zone of GPCR) (is positioned at the TM6 zone of GPCR by conservative proline(Pro) (or its endogenous conservative surrogate) residue thus, near the TM6/IC3 intersection) undergo mutation, more preferably sport L-Ala, Histidine, arginine or lysine residue, most preferably sport lysine residue.

1.Transformer Sited-Directed ^TMMutagenesis

Utilize Transformer Sited-Directed ^TMMutagenesis kit is by businessman's explanation, and preparing non-endogenous people GPCR by people GPCR can be achieved.Use two mutagenic primers, at first preferably produce the Methionin mutagenic oligonucleotide of lysine mutation, and a selective marker oligonucleotide.Reason for convenience, the codon mutation that mixes among the people GPCR marks (table D) with standard manner:

Table D

The acceptor title	Codon mutation
The acceptor title	Codon mutation	hRUP8	V274K
hRUP9	T249K	hRUP8	V274K
hRUP9	T249K	hRUP10	R232K
hRUP11	M294K	hRUP10	R232K
hRUP11	M294K	hRUP12	F220K
hRUP16	A238K	hRUP12	F220K
hRUP16	A238K	hRUP17	Y215K
hRUP18	L294K	hRUP17	Y215K
hRUP18	L294K	hRUP19	T219K
hRUP20	K248A K248H K248R	hRUP19	T219K
hRUP20	K248A K248H K248R	hRUP21	R240K
hRUP22	Y222K	hRUP21	R240K
hRUP22	Y222K	hRUP24	A245K
hRUP25	I230K	hRUP24	A245K
hRUP25	I230K	hRUP26	V285K
hRUP27	T248K	hRUP26	V285K

2.QuikChange ^TMSite-Directed ^TMMutagenesis

Utilize QuikChange ^TMSited-Directed ^TMMutagenesis kit (Stratagene is by businessman's explanation), preparing non-endogenous people GPCR can be achieved.Be template preferably and use two mutagenic primers, equally preferably a Methionin mutagenic oligonucleotide and a selective marker oligonucleotide (carrying in the test kit) with endogenous GPCR.Reason for convenience, the codon mutation and the discrete oligonucleotide that mix among the new people GPCR mark (table E) with standard manner:

Table E

The acceptor title	Codon mutation	5 '-3 ' direction (justice is arranged), (SEQ.ID.NO.) mutant nucleotide sequence underscore	5 '-3 ' direction (antisense) (SEQ.ID.NO.)	Cycling condition, (divide ' second ") from 2-4 step circulation 16 times
The acceptor title	Codon mutation		5 '-3 ' direction (antisense) (SEQ.ID.NO.)		hRUP 13	A268 K	GGGGAGGGAAAGCA AAGGTGGTCCTCCTG G(81)	CCAGGAGAACCAC C TTTGCTTTCCCTCCC C(82)	98°2’ 98°30” 56℃ 30” 72°11’40” 72°5’
hRUP 14	L246 K	CAGGAAGGCA AAGA CCACCATCATCATC(8 5)	GATGATGATGGTGG T CTTTGCCTTCCTG(8 6)	98°2’ 98°30” 55℃ 30” 72°11’40” 72°5’	hRUP 13	A268 K	GGGGAGGGAAAGCA AAGGTGGTCCTCCTG G(81)	CCAGGAGAACCAC C TTTGCTTTCCCTCCC C(82)	98°2’ 98°30” 56℃ 30” 72°11’40” 72°5’
hRUP 14	L246 K	CAGGAAGGCA AAGA CCACCATCATCATC(8 5)	GATGATGATGGTGG T CTTTGCCTTCCTG(8 6)	98°2’ 98°30” 55℃ 30” 72°11’40” 72°5’	hRUP 15	A398 K	CCAGTGCAAAGCT AA GAAAGTGATCTTC(89 )	GAAGATCACTTT CT TAGCTTTGCACTGG( 90)	98°2’ 98°30” 55℃ 30” 72°11’40” 72°5’
hRUP 23	W275 K	GCCGCCACCGCGCC A AGAGGAAGATTGGC( 93)	GCCAATCTTCCT CTTGGCGCGGTGGCGGC (94)	98°2’ 98°30” 56℃ 30” 72°11’40” 72°5’	hRUP 15	A398 K	CCAGTGCAAAGCT AA GAAAGTGATCTTC(89 )	GAAGATCACTTT CT TAGCTTTGCACTGG( 90)	98°2’ 98°30” 55℃ 30” 72°11’40” 72°5’

To non-endogenous people GPCR order-checking, the nucleic acid and the aminoacid sequence that obtain and confirm are listed in the present patent application file appended " sequence table " then, and with following table F as summary.

Table F

Non-endogenous people GPCR	The nucleotide sequence table	The aminoacid sequence table
Non-endogenous people GPCR	The nucleotide sequence table	The aminoacid sequence table	hRUP13	SEQ.ID.NO.：83	SEQ.ID.NO.：84
hRUP14	SEQ.ID.NO.：87	SEQ.ID.NO.：88	hRUP13	SEQ.ID.NO.：83	SEQ.ID.NO.：84
hRUP14	SEQ.ID.NO.：87	SEQ.ID.NO.：88	hRUP15	SEQ.ID.NO.：91	SEQ.ID.NO.：92
hRUP23	SEQ.ID.NO.：95	SEQ.ID.NO.：96	hRUP15	SEQ.ID.NO.：91	SEQ.ID.NO.：92

Embodiment 3

Expression of receptor

Although there is various kinds of cell to can be used for protein expression in the art, what most preferably use is mammalian cell.It is predicted, its fundamental cause is a practicality, promptly for example express the application of the yeast cell of GPCR, might be incorporated into a kind of nonmammalian cell in the program, this cell may be not (in fact, for yeast, be not) comprise coupled receptor, genetic mechanism and Secretory Pathway, and these are used for mammlian system through evolving.Therefore, the result who in the nonmammalian cell, obtains, although it is come in handy, also preferred not as the result who from mammalian cell, obtains.In mammalian cell, COS-7,293 and the 293T cell be particularly preferred, although the specific mammalian cell of using can be determined by technician's special needs.

A. transient transfection

First day, with 6 * 10 ⁶Individual 293 cell inoculations are to the culture plate of 10cm.Second day, prepare two test tubes (ratio is that every plate is used for a test tube): prepare test tube A at the DMEM of 0.5ml serum-free (Gibco BRL) by mixing 4 μ g DNA (for example pCMV carrier, have the pCMV carrier of receptor cdna etc.); In 0.5ml serum-free DMEM, prepare test tube B by mixing 24 μ l lipofectamine (Gibco BRL).Test tube A and the B mixing (several times) of inclining mutually, at room temperature incubation 30-45 minute then.Composition is called as " transfection group compound ".Plant the 293T cell that and wash, add the DMEM of 5ml serum-free then with 1XPBS.1ml transfection group compound joined in the cell go, then at 37 ℃/5%CO ₂Following incubation 4 hours.Then remove the transfection group compound, add the DMEM/10% foetal calf serum of 10ml then by suction.Then cell is at 37 ℃/5%CO ₂Incubation.Harvested cell and be used for analyzing after 48 hours.

B. stable clone: Gs fusion rotein

With about 12 * 10 ⁶Individual 293 cell inoculations are on the tissue culture plate of 15cm.Containing 10% foetal calf serum and 1% Sodium.alpha.-ketopropionate, L-glutaminate is grown in the high dextrose culture-medium of antibiotic DME.Behind the 293 cell bed boards 24 hours its converge level and reach～80%, cell carries out transfection with 12 μ gDNA.This 12 μ gDNA mixes with the high dextrose culture-medium of DME of 60 μ l lipofectamine and 2ml serum-free.From culture plate, inhale and remove substratum, once with serum free medium rinse cell.DNA, the mixture of lipofectamine and substratum and 10ml serum free medium together add flat board.Cultivate after 4-5 hour for 37 ℃, inhale and remove substratum, add the substratum that 25ml contains serum.After the transfection 24 hours, inhale again and remove substratum, add the fresh blood serum medium that contains.After the transfection 48 hours, inhale and remove substratum, add the substratum that contains Geneticin (G418 medicine, final concentration are 500 μ g/ml).At this moment the positive transfectional cell that contains the G418 resistant gene is selected.Per 4 to 5 hours replacement one subcultures during selection.In the chosen process, the cell growth also produces stable groups of cells, or stable clonal selection is carried out in cell fission.

Embodiment 4

Determine the analysis of non-endogenous GPCR constitutive activity

Several different methods can be used to assess the constitutive activity of non-endogenous people GPCR, and following is illustrative for example; Those of ordinary skill in the art has the ability to determine those to their the most useful required technology.

1. cytolemma binding analysis: [ ³⁵S] GTP γ S analysis

When g protein coupled receptor in its active condition, and as part in conjunction with or as the composing type activatory as a result the time, acceptor and G albumen coupling and stimulate the release of GDP and thereafter GTP combine with G is proteic.The α subunit of G albumen-receptor complex as the GTP enzyme and at leisure hydrolysis GTP be GDP, inactivation takes place usually at this acceptor.The composing type activated receptor continues GDP is converted into GTP.The GTP analogue of non-hydrolysable [ ³⁵S] GTP γ S, can be used to show [ ³⁵S] GTP γ S combines with the enhanced of the film of expressing the composing type activated receptor.Use [ ³⁵S] GTP γ S in conjunction with measuring composing type activatory advantage is: (a) it is blanket to all g protein coupled receptors; (b) its adjacent cells film surface, less herein may choosing runs into the molecule that influences the reaction of intracellular level connection.

This test utilize g protein coupled receptor stimulation [ ³⁵S] GTP γ S and the cytolemma bonded ability of expressing associated receptor.Therefore this mensuration can be used for the Direct Recognition method and removes to screen candidate compound at known, orphan and composing type activation g protein coupled receptor.This mensuration is general and can be used for drug discovery at all g protein coupled receptors.

[ ³⁵S] GTP γ S test: at 20mM HEPES, 1 to about 20mM MgCl ₂(although 20mM be preferred, this dosage can be adjusted at result's optimization), pH7.4, contain 0.3 and 1.2nM between [ ³⁵S] GTP γ S (although 1.2 be preferred, this dosage can be adjusted at result's optimum), 12.5 to 75 μ g membranins (for example, express 293 cells of Gs fusion rotein; This dosage can be optimization adjustment) and the binding buffer liquid of 10 μ M GDP (this dosage can be transformed at result's optimization) in incubation one hour.Then add the wheat germ agglutinin globule (25 μ l, Amersham), composition is incubation 30 minutes more at room temperature, test tube is under 1500 * g, room temperature centrifugal 5 minutes then, and counts on scintillometer.

2. adenylate cyclase

Design is used for carrying out the Flash Plate based on the mensuration of cell ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Catalog number (Cat.No.) SMP004A) is modified to be applied to unprocessed plasma membrane.Scintillator bag tegillum is contained in the hole of flicker plate, wherein contains the specific antibody of discerning cAMP.The cAMP that produces in the hole can pass through directly and radioactivity cAMP tracer is competed with the cAMP antibodies and by quantitative.Be the Short Description that cAMP level in the whole cell of measuring expressed receptor is changed program below.

About 24 hours results cells transfected behind transient transfection.Careful suction nutrient solution also discards.Light and slow adding 10ml PBS in each Tissue Culture Dish, suction carefully then.Every plate adds 1ml Sigma cell dissociation damping fluid and 3ml PBS.With cell sucking-off from flat board, the cell suspension of results is added 50 milliliters tapered centrifuge tube, centrifugal 5 minutes then at room temperature 1100rpm.With the PBS of appropriate amount re-suspended cell precipitation (the about 3ml of every plate) carefully.Cell is counted with hematimeter, and adjusts to suitable cell quantity (final volume is about every hole 50 μ l) with extra PBS.

According to the instruction of manufacturers preparation and keep the cAMP standard substance and detect damping fluid (contain 1 μ Ci tracer [ ¹²⁵I cAMP (50 μ l)] 11ml detect damping fluid).For the analysis buffer of screening usefulness by prepared fresh, it contains 50 μ l stimulates damping fluid, the cell of the experimental compound of 3 μ l (final concentration is 12mM) and 50 μ l; Analysis buffer can be stored standby on ice.At first in suitable hole, add 50 μ l the cAMP standard substance, then add 50 μ l PBSA to hole H-11 and H-12.Adding 50 μ l in all holes stimulates damping fluid, and use can disperse the fall needle tool of liquid of 3 μ l compounds that DMSO (or selected candidate compound) is added in the suitable hole, and the final concentration of experimental compound is 12mM, and the experiment cumulative volume is 100 μ l.Then cell is added in the hand-hole, incubated at room temperature 60 minutes adds the detection mixture that 100 μ l contain spike cAMP then.Culture plate is followed incubation 2 hours again, then with the numeration of Wallac MicroBeta liquid flashing counting device.The numerical value in cAMP/ hole is from standard cAMP curve extrapolation, and this curve is included within each assay plate.

3.Gi the cAMP based on cell of coupling target GPCR analyzes

TSHR is a kind of Gs link coupled GPCR, causes the accumulation of cAMP when activation.Amino-acid residue 623 sudden changes (that is, alanine residue being become the Isoleucine residue) can be made the activation of TSHR composing type.Expection Gi coupled receptor suppresses adenylate cyclase, and therefore reduces the level of cAMP, thereby makes the assessment of cAMP level difficult.Detection is as the reduction of the cAMP output of composing type activation Gi coupled receptor indication, a kind of effective means realizes by cotransfection, preferred, be by being " signal enhanser " with non-endogenous composing type activation TSHR (TSHR-A623I) (or an endogenous composing type activatory Gs coupled receptor), with Gi link coupled target GPCR cotransfection, establish the datum-plane of cAMP.After producing non-endogenous Gi coupled receptor, non-endogenous target GPCR and signal enhanser cotransfection, and be used as the material of screening.When using cAMP to analyze, we will utilize this method to produce signal effectively; This method is preferably used for the Direct Recognition at the candidate compound of Gi coupled receptor.Be noted that for Gi coupling GPCR when using this method, the inverse agonist of this target GPCR can increase the cAMP signal and agonist can reduce the cAMP signal.

First day, 293 and 293 cells were with every hole 2 * 10 ⁴The density of individual cell is planted on plate.Second day, prepare two test tubes (ratio is that every plate is used for a test tube): by mixing altogether each receptor dna 2 μ g of 4 μ gDNA (pCMV carrier for example; The pCMV carrier (TSHR-A623I) that has mutation T HSR; TSHR-A623 and GPCR etc.) (Irvine Scientific, Irvine prepare test tube A in CA) at the DMEM of 1.2ml serum-free; In 1.2ml serum-free DMEM, prepare test tube B by mixing 120 μ llipofectamine (Gibco BRL).Test tube A and the B mixing (several times) of inclining mutually, at room temperature incubation 30-45 minute then.Composition is called as " transfection group compound ".Plant 293 cells that and wash, add the DMEM of 10ml serum-free then with 1XPBS.2.4ml transfection group compound joined in the cell go, then at 37 ℃/5%CO ₂Following incubation 4 hours.Then remove the transfection group compound, add the DMEM/10% foetal calf serum of 25ml then by suction.Then cell is at 37 ℃/5%CO ₂Incubation.Harvested cell and be used for analyzing after 24 hours.

Design is used for the Flash Plate that measures based on cell ^TMAdenylate cyclase enzyme reagent kit (New England Nuclear; Catalog number (Cat.No.) SMP004A), can be modified to be applied to unprocessed plasma membrane according to those skilled in the art's needs.Scintillator bag tegillum is contained in the hole of flicker plate, wherein contains the specific antibody of discerning cAMP.The cAMP that produces in the hole passes through directly and radioactivity cAMP tracer is competed with the cAMP antibodies and by quantitative.Be to measuring the Short Description of cAMP level variation program in the full cell of expressed receptor below.

Behind transient transfection about 24 hours, the results cells transfected.Careful suction nutrient solution also discards.Light and slow adding 10ml PBS in each Tissue Culture Dish.Every plate adds 1ml Sigma cell dissociation damping fluid and 3ml PBS.With cell sucking-off from flat board, the cell suspension of results is added 50 milliliters tapered centrifuge tube, centrifugal 5 minutes then at room temperature 1100rpm.With the PBS of appropriate amount re-suspended cell precipitation (the about 3ml of every plate) carefully.Count with hematimeter, and adjust to suitable cell quantity (final volume is about every hole 50 μ l) with extra PBS.

According to the instruction of manufacturers preparation and keep the cAMP standard substance and detect damping fluid (contain 1 μ Ci tracer [ ¹²⁵I cAMP (50 μ l)] 11ml detect damping fluid).For the analysis buffer of screening usefulness by prepared fresh, it contains 50 μ l stimulates damping fluid, the cell of the experimental compound of 3 μ l (final concentration is 12mM) and 50 μ l; Analysis buffer can be stored standby on ice.At first in suitable hole, add 50 μ l the cAMP standard substance, then add 50 μ l PBSA to hole H-11 and H-12.Adding 50 μ l in all holes stimulates damping fluid, and use can disperse the needle set of 3 μ l mixed solutions that selected compound (as TSH) is added in the suitable hole, and the final concentration of experimental compound is 12 μ M, and the experiment cumulative volume is 100 μ l.Then cell is added in the hand-hole, incubated at room temperature 60 minutes adds the detection mixture that 100 μ l contain spike cAMP then.Culture plate is followed incubation 2 hours again, then with the numeration of Wallac MicroBeta liquid flashing counting device.The numerical value in cAMP/ hole is from standard cAMP curve extrapolation, and this curve is included within each assay plate.

4. based on the mensuration of reporter gene

A.CRE-Luc reporter-gene assays (acceptor that Gs is relevant)

293 and the 293T cell with every hole 2 * 10 ⁴The density of individual cell is planted on 96 orifice plates, carried out transfection by businessman explanation with Lipofectamine reagent (BRL) in second day, it is as follows that per 6 hole transfections prepare the DNA/ lipid mixt: the 260ng plasmid DNA among the 100 μ l DMEM leniently with 100 μ l DMEM in 2 μ l lipids mixed (the 260ng plasmid DNA is made up of following, 200ng8 * CRE-Luc reporter plasmid, 50ng comprises the pCMV of endogenous recipient or non-endogenous recipient, or independent pCMV, and 10ng GPRS expression plasmid (GPRS is in pcDNA3 (Invitrogen))).Being prepared as follows of 8 * CRE-Luc reporter plasmid: (71/+51) in the promotor of the BglV-HindIII site of p β gal-underlying carrier (Clontech) clone mouse somatostatin, obtain SRIF-β-gal carrier, the cAMP response elements of 8 copies by PCR by adenovirus template AdpCF126CCRE8 obtain (referring to, 7 people's gene therapies (Human GeneTherapy) 1883 (1996)), it is cloned in the Kpn-BglV site of SRIF-β-gal carrier, produce 8 * CRE-β-gal report carrier, with the beta-galactosidase gene in luciferase gene replacement 8 * CRE-β-gal report carrier, produce 8 * CRE-Luc reporter plasmid, described luciferase gene is taken from the HindIII-BamHI site of pGL3-underlying carrier (Promega).Place after 30 minutes under the room temperature, the DNA/ lipid mixt dilutes with 400 μ l DMEM, and every hole adds the mixture of 100 μ l dilution, cultivates to add the DMEM that 100 μ l contain 10%FCS after 4 hours in every hole in cell culture incubator.Second day, the every hole of cells transfected changed the DMEM that 200 μ l contain 10%FCS into, after 8 hours, cleaned once with PBS, and each sample well changes 100 μ l into and do not contain phenol red DMEM.Next day is by the explanation LucLite of businessman ^TMReporter gene assay kit (Packard) is measured uciferase activity and at 1450 MicroBeta ^TMFlicker luminescent counter (Wallac) is gone up reading.

The b.AP1 reporter gene is analyzed (Gq associated receptor)

Measuring Gq stimulates the method that relies on to depend on the known characteristic of Phospholipase C that Gq relies on, and promptly it can cause the gene activation that contains the AP1 element in its promotor.According to the said program of above-mentioned CREB reporter-gene assays, use Pathdetect ^TM(Stratagene Catalogue#219073), wherein just changes the component of calcium phosphate precipitation into 410ng pAP1-Luc, 80ng pCMV-expression of receptor plasmid and 20ng CMV-SEAP to AP-1 cis-Reporting System.

The c.SRF-Luc reporter gene is analyzed (Gq associated receptor)

The method that detecting Gq stimulates relies on the known features of Gq dependency Phospholipase C, causes the gene activation that comprises serum response factor in the promotor.Pathdetect ^TMSRF-Luc reporting system (Stratagene) can be used to analyze Gq link coupled activity, such as analyze Gq link coupled activity in the COS7 cell.Use Mammalian Transfection ^TMTest kit (Stratagene, Catalogue#200285), with various plasmids in this system and the coding indicated is endogenous or the expression plasmid of non-endogenous GPCR, by businessman's explanation transfectional cell.Say 410ng SRF-Luc, 80ng pCMV expression of receptor plasmid and the 20ng CMV-SEAP (expression plasmid of secreting alkaline phosphorus phytase simply; Measure the difference of transfectional cell substratum neutral and alkali phosphatase activity) with control sample room transfection efficiency, explanation and calcium phosphate precipitation according to each businessman are combined, the throw out of half is assigned in 3 holes of 96 orifice plates equally, and cell was kept in serum free medium 24 hours.On request, in the end 5 hour cells are cultivated with 1 μ M Angiotensin, and dissolved cell uses LucLite then ^TM(Packard is cat#6016911) with " Trilax1450 Microbeta " liquid scintillation luminescent counter (Wallac), by the plain enzymic activity of the explanation analysis of fluorescence of each businessman, data GraphPad Prism for test kit ^TM2.0a software analysis (GraphPadSoftware Inc.).

D. IP in the born of the same parents ₃(Gq associated receptor) analyzed in accumulation

At first day, the cell that contains this receptor (endogenous and/or non-endogenous) was inoculated on 24 well culture plates, generally is 1 * 10 ⁵Cells/well (though this number also can be optimized).At second day transfectional cell, at first be blended in 0.25 μ g DNA and 2 μ l lipofectamine in 50 μ l/ hole serum-free DMEM among the 50 μ l/ hole serum-free DMEM.Mixing solutions is also at room temperature incubation 15-30 minute lightly.With 0.5ml PBS washed cell, 400 μ l serum free mediums are mixed with transfection media and be added in the cell.Then at 37 ℃/5%CO ₂Following incubation cell 3-4 hour is removed transfection media again, replaces with the conventional substratum in 1ml/ hole.At the 3rd day, use ³H-inositol labeled cell.Speak briefly, remove substratum, cell washs with 0.5ml PBS, and then adding the 0.5ml/ hole does not have inositol/serum free medium (GIBCO BRL) and 0.25 μ Ci/ hole ³The H-inositol is at 37 ℃/5%CO ₂Following incubation cell 16-18 hour.At the 4th day, with 0.5ml PBS washed cell, add the 0.45ml test medium, wherein contain and have or not the inositol/serum free medium 10 μ M 10mM of pargyline lithium chlorides or 0.4ml test medium and 50 μ l, 10 * ketaserin (ket) to obtain the final concentration of 10 μ M.Then in 37 ℃ of incubation cells 30 minutes.With 0.5ml PBS washed cell, add 200 μ l/ holes fresh/ice-cold stop buffer (1M KOH, 18mM Sodium Tetraborate, 3.8mM EDTA).Solution was placed on ice 5-10 minute or dissolved up to cell, use then 200 μ l fresh/neutralization of ice-cold neutralizer (7.5%HCl).Then lysate is transferred in the 1.5ml centrifuge tube, added 1ml/ pipe chloroform/methanol (1: 2).Make 15 seconds of solution vortex then, sample on the upper strata to Biorad AG1-X8 ^TMAnionite-exchange resin (100-200 order).At first, resin washes with water with the ratio of 1: 1.25 W/V, and Xiang Zhuzhong loads the upper solution of 0.9ml.Sodium Tetraborate/60mM sodium formiate washing pillar with 10ml 5mM inositol and 10ml 5mM.Triphosphoinositol is gone into liquid by elution and is dodged in the pipe, wherein contains 10ml liquid and dodges the chicken tail, and it has 2ml0.1M formic acid/1M ammonium formiate.By washing and use ddH with 10ml 0.1M formic acid/3M ammonium formiate ₂The O washed twice exchange column of regenerating, pillar are stored in 4 ℃ the water.

Table G below typical results is listed in:

Table G

N/A=does not use

Shown in above embodiment 4 (1), the typical consequence of the moulding activated analysis of GTP γ S test set is the Gs that utilizes on people RUP13 and the people RUP15: fusion protein construct is achieved.Listed the difference between signal that this analysis produced and signal among the following table H:

Table H

Embodiment 5

The preparation of fusion rotein

The a.GPCR:Gs fusion constructs

Following the finishing of design of composing type activatory GPCR-G albumen fusion constructs: build rat G egg Gs α (long formula; Ltoch, H. etc., 83 PNAS 3776 (1986)) 5 ' and 3 ' end, make it to comprise HindIII (5 '-AAGCTT-3 ') sequence.Correct sequence (flanking sequence that comprises HindIII) is carried out subclone by the HindIII restriction site that utilizes this carrier after confirming, with complete sequence insert pcDNA3.1 (-) (Invitrogen, cat.no.V795-20).Subclone enters pcDNA3.1 (-) afterwards, confirms the correct direction of Gs α sequence, confirms to have on the HindIII sequence this modified pcDNA3.1 (-) of rat Gs α gene then, and this carrier can be used as " general " Gs α protein carrier now.A series of known restriction sites are contained in the upstream that pcDNA3.1 (-) is loaded in the HindIII site, can advantageously insert the encoding sequence of endogenous composing type activation GPCR like this in Gs albumen upstream.Can use the same method and create other " general " G protein carrier, certainly, can to use other carrier commercially available or well known by persons skilled in the art----important criterion are upstreams of the GPCR sequence G of place protein sequence and meet frame with the G protein sequence.

RUP13 for following typical gpcr fusion protein, has realized the fusion with Gs α through the Gs coupling.

Being prepared as follows of RUP13-Gs alpha fusion protein construct:

Design of primers is as follows:

5 '-gatc[TCTAGAAT] GGAGTCCTCACCCATCCCCCAG-3 ' (SEQ.ID.NO.:97; Justice is arranged)

5 '-gcta[GATATC] CGTGACTCCAGCCGGGGTGAGGCGGC-3 ' (SEQ.ID.NO.:98, antisense)

The Nucleotide of small letter is included in the restriction site (square brackets) between G albumen and RUP13 as introns, have justice and antisense primer to comprise XbaI and EcoRV restriction site respectively, introns (restriction site is had contribution) are present between this G albumen and the RUP15.

Use PCR then and obtain each receptor sequence, be used for the fusion of top disclosed Gs α universal support, each PCR uses following program: 100ng RUP15 cDNA is added in the different pipes, wherein each pipe contains each primer (justice and antisense are arranged) 2 μ l, 3 μ l 10mM dNTP, 10 μ l10 * TaqPlus ^TMAccurate damping fluid, 1 μ l TaqPlus ^TMAccurately polysaccharase (Stratagene:#600211), and 80 μ l water.The temperature of reaction and the cycling time that are used for RUP15 are as follows: under 94 ℃, carried out 1 minute, carry out then 94 ℃ 30 seconds; 62 ℃ 20 seconds; 72 ℃ 1 minute 40 seconds; Carried out under 72 ℃ 5 minutes, and went on foot the 4th step circulation 35 times from the 2nd.The PCR product is walked 1% sepharose, purifying (data not shown) then, and purified product is cut with the EcoRV enzyme of XbaI, and required insertion fragment is purified to be connected in the Gs universal support separately restriction site.Transforming the back separates positive colony and cuts affirmation with the Restriction Enzyme enzyme; Realize expressing by following program with 293 cells, each positive colony of RUP15-Gs fusion rotein is checked order to confirm exactness.(nucleotide sequence is seen SEQ.ID.NO.:99, and aminoacid sequence is seen SEQ.ID.NO.:100).

RUP15 for following typical gpcr fusion protein, has realized the fusion with Gs α through the Gs coupling.

Being prepared as follows of RUP15-Gs alpha fusion protein construct:

Design of primers is as follows:

5 '-TCTAGAATGACGTCCACCTGCACCAACAGC-3 ' (SEQ.ID.NO.:101; Justice is arranged)

5 '-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3 ' (SEQ.ID.NO.:102, antisense)

The Nucleotide of small letter is included in the restriction site between G albumen and RUP15 as introns, has justice and antisense primer to comprise EcoRV and XbaI restriction site respectively, and introns (restriction site is had contribution) are present between this G albumen and the RUP15.

Use PCR then and obtain each receptor sequence, be used for the fusion of top disclosed Gs α universal support, each PCR uses following program: 100ng RUP15 cDNA is added in the different pipes, wherein each pipe contains each primer (justice and antisense are arranged) 2 μ l, 3 μ l 10mM dNTP, 10 μ l10 * TaqPlus ^TMAccurate damping fluid, 1 μ l TaqPlus ^TMAccurately polysaccharase (Stratagene:#600211), and 80 μ l water.The temperature of reaction and the cycling time that are used for RUP15 are as follows: under 94 ℃, carried out 1 minute, carry out then 94 ℃ 30 seconds; 62 ℃ 20 seconds; 72 ℃ 1 minute 40 seconds; Carried out under 72 ℃ 5 minutes, and went on foot the 4th step circulation 35 times from the 2nd.The PCR product is walked 1% sepharose, then purifying (data not shown).Digest purified product.Purified product is with EcoRV and XbaI enzyme cutting, and required insertion fragment is purified to be connected in the Gs universal support separately restriction site.Transforming the back separates positive colony and cuts affirmation with the Restriction Enzyme enzyme; Realize expressing by following program with 293 cells, each positive colony of RUP15-Gs fusion rotein is checked order to confirm exactness.(nucleotide sequence is seen SEQ.ID.NO.:103, and aminoacid sequence is seen SEQ.ID.NO.:104).

B.Gq (6 aminoacid deletion)/Gi fusion constructs

The design of Gq (disappearance)/Gi fusion constructs is as follows: 6 amino acid of N-terminal (amino acid of from 2 to 7 of G α q subunit, sequence is TLESIM (SEQ.ID.NO.:129)) lacked, (sequence is EYNLV to 5 amino acid of its C-terminal, SEQ.ID.NO.:130) (sequence is DCGLF, SEQ.ID.NO.:131) substitutes by the proteic corresponding amino acid of G α i.This fusion constructs carries out the PCR clone with following primer:

5 '-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3 ' (SEQ.ID.NO.:132) and

5′-gatcggatccTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3′(SEQ.ID.NO.：133)

And the plasmid 63313 that has the mouse wild-type G α q of hemagglutinin label is template.The Nucleotide of small letter is introns.

Use the accurate archaeal dna polymerase of TaqPlus (Stratagene) to carry out following cyclic amplification reaction: 95 ℃ 2 minutes; 95 ℃ 20 seconds; 56 ℃ 20 seconds; 72 ℃ 2 minutes; Carried out under 72 ℃ 7 minutes, and went on foot the 4th step circulation 35 times from the 2nd.The PCR product cloning arrives in the pCRII-TOPO carrier (Invitrogen), and checks order with ABI Big Dye Terminator test kit (P.E.Biosystem).From the TOPO clone's who contains this fusion constructs sequence insertion fragment,, can transfer to the HindIII/BamHI site of expression vector pcDNA3.1 (+) by two step clones with being shuttled back and forth.

Embodiment 6

The tissue distribution of disclosed people GPCR: RT-PCR

Use the RT-PCR conclusive evidence and whether express, and several new distribution of people GPCR in tissue.The oligonucleotide that uses is that GPCR is specific, with the multiple tissue cDNA of people as template (MTC, Clontech).Use Taq DNA polymerase (Stratagene) in 40 μ l reaction solutions, to increase, use according to manufacturer's explanation.20 μ l reaction solutions are carried out 1.5% agarose gel electrophoresis, analyze the RT-PCR product.Following table J has listed acceptor, the primer of circulating reaction condition and use.

Table J

Acceptor specy	Cycling condition (divide ' second ") from 2-4 step circulation 30 times	5 ' primer (SEQ.ID.NO)	3 ' primer (SEQ.ID.NO)	The DNA fragment	Tissue expression
Acceptor specy		5 ' primer (SEQ.ID.NO)	3 ' primer (SEQ.ID.NO)	The DNA fragment	Tissue expression	hRUP10	94°30” 94°10” 62°20” 72°1’ 72°7’ ^*2-4 step circulation 35 times	CATGTATGC CAGCGTCCT GCTCC(105)	GCTATGCCTG AAGCCAGTC TTGTG(106)	730bp	Kidney, white corpuscle, liver, placenta, spleen
hRUP11	94°2’ 94°15” 67°15” 72°45” 72°5’	GCACCTGCT CCTGAGCAC CTTCTCC(107)	CACAGCGCT GCAGCCCTG CAGCTGGC(108)	630bp	Liver, kidney, pancreas, colon small intestine, spleen, and prostate gland	hRUP10		CATGTATGC CAGCGTCCT GCTCC(105)	GCTATGCCTG AAGCCAGTC TTGTG(106)	730bp	Kidney, white corpuscle, liver, placenta, spleen
hRUP11	94°2’ 94°15” 67°15” 72°45” 72°5’	GCACCTGCT CCTGAGCAC CTTCTCC(107)	CACAGCGCT GCAGCCCTG CAGCTGGC(108)	630bp		hRUP12	94°2’ 94°15” 66°15” 72°45” 72°5’	CCAGTGATG ACTCTGTCC AGCCTG(109)	CAGACACTT GGCAGGGAC GAGGTG(110)	490bp	Brain, heart, kidney, colon, white corpuscle, pancreas, prostate gland, small intestine, spleen, testis, and thymus gland
hRUP13	94°1’ 94°15” 68°20” 72°1’45” 72°5’	CTTGTGGTCT ACTGCAGCA TGTTCCG(111)	CATAACCCTC CGAGTGTCC AGCGGC(112)	700bp	Placenta and lung	hRUP12	94°2’ 94°15” 66°15” 72°45” 72°5’	CCAGTGATG ACTCTGTCC AGCCTG(109)	CAGACACTT GGCAGGGAC GAGGTG(110)	490bp

hRUP14	94°1’ 94°15” 68°20” 72°1’45” 72°5’	ATGGATCCT TATCATGGC TTCCTC(113)	CAAGAACAG GTCTCATCTA AGAGCTCC(114)	700bp	Unknown
hRUP14	94°1’ 94°15” 68°20” 72°1’45” 72°5’	ATGGATCCT TATCATGGC TTCCTC(113)	CAAGAACAG GTCTCATCTA AGAGCTCC(114)	700bp	Unknown	hRUP16	94°30” 94°5” 69°15” 72°30” 72°5’	CTCTGATGC CATCTGCTG GATTCCTG(11 5)	GTAGTCCACT GAAAGTCCA GTGATCC(116)	370bp	Fetal brain, fetal kidney, and fetal skeletal muscle
hRUP18	94°2’ 94°15” 60°20” 72°1’ 72°5’	TGGTGGCGA TGGCCAACA GCGCTC(117)	GTTGCGCCTT AGCGACAGA TGACC(118)	330bp	Pancreas	hRUP16	94°30” 94°5” 69°15” 72°30” 72°5’	CTCTGATGC CATCTGCTG GATTCCTG(11 5)	GTAGTCCACT GAAAGTCCA GTGATCC(116)	370bp	Fetal brain, fetal kidney, and fetal skeletal muscle
hRUP18	94°2’ 94°15” 60°20” 72°1’ 72°5’	TGGTGGCGA TGGCCAACA GCGCTC(117)	GTTGCGCCTT AGCGACAGA TGACC(118)	330bp	Pancreas	hRUP21	94°1’ 94°15” 56°20” 72°40” ^*2-3 step circulation 30 times	TCAACCTGT ATAGCAGCA TCCTC(119)	AAGGAGTAG CAGAATGGT TAGCC(120)		Kidney, lung and testis
hRUP22	94°30” 94°15” 69°20” 72°40” ^*2-3 step circulation 30 times	GACACCTGT CAGCGGTCG TGTGTG(121)	CTGATGGAA GTAGAGGCT GTCCATCTC (122)		Testis, thymus gland and spleen	hRUP21	94°1’ 94°15” 56°20” 72°40” ^*2-3 step circulation 30 times	TCAACCTGT ATAGCAGCA TCCTC(119)	AAGGAGTAG CAGAATGGT TAGCC(120)		Kidney, lung and testis

hRUP23	94°2’ 94°15” 60°20” 72°1’ 72°5’	GCGCTGAGC GCAGACCAG TGGCTG(123)	CACGGTGAC GAAGGGCAC GAGCTC(124)	520bp	Placenta
hRUP23	94°2’ 94°15” 60°20” 72°1’ 72°5’	GCGCTGAGC GCAGACCAG TGGCTG(123)	CACGGTGAC GAAGGGCAC GAGCTC(124)	520bp	Placenta	hRUP26	94°2’ 94°15” 65°20” 72°1’ 72°5’	AGCCATCCC TGCCAGGAA GCATGG(125)	CCAGGTAGG TGTGCAGCA CAATGGC(126)	470bp	Pancreas
hRUP27	94°30” 94°10” 55°20” 72°1’ 72°3’ ^*2-4 step circulation 35 times	CTGTTCAAC AGGGCTGGT TGGCAAC(127)	ATCATGTCTA GACTCATGGT GATCC(128)	890bp	Brain	hRUP26	94°2’ 94°15” 65°20” 72°1’ 72°5’	AGCCATCCC TGCCAGGAA GCATGG(125)	CCAGGTAGG TGTGCAGCA CAATGGC(126)	470bp	Pancreas

Embodiment 7

Rules: Direct Recognition inverse agonist and agonist

A:[ ³⁵S] GTP γ S analysis

Though we are used for Direct Recognition (for example) as the candidate compound as inverse agonist with the GPCR of endogenous constitutive activity, owing to not exclusively understand, the error in analyzing can become and increase the weight of.So, as above disclosed gpcr fusion protein is also preferably used with non-endogenous composing type activatory GPCR.We have determined to use such albumen, and error seems basicly stable in analyzing, and therefore obtain effective signal to noise ratio.This helps candidate compound is discerned more fully.Therefore, for Direct Recognition, the reasonable gpcr fusion protein that is to use, and, preferably use following analysis rules in the time spent.

1. membrane prepare

Comprise the film of purpose constitutive activity orphan gpcr fusion protein and be used for the film of Direct Recognition, preferably be prepared as follows as follows as the candidate compound of inverse agonist, agonist or partial agonist:

A. material

" film extraction damping fluid " forms pH7.4 by 20mM HEPES and 10mM EDTA; " film cleaning buffer solution " forms pH7.4 by 20mM HEPES and 0.1mM EDTA; " binding buffer liquid " is by 20mM HEPES, 100mM NaCl and 10mM MgCl ₂Form pH7.4.

B. step

All material all places on ice in the whole process.At first, substratum inhaled from the monolayer cell that converges go, clean with the cold PBS of 10ml subsequently, sop up subsequently, after this 5ml film being extracted damping fluid is added on the extraction cell, subsequently cell extract is transferred in the 50ml centrifuge tube (4 ℃ down 20, centrifugal 17 minutes of 000rpm), sucking-off supernatant liquor after this is resuspended in 30ml film cleaning buffer solution with precipitation, 4 ℃ following 20 subsequently, centrifugal 17 minutes of 000rpm, sucking-off supernatant liquor then, precipitation is resuspended in the binding buffer liquid, uses Brinkman polytron then ^TMHomogenizer carries out homogenizing (15-20 high vibration second is up to all material place suspended state), is referred to as " membranin " herein.

2.Bradford analysis of protein

After the homogenizing, the protein concentration of these films is determined with the Bradford analysis of protein.(albumen can be diluted to about 1.5mg/ml, and equal portions packing and freezing (80 ℃) are standby; Under the freezing state, used rules are as follows: with dissolving under the refrigerated membranin room temperature, used the vortex vibrator subsequently the same day of analysis, and used polytron then with about 12 * 1000rpm homogenizing about 5～10 seconds; Note that for repeatedly preparation, should thoroughly clean homogenizer between the homogenizing of different prepared products.

A. material

Binding buffer liquid (as above); The Bradford staining reagent, Bradford protein standard thing, by businessman explanation use (Biorad, cat.no.500-0006).

B. step

Prepare two test tubes, one contains film, a conduct " blank " contrast.Every pipe 800 μ l binding buffer liquid of packing into, afterwards 10 μ l Bradford protein standard things (1mg/ml) are added in each test tube, 10 μ l membranins only are added in the test tube and (do not add in the blank pipe) then, 200 μ l Bradford staining reagents are added in each test tube after this, each test tube vibrates through vortex subsequently, after 5 minutes, and test tube vortex vibration once more, in cuvette, cuvette is with CECIL 3041 spectrophotometers reading under 595 wavelength then with wherein material transfer.

3. Direct Recognition analysis

A. material

(Sigma cat.no.G.7127) forms the GDP damping fluid, does a series of dilutions to obtain 0.2 μ M GDP (final concentration of GDP is 0.1 μ M GDP in every hole) with binding buffer liquid subsequently by 37.5ml binding buffer liquid and 2mg GDP; A kind of candidate compound is contained in every hole, and final volume is 200 μ l, and 100 μ l GDP damping fluids (final concentration 0.1 μ M GDP) are wherein arranged, and 50 μ l are suspended in the membranin in the binding buffer liquid, and 50 μ l be dissolved in the binding buffer liquid [ ³⁵S] GTP γ S (0.6nM) (every 10ml binding buffer liquid 2.5 μ l[ ³⁵S] GTP γ S).

B. step

Candidate compound is most with 96-orifice plate screening (can be freezing at-80 ℃), membranin (or is had film except the expression vector of gpcr fusion protein, in contrast) homogenizing is determined protein concentration with above-mentioned Bradford analysis of protein then to suspended state simply.Membranin (and contrast) is diluted to 0.25mg/ml (final analysis concentration, 12.5 μ g/ holes) with binding buffer liquid then, and after this, 100 μ l GDP damping fluids are added to Wallac Scintistrip ^TM(Wallac) in each sample well, 5 μ l candidate compounds are transferred in these sample wells (5 μ l are 1: 40 ratio thereby the final screening concentration 10 μ M of candidate compound) with 5 μ l needle sets in 200 μ l bulk analysis volumes then.Have, for avoiding polluting, after each transfer step, needle set should clean three times again: comprise water (1X), ethanol (1X) and the unnecessary liquid of water (2X)----get rid of after each the cleaning, with paper and kimwipe drying.50 μ l membranins are added to (film of not being with gpcr fusion protein is contained in the control sample hole) in each sample well after this, educated in advance under the room temperature 5-10 minute, and after this, in the 50 μ l binding buffer liquid [ ³⁵S] GTP γ S (0.6nM) is added in each sample well, subsequently under the room temperature on shaking table incubation (have, in the present embodiment, culture plate covered with tinsel again in 60 minutes.Then plate is rotated 15 minutes termination analysis down with 4000RPM22 ℃, with 8 road arms exhaustion plates, cover then, last plate Wallac 1450 readings that are arranged on " Prot.#37 " shelves (complying with each businessman's explanation) with plate.

B. encircling AMP analyzes

The analytical procedure of another kind of Direct Recognition candidate compound is based on the analysis of cyclase.Except Direct Recognition, this method also can be used as independently method, confirm above-mentioned [ ³⁵S] GTP γ S analyzes the result of gained.

Preferably use through improved Flash Plate ^TMAdenylate cyclase enzyme reagent kit (NewEngland Nuclear; Cat.no.SMP004A), go out inverse agonist and the agonist that candidate compound is composing type activatory orphan GPCR by following rules Direct Recognition.

Collect transfectional cell in about three days after the transfection, containing 20mM HEPES (pH7.4) and 10mM MgCl ₂Damping fluid in, prepare film by the cell homogenizing that will suspend, use Brinkman Polytron ^TMCarry out homogenizing on ice about 10 seconds, 4 ℃ following 49 of the homogenate that produces, centrifugal 15 minutes of 000 * g, the precipitation of Chan Shenging is resuspended in the damping fluid that contains 20mM HEPES (pH7.4) and 0.1mM EDTA then, homogenizing 10 seconds, 4 ℃ were descended 49,000 * g centrifugal 15 minutes subsequently, and the precipitation of generation-80 ℃ preservation is standby.Direct Recognition screening same day is deposited in film under the room temperature and slowly melts, and is resuspended in and contains 20mM HEPES (pH7.4) and 10mMMgCl ₂Damping fluid in, produce the final protein concentration (resuspended film places standby on ice) of 0.6mg/ml.

CAMP standard substance and mensuration damping fluid (2 μ Ci tracers [ ¹²⁵ I cAMP 100 μ l] be added in the 11ml mensuration damping fluid), according to businessman's explanation preparation and keeping.Prepare fresh analysis buffer and be used for screening, wherein comprise 20mM HEPES (pH7.4), 10mM MgCl ₂, 20mM phosphocreatine (Sigma), 0.1 unit/ml creatine phosphokinase (Sigma), 50 μ M GTP (Sigma), and 0.2mM ATP (Sigma); Analysis buffer can be preserved standby on ice.

Preferably the candidate compound (as freezing, melting under the room temperature) of above identification is added in the sample well of 96 orifice plates (3 μ l/ holes, the analysis final concentration of 12 μ M), lumps together with 40 μ l membranins (30 μ g/ hole) and 50 μ l analysis buffer.With this mixture incubation 30 minutes at room temperature, shake gently simultaneously then.

After incubation was finished, 100 μ l measured damping fluid and are added in each sample well, place subsequently 2～4 hours, and plate is at Wallac MicroBeta then ^TMRead in the plate device with " Prot.#31 " shelves counting (according to each businessman's description operation).

In Figure 12, shown a representative screening analysis dull and stereotyped (96 orifice plate).Represent in each hole the result of the RUP13-Gs alpha fusion protein construct of preparation in the different compounds and above embodiment 5 (a) for every.Typical consequence among Figure 12 also provides standard deviation, its acquisition is based on each dull and stereotyped mean value (" m "), mean value is together with the inverse agonist of two picked at random conducts " guide " from the preliminary screening process, comprise and select such candidate compound that its reaction reduced rate is at least the standard deviation that the average response value deducts twice.Opposite, from the preliminary screening process, preferentially choose agonist arbitrarily as " guide ", comprise and select such candidate compound that its reaction increment rate is at least the standard deviation that the average response value adds twice.Based on above select procedure, the candidate compound in the following hole can be respectively as the possible inverse agonist (compd A) of RUP13 among hole A2 and the G9 and agonist (compd B) by Direct Recognition.Referring to Figure 12.For the sake of clarity specify: these compounds under the situation of unknown this GPCR endogenous ligands by Direct Recognition.Based on function of receptors but not the analytical technology of the binding affinity of compound, we can confirm to reduce the active compound of function of receptors (compd A), also can confirm to increase the active compound of function of receptors (compd B) by this.In the location of lung (seeing, for example hRUP13 and the hRUP21 among the embodiment 6), can invent the medicament that treatment lung cancer is had potential curative effect based on these acceptors.

Each document of mentioning in the present patent application file comprises common pending application and related application, and unless otherwise specified, it is for referencial use all to introduce the application in full with it.The modification of carrying out at disclosed invention in those skilled in the art understand scope and extending all within the above-mentioned scope that openly reaches claims.

Though the purpose that it is endogenous and non-endogenous GPCR that those of ordinary skill in the art can obtain many different carriers is used, and preferably uses the pCMV carrier.The microbial preservation budapest treaty that is used for patented procedure according to international recognition, this carrier is deposited in (ATCC) (University Blvd of American type culture collection (American Type Culture Collection) on October 13rd, 1998, Manassas, VA20110-2209 USA7).Its DNA is through ATCC mensuration and through confirming to be in existing state, and ATCC is that pCMV has provided following preserving number ATCC#203351.

Sequence table

＜110〉Arena Pharmaceuticals, Inc (Arena Pharmaceuticals, Inc.)

Chen Ruoping (Ruoping CHEN); Deng Hang (Huong T.DANG); Kevin P Loews (Kevin P.LOWITZ)

＜120〉non-endogenous composing type activatory human G protein-coupled receptor

(Non-Endogenous，Constitutively Activated Human G Protein-Coupled Receptors)

<130>SPI064359-47

<150>60/166,088

<151>1999-11-17

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<160>133

<170>PatentIn version 3.1

<210>1

<211>1155

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

atggcagccc agaatggaaa caccagtttc acacccaact ttaatccacc ccaagaccat 60

gcctcctccc tctcctttaa cttcagttat ggtgattatg acctccctat ggatgaggat 120

gaggacatga ccaagacccg gaccttcttc gcagccaaga tcgtcattgg cattgcactg 180

gcaggcatca tgctggtctg cggcatcggt aactttgtct ttatcgctgc cctcacccgc 240

tataagaagt tgcgcaacct caccaatctg ctcattgcca acctggccat ctccgacttc 300

ctggtggcca tcatctgctg ccccttcgag atggactact acgtggtacg gcagctctcc 360

tgggagcatg gccacgtgct ctgtgcctcc gtcaactacc tgcgcaccgt ctccctctac 420

gtctccacca atgccttgct ggccattgcc attgacagat atctcgccat cgttcacccc 480

ttgaaaccac ggatgaatta tcaaacggcc tccttcctga tcgccttggt ctggatggtg 540

tccattctca ttgccatccc atcggcttac tttgcaacag aaacggtcct ctttattgtc 600

aagagccagg agaagatctt ctgtggccag atctggcctg tggatcagca gctctactac 660

aagtcctact tcctcttcat ctttggtgtc gagttcgtgg gccctgtggt caccatgacc 720

ctgtgctatg ccaggatctc ccgggagctc tggttcaagg cagtccctgg gttccagacg 780

gagcagattc gcaagcggct gcgctgccgc aggaagacgg tcctggtgct catgtgcatt 840

ctcacggcct atgtgctgtg ctgggcaccc ttctacggtt tcaccatcgt tcgtgacttc 900

ttccccactg tgttcgtgaa ggaaaagcac tacctcactg ccttctacgt ggtcgagtgc 960

atcgccatga gcaacagcat gatcaacacc gtgtgcttcg tgacggtcaa gaacaacacc 1020

atgaagtact tcaagaagat gatgctgctg cactggcgtc cctcccagcg ggggagcaag 1080

tccagtgctg accttgacct cagaaccaac ggggtgccca ccacagaaga ggtggactgt 1140

atcaggctga agtga 1155

<210>2

<211>384

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Ala Ala Gln Asn Gly Asn Thr Ser Phe Thr Pro Asn Phe Asn Pro

1 5 10 15

Pro Gln Asp His Ala Ser Ser Leu Ser Phe Asn Phe Ser Tyr Gly Asp

20 25 30

Tyr Asp Leu Pro Met Asp Glu Asp Glu Asp Met Thr Lys Thr Arg Thr

35 40 45

Phe Phe Ala Ala Lys Ile Val Ile Gly Ile Ala Leu Ala Gly Ile Met

50 55 60

Leu Val Cys Gly Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Thr Arg

65 70 75 80

Tyr Lys Lys Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala

85 90 95

Ile Ser Asp Phe Leu Val Ala Ile Ile Cys Cys Pro Phe Glu Met Asp

100 105 110

Tyr Tyr Val Val Arg Gln Leu Ser Trp Glu His Gly His Val Leu Cys

115 120 125

Ala Ser Val Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val Ser Thr Asn

130 135 140

Ala Leu Leu Ala Ile Ala Ile Asp Arg Tyr Leu Ala Ile Val His Pro

145 150 155 160

Leu Lys Pro Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile Ala Leu

165 170 175

Val Trp Met Val Ser Ile Leu Ile Ala Ile Pro Ser Ala Tyr Phe Ala

180 185 190

Thr Glu Thr Val Leu Phe Ile Val Lys Ser Gln Glu Lys Ile Phe Cys

195 200 205

Gly Gln Ile Trp Pro Val Asp Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe

210 215 220

Leu Phe Ile Phe Gly Val Glu Phe Val Gly Pro Val Val Thr Met Thr

225 230 235 240

Leu Cys Tyr Ala Arg Ile Ser Arg Glu Leu Trp Phe Lys Ala Val Pro

245 250 255

Gly Phe Gln Thr Glu Gln Ile Arg Lys Arg Leu Arg Cys Arg Arg Lys

260 265 270

Thr Val Leu Val Leu Met Cys Ile Leu Thr Ala Tyr Val Leu Cys Trp

275 280 285

Ala Pro Phe Tyr Gly Phe Thr Ile Val Arg Asp Phe Phe Pro Thr Val

290 295 300

Phe Val Lys Glu Lys His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys

305 310 315 320

Ile Ala Met Ser Asn Ser Met Ile Asn Thr Val Cys Phe Val Thr Val

325 330 335

Lys Asn Asn Thr Met Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp

340 345 350

Arg Pro Ser Gln Arg Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg

355 360 365

Thr Asn Gly Val Pro Thr Thr Glu Glu Val Asp Cys Ile Arg Leu Lys

370 375 380

<210>3

<211>1260

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

atgctggcag ctgcctttgc agactctaac tccagcagca tgaatgtgtc ctttgctcac 60

ctccactttg ccggagggta cctgccctct gattcccagg actggagaac catcatcccg 120

gctctcttgg tggctgtctg cctggtgggc ttcgtgggaa acctgtgtgt gattggcatc 180

ctccttcaca atgcttggaa aggaaagcca tccatgatcc actccctgat tctgaatctc 240

agcctggctg atctctccct cctgctgttt tctgcaccta tccgagctac ggcgtactcc 300

aaaagtgttt gggatctagg ctggtttgtc tgcaagtcct ctgactggtt tatccacaca 360

tgcatggcag ccaagagcct gacaatcgtt gtggtggcca aagtatgctt catgtatgca 420

agtgacccag ccaagcaagt gagtatccac aactacacca tctggtcagt gctggtggcc 480

atctggactg tggctagcct gttacccctg ccggaatggt tctttagcac catcaggcat 540

catgaaggtg tggaaatgtg cctcgtggat gtaccagctg tggctgaaga gtttatgtcg 600

atgtttggta agctctaccc actcctggca tttggccttc cattattttt tgccagcttt 660

tatttctgga gagcttatga ccaatgtaaa aaacgaggaa ctaagactca aaatcttaga 720

aaccagatac gctcaaagca agtcacagtg atgctgctga gcattgccat catctctgct 780

ctcttgtggc tccccgaatg ggtagcttgg ctgtgggtat ggcatctgaa ggctgcaggc 840

ccggccccac cacaaggttt catagccctg tctcaagtct tgatgttttc catctcttca 900

gcaaatcctc tcatttttct tgtgatgtcg gaagagttca gggaaggctt gaaaggtgta 960

tggaaatgga tgataaccaa aaaacctcca actgtctcag agtctcagga aacaccagct 1020

ggcaactcag agggtcttcc tgacaaggtt ccatctccag aatccccagc atccatacca 1080

gaaaaagaga aacccagctc tccctcctct ggcaaaggga aaactgagaa ggcagagatt 1140

cccatccttc ctgacgtaga gcagttttgg catgagaggg acacagtccc ttctgtacag 1200

gacaatgacc ctatcccctg ggaacatgaa gatcaagaga caggggaagg tgttaaatag 1260

<210>4

<211>419

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Met Leu Ala Ala Ala Phe Ala Asp Ser Asn Ser Ser Ser Met Asn Val

1 5 10 15

Ser Phe Ala His Leu His Phe Ala Gly Gly Tyr Leu Pro Ser Asp Ser

20 25 30

Gln Asp Trp Arg Thr Ile Ile Pro Ala Leu Leu Val Ala Val Cys Leu

35 40 45

Val Gly Phe Val Gly Asn Leu Cys Val Ile Gly Ile Leu Leu His Asn

50 55 60

Ala Trp Lys Gly Lys Pro Ser Met Ile His Ser Leu Ile Leu Asn Leu

65 70 75 80

Ser Leu Ala Asp Leu Ser Leu Leu Leu Phe Ser Ala Pro Ile Arg Ala

85 90 95

Thr Ala Tyr Ser Lys Ser Val Trp Asp Leu Gly Trp Phe Val Cys Lys

100 105 110

Ser Ser Asp Trp Phe Ile His Thr Cys Met Ala Ala Lys Ser Leu Thr

115 120 125

Ile Val Val Val Ala Lys Val Cys Phe Met Tyr Ala Ser Asp Pro Ala

130 135 140

Lys Gln Val Ser Ile His Asn Tyr Thr Ile Trp Ser Val Leu Val Ala

145 150 155 160

Ile Trp Thr Val Ala Ser Leu Leu Pro Leu Pro Glu Trp Phe Phe Ser

165 170 175

Thr Ile Arg His His Glu Gly Val Glu Met Cys Leu Val Asp Val Pro

180 185 190

Ala Val Ala Glu Glu Phe Met Ser Met Phe Gly Lys Leu Tyr Pro Leu

195 200 205

Leu Ala Phe Gly Leu Pro Leu Phe Phe Ala Ser Phe Tyr Phe Trp Arg

210 215 220

Ala Tyr Asp Gln Cys Lys Lys Arg Gly Thr Lys Thr Gln Asn Leu Arg

225 230 235 240

Asn Gln Ile Arg Ser Lys Gln Val Thr Val Met Leu Leu Ser Ile Ala

245 250 255

Ile Ile Ser Ala Leu Leu Trp Leu Pro Glu Trp Val Ala Trp Leu Trp

260 265 270

Val Trp His Leu Lys Ala Ala Gly Pro Ala Pro Pro Gln Gly Phe Ile

275 280 285

Ala Leu Ser Gln Val Leu Met Phe Ser Ile Ser Ser Ala Asn Pro Leu

290 295 300

Ile Phe Leu Val Met Ser Glu Glu Phe Arg Glu Gly Leu Lys Gly Val

305 310 315 320

Trp Lys Trp Met Ile Thr Lys Lys Pro Pro Thr Val Ser Glu Ser Gln

325 330 335

Glu Thr Pro Ala Gly Asn Ser Glu Gly Leu Pro Asp Lys Val Pro Ser

340 345 350

Pro Glu Ser Pro Ala Ser Ile Pro Glu Lys Glu Lys Pro Ser Ser Pro

355 360 365

Ser Ser Gly Lys Gly Lys Thr Glu Lys Ala Glu Ile Pro Ile Leu Pro

370 375 380

Asp Val Glu Gln Phe Trp His Glu Arg Asp Thr Val Pro Ser Val Gln

385 390 395 400

Asp Asn Asp Pro Ile Pro Trp Glu His Glu Asp Gln Glu Thr Gly Glu

405 410 415

Gly Val Lys

<210>5

<211>1014

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

atggggaacg attctgtcag ctacgagtat ggggattaca gcgacctctc ggaccgccct 60

gtggactgcc tggatggcgc ctgcctggcc atcgacccgc tgcgcgtggc cccgctccca 120

ctgtatgccg ccatcttcct ggtgggggtg ccgggcaatg ccatggtggc ctgggtggct 180

gggaaggtgg cccgccggag ggtgggtgcc acctggttgc tccacctggc cgtggcggat 240

ttgctgtgct gtttgtctct gcccatcctg gcagtgccca ttgcccgtgg aggccactgg 300

ccgtatggtg cagtgggctg tcgggcgctg ccctccatca tcctgctgac catgtatgcc 360

agcgtcctgc tcctggcagc tctcagtgcc gacctctgct tcctggctct cgggcctgcc 420

tggtggtcta cggttcagcg ggcgtgcggg gtgcaggtgg cctgtggggc agcctggaca 480

ctggccttgc tgctcaccgt gccctccgcc atctaccgcc ggctgcacca ggagcacttc 540

ccagcccggc tgcagtgtgt ggtggactac ggcggctcct ccagcaccga gaatgcggtg 600

actgccatcc ggtttctttt tggcttcctg gggcccctgg tggccgtggc cagctgccac 660

agtgccctcc tgtgctgggc agcccgacgc tgccggccgc tgggcacagc cattgtggtg 720

gggttttttg tctgctgggc accctaccac ctgctggggc tggtgctcac tgtggcggcc 780

ccgaactccg cactcctggc cagggccctg cgggctgaac ccctcatcgt gggccttgcc 840

ctcgctcaca gctgcctcaa tcccatgctc ttcctgtatt ttgggagggc tcaactccgc 900

cggtcactgc cagctgcctg tcactgggcc ctgagggagt cccagggcca ggacgaaagt 960

gtggacagca agaaatccac cagccatgac ctggtctcgg agatggaggt gtag 1014

<210>6

<211>337

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Met Gly Asn Asp Ser Val Ser Tyr Glu Tyr Gly Asp Tyr Ser Asp Leu

1 5 10 15

Ser Asp Arg Pro Val Asp Cys Leu Asp Gly Ala Cys Leu Ala Ile Asp

20 25 30

Pro Leu Arg Val Ala Pro Leu Pro Leu Tyr Ala Ala Ile Phe Leu Val

35 40 45

Gly Val Pro Gly Asn Ala Met Val Ala Trp Val Ala Gly Lys Val Ala

50 55 60

Arg Arg Arg Val Gly Ala Thr Trp Leu Leu His Leu Ala Val Ala Asp

65 70 75 80

Leu Leu Cys Cys Leu Ser Leu Pro Ile Leu Ala Val Pro Ile Ala Arg

85 90 95

Gly Gly His Trp Pro Tyr Gly Ala Val Gly Cys Arg Ala Leu Pro Ser

100 105 110

Ile Ile Leu Leu Thr Met Tyr Ala Ser Val Leu Leu Leu Ala Ala Leu

115 120 125

Ser Ala Asp Leu Cys Phe Leu Ala Leu Gly Pro Ala Trp Trp Ser Thr

130 135 140

Val Gln Arg Ala Cys Gly Val Gln Val Ala Cys Gly Ala Ala Trp Thr

145 150 155 160

Leu Ala Leu Leu Leu Thr Val Pro Ser Ala Ile Tyr Arg Arg Leu His

165 170 175

Gln Glu His Phe Pro Ala Arg Leu Gln Cys Val Val Asp Tyr Gly Gly

180 185 190

Ser Ser Ser Thr Glu Asn Ala Val Thr Ala Ile Arg Phe Leu Phe Gly

195 200 205

Phe Leu Gly Pro Leu Val Ala Val Ala Ser Cys His Ser Ala Leu Leu

210 215 220

Cys Trp Ala Ala Arg Arg Cys Arg Pro Leu Gly Thr Ala Ile Val Val

225 230 235 240

Gly Phe Phe Val Cys Trp Ala Pro Tyr His Leu Leu Gly Leu Val Leu

245 250 255

Thr Val Ala Ala Pro Asn Ser Ala Leu Leu Ala Arg Ala Leu Arg Ala

260 265 270

Glu Pro Leu Ile Val Gly Leu Ala Leu Ala His Ser Cys Leu Asn Pro

275 280 285

Met Leu Phe Leu Tyr Phe Gly Arg Ala Gln Leu Arg Arg Ser Leu Pro

290 295 300

Ala Ala Cys His Trp Ala Leu Arg Glu Ser Gln Gly Gln Asp Glu Ser

305 310 315 320

Val Asp Ser Lys Lys Ser Thr Ser His Asp Leu Val Ser Glu Met Glu

325 330 335

Val

<210>7

<211>1272

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>7

atgttgtgtc accgtggtgg ccagctgata gtgccaatca tcccactttg ccctgagcac 60

tcctgcaggg gtagaagact ccagaacctt ctctcaggcc catggcccaa gcagcccatg 120

gaacttcata acctgagctc tccatctccc tctctctcct cctctgttct ccctccctcc 180

ttctctccct caccctcctc tgctccctct gcctttacca ctgtgggggg gtcctctgga 240

gggccctgcc accccacctc ttcctcgctg gtgtctgcct tcctggcacc aatcctggcc 300

ctggagtttg tcctgggcct ggtggggaac agtttggccc tcttcatctt ctgcatccac 360

acgcggccct ggacctccaa cacggtgttc ctggtcagcc tggtggccgc tgacttcctc 420

ctgatcagca acctgcccct ccgcgtggac tactacctcc tccatgagac ctggcgcttt 480

ggggctgctg cctgcaaagt caacctcttc atgctgtcca ccaaccgcac ggccagcgtt 540

gtcttcctca cagccatcgc actcaaccgc tacctgaagg tggtgcagcc ccaccacgtg 600

ctgagccgtg cttccgtggg ggcagctgcc cgggtggccg ggggactctg ggtgggcatc 660

ctgctcctca acgggcacct gctcctgagc accttctccg gcccctcctg cctcagctac 720

agggtgggca cgaagccctc ggcctcgctc cgctggcacc aggcactgta cctgctggag 780

ttcttcctgc cactggcgct catcctcttt gctattgtga gcattgggct caccatccgg 840

aaccgtggtc tgggcgggca ggcaggcccg cagagggcca tgcgtgtgct ggccatggtg 900

gtggccgtct acaccatctg cttcttgccc agcatcatct ttggcatggc ttccatggtg 960

gctttctggc tgtccgcctg ccgatccctg gacctctgca cacagctctt ccatggctcc 1020

ctggccttca cctacctcaa cagtgtcctg gaccccgtgc tctactgctt ctctagcccc 1080

aacttcctcc accagagccg ggccttgctg ggcctcacgc ggggccggca gggcccagtg 1140

agcgacgaga gctcctacca accctccagg cagtggcgct accgggaggc ctctaggaag 1200

gcggaggcca tagggaagct gaaagtgcag ggcgaggtct ctctggaaaa ggaaggctcc 1260

tcccagggct ga 1272

<210>8

<211>423

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Met Leu Cys His Arg Gly Gly Gln Leu Ile Val Pro Ile Ile Pro Leu

1 5 10 15

Cys Pro Glu His Ser Cys Arg Gly Arg Arg Leu Gln Asn Leu Leu Ser

20 25 30

Gly Pro Trp Pro Lys Gln Pro Met Glu Leu His Asn Leu Ser Ser Pro

35 40 45

Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro Ser

50 55 60

Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser Gly

65 70 75 80

Gly Pro Cys His Pro Thr Ser Ser Ser Leu Val Ser Ala Phe Leu Ala

85 90 95

Pro Ile Leu Ala Leu Glu Phe Val Leu Gly Leu Val Gly Asn Ser Leu

100 105 110

Ala Leu Phe Ile Phe Cys Ile His Thr Arg Pro Trp Thr Ser Asn Thr

115 120 125

Val Phe Leu Val Ser Leu Val Ala Ala Asp Phe Leu Leu Ile Ser Asn

130 135 140

Leu Pro Leu Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg Phe

145 150 155 160

Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn Arg

165 170 175

Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr Leu

180 185 190

Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser Val Gly Ala

195 200 205

Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly Ile Leu Leu Leu Asn

210 215 220

Gly His Leu Leu Leu Ser Thr Phe Ser Gly Pro Ser Cys Leu Ser Tyr

225 230 235 240

Arg Val Gly Thr Lys Pro Ser Ala Ser Leu Arg Trp His Gln Ala Leu

245 250 255

Tyr Leu Leu Glu Phe Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala Ile

260 265 270

Val Ser Ile Gly Leu Thr Ile Arg Asn Arg Gly Leu Gly Gly Gln Ala

275 280 285

Gly Pro Gln Arg Ala Met Arg Val Leu Ala Met Val Val Ala Val Tyr

290 295 300

Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met Val

305 310 315 320

Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu Cys Thr Gln Leu

325 330 335

Phe His Gly Ser Leu Ala Phe Thr Tyr Leu Asn Ser Val Leu Asp Pro

340 345 350

Val Leu Tyr Cys Phe Ser Ser Pro Asn Phe Leu His Gln Ser Arg Ala

355 360 365

Leu Leu Gly Leu Thr Arg Gly Arg Gln Gly Pro Val Ser Asp Glu Ser

370 375 380

Ser Tyr Gln Pro Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg Lys

385 390 395 400

Ala Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu Glu

405 410 415

Lys Glu Gly Ser Ser Gln Gly

420

<210>9

<211>966

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>9

atgaaccaga ctttgaatag cagtgggacc gtggagtcag ccctaaacta ttccagaggg 60

agcacagtgc acacggccta cctggtgctg agctccctgg ccatgttcac ctgcctgtgc 120

gggatggcag gcaacagcat ggtgatctgg ctgctgggct ttcgaatgca caggaacccc 180

ttctgcatct atatcctcaa cctggcggca gccgacctcc tcttcctctt cagcatggct 240

tccacgctca gcctggaaac ccagcccctg gtcaatacca ctgacaaggt ccacgagctg 300

atgaagagac tgatgtactt tgcctacaca gtgggcctga gcctgctgac ggccatcagc 360

acccagcgct gtctctctgt cctcttccct atctggttca agtgtcaccg gcccaggcac 420

ctgtcagcct gggtgtgtgg cctgctgtgg acactctgtc tcctgatgaa cgggttgacc 480

tcttccttct gcagcaagtt cttgaaattc aatgaagatc ggtgcttcag ggtggacatg 540

gtccaggccg ccctcatcat gggggtctta accccagtga tgactctgtc cagcctgacc 600

ctctttgtct gggtgcggag gagctcccag cagtggcggc ggcagcccac acggctgttc 660

gtggtggtcc tggcctctgt cctggtgttc ctcatctgtt ccctgcctct gagcatctac 720

tggtttgtgc tctactggtt gagcctgccg cccgagatgc aggtcctgtg cttcagcttg 780

tcacgcctct cctcgtccgt aagcagcagc gccaaccccg tcatctactt cctggtgggc 840

agccggagga gccacaggct gcccaccagg tccctgggga ctgtgctcca acaggcgctt 900

cgcgaggagc ccgagctgga aggtggggag acgcccaccg tgggcaccaa tgagatgggg 960

gcttga 966

<210>10

<211>321

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>10

Met Asn Gln Thr Leu Asn Ser Ser Gly Thr Val Glu Ser Ala Leu Asn

1 5 10 15

Tyr Ser Arg Gly Ser Thr Val His Thr Ala Tyr Leu Val Leu Ser Ser

20 25 30

Leu Ala Met Phe Thr Cys Leu Cys Gly Met Ala Gly Asn Ser Met Val

35 40 45

Ile Trp Leu Leu Gly Phe Arg Met His Arg Asn Pro Phe Cys Ile Tyr

50 55 60

Ile Leu Asn Leu Ala Ala Ala Asp Leu Leu Phe Leu Phe Ser Met Ala

65 70 75 80

Ser Thr Leu Ser Leu Glu Thr Gln Pro Leu Val Asn Thr Thr Asp Lys

85 90 95

Val His Glu Leu Met Lys Arg Leu Met Tyr Phe Ala Tyr Thr Val Gly

100 105 110

Leu Ser Leu Leu Thr Ala Ile Ser Thr Gln Arg Cys Leu Ser Val Leu

115 120 125

Phe Pro Ile Trp Phe Lys Cys His Arg Pro Arg His Leu Ser Ala Trp

130 135 140

Val Cys Gly Leu Leu Trp Thr Leu Cys Leu Leu Met Asn Gly Leu Thr

145 150 155 160

Ser Ser Phe Cys Ser Lys Phe Leu Lys Phe Asn Glu Asp Arg Cys Phe

165 170 175

Arg Val Asp Met Val Gln Ala Ala Leu Ile Met Gly Val Leu Thr Pro

180 185 190

Val Met Thr Leu Ser Ser Leu Thr Leu Phe Val Trp Val Arg Arg Ser

195 200 205

Ser Gln Gln Trp Arg Arg Gln Pro Thr Arg Leu Phe Val Val Val Leu

210 215 220

Ala Ser Val Leu Val Phe Leu Ile Cys Ser Leu Pro Leu Ser Ile Tyr

225 230 235 240

Trp Phe Val Leu Tyr Trp Leu Ser Leu Pro Pro Glu Met Gln Val Leu

245 250 255

Cys Phe Ser Leu Ser Arg Leu Ser Ser Ser Val Ser Ser Ser Ala Asn

260 265 270

Pro Val Ile Tyr Phe Leu Val Gly Ser Arg Arg Ser His Arg Leu Pro

275 280 285

Thr Arg Ser Leu Gly Thr Val Leu Gln Gln Ala Leu Arg Glu Glu Pro

290 295 300

Glu Leu Glu Gly Gly Glu Thr Pro Thr Val Gly Thr Asn Glu Met Gly

305 310 315 320

Ala

<210>11

<211>1356

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>11

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcagcagt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcatga 1356

<210>12

<211>451

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>12

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser

450

<210>13

<211>1041

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>13

atggagagaa aatttatgtc cttgcaacca tccatctccg tatcagaaat ggaaccaaat 60

ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120

ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180

tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat gctaaatctg 240

gccatttcag atctcctgtt cataagcacg cttcccttca gggctgacta ttatcttaga 300

ggctccaatt ggatatttgg agacctggcc tgcaggatta tgtcttattc cttgtatgtc 360

aacatgtaca gcagtattta tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420

gttcacccct ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480

atcatatgga tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag 540

aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc 600

atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact cagcatctgt 660

tatctgctga tcattcgggt tctgttaaaa gtggaggtcc cagaatcggg gctgcgggtt 720

tctcacagga aggcactgac caccatcatc atcaccttga tcatcttctt cttgtgtttc 780

ctgccctatc acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840

gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900

aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa gtctgcactc 960

agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag tgtgtggttg 1020

agaaaggaaa caagagtata a 1041

<210>14

<211>346

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>14

Met Glu Arg Lys Phe Met Ser Leu Gln Pro Ser Ile Ser Val Ser Glu

1 5 10 15

Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser Arg Asn Cys Thr

20 25 30

Ile Glu Asn Phe Lys Arg Glu Phe Phe Pro Ile Val Tyr Leu Ile Ile

35 40 45

Phe Phe Trp Gly Val Leu Gly Asn Gly Leu Ser Ile Tyr Val Phe Leu

50 55 60

Gln Pro Tyr Lys Lys Ser Thr Ser Val Asn Val Phe Met Leu Asn Leu

65 70 75 80

Ala Ile Ser Asp Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp

85 90 95

Tyr Tyr Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg

100 105 110

Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe

115 120 125

Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His Pro Phe

130 135 140

Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile Leu Cys Gly

145 150 155 160

Ile Ile Trp Ile Leu Ile Met Ala Ser Ser Ile Met Leu Leu Asp Ser

165 170 175

Gly Ser Glu Gln Asn Gly Ser Val Thr Ser Cys Leu Glu Leu Asn Leu

180 185 190

Tyr Lys Ile Ala Lys Leu Gln Thr Met Asn Tyr Ile Ala Leu Val Val

195 200 205

Gly Cys Leu Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile

210 215 220

Ile Arg Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val

225 230 235 240

Ser His Arg Lys Ala Leu Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe

245 250 255

Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His Leu Thr

260 265 270

Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu His Lys Ala Leu Val

275 280 285

Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala Cys Phe Asn Pro Leu Leu

290 295 300

Tyr Tyr Phe Ala Gly Glu Asn Phe Lys Asp Arg Leu Lys Ser Ala Leu

305 310 315 320

Arg Lys Gly His Pro Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val

325 330 335

Ser Val Trp Leu Arg Lys Glu Thr Arg Val

340 345

<210>15

<211>1527

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>15

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tccttga 1527

<210>16

<211>508

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>16

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 140

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro

500 505

<210>17

<211>1068

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>17

atgcccttga cggacggcat ttcttcattt gaggacctct tggctaacaa tatcctcaga 60

atatttgtct gggttatagc tttcattacc tgctttggaa atctttttgt cattggcatg 120

agatctttca ttaaagctga aaatacaact cacgctatgt ccatcaaaat cctttgttgc 180

gctgattgcc tgatgggtgt ttacttgttc tttgttggca ttttcgatat aaaataccga 240

gggcagtatc agaagtatgc cttgctgtgg atggagagcg tgcagtgccg cctcatgggg 300

ttcctggcca tgctgtccac cgaagtctct gttctgctac tgacctactt gactttggag 360

aagttcctgg tcattgtctt ccccttcagt aacattcgac ctggaaaacg gcagacctca 420

gtcatcctca tttgcatctg gatggcggga tttttaatag ctgtaattcc attttggaat 480

aaggattatt ttggaaactt ttatgggaaa aatggagtat gtttcccact ttattatgac 540

caaacagaag atattggaag caaagggtat tctcttggaa ttttcctagg tgtgaacttg 600

ctggcttttc tcatcattgt gttttcctat attactatgt tctgttccat tcaaaaaacc 660

gccttgcaga ccacagaagt aaggaattgt tttggaagag aggtggctgt tgcaaatcgt 720

ttctttttta tagtgttctc tgatgccatc tgctggattc ctgtatttgt agttaaaatc 780

ctttccctct tccgggtgga aataccagac acaatgactt cctggatagt gatttttttc 840

cttccagtta acagtgcttt gaatccaatc ctctatactc tcacaaccaa cttttttaag 900

gacaagttga aacagctgct gcacaaacat cagaggaaat caattttcaa aattaaaaaa 960

aaaagtttat ctacatccat tgtgtggata gaggactcct cttccctgaa acttggggtt 1020

ttgaacaaaa taacacttgg agacagtata atgaaaccag tttcctag 1068

<210>18

<211>355

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>18

Met Pro Leu Thr Asp Gly Ile Ser Ser Phe Glu Asp Leu Leu Ala Asn

1 5 10 15

Asn Ile Leu Arg Ile Phe Val Trp Val Ile Ala Phe Ile Thr Cys Phe

20 25 30

Gly Asn Leu Phe Val Ile Gly Met Arg Ser Phe Ile Lys Ala Glu Asn

35 40 45

Thr Thr His Ala Met Ser Ile Lys Ile Leu Cys Cys Ala Asp Cys Leu

50 55 60

Met Gly Val Tyr Leu Phe Phe Val Gly Ile Phe Asp Ile Lys Tyr Arg

65 70 75 80

Gly Gln Tyr Gln Lys Tyr Ala Leu Leu Trp Met Glu Ser Val Gln Cys

85 90 95

Arg Leu Met Gly Phe Leu Ala Met Leu Ser Thr Glu Val Ser Val Leu

100 105 110

Leu Leu Thr Tyr Leu Thr Leu Glu Lys Phe Leu Val Ile Val Phe Pro

115 120 125

Phe Ser Asn Ile Arg Pro Gly Lys Arg Gln Thr Ser Val Ile Leu Ile

130 135 140

Cys Ile Trp Met Ala Gly Phe Leu Ile Ala Val Ile Pro Phe Trp Asn

145 150 155 160

Lys Asp Tyr Phe Gly Asn Phe Tyr Gly Lys Asn Gly Val Cys Phe Pro

165 170 175

Leu Tyr Tyr Asp Gln Thr Glu Asp Ile Gly Ser Lys Gly Tyr Ser Leu

180 185 190

Gly Ile Phe Leu Gly Val Asn Leu Leu Ala Phe Leu Ile Ile Val Phe

195 200 205

Ser Tyr Ile Thr Met Phe Cys Ser Ile Gln Lys Thr Ala Leu Gln Thr

210 215 220

Thr Glu Val Arg Asn Cys Phe Gly Arg Glu Val Ala Val Ala Asn Arg

225 230 235 240

Phe Phe Phe Ile Val Phe Ser Asp Ala Ile Cys Trp Ile Pro Val Phe

245 250 255

Val Val Lys Ile Leu Ser Leu Phe Arg Val Glu Ile Pro Asp Thr Met

260 265 270

Thr Ser Trp Ile Val Ile Phe Phe Leu Pro Val Asn Ser Ala Leu Asn

275 280 285

Pro Ile Leu Tyr Thr Leu Thr Thr Asn Phe Phe Lys Asp Lys Leu Lys

290 295 300

Gln Leu Leu His Lys His Gln Arg Lys Ser Ile Phe Lys Ile Lys Lys

305 310 315 320

Lys Ser Leu Ser Thr Ser Ile Val Trp Ile Glu Asp Ser Ser Ser Leu

325 330 335

Lys Leu Gly Val Leu Asn Lys Ile Thr Leu Gly Asp Ser Ile Met Lys

340 345 350

Pro Val Ser

355

<210>19

<211>969

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>19

atggatccaa ccatctcaac cttggacaca gaactgacac caatcaacgg aactgaggag 60

actctttgct acaagcagac cttgagcctc acggtgctga cgtgcatcgt ttcccttgtc 120

gggctgacag gaaacgcagt tgtgctctgg ctcctgggct gccgcatgcg caggaacgcc 180

ttctccatct acatcctcaa cttggccgca gcagacttcc tcttcctcag cggccgcctt 240

atatattccc tgttaagctt catcagtatc ccccatacca tctctaaaat cctctatcct 300

gtgatgatgt tttcctactt tgcaggcctg agctttctga gtgccgtgag caccgagcgc 360

tgcctgtccg tcctgtggcc catctggtac cgctgccacc gccccacaca cctgtcagcg 420

gtggtgtgtg tcctgctctg ggccctgtcc ctgctgcgga gcatcctgga gtggatgtta 480

tgtggcttcc tgttcagtgg tgctgattct gcttggtgtc aaacatcaga tttcatcaca 540

gtcgcgtggc tgattttttt atgtgtggtt ctctgtgggt ccagcctggt cctgctgatc 600

aggattctct gtggatcccg gaagataccg ctgaccaggc tgtacgtgac catcctgctc 660

acagtactgg tcttcctcct ctgtggcctg ccctttggca ttcagttttt cctattttta 720

tggatccacg tggacaggga agtcttattt tgtcatgttc atctagtttc tattttcctg 780

tccgctctta acagcagtgc caaccccatc atttacttct tcgtgggctc ctttaggcag 840

cgtcaaaata ggcagaacct gaagctggtt ctccagaggg ctctgcagga cgcgtctgag 900

gtggatgaag gtggagggca gcttcctgag gaaatcctgg agctgtcggg aagcagattg 960

gagcagtga 969

<210>20

<211>322

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>20

Met Asp Pro Thr Ile Ser Thr Leu Asp Thr Glu Leu Thr Pro Ile Asn

1 5 10 15

Gly Thr Glu Glu Thr Leu Cys Tyr Lys Gln Thr Leu Ser Leu Thr Val

20 25 30

Leu Thr Cys Ile Val Ser Leu Val Gly Leu Thr Gly Asn Ala Val Val

35 40 45

Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Phe Ser Ile Tyr

50 55 60

Ile Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser Gly Arg Leu

65 70 75 80

Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile Pro His Thr Ile Ser Lys

85 90 95

Ile Leu Tyr Pro Val Met Met Phe Ser Tyr Phe Ala Gly Leu Ser Phe

100 105 110

Leu Ser Ala Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile

115 120 125

Trp Tyr Arg Cys His Arg Pro Thr His Leu Ser Ala Val Val Cys Val

130 135 140

Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Leu

145 150 155 160

Cys Gly Phe Leu Phe Ser Gly Ala Asp Ser Ala Trp Cys Gln Thr Ser

165 170 175

Asp Phe Ile Thr Val Ala Trp Leu Ile Phe Leu Cys Val Val Leu Cys

180 185 190

Gly Ser Ser Leu Val Leu Leu Ile Arg Ile Leu Cys Gly Ser Arg Lys

195 200 205

Ile Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu Val

210 215 220

Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Phe Phe Leu Phe Leu

225 230 235 240

Trp Ile His Val Asp Arg Glu Val Leu Phe Cys His Val His Leu Val

245 250 255

Ser Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr

260 265 270

Phe Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys

275 280 285

Leu Val Leu Gln Arg Ala Leu Gln Asp Ala Ser Glu Val Asp Glu Gly

290 295 300

Gly Gly Gln Leu Pro Glu Glu Ile Leu Glu Leu Ser Gly Ser Arg Leu

305 310 315 320

Glu Gln

<210>21

<211>1305

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>21

atggaggatc tctttagccc ctcaattctg ccgccggcgc ccaacatttc cgtgcccatc 60

ttgctgggct ggggtctcaa cctgaccttg gggcaaggag cccctgcctc tgggccgccc 120

agccgccgcg tccgcctggt gttcctgggg gtcatcctgg tggtggcggt ggcaggcaac 180

accacagtgc tgtgccgcct gtgcggcggc ggcgggccct gggcgggccc caagcgtcgc 240

aagatggact tcctgctggt gcagctggcc ctggcggacc tgtacgcgtg cgggggcacg 300

gcgctgtcac agctggcctg ggaactgctg ggcgagcccc gcgcggccac gggggacctg 360

gcgtgccgct tcctgcagct gctgcaggca tccgggcggg gcgcctcggc ccacctcgtg 420

gtgctcatcg ccctcgagcg ccggcgcgcg gtgcgtcttc cgcacggccg gccgctgccc 480

gcgcgtgccc tcgccgccct gggctggctg ctggcactgc tgctggcgct gcccccggcc 540

ttcgtggtgc gcggggactc cccctcgccg ctgccgccgc cgccgccgcc aacgtccctg 600

cagccaggcg cgcccccggc cgcccgcgcc tggccggggg agcgtcgctg ccacgggatc 660

ttcgcgcccc tgccgcgctg gcacctgcag gtctacgcgt tctacgaggc cgtcgcgggc 720

ttcgtcgcgc ctgttacggt cctgggcgtc gcttgcggcc acctactctc cgtctggtgg 780

cggcaccggc cgcaggcccc cgcggctgca gcgccctggt cggcgagccc aggtcgagcc 840

cctgcgccca gcgcgctgcc ccgcgccaag gtgcagagcc tgaagatgag cctgctgctg 900

gcgctgctgt tcgtgggctg cgagctgccc tactttgccg cccggctggc ggccgcgtgg 960

tcgtccgggc ccgcgggaga ctgggaggga gagggcctgt cggcggcgct gcgcgtggtg 1020

gcgatggcca acagcgctct caatcccttc gtctacctct tcttccaggc gggcgactgc 1080

cggctccggc gacagctgcg gaagcggctg ggctctctgt gctgcgcgcc gcagggaggc 1140

gcggaggacg aggaggggcc ccggggccac caggcgctct accgccaacg ctggccccac 1200

cctcattatc accatgctcg gcgggaaccg ctggacgagg gcggcttgcg cccaccccct 1260

ccgcgcccca gacccctgcc ttgctcctgc gaaagtgcct tctag 1305

<210>22

<211>434

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>22

Met Glu Asp Leu Phe Ser Pro Ser Ile Leu Pro Pro Ala Pro Asn Ile

1 5 10 15

Ser Val Pro Ile Leu Leu Gly Trp Gly Leu Asn Leu Thr Leu Gly Gln

20 25 30

Gly Ala Pro Ala Ser Gly Pro Pro Ser Arg Arg Val Arg Leu Val Phe

35 40 45

Leu Gly Val Ile Leu Val Val Ala Val Ala Gly Asn Thr Thr Val Leu

50 55 60

Cys Arg Leu Cys Gly Gly Gly Gly Pro Trp Ala Gly Pro Lys Arg Arg

65 70 75 80

Lys Met Asp Phe Leu Leu Val Gln Leu Ala Leu Ala Asp Leu Tyr Ala

85 90 95

Cys Gly Gly Thr Ala Leu Ser Gln Leu Ala Trp Glu Leu Leu Gly Glu

100 105 110

Pro Arg Ala Ala Thr Gly Asp Leu Ala Cys Arg Phe Leu Gln Leu Leu

115 120 125

Gln Ala Ser Gly Arg Gly Ala Ser Ala His Leu Val Val Leu Ile Ala

130 135 140

Leu Glu Arg Arg Arg Ala Val Arg Leu Pro His Gly Arg Pro Leu Pro

145 150 155 160

Ala Arg Ala Leu Ala Ala Leu Gly Trp Leu Leu Ala Leu Leu Leu Ala

165 170 175

Leu Pro Pro Ala Phe Val Val Arg Gly Asp Ser Pro Ser Pro Leu Pro

180 185 190

Pro Pro Pro Pro Pro Thr Ser Leu Gln Pro Gly Ala Pro Pro Ala Ala

195 200 205

Arg Ala Trp Pro Gly Glu Arg Arg Cys His Gly Ile Phe Ala Pro Leu

210 215 220

Pro Arg Trp His Leu Gln Val Tyr Ala Phe Tyr Glu Ala Val Ala Gly

225 230 235 240

Phe Val Ala Pro Val Thr Val Leu Gly Val Ala Cys Gly His Leu Leu

245 250 255

Ser Val Trp Trp Arg His Arg Pro Gln Ala Pro Ala Ala Ala Ala Pro

260 265 270

Trp Ser Ala Ser Pro Gly Arg Ala Pro Ala Pro Ser Ala Leu Pro Arg

275 280 285

Ala Lys Val Gln Ser Leu Lys Met Ser Leu Leu Leu Ala Leu Leu Phe

290 295 300

Val Gly Cys Glu Leu Pro Tyr Phe Ala Ala Arg Leu Ala Ala Ala Trp

305 310 315 320

Ser Ser Gly Pro Ala Gly Asp Trp Glu Gly Glu Gly Leu Ser Ala Ala

325 330 335

Leu Arg Val Val Ala Met Ala Asn Ser Ala Leu Asn Pro Phe Val Tyr

340 345 350

Leu Phe Phe Gln Ala Gly Asp Cys Arg Leu Arg Arg Gln Leu Arg Lys

355 360 365

Arg Leu Gly Ser Leu Cys Cys Ala Pro Gln Gly Gly Ala Glu Asp Glu

370 375 380

Glu Gly Pro Arg Gly His Gln Ala Leu Tyr Arg Gln Arg Trp Pro His

385 390 395 400

Pro His Tyr His His Ala Arg Arg Glu Pro Leu Asp Glu Gly Gly Leu

405 410 415

Arg Pro Pro Pro Pro Arg Pro Arg Pro Leu Pro Cys Ser Cys Glu Ser

420 425 430

Ala Phe

<210>23

<211>1041

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>23

atgtacaacg ggtcgtgctg ccgcatcgag ggggacacca tctcccaggt gatgccgccg 60

ctgctcattg tggcctttgt gctgggcgca ctaggcaatg gggtcgccct gtgtggtttc 120

tgcttccaca tgaagacctg gaagcccagc actgtttacc ttttcaattt ggccgtggct 180

gatttcctcc ttatgatctg cctgcctttt cggacagact attacctcag acgtagacac 240

tgggcttttg gggacattcc ctgccgagtg gggctcttca cgttggccat gaacagggcc 300

gggagcatcg tgttccttac ggtggtggct gcggacaggt atttcaaagt ggtccacccc 360

caccacgcgg tgaacactat ctccacccgg gtggcggctg gcatcgtctg caccctgtgg 420

gccctggtca tcctgggaac agtgtatctt ttgctggaga accatctctg cgtgcaagag 480

acggccgtct cctgtgagag cttcatcatg gagtcggcca atggctggca tgacatcatg 540

ttccagctgg agttctttat gcccctcggc atcatcttat tttgctcctt caagattgtt 600

tggagcctga ggcggaggca gcagctggcc agacaggctc ggatgaagaa ggcgacccgg 660

ttcatcatgg tggtggcaat tgtgttcatc acatgctacc tgcccagcgt gtctgctaga 720

ctctatttcc tctggacggt gccctcgagt gcctgcgatc cctctgtcca tggggccctg 780

cacataaccc tcagcttcac ctacatgaac agcatgctgg atcccctggt gtattatttt 840

tcaagcccct cctttcccaa attctacaac aagctcaaaa tctgcagtct gaaacccaag 900

cagccaggac actcaaaaac acaaaggccg gaagagatgc caatttcgaa cctcggtcgc 960

aggagttgca tcagtgtggc aaatagtttc caaagccagt ctgatgggca atgggatccc 1020

cacattgttg agtggcactg a 1041

<210>24

<211>346

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>24

Met Tyr Asn Gly Ser Cys Cys Arg Ile Glu Gly Asp Thr Ile Ser Gln

1 5 10 15

Val Met Pro Pro Leu Leu Ile Val Ala Phe Val Leu Gly Ala Leu Gly

20 25 30

Asn Gly Val Ala Leu Cys Gly Phe Cys Phe His Met Lys Thr Trp Lys

35 40 45

Pro Ser Thr Val Tyr Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu

50 55 60

Met Ile Cys Leu Pro Phe Arg Thr Asp Tyr Tyr Leu Arg Arg Arg His

65 70 75 80

Trp Ala Phe Gly Asp Ile Pro Cys Arg Val Gly Leu Phe Thr Leu Ala

85 90 95

Met Asn Arg Ala Gly Ser Ile Val Phe Leu Thr Val Val Ala Ala Asp

100 105 110

Arg Tyr Phe Lys Val Val His Pro His His Ala Val Asn Thr Ile Ser

115 120 125

Thr Arg Val Ala Ala Gly Ile Val Cys Thr Leu Trp Ala Leu Val Ile

130 135 140

Leu Gly Thr Val Tyr Leu Leu Leu Glu Asn His Leu Cys Val Gln Glu

145 150 155 160

Thr Ala Val Ser Cys Glu Ser Phe Ile Met Glu Ser Ala Asn Gly Trp

165 170 175

His Asp Ile Met Phe Gln Leu Glu Phe Phe Met Pro Leu Gly Ile Ile

180 185 190

Leu Phe Cys Ser Phe Lys Ile Val Trp Ser Leu Arg Arg Arg Gln Gln

195 200 205

Leu Ala Arg Gln Ala Arg Met Lys Lys Ala Thr Arg Phe Ile Met Val

210 215 220

Val Ala Ile Val Phe Ile Thr Cys Tyr Leu Pro Ser Val SerAla Arg

225 230 235 240

Leu Tyr Phe Leu Trp Thr Val Pro Ser Ser Ala Cys Asp Pro Ser Val

245 250 255

His Gly Ala Leu His Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met

260 265 270

Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Lys Phe

275 280 285

Tyr Asn Lys Leu Lys Ile Cys Ser Leu Lys Pro Lys Gln Pro Gly His

290 295 300

Ser Lys Thr Gln Arg Pro Glu Glu Met Pro Ile Ser Asn Leu Gly Arg

305 310 315 320

Arg Ser Cys Ile Ser Val Ala Asn Ser Phe Gln Ser Gln Ser Asp Gly

325 330 335

Gln Trp Asp Pro His Ile Val Glu Trp His

340 345

<210>25

<211>1011

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>25

atgaacaaca atacaacatg tattcaacca tctatgatct cttccatggc tttaccaatc 60

atttacatcc tcctttgtat tgttggtgtt tttggaaaca ctctctctca atggatattt 120

ttaacaaaaa taggtaaaaa aacatcaacg cacatctacc tgtcacacct tgtgactgca 180

aacttacttg tgtgcagtgc catgcctttc atgagtatct atttcctgaa aggtttccaa 240

tgggaatatc aatctgctca atgcagagtg gtcaattttc tgggaactct atccatgcat 300

gcaagtatgt ttgtcagtct cttaatttta agttggattg ccataagccg ctatgctacc 360

ttaatgcaaa aggattcctc gcaagagact acttcatgct atgagaaaat attttatggc 420

catttactga aaaaatttcg ccagcccaac tttgctagaa aactatgcat ttacatatgg 480

ggagttgtac tgggcataat cattccagtt accgtatact actcagtcat agaggctaca 540

gaaggagaag agagcctatg ctacaatcgg cagatggaac taggagccat gatctctcag 600

attgcaggtc tcattggaac cacatttatt ggattttcct ttttagtagt actaacatca 660

tactactctt ttgtaagcca tctgagaaaa ataagaacct gtacgtccat tatggagaaa 720

gatttgactt acagttctgt gaaaagacat cttttggtca tccagattct actaatagtt 780

tgcttccttc cttatagtat ttttaaaccc attttttatg ttctacacca aagagataac 840

tgtcagcaat tgaattattt aatagaaaca aaaaacattc tcacctgtct tgcttcggcc 900

agaagtagca cagaccccat tatatttctt ttattagata aaacattcaa gaagacacta 960

tataatctct ttacaaagtc taattcagca catatgcaat catatggttg a 1011

<210>26

<211>336

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>26

Met Asn Asn Asn Thr Thr Cys Ile Gln Pro Ser Met Ile Ser Ser Met

1 5 10 15

Ala Leu Pro Ile Ile Tyr Ile Leu Leu Cys Ile Val Gly Val Phe Gly

20 25 30

Asn Thr Leu Ser Gln Trp Ile Phe Leu Thr Lys Ile Gly Lys Lys Thr

35 40 45

Ser Thr His Ile Tyr Leu Ser His Leu Val Thr Ala Asn Leu Leu Val

50 55 60

Cys Ser Ala Met Pro Phe Met Ser Ile Tyr Phe Leu Lys Gly Phe Gln

65 70 75 80

Trp Glu Tyr Gln Ser Ala Gln Cys Arg Val Val Asn Phe Leu Gly Thr

85 90 95

Leu Ser Met His Ala Ser Met Phe Val Ser Leu Leu Ile Leu Ser Trp

100 105 110

Ile Ala Ile Ser Arg Tyr Ala Thr Leu Met Gln Lys Asp Ser Ser Gln

115 120 125

Glu Thr Thr Ser Cys Tyr Glu Lys Ile Phe Tyr Gly His Leu Leu Lys

130 135 140

Lys Phe Arg Gln Pro Asn Phe Ala Arg Lys Leu Cys Ile Tyr Ile Trp

145 150 155 160

Gly Val Val Leu Gly Ile Ile Ile Pro Val Thr Val Tyr Tyr Ser Val

165 170 175

Ile Glu Ala Thr Glu Gly Glu Glu Ser Leu Cys Tyr Asn Arg Gln Met

180 185 190

Glu Leu Gly Ala Met Ile Ser Gln Ile Ala Gly Leu Ile Gly Thr Thr

195 200 205

Phe Ile Gly Phe Ser Phe Leu Val Val Leu Thr Ser Tyr Tyr Ser Phe

210 215 220

Val Ser His Leu Arg Lys Ile Arg Thr Cys Thr Ser Ile Met Glu Lys

225 230 235 240

Asp Leu Thr Tyr Ser Ser Val Lys Arg His Leu Leu Val Ile Gln Ile

245 250 255

Leu Leu Ile Val Cys Phe Leu Pro Tyr Ser Ile Phe Lys Pro Ile Phe

260 265 270

Tyr Val Leu His Gln Arg Asp Asn Cys Gln Gln Leu Asn Tyr Leu Ile

275 280 285

Glu Thr Lys Asn Ile Leu Thr Cys Leu Ala Ser Ala Arg Ser Ser Thr

290 295 300

Asp Pro Ile Ile Phe Leu Leu Leu Asp Lys Thr Phe Lys Lys Thr Leu

305 310 315 320

Tyr Asn Leu Phe Thr Lys Ser Asn Ser Ala His Met Gln Ser Tyr Gly

325 330 335

<210>27

<211>1014

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>27

atgaatgagc cactagacta tttagcaaat gcttctgatt tccccgatta tgcagctgct 60

tttggaaatt gcactgatga aaacatccca ctcaagatgc actacctccc tgttatttat 120

ggcattatct tcctcgtggg atttccaggc aatgcagtag tgatatccac ttacattttc 180

aaaatgagac cttggaagag cagcaccatc attatgctga acctggcctg cacagatctg 240

ctgtatctga ccagcctccc cttcctgatt cactactatg ccagtggcga aaactggatc 300

tttggagatt tcatgtgtaa gtttatccgc ttcagcttcc atttcaacct gtatagcagc 360

atcctcttcc tcacctgttt cagcatcttc cgctactgtg tgatcattca cccaatgagc 420

tgcttttcca ttcacaaaac tcgatgtgca gttgtagcct gtgctgtggt gtggatcatt 480

tcactggtag ctgtcattcc gatgaccttc ttgatcacat caaccaacag gaccaacaga 540

tcagcctgtc tcgacctcac cagttcggat gaactcaata ctattaagtg gtacaacctg 600

attttgactg caactacttt ctgcctcccc ttggtgatag tgacactttg ctataccacg 660

attatccaca ctctgaccca tggactgcaa actgacagct gccttaagca gaaagcacga 720

aggctaacca ttctgctact ccttgcattt tacgtatgtt ttttaccctt ccatatcttg 780

agggtcattc ggatcgaatc tcgcctgctt tcaatcagtt gttccattga gaatcagatc 840

catgaagctt acatcgtttc tagaccatta gctgctctga acacctttgg taacctgtta 900

ctatatgtgg tggtcagcga caactttcag caggctgtct gctcaacagt gagatgcaaa 960

gtaagcggga accttgagca agcaaagaaa attagttact caaacaaccc ttga 1014

<210>28

<211>337

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>28

Met Asn Glu Pro Leu Asp Tyr Leu Ala Asn Ala Ser Asp Phe Pro Asp

1 5 10 15

Tyr Ala Ala Ala Phe Gly Asn Cys Thr Asp Glu Asn Ile Pro Leu Lys

20 25 30

Met His Tyr Leu Pro Val Ile Tyr Gly Ile Ile Phe Leu Val Gly Phe

35 40 45

Pro Gly Asn Ala Val Val Ile Ser Thr Tyr Ile Phe Lys Met Arg Pro

50 55 60

Trp Lys Ser Ser Thr Ile Ile Met Leu Asn Leu Ala Cys Thr Asp Leu

65 70 75 80

Leu Tyr Leu Thr Ser Leu Pro Phe Leu Ile His Tyr Tyr Ala Ser Gly

85 90 95

Glu Asn Trp Ile Phe Gly Asp Phe Met Cys Lys Phe Ile Arg Phe Ser

100 105 110

Phe His Phe Asn Leu Tyr Ser Ser Ile Leu Phe Leu Thr Cys Phe Ser

115 120 125

Ile Phe Arg Tyr Cys Val Ile Ile His Pro Met Ser Cys Phe Ser Ile

130 135 140

His Lys Thr Arg Cys Ala Val Val Ala Cys Ala Val Val Trp Ile Ile

145 150 155 160

Ser Leu Val Ala Val Ile Pro Met Thr Phe Leu Ile Thr Ser Thr Asn

165 170 175

Arg Thr Asn Arg Ser Ala Cys Leu Asp Leu Thr Ser Ser Asp Glu Leu

180 185 190

Asn Thr Ile Lys Trp Tyr Asn Leu Ile Leu Thr Ala Thr Thr Phe Cys

195 200 205

Leu Pro Leu Val Ile Val Thr Leu Cys Tyr Thr Thr Ile Ile His Thr

210 215 220

Leu Thr His Gly Leu Gln Thr Asp Ser Cys Leu Lys Gln Lys Ala Arg

225 230 235 240

Arg Leu Thr Ile Leu Leu Leu Leu Ala Phe Tyr Val Cys Phe Leu Pro

245 250 255

Phe His Ile Leu Arg Val Ile Arg Ile Glu Ser Arg Leu Leu Ser Ile

260 265 270

Ser Cys Ser Ile Glu Asn Gln Ile His Glu Ala Tyr Ile Val Ser Arg

275 280 285

Pro Leu Ala Ala Leu Asn Thr Phe Gly Asn Leu Leu Leu Tyr Val Val

290 295 300

Val Ser Asp Asn Phe Gln Gln Ala Val Cys Ser Thr Val Arg Cys Lys

305 310 315 320

Val Ser Gly Asn Leu Glu Gln Ala Lys Lys Ile Ser Tyr Ser Asn Asn

325 330 335

Pro

<210>29

<211>993

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>29

atggatccaa ccaccccggc ctggggaaca gaaagtacaa cagtgaatgg aaatgaccaa 60

gcccttcttc tgctttgtgg caaggagacc ctgatcccgg tcttcctgat ccttttcatt 120

gccctggtcg ggctggtagg aaacgggttt gtgctctggc tcctgggctt ccgcatgcgc 180

aggaacgcct tctctgtcta cgtcctcagc ctggccgggg ccgacttcct cttcctctgc 240

ttccagatta taaattgcct ggtgtacctc agtaacttct tctgttccat ctccatcaat 300

ttccctagct tcttcaccac tgtgatgacc tgtgcctacc ttgcaggcct gagcatgctg 360

agcaccgtca gcaccgagcg ctgcctgtcc gtcctgtggc ccatctggta tcgctgccgc 420

cgccccagac acctgtcagc ggtcgtgtgt gtcctgctct gggccctgtc cctactgctg 480

agcatcttgg aagggaagtt ctgtggcttc ttatttagtg atggtgactc tggttggtgt 540

cagacatttg atttcatcac tgcagcgtgg ctgatttttt tattcatggt tctctgtggg 600

tccagtctgg ccctgctggt caggatcctc tgtggctcca ggggtctgcc actgaccagg 660

ctgtacctga ccatcctgct cacagtgctg gtgttcctcc tctgcggcct gccctttggc 720

attcagtggt tcctaatatt atggatctgg aaggattctg atgtcttatt ttgtcatatt 780

catccagttt cagttgtcct gtcatctctt aacagcagtg ccaaccccat catttacttc 840

ttcgtgggct cttttaggaa gcagtggcgg ctgcagcagc cgatcctcaa gctggctctc 900

cagagggctc tgcaggacat tgctgaggtg gatcacagtg aaggatgctt ccgtcagggc 960

accccggaga tgtcgagaag cagtctggtg tag 993

<210>30

<211>330

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>30

Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn

1 5 10 15

Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile

20 25 30

Pro Val Phe Leu Ile Leu Phe Ile Ala Leu Val Gly Leu Val Gly Asn

35 40 45

Gly Phe Val Leu Trp Leu Leu Gly Phe Arg Met Arg Arg Asn Ala Phe

50 55 60

Ser Val Tyr Val Leu Ser Leu Ala Gly Ala Asp Phe Leu Phe Leu Cys

65 70 75 80

Phe Gln Ile Ile Asn Cys Leu Val Tyr Leu Ser Asn Phe Phe Cys Ser

85 90 95

Ile Ser Ile Asn Phe Pro Ser Phe Phe Thr Thr Val Met Thr Cys Ala

100 105 110

Tyr Leu Ala Gly Leu Ser Met Leu Ser Thr Val Ser Thr Glu Arg Cys

115 120 125

Leu Ser Val Leu Trp Pro Ile Trp Tyr Arg Cys Arg Arg Pro Arg His

130 135 140

Leu Ser Ala Val Val Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Leu

145 150 155 160

Ser Ile Leu Glu Gly Lys Phe Cys Gly Phe Leu Phe Ser Asp Gly Asp

165 170 175

Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile

180 185 190

Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg

195 200 205

Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr

210 215 220

Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly

225 230 235 240

Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu

245 250 255

Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser

260 265 270

Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln

275 280 285

Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala Leu

290 295 300

Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe Arg Gln Gly

305 310 315 320

Thr Pro Glu Met Ser Arg Ser Ser Leu Val

325 330

<210>31

<211>1092

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>31

atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg 60

ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120

tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc tgctggcggc gctggacatg 180

cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa 240

gtcattggct tcctggacac cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300

agcgcagacc agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360

cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420

gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480

ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540

ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc 600

agccactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660

caccccagtg tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720

aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc 780

aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agtggggcat cctcagcaag 840

tgcctgacct acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900

ttccgccaag tcctggccgg catggtgcac cggctgctga agagaacccc gcgcccagca 960

tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020

ccgcgcccag cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080

cagacacact ga 1092

<210>32

<211>363

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>32

Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu

1 5 10 15

Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr

20 25 30

Ser Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu

35 40 45

Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu

50 55 60

Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala Cys Gln

65 70 75 80

Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn Ala Ala Leu Ser

85 90 95

Val Ala Ala Leu Ser Ala Asp Gln Trp Leu Ala Val Gly Phe Pro Leu

100 105 110

Arg Tyr Ala Gly Arg Leu Arg Pro Arg Tyr Ala Gly Leu Leu Leu Gly

115 120 125

Cys Ala Trp Gly Gln Ser Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys

130 135 140

Ser Trp Leu Gly Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu

145 150 155 160

Pro Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu

165 170 175

His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser

180 185 190

Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met Asp Thr

195 200 205

Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu His Pro Ser Val

210 215 220

Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr

225 230 235 240

Arg Lys Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala Pro

245 250 255

Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn

260 265 270

Ala Gln Trp Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val

275 280 285

Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val

290 295 300

Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala

305 310 315 320

Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val His Gln Leu

325 330 335

Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His Asn Gly Ser Val Asp

340 345 350

Thr Glu Asn Asp Ser Cys Leu Gln Gln Thr His

355 360

<210>33

<211>1125

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>33

atgcccacac tcaatacttc tgcctctcca cccacattct tctgggccaa tgcctccgga 60

ggcagtgtgc tgagtgctga tgatgctccg atgcctgtca aattcctagc cctgaggctc 120

atggttgccc tggcctatgg gcttgtgggg gccattggct tgctgggaaa tttggcggtg 180

ctgtgggtac tgagtaactg tgcccggaga gcccctggcc caccttcaga caccttcgtc 240

ttcaacctgg ctctggcgga cctgggactg gcactcactc tccccttttg ggcagccgag 300

tcggcactgg actttcactg gcccttcgga ggtgccctct gcaagatggt tctgacggcc 360

actgtcctca acgtctatgc cagcatcttc ctcatcacag cgctgagcgt tgctcgctac 420

tgggtggtgg ccatggctgc ggggccaggc acccacctct cactcttctg ggcccgaata 480

gccaccctgg cagtgtgggc ggcggctgcc ctggtgacgg tgcccacagc tgtcttcggg 540

gtggagggtg aggtgtgtgg tgtgcgcctt tgcctgctgc gtttccccag caggtactgg 600

ctgggggcct accagctgca gagggtggtg ctggctttca tggtgccctt gggcgtcatc 660

accaccagct acctgctgct gctggccttc ctgcagcggc ggcaacggcg gcggcaggac 720

agcagggtcg tggcccgctc tgtccgcatc ctggtggctt ccttcttcct ctgctggttt 780

cccaaccatg tggtcactct ctggggtgtc ctggtgaagt ttgacctggt gccctggaac 840

agtactttct atactatcca gacgtatgtc ttccctgtca ctacttgctt ggcacacagc 900

aatagctgcc tcaaccctgt gctgtactgt ctcctgaggc gggagccccg gcaggctctg 960

gcaggcacct tcagggatct gcggtcgagg ctgtggcccc agggcggagg ctgggtgcaa 1020

caggtggccc taaagcaggt aggcaggcgg tgggtcgcaa gcaacccccg ggagagccgc 1080

ccttctaccc tgctcaccaa cctggacaga gggacacccg ggtga 1125

<210>34

<211>374

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>34

Met Pro Thr Leu Asn Thr Ser Ala Ser Pro Pro Thr Phe Phe Trp Ala

1 5 10 15

Asn Ala Ser Gly Gly Ser Val Leu Ser Ala Asp Asp Ala Pro Met Pro

20 25 30

Val Lys Phe Leu Ala Leu Arg Leu Met Val Ala Leu Ala Tyr Gly Leu

35 40 45

Val Gly Ala Ile Gly Leu Leu Gly Asn Leu Ala Val Leu Trp Val Leu

50 55 60

Ser Asn Cys Ala Arg Arg Ala Pro Gly Pro Pro Ser Asp Thr Phe Val

65 70 75 80

Phe Asn Leu Ala Leu Ala Asp Leu Gly Leu Ala Leu Thr Leu Pro Phe

85 90 95

Trp Ala Ala Glu Ser Ala Leu Asp Phe His Trp Pro Phe Gly Gly Ala

100 105 110

Leu Cys Lys Met Val Leu Thr Ala Thr Val Leu Asn Val Tyr Ala Ser

115 120 125

Ile Phe Leu Ile Thr Ala Leu Ser Val Ala Arg Tyr Trp Val Val Ala

130 135 140

Met Ala Ala Gly Pro Gly Thr His Leu Ser Leu Phe Trp Ala Arg Ile

145 150 155 160

Ala Thr Leu Ala Val Trp Ala Ala Ala Ala Leu Val Thr Val Pro Thr

165 170 175

Ala Val Phe Gly Val Glu Gly Glu Val Cys Gly Val Arg Leu Cys Leu

180 185 190

Leu Arg Phe Pro Ser Arg Tyr Trp Leu Gly Ala Tyr Gln Leu Gln Arg

195 200 205

Val Val Leu Ala Phe Met Val Pro Leu Gly Val Ile Thr Thr Ser Tyr

210 215 220

Leu Leu Leu Leu Ala Phe Leu Gln Arg Arg Gln Arg Arg Arg Gln Asp

225 230 235 240

Ser Arg Val Val Ala Arg Ser Val Arg Ile Leu Val Ala Ser Phe Phe

245 250 255

Leu Cys Trp Phe Pro Asn His Val Val Thr Leu Trp Gly Val Leu Val

260 265 270

Lys Phe Asp Leu Val Pro Trp Asn Ser Thr Phe Tyr Thr Ile Gln Thr

275 280 285

Tyr Val Phe Pro Val Thr Thr Cys Leu Ala His Ser Asn Ser Cys Leu

290 295 300

Asn Pro Val Leu Tyr Cys Leu Leu Arg Arg Glu Pro Arg Gln Ala Leu

305 310 315 320

Ala Gly Thr Phe Arg Asp Leu Arg Ser Arg Leu Trp Pro Gln Gly Gly

325 330 335

Gly Trp Val Gln Gln Val Ala Leu Lys Gln Val Gly Arg Arg Trp Val

340 345 350

Ala Ser Asn Pro Arg Glu Ser Arg Pro Ser Thr Leu Leu Thr Asn Leu

355 360 365

Asp Arg Gly Thr Pro Gly

370

<210>35

<211>1092

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>35

atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg 60

ttccgagatg acttcattgt caaggtgttg ccgccggtgt tggggctgga gtttatcttc 120

gggcttctgg gcaatggcct tgccctgtgg attttctgtt tccacctcaa gtcctggaaa 180

tccagccgga ttttcctgtt caacctggca gtggctgact ttctactgat catctgcctg 240

cccttcctga tggacaacta tgtgaggcgt tgggactgga agtttgggga catcccttgc 300

cggctgatgc tcttcatgtt ggctatgaac cgccagggca gcatcatctt cctcacggtg 360

gtggcggtag acaggtattt ccgggtggtc catccccacc acgccctgaa caagatctcc 420

aatcggacag cagccatcat ctcttgcctt ctgtggggca tcactattgg cctgacagtc 480

cacctcctga agaagaagat gccgatccag aatggcggtg caaatttgtg cagcagcttc 540

agcatctgcc ataccttcca gtggcacgaa gccatgttcc tcctggagtt cttcctgccc 600

ctgggcatca tcctgttctg ctcagccaga attatctgga gcctgcggca gagacaaatg 660

gaccggcatg ccaagatcaa gagagccatc accttcatca tggtggtggc catcgtcttt 720

gtcatctgct tccttcccag cgtggttgtg cggatccgca tcttctggct cctgcacact 780

tcgggcacgc agaattgtga agtgtaccgc tcggtggacc tggcgttctt tatcactctc 840

agcttcacct acatgaacag catgctggac cccgtggtgt actacttctc cagcccatcc 900

tttcccaact tcttctccac tttgatcaac cgctgcctcc agaggaagat gacaggtgag 960

ccagataata accgcagcac gagcgtcgag ctcacagggg accccaacaa aaccagaggc 1020

gctccagagg cgttaatggc caactccggt gagccatgga gcccctctta tctgggccca 1080

acctctcctt aa 1092

<210>36

<211>363

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>36

Met Asn Arg His His Leu Gln Asp His Phe Leu Glu Ile Asp Lys Lys

1 5 10 15

Asn Cys Cys Val Phe Arg Asp Asp Phe Ile Val Lys Val Leu Pro Pro

20 25 30

Val Leu Gly Leu Glu Phe Ile Phe Gly Leu Leu Gly Asn Gly Leu Ala

35 40 45

Leu Trp Ile Phe Cys Phe His Leu Lys Ser Trp Lys Ser Ser Arg Ile

50 55 60

Phe Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu Ile Ile Cys Leu

65 70 75 80

Pro Phe Leu Met Asp Asn Tyr Val Arg Arg Trp Asp Trp Lys Phe Gly

85 90 95

Asp Ile Pro Cys Arg Leu Met Leu Phe Met Leu Ala Met Asn Arg Gln

100 105 110

Gly Ser Ile Ile Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg

115 120 125

Val Val His Pro His His Ala Leu Asn Lys Ile Ser Asn Arg Thr Ala

130 135 140

Ala Ile Ile Ser Cys Leu Leu Trp Gly Ile Thr Ile Gly Leu Thr Val

145 150 155 160

His Leu Leu Lys Lys Lys Met Pro Ile Gln Asn Gly Gly Ala Asn Leu

165 170 175

Cys Ser Ser Phe Ser Ile Cys His Thr Phe Gln Trp His Glu Ala Met

180 185 190

Phe Leu Leu Glu Phe Phe Leu Pro Leu Gly Ile Ile Leu Phe Cys Ser

195 200 205

Ala Arg Ile Ile Trp Ser Leu Arg Gln Arg Gln Met Asp Arg His Ala

210 215 220

Lys Ile Lys Arg Ala Ile Thr Phe Ile Met Val Val Ala Ile Val Phe

225 230 235 240

Val Ile Cys Phe Leu Pro Ser Val Val Val Arg Ile Arg Ile Phe Trp

245 250 255

Leu Leu His Thr Ser Gly Thr Gln Asn Cys Glu Val Tyr Arg Ser Val

260 265 270

Asp Leu Ala Phe Phe Ile Thr Leu Ser Phe Thr Tyr Met Asn Ser Met

275 280 285

Leu Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Asn Phe

290 295 300

Phe Ser Thr Leu Ile Asn Arg Cys Leu Gln Arg Lys Met Thr Gly Glu

305 310 315 320

Pro Asp Asn Asn Arg Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn

325 330 335

Lys Thr Arg Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro

340 345 350

Trp Ser Pro Ser Tyr Leu Gly Pro Thr Ser Pro

355 360

<210>37

<211>1044

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>37

atgggggatg agctggcacc ttgccctgtg ggcactacag cttggccggc cctgatccag 60

ctcatcagca agacaccctg catgccccaa gcagccagca acacttcctt gggcctgggg 120

gacctcaggg tgcccagctc catgctgtac tggcttttcc ttccctcaag cctgctggct 180

gcagccacac tggctgtcag ccccctgctg ctggtgacca tcctgcggaa ccaacggctg 240

cgacaggagc cccactacct gctcccggct aacatcctgc tctcagacct ggcctacatt 300

ctcctccaca tgctcatctc ctccagcagc ctgggtggct gggagctggg ccgcatggcc 360

tgtggcattc tcactgatgc tgtcttcgcc gcctgcacca gcaccatcct gtccttcacc 420

gccattgtgc tgcacaccta cctggcagtc atccatccac tgcgctacct ctccttcatg 480

tcccatgggg ctgcctggaa ggcagtggcc ctcatctggc tggtggcctg ctgcttcccc 540

acattcctta tttggctcag caagtggcag gatgcccagc tggaggagca aggagcttca 600

tacatcctac caccaagcat gggcacccag ccgggatgtg gcctcctggt cattgttacc 660

tacacctcca ttctgtgcgt tctgttcctc tgcacagctc tcattgccaa ctgtttctgg 720

aggatctatg cagaggccaa gacttcaggc atctgggggc agggctattc ccgggccagg 780

ggcaccctgc tgatccactc agtgctgatc acattgtacg tgagcacagg ggtggtgttc 840

tccctggaca tggtgctgac caggtaccac cacattgact ctgggactca cacatggctc 900

ctggcagcta acagtgaggt actcatgatg cttccccgtg ccatgctccc atacctgtac 960

ctgctccgct accggcagct gttgggcatg gtccggggcc acctcccatc caggaggcac 1020

caggccatct ttaccatttc ctag 1044

<210>38

<211>347

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>38

Met Gly Asp Glu Leu Ala Pro Cys Pro Val Gly Thr Thr Ala Trp Pro

1 5 10 15

Ala Leu Ile Gln Leu Ile Ser Lys Thr Pro Cys Met Pro Gln Ala Ala

20 25 30

Ser Asn Thr Ser Leu Gly Leu Gly Asp Leu Arg Val Pro Ser Ser Met

35 40 45

Leu Tyr Trp Leu Phe Leu Pro Ser Ser Leu Leu Ala Ala Ala Thr Leu

50 55 60

Ala Val Ser Pro Leu Leu Leu Val Thr Ile Leu Arg Asn Gln Arg Leu

65 70 75 80

Arg Gln Glu Pro His Tyr Leu Leu Pro Ala Asn Ile Leu Leu Ser Asp

85 90 95

Leu Ala Tyr Ile Leu Leu His Met Leu Ile Ser Ser Ser Ser Leu Gly

100 105 110

Gly Trp Glu Leu Gly Arg Met Ala Cys Gly Ile Leu Thr Asp Ala Val

115 120 125

Phe Ala Ala Cys Thr Ser Thr Ile Leu Ser Phe Thr Ala Ile Val Leu

130 135 140

His Thr Tyr Leu Ala Val Ile His Pro Leu Arg Tyr Leu Ser Phe Met

145 150 155 160

Ser His Gly Ala Ala Trp Lys Ala Val Ala Leu Ile Trp Leu Val Ala

165 170 175

Cys Cys Phe Pro Thr Phe Leu Ile Trp Leu Ser Lys Trp Gln Asp Ala

180 185 190

Gln Leu Glu Glu Gln Gly Ala Ser Tyr Ile Leu Pro Pro Ser Met Gly

195 200 205

Thr Gln Pro Gly Cys Gly Leu Leu Val Ile Val Thr Tyr Thr Ser Ile

210 215 220

Leu Cys Val Leu Phe Leu Cys Thr Ala Leu Ile Ala Asn Cys Phe Trp

225 230 235 240

Arg Ile Tyr Ala Glu Ala Lys Thr Ser Gly Ile Trp Gly Gln Gly Tyr

245 250 255

Ser Arg Ala Arg Gly Thr Leu Leu Ile His Ser Val Leu Ile Thr Leu

260 265 270

Tyr Val Ser Thr Gly Val Val Phe Ser Leu Asp Met Val Leu Thr Arg

275 280 285

Tyr His His Ile Asp Ser Gly Thr His Thr Trp Leu Leu Ala Ala Asn

290 295 300

Ser Glu Val Leu Met Met Leu Pro Arg Ala Met Leu Pro Tyr Leu Tyr

305 310 315 320

Leu Leu Arg Tyr Arg Gln Leu Leu Gly Met Val Arg Gly His Leu Pro

325 330 335

Ser Arg Arg His Gln Ala Ile Phe Thr Ile Ser

340 345

<210>39

<211>1023

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>39

atgaatccat ttcatgcatc ttgttggaac acctctgccg aacttttaaa caaatcctgg 60

aataaagagt ttgcttatca aactgccagt gtggtagata cagtcatcct cccttccatg 120

attgggatta tctgttcaac agggctggtt ggcaacatcc tcattgtatt cactataata 180

agatccagga aaaaaacagt ccctgacatc tatatctgca acctggctgt ggctgatttg 240

gtccacatag ttggaatgcc ttttcttatt caccaatggg cccgaggggg agagtgggtg 300

tttggggggc ctctctgcac catcatcaca tccctggata cttgtaacca atttgcctgt 360

agtgccatca tgactgtaat gagtgtggac aggtactttg ccctcgtcca accatttcga 420

ctgacacgtt ggagaacaag gtacaagacc atccggatca atttgggcct ttgggcagct 480

tcctttatcc tggcattgcc tgtctgggtc tactcgaagg tcatcaaatt taaagacggt 540

gttgagagtt gtgcttttga tttgacatcc cctgacgatg tactctggta tacactttat 600

ttgacgataa caactttttt tttccctcta cccttgattt tggtgtgcta tattttaatt 660

ttatgctata cttgggagat gtatcaacag aataaggatg ccagatgctg caatcccagt 720

gtaccaaaac agagagtgat gaagttgaca aagatggtgc tggtgctggt ggtagtcttt 780

atcctgagtg ctgcccctta tcatgtgata caactggtga acttacagat ggaacagccc 840

acactggcct tctatgtggg ttattacctc tccatctgtc tcagctatgc cagcagcagc 900

attaaccctt ttctctacat cctgctgagt ggaaatttcc agaaacgtct gcctcaaatc 960

caaagaagag cgactgagaa ggaaatcaac aatatgggaa acactctgaa atcacacttt 1020

tag 1023

<210>40

<211>340

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>40

Met Asn Pro Phe His Ala Ser Cys Trp Asn Thr Ser Ala Glu Leu Leu

1 5 10 15

Asn Lys Ser Trp Asn Lys Glu Phe Ala Tyr Gln Thr Ala Ser Val Val

20 25 30

Asp Thr Val Ile Leu Pro Ser Met Ile Gly Ile Ile Cys Ser Thr Gly

35 40 45

Leu Val Gly Asn Ile Leu Ile Val Phe Thr Ile Ile Arg Ser Arg Lys

50 55 60

Lys Thr Val Pro Asp Ile Tyr Ile Cys Asn Leu Ala Val Ala Asp Leu

65 70 75 80

Val His Ile Val Gly Met Pro Phe Leu Ile His Gln Trp Ala Arg Gly

85 90 95

Gly Glu Trp Val Phe Gly Gly Pro Leu Cys Thr Ile Ile Thr Ser Leu

100 105 110

Asp Thr Cys Asn Gln Phe Ala Cys Ser Ala Ile Met Thr Val Met Ser

115 120 125

Val Asp Arg Tyr Phe Ala Leu Val Gln Pro Phe Arg Leu Thr Arg Trp

130 135 140

Arg Thr Arg Tyr Lys Thr Ile Arg Ile Asn Leu Gly Leu Trp Ala Ala

145 150 155 160

Ser Phe Ile Leu Ala Leu Pro Val Trp Val Tyr Ser Lys Val Ile Lys

165 170 175

Phe Lys Asp Gly Val Glu Ser Cys Ala Phe Asp Leu Thr Ser Pro Asp

180 185 190

Asp Val Leu Trp Tyr Thr Leu Tyr Leu Thr Ile Thr Thr Phe Phe Phe

195 200 205

Pro Leu Pro Leu Ile Leu Val Cys Tyr Ile Leu Ile Leu Cys Tyr Thr

210 215 220

Trp Glu Met Tyr Gln Gln Asn Lys Asp Ala Arg Cys Cys Asn Pro Ser

225 230 235 240

Val Pro Lys Gln Arg Val Met Lys Leu Thr Lys Met Val Leu Val Leu

245 250 255

Val Val Val Phe Ile Leu Ser Ala Ala Pro Tyr His Val Ile Gln Leu

260 265 270

Val Asn Leu Gln Met Glu Gln Pro Thr Leu Ala Phe Tyr Val Gly Tyr

275 280 285

Tyr Leu Ser Ile Cys Leu Ser Tyr Ala Ser Ser Ser Ile Asn Pro Phe

290 295 300

Leu Tyr Ile Leu Leu Ser Gly Asn Phe Gln Lys Arg Leu Pro Gln Ile

305 310 315 320

Gln Arg Arg Ala Thr Glu Lys Glu Ile Asn Asn Met Gly Asn Thr Leu

325 330 335

Lys Ser His Phe

340

<210>41

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>41

cttgcagaca tcaccatggc agcc 24

<210>42

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>42

gtgatgctct gagtactgga ctgg 24

<210>43

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>43

gaagctgtga agagtgatgc 20

<210>44

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>44

gtcagcaata ttgataagca gcag 24

<210>45

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>45

ccatggggaa cgattctgtc agctacg 27

<210>46

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>46

gctatgcctg aagccagtct tgtg 24

<210>47

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>47

ccaggatgtt gtgtcaccgt ggtggc 26

<210>48

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>48

cacagcgctg cagccctgca gctggc 26

<210>49

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>49

cttcctctcg tagggatgaa ccagac 26

<210>50

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>50

ctcgcacagg tgggaagcac ctgtgg 26

<210>51

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>51

gcctgtgaca ggaggtaccc tgg 23

<210>52

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>52

catatccctc cgagtgtcca gcggc 25

<210>53

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>53

gcatggagag aaaatttatg tccttgcaac c 31

<210>54

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>54

caagaacagg tctcatctaa gagctcc 27

<210>55

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>55

gctgttgcca tgacgtccac ctgcac 26

<210>56

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>56

ggacagttca aggtttgcct tagaac 26

<210>57

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>57

ctttcgatac tgctcctatg ctc 23

<210>58

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>58

gtagtccact gaaagtccag tgatcc 26

<210>59

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>59

tttctgagca tggatccaac catctc 26

<210>60

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>60

ctgtctgaca gggcagaggc tcttc 25

<210>61

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>61

ggaactcgta tagacccagc gtcgctcc 28

<210>62

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>62

ggaggttgcg ccttagcgac agatgacc 28

<210>63

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>63

ctgcacccgg acacttgctc tg 22

<210>64

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>64

gtctgcttgt tcagtgccac tcaac 25

<210>65

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>65

tatctgcaat tctattctag ctcctg 26

<210>66

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>66

tgtccctaat aaagtcacat gaatgc 26

<210>67

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>67

ggagacaacc atgaatgagc cac 23

<210>68

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>68

tatttcaagg gttgtttgag taac 24

<210>69

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>69

ggcaccagtg gaggttttct gagcatg 27

<210>70

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>70

ctgatggaag tagaggctgt ccatctc 27

<210>71

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>71

cctggcgagc cgctagcgcc atg 23

<210>72

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>72

atgagccctg ccaggccctc agt 23

<210>73

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>73

ctgcgatgcc cacactcaat acttctg 27

<210>74

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>74

aaggatccta cacttggtgg atctcag 27

<210>75

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>75

gctggagcat tcactaggcg ag 22

<210>76

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>76

agatcctggt tcttggtgac aatg 24

<210>77

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>77

agcca tccct gccaggaagc atgg 24

<210>78

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>78

ccagactgtg gactcaagaa ctctagg 27

<210>79

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>79

agtccacgaa caatgaatcc atttcatg 28

<210>80

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>80

atcatgtcta gactcatggt gatcc 25

<210>81

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>81

ggggagggaa agcaaaggtg gtcctcctgg 30

<210>82

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>82

ccaggagaac cacctttgct ttccctcccc 30

<210>83

<211>1356

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>83

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcaaaggt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcatga 1356

<210>84

<211>451

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>84

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Lys Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser

450

<210>85

<211>28

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>85

caggaaggca aagaccacca tcatcatc 28

<210>86

<211>28

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>86

gatgatgatg gtggtctttg ccttcctg 28

<210>87

<211>1041

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>87

atggagagaa aatttatgtc cttgcaacca tccatctccg tatcagaaat ggaaccaaat 60

ggcaccttca gcaataacaa cagcaggaac tgcacaattg aaaacttcaa gagagaattt 120

ttcccaattg tatatctgat aatatttttc tggggagtct tgggaaatgg gttgtccata 180

tatgttttcc tgcagcctta taagaagtcc acatctgtga acgttttcat gctaaatctg 240

gccatttcag atctcctgtt cataagcacg cttcccttca gggctgacta ttatcttaga 300

ggctccaatt ggatatttgg agacctggcc tgcaggatta tgtcttattc cttgtatgtc 360

aacatgtaca gcagtattta tttcctgacc gtgctgagtg ttgtgcgttt cctggcaatg 420

gttcacccct ttcggcttct gcatgtcacc agcatcagga gtgcctggat cctctgtggg 480

atcatatgga tccttatcat ggcttcctca ataatgctcc tggacagtgg ctctgagcag 540

aacggcagtg tcacatcatg cttagagctg aatctctata aaattgctaa gctgcagacc 600

atgaactata ttgccttggt ggtgggctgc ctgctgccat ttttcacact cagcatctgt 660

tatctgctga tcattcgggt tctgttaaaa gtggaggtcc cagaatcggg gctgcgggtt 720

tctcacagga aggcaaagac caccatcatc atcaccttga tcatcttctt cttgtgtttc 780

ctgccctatc acacactgag gaccgtccac ttgacgacat ggaaagtggg tttatgcaaa 840

gacagactgc ataaagcttt ggttatcaca ctggccttgg cagcagccaa tgcctgcttc 900

aatcctctgc tctattactt tgctggggag aattttaagg acagactaaa gtctgcactc 960

agaaaaggcc atccacagaa ggcaaagaca aagtgtgttt tccctgttag tgtgtggttg 1020

agaaaggaaa caagagtata a 1041

<210>88

<211>346

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>88

Met Glu Arg Lys Phe MetSer Leu Gln Pro Ser Ile Ser Val Ser Glu

1 5 10 15

Met Glu Pro Asn Gly Thr Phe Ser Asn Asn Asn Ser Arg Asn Cys Thr

20 25 30

Ile Glu Asn Phe Lys Arg Glu Phe Phe Pro Ile Val Tyr Leu Ile Ile

35 40 45

Phe Phe Trp Gly Val Leu Gly Asn Gly Leu Ser Ile Tyr Val Phe Leu

50 55 60

Gln Pro Tyr Lys Lys Ser Thr Ser Val Asn Val Phe Met Leu Asn Leu

65 70 75 80

Ala Ile Ser Asp Leu Leu Phe Ile Ser Thr Leu Pro Phe Arg Ala Asp

85 90 95

Tyr Tyr Leu Arg Gly Ser Asn Trp Ile Phe Gly Asp Leu Ala Cys Arg

100 105 110

Ile Met Ser Tyr Ser Leu Tyr Val Asn Met Tyr Ser Ser Ile Tyr Phe

115 120 125

Leu Thr Val Leu Ser Val Val Arg Phe Leu Ala Met Val His Pro Phe

130 135 140

Arg Leu Leu His Val Thr Ser Ile Arg Ser Ala Trp Ile Leu Cys Gly

145 150 155 160

Ile Ile Trp Ile Leu Ile Met Ala Ser Ser Ile Met Leu Leu Asp Ser

165 170 175

Gly Ser Glu Gln Asn Gly Ser Val Thr Ser Cys Leu Glu Leu Asn Leu

180 185 190

Tyr Lys Ile Ala Lys Leu Gln Thr Met Asn Tyr Ile Ala Leu Val Val

195 200 205

Gly Cys Leu Leu Pro Phe Phe Thr Leu Ser Ile Cys Tyr Leu Leu Ile

210 215 220

Ile Arg Val Leu Leu Lys Val Glu Val Pro Glu Ser Gly Leu Arg Val

225 230 235 240

Ser His Arg Lys Ala Lys Thr Thr Ile Ile Ile Thr Leu Ile Ile Phe

245 250 255

Phe Leu Cys Phe Leu Pro Tyr His Thr Leu Arg Thr Val His Leu Thr

260 265 270

Thr Trp Lys Val Gly Leu Cys Lys Asp Arg Leu His Lys Ala Leu Val

275 280 285

Ile Thr Leu Ala Leu Ala Ala Ala Asn Ala Cys Phe Asn Pro Leu Leu

290 295 300

Tyr Tyr Phe Ala Gly Glu Asn Phe Lys Asp Arg Leu Lys Ser Ala Leu

305 310 315 320

Arg Lys Gly His Pro Gln Lys Ala Lys Thr Lys Cys Val Phe Pro Val

325 330 335

Ser Val Trp Leu Arg Lys Glu Thr Arg Val

340 345

<210>89

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400)89

ccagtgcaaa gctaagaaag tgatcttc 28

<210>90

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>90

gaagatcact ttcttagctt tgcactgg 28

<210>91

<211>1527

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>91

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc taagaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tccttga 1527

<210>92

<211>508

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>92

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 140

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Lys Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro

500 505

<210>93

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>93

gccgccaccg cgccaagagg aagattggc 29

<210>94

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>94

gccaatcttc ctcttggcgc ggtggcggc 29

<210>95

<211>1092

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>95

atgggccccg gcgaggcgct gctggcgggt ctcctggtga tggtactggc cgtggcgctg 60

ctatccaacg cactggtgct gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120

tcaggcgtcc tcctggtgaa tctgtcgctg ggccacctgc tgctggcggc gctggacatg 180

cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa 240

gtcattggct tcctggacac cttcctggcg tccaacgcgg cgctgagcgt ggcggcgctg 300

agcgcagacc agtggctggc agtgggcttc ccactgcgct acgccggacg cctgcgaccg 360

cgctatgccg gcctgctgct gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420

gcacttggct gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480

ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540

ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc 600

agccactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct cgccgacctg 660

caccccagtg tgcggcagcg ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720

aggaagattg gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc 780

aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agaagggcat cctcagcaag 840

tgcctgacct acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900

ttccgccaag tcctggccgg catggtgcac cggctgctga agagaacccc gcgcccagca 960

tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020

ccgcgcccag cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag 1080

cagacacact ga 1092

<210>96

<211>363

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>96

Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu

1 5 10 15

Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala Tyr

20 25 30

Ser Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu Val Asn Leu

35 40 45

Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met Pro Phe Thr Leu

50 55 60

Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala Pro Gly Ala Cys Gln

65 70 75 80

Val Ile Gly Phe Leu Asp Thr Phe Leu Ala Ser Asn Ala Ala Leu Ser

85 90 95

Val Ala Ala Leu Ser Ala Asp Gln Trp Leu Ala Val Gly Phe Pro Leu

100 105 110

Arg Tyr Ala Gly Arg Leu Arg Pro Arg Tyr Ala Gly Leu Leu Leu Gly

115 120 125

Cys Ala Trp Gly Gln Ser Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys

130 135 140

Ser Trp Leu Gly Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu

145 150 155 160

Pro Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu

165 170 175

His Ala Val Gly Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser

180 185 190

Leu Gln Val His Arg Val Ala Arg Ser His Cys Gln Arg Met Asp Thr

195 200 205

Val Thr Met Lys Ala Leu Ala Leu Leu Ala Asp Leu His Pro Ser Val

210 215 220

Arg Gln Arg Cys Leu Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr

225 230 235 240

Arg Lys Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala Pro

245 250 255

Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn

260 265 270

Ala Gln Lys Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala Val

275 280 285

Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe Arg Gln Val

290 295 300

Leu Ala Gly Met Val His Arg Leu Leu Lys Arg Thr Pro Arg Pro Ala

305 310 315 320

Ser Thr His Asp Ser Ser Leu Asp Val Ala Gly Met Val His Gln Leu

325 330 335

Leu Lys Arg Thr Pro Arg Pro Ala Ser Thr His Asn Gly Ser Val Asp

340 345 350

Thr Glu Asn Asp Ser Cys Leu Gln Gln Thr His

355 360

<210>97

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>97

gatctctaga atggagtcct cacccatccc ccag 34

<210>98

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>98

gatcgatatc cgtgactcca gccggggtga ggcggc 36

<210>99

<211>2610

<212>DNA

＜213〉homo sapiens (Homo sapiens) and rat

<400>99

atggagtcct cacccatccc ccagtcatca gggaactctt ccactttggg gagggtccct 60

caaaccccag gtccctctac tgccagtggg gtcccggagg tggggctacg ggatgttgct 120

tcggaatctg tggccctctt cttcatgctc ctgctggact tgactgctgt ggctggcaat 180

gccgctgtga tggccgtgat cgccaagacg cctgccctcc gaaaatttgt cttcgtcttc 240

cacctctgcc tggtggacct gctggctgcc ctgaccctca tgcccctggc catgctctcc 300

agctctgccc tctttgacca cgccctcttt ggggaggtgg cctgccgcct ctacttgttt 360

ctgagcgtgt gctttgtcag cctggccatc ctctcggtgt cagccatcaa tgtggagcgc 420

tactattacg tagtccaccc catgcgctac gaggtgcgca tgacgctggg gctggtggcc 480

tctgtgctgg tgggtgtgtg ggtgaaggcc ttggccatgg cttctgtgcc agtgttggga 540

agggtctcct gggaggaagg agctcccagt gtccccccag gctgttcact ccagtggagc 600

cacagtgcct actgccagct ttttgtggtg gtctttgctg tcctttactt tctgttgccc 660

ctgctcctca tacttgtggt ctactgcagc atgttccgag tggcccgcgt ggctgccatg 720

cagcacgggc cgctgcccac gtggatggag acaccccggc aacgctccga atctctcagc 780

agccgctcca cgatggtcac cagctcgggg gccccccaga ccaccccaca ccggacgttt 840

gggggaggga aagcagcagt ggttctcctg gctgtggggg gacagttcct gctctgttgg 900

ttgccctact tctctttcca cctctatgtt gccctgagtg ctcagcccat ttcaactggg 960

caggtggaga gtgtggtcac ctggattggc tacttttgct tcacttccaa ccctttcttc 1020

tatggatgtc tcaaccggca gatccggggg gagctcagca agcagtttgt ctgcttcttc 1080

aagccagctc cagaggagga gctgaggctg cctagccggg agggctccat tgaggagaac 1140

ttcctgcagt tccttcaggg gactggctgt ccttctgagt cctgggtttc ccgaccccta 1200

cccagcccca agcaggagcc acctgctgtt gactttcgaa tcccaggcca gatagctgag 1260

gagacctctg agttcctgga gcagcaactc accagcgaca tcatcatgtc agacagctac 1320

ctccgtcctg ccgcctcacc ccggctggag tcagcgatat ctgcagaatt ccaccacact 1380

ggactagtgg atccgagctc ggtaccaagc ttgggctgca ggtcgatggg ctgcctcggc 1440

aacagtaaga ccgaggacca gcgcaacgag gagaaggcgc agcgcgaggc caacaaaaag 1500

atcgagaagc agctgcagaa ggacaagcag gtctaccggg ccacgcaccg cctgctgctg 1560

ctgggtgctg gagagtctgg caaaagcacc attgtgaagc agatgaggat cctacatgtt 1620

aatgggttta acggagaggg cggcgaagag gacccgcagg ctgcaaggag caacagcgat 1680

ggtgagaagg ccaccaaagt gcaggacatc aaaaacaacc tgaaggaggc cattgaaacc 1740

attgtggccg ccatgagcaa cctggtgccc cccgtggagc tggccaaccc tgagaaccag 1800

ttcagagtgg actacattct gagcgtgatg aacgtgccaa actttgactt cccacctgaa 1860

ttctatgagc atgccaaggc tctgtgggag gatgagggag ttcgtgcctg ctacgagcgc 1920

tccaacgagt accagctgat cgactgtgcc cagtacttcc tggacaagat tgatgtgatc 1980

aagcaggccg actacgtgcc aagtgaccag gacctgcttc gctgccgcgt cctgacctct 2040

ggaatctttg agaccaagtt ccaggtggac aaagtcaact tccacatgtt cgatgtgggc 2100

ggccagcgcg atgaacgccg caagtggatc cagtgcttca atgatgtgac tgccatcatc 2160

ttcgtggtgg ccagcagcag ctacaacatg gtcatccggg aggacaacca gaccaaccgt 2220

ctgcaggagg ctctgaacct cttcaagagc atctggaaca acagatggct gcgtaccatc 2280

tctgtgatcc tcttcctcaa caagcaagat ctgcttgctg agaaggtcct cgctgggaaa 2340

tcgaagattg aggactactt tccagagttc gctcgctaca ccactcctga ggatgcgact 2400

cccgagcccg gagaggaccc acgcgtgacc cgggccaagt acttcatccg ggatgagttt 2460

ctgagaatca gcactgctag tggagatgga cgtcactact gctaccctca ctttacctgc 2520

gccgtggaca ctgagaacat ccgccgtgtc ttcaacgact gccgtgacat catccagcgc 2580

atgcatcttc gccaatacga gctgctctaa 2610

<210>100

<211>869

<212>PRT

＜213〉homo sapiens (Homo sapiens) and rat

<400>100

Met Glu Ser Ser Pro Ile Pro Gln Ser Ser Gly Asn Ser Ser Thr Leu

1 5 10 15

Gly Arg Val Pro Gln Thr Pro Gly Pro Ser Thr Ala Ser Gly Val Pro

20 25 30

Glu Val Gly Leu Arg Asp Val Ala Ser Glu Ser Val Ala Leu Phe Phe

35 40 45

Met Leu Leu Leu Asp Leu Thr Ala Val Ala Gly Asn Ala Ala Val Met

50 55 60

Ala Val Ile Ala Lys Thr Pro Ala Leu Arg Lys Phe Val Phe Val Phe

65 70 75 80

His Leu Cys Leu Val Asp Leu Leu Ala Ala Leu Thr Leu Met Pro Leu

85 90 95

Ala Met Leu Ser Ser Ser Ala Leu Phe Asp His Ala Leu Phe Gly Glu

100 105 110

Val Ala Cys Arg Leu Tyr Leu Phe Leu Ser Val Cys Phe Val Ser Leu

115 120 125

Ala Ile Leu Ser Val Ser Ala Ile Asn Val Glu Arg Tyr Tyr Tyr Val

130 135 140

Val His Pro Met Arg Tyr Glu Val Arg Met Thr Leu Gly Leu Val Ala

145 150 155 160

Ser Val Leu Val Gly Val Trp Val Lys Ala Leu Ala Met Ala Ser Val

165 170 175

Pro Val Leu Gly Arg Val Ser Trp Glu Glu Gly Ala Pro Ser Val Pro

180 185 190

Pro Gly Cys Ser Leu Gln Trp Ser His Ser Ala Tyr Cys Gln Leu Phe

195 200 205

Val Val Val Phe Ala Val Leu Tyr Phe Leu Leu Pro Leu Leu Leu Ile

210 215 220

Leu Val Val Tyr Cys Ser Met Phe Arg Val Ala Arg Val Ala Ala Met

225 230 235 240

Gln His Gly Pro Leu Pro Thr Trp Met Glu Thr Pro Arg Gln Arg Ser

245 250 255

Glu Ser Leu Ser Ser Arg Ser Thr Met Val Thr Ser Ser Gly Ala Pro

260 265 270

Gln Thr Thr Pro His Arg Thr Phe Gly Gly Gly Lys Ala Ala Val Val

275 280 285

Leu Leu Ala Val Gly Gly Gln Phe Leu Leu Cys Trp Leu Pro Tyr Phe

290 295 300

Ser Phe His Leu Tyr Val Ala Leu Ser Ala Gln Pro Ile Ser Thr Gly

305 310 315 320

Gln Val Glu Ser Val Val Thr Trp Ile Gly Tyr Phe Cys Phe Thr Ser

325 330 335

Asn Pro Phe Phe Tyr Gly Cys Leu Asn Arg Gln Ile Arg Gly Glu Leu

340 345 350

Ser Lys Gln Phe Val Cys Phe Phe Lys Pro Ala Pro Glu Glu Glu Leu

355 360 365

Arg Leu Pro Ser Arg Glu Gly Ser Ile Glu Glu Asn Phe Leu Gln Phe

370 375 380

Leu Gln Gly Thr Gly Cys Pro Ser Glu Ser Trp Val Ser Arg Pro Leu

385 390 395 400

Pro Ser Pro Lys Gln Glu Pro Pro Ala Val Asp Phe Arg Ile Pro Gly

405 410 415

Gln Ile Ala Glu Glu Thr Ser Glu Phe Leu Glu Gln Gln Leu Thr Ser

420 425 430

Asp Ile Ile Met Ser Asp Ser Tyr Leu Arg Pro Ala Ala Ser Pro Arg

435 440 445

Leu Glu Ser Ala Ile Ser Ala Glu Phe His His Thr Gly Leu Val Asp

450 455 460

Pro Ser Ser Val Pro Ser Leu Gly Cys Arg Ser Met Gly Cys Leu Gly

465 470 475 480

Asn Ser Lys Thr Glu Asp Gln Arg Asn Glu Glu Lys Ala Gln Arg Glu

485 490 495

Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys Asp Lys Gln Val Tyr

500 505 510

Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys

515 520 525

Ser Thr Ile Val Lys Gln Met Arg Ile Leu His Val Asn Gly Phe Asn

530 535 540

Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala Arg Ser Asn Ser Asp

545 550 555 560

Gly Glu Lys Ala Thr Lys Val Gln Asp Ile Lys Asn Asn Leu Lys Glu

565 570 575

Ala Ile Glu Thr Ile Val Ala Ala Met Ser Asn Leu Val Pro Pro Val

580 585 590

Glu Leu Ala Asn Pro Glu Asn Gln Phe Arg Val Asp Tyr Ile Leu Ser

595 600 605

Val Met Asn Val Pro Asn Phe Asp Phe Pro Pro Glu Phe Tyr Glu His

610 615 620

Ala Lys Ala Leu Trp Glu Asp Glu Gly Val Arg Ala Cys Tyr Glu Arg

625 630 635 640

Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln Tyr Phe Leu Asp Lys

645 650 655

Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro Ser Asp Gln Asp Leu

660 665 670

Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe Glu Thr Lys Phe Gln

675 680 685

Val Asp Lys Val Asn Phe His Met Phe Asp Val Gly Gly Gln Arg Asp

690 695 700

Glu Arg Arg Lys Trp Ile Gln Cys Phe Asn Asp Val Thr Ala Ile Ile

705 710 715 720

Phe Val Val Ala Ser Ser Ser Tyr Asn Met Val Ile Arg Glu Asp Asn

725 730 735

Gln Thr Asn Arg Leu Gln Glu Ala Leu Asn Leu Phe Lys Ser Ile Trp

740 745 750

Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile Leu Phe Leu Asn Lys

755 760 765

Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly Lys Ser Lys Ile Glu

770 775 780

Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr Pro Glu Asp Ala Thr

785 790 795 800

Pro Glu Pro Gly Glu Asp Pro Arg Val Thr Arg Ala Lys Tyr Phe Ile

805 810 815

Arg Asp Glu Phe Leu Arg Ile Ser Thr Ala Ser Gly Asp Gly Arg His

820 825 830

Tyr Cys Tyr Pro His Phe Thr Cys Ala Val Asp Thr Glu Asn Ile Arg

835 840 845

Arg Val Phe Asn Asp Cys Arg Asp Ile Ile Gln Arg Met His Leu Arg

850 855 860

Gln Tyr Glu Leu Leu

865

<210>101

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>101

tctagaatga cgtccacctg caccaacagc 30

<210>102

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>102

gatatcgcag gaaaagtagc agaatcgtag gaag 34

<210>103

<211>2781

<212>DNA

＜213〉homo sapiens (Homo sapiens) and rat

<400>103

atgacgtcca cctgcaccaa cagcacgcgc gagagtaaca gcagccacac gtgcatgccc 60

ctctccaaaa tgcccatcag cctggcccac ggcatcatcc gctcaaccgt gctggttatc 120

ttcctcgccg cctctttcgt cggcaacata gtgctggcgc tagtgttgca gcgcaagccg 180

cagctgctgc aggtgaccaa ccgttttatc tttaacctcc tcgtcaccga cctgctgcag 240

atttcgctcg tggccccctg ggtggtggcc acctctgtgc ctctcttctg gcccctcaac 300

agccacttct gcacggccct ggttagcctc acccacctgt tcgccttcgc cagcgtcaac 360

accattgtcg tggtgtcagt ggatcgctac ttgtccatca tccaccctct ctcctacccg 420

tccaagatga cccagcgccg cggttacctg ctcctctatg gcacctggat tgtggccatc 480

ctgcagagca ctcctccact ctacggctgg ggccaggctg cctttgatga gcgcaatgct 540

ctctgctcca tgatctgggg ggccagcccc agctacacta ttctcagcgt ggtgtccttc 600

atcgtcattc cactgattgt catgattgcc tgctactccg tggtgttctg tgcagcccgg 660

aggcagcatg ctctgctgta caatgtcaag agacacagct tggaagtgcg agtcaaggac 720

tgtgtggaga atgaggatga agagggagca gagaagaagg aggagttcca ggatgagagt 780

gagtttcgcc gccagcatga aggtgaggtc aaggccaagg agggcagaat ggaagccaag 840

gacggcagcc tgaaggccaa ggaaggaagc acggggacca gtgagagtag tgtagaggcc 900

aggggcagcg aggaggtcag agagagcagc acggtggcca gcgacggcag catggagggt 960

aaggaaggca gcaccaaagt tgaggagaac agcatgaagg cagacaaggg tcgcacagag 1020

gtcaaccagt gcagcattga cttgggtgaa gatgacatgg agtttggtga agacgacatc 1080

aatttcagtg aggatgacgt cgaggcagtg aacatcccgg agagcctccc acccagtcgt 1140

cgtaacagca acagcaaccc tcctctgccc aggtgctacc agtgcaaagc tgctaaagtg 1200

atcttcatca tcattttctc ctatgtgcta tccctggggc cctactgctt tttagcagtc 1260

ctggccgtgt gggtggatgt cgaaacccag gtaccccagt gggtgatcac cataatcatc 1320

tggcttttct tcctgcagtg ctgcatccac ccctatgtct atggctacat gcacaagacc 1380

attaagaagg aaatccagga catgctgaag aagttcttct gcaaggaaaa gcccccgaaa 1440

gaagatagcc acccagacct gcccggaaca gagggtggga ctgaaggcaa gattgtccct 1500

tcctacgatt ctgctacttt tcctgcgata tctgcagaat tccaccacac tggactagtg 1560

gatccgagct cggtaccaag cttgggctgc aggtcgatgg gctgcctcgg caacagtaag 1620

accgaggacc agcgcaacga ggagaaggcg cagcgcgagg ccaacaaaaa gatcgagaag 1680

cagctgcaga aggacaagca ggtctaccgg gccacgcacc gcctgctgct gctgggtgct 1740

ggagagtctg gcaaaagcac cattgtgaag cagatgagga tcctacatgt taatgggttt 1800

aacggagagg gcggcgaaga ggacccgcag gctgcaagga gcaacagcga tggtgagaag 1860

gccaccaaag tgcaggacat caaaagcaac ctgaaggagg ccattgaaac cattgtggcc 1920

gccatgagca acctggtgcc ccccgtggag ctggccaacc ctgagaacca gttcagagtg 1980

gactacattc tgagcgtgat gaacgtgcca aactttgact tcccacctga attctatgag 2040

catgccaagg ctctgtggga ggatgaggga gttcgtgcct gctacgagcg ctccaacgag 2100

taccagctga tcgactgtgc ccagtacttc ctggacaaga ttgatgtgat caagcaggcc 2160

gactacgtgc caagtgacca ggacctgctt cgctgccgcg tcctgacctc tggaatcttt 2220

gagaccaagt tccaggtgga caaagtcaac ttccacatgt tcgatgtggg cggccagcgc 2280

gatgaacgcc gcaagtggat ccagtgcttc aatgatgtga ctgccatcat cttcgtggtg 2340

gccagcagca gctacaacat ggtcatccgg gaggacaacc agaccaaccg tctgcaggag 2400

gctctgaacc tcttcaagag catctggaac aacagatggc tgcgtaccat ctctgtgatc 2460

ctcttcctca acaagcaaga tctgcttgct gagaaggtcc tcgctgggaa atcgaagatt 2520

gaggactact ttccagagtt cgctcgctac accactcctg aggatgcgac tcccgagccc 2580

ggagaggacc cacgcgtgac ccgggccaag tacttcatcc gggatgagtt tctgagaatc 2640

agcactgcta gtggagatgg acgtcactac tgctaccctc actttacctg cgccgtggac 2700

actgagaaca tccgccgtgt cttcaacgac tgccgtgaca tcatccagcg catgcatctt 2760

cgccaatacg agctgctcta a 2781

<210>104

<211>926

<212>PRT

＜213〉homo sapiens (Homo sapiens) and rat

<400>104

Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His

1 5 10 15

Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20 25 30

Ile Arg Ser Thr Val Leu Val Ile Phe Leu Ala Ala Ser Phe Val Gly

35 40 45

Asn Ile Val Leu Ala Leu Val Leu Gln Arg Lys Pro Gln Leu Leu Gln

50 55 60

Val Thr Asn Arg Phe Ile Phe Asn Leu Leu Val Thr Asp Leu Leu Gln

65 70 75 80

Ile Ser Leu Val Ala Pro Trp Val Val Ala Thr Ser Val Pro Leu Phe

85 90 95

Trp Pro Leu Asn Ser His Phe Cys Thr Ala Leu Val Ser Leu Thr His

100 105 110

Leu Phe Ala Phe Ala Ser Val Asn Thr Ile Val Val Val Ser Val Asp

115 120 125

Arg Tyr Leu Ser Ile Ile His Pro Leu Ser Tyr Pro Ser Lys Met Thr

130 135 140

Gln Arg Arg Gly Tyr Leu Leu Leu Tyr Gly Thr Trp Ile Val Ala Ile

145 150 155 160

Leu Gln Ser Thr Pro Pro Leu Tyr Gly Trp Gly Gln Ala Ala Phe Asp

165 170 175

Glu Arg Asn Ala Leu Cys Ser Met Ile Trp Gly Ala Ser Pro Ser Tyr

180 185 190

Thr Ile Leu Ser Val Val Ser Phe Ile Val Ile Pro Leu Ile Val Met

195 200 205

Ile Ala Cys Tyr Ser Val Val Phe Cys Ala Ala Arg Arg Gln His Ala

210 215 220

Leu Leu Tyr Asn Val Lys Arg His Ser Leu Glu Val Arg Val Lys Asp

225 230 235 240

Cys Val Glu Asn Glu Asp Glu Glu Gly Ala Glu Lys Lys Glu Glu Phe

245 250 255

Gln Asp Glu Ser Glu Phe Arg Arg Gln His Glu Gly Glu Val Lys Ala

260 265 270

Lys Glu Gly Arg Met Glu Ala Lys Asp Gly Ser Leu Lys Ala Lys Glu

275 280 285

Gly Ser Thr Gly Thr Ser Glu Ser Ser Val Glu Ala Arg Gly Ser Glu

290 295 300

Glu Val Arg Glu Ser Ser Thr Val Ala Ser Asp Gly Ser Met Glu Gly

305 310 315 320

Lys Glu Gly Ser Thr Lys Val Glu Glu Asn Ser Met Lys Ala Asp Lys

325 330 335

Gly Arg Thr Glu Val Asn Gln Cys Ser Ile Asp Leu Gly Glu Asp Asp

340 345 350

Met Glu Phe Gly Glu Asp Asp Ile Asn Phe Ser Glu Asp Asp Val Glu

355 360 365

Ala Val Asn Ile Pro Glu Ser Leu Pro Pro Ser Arg Arg Asn Ser Asn

370 375 380

Ser Asn Pro Pro Leu Pro Arg Cys Tyr Gln Cys Lys Ala Ala Lys Val

385 390 395 400

Ile Phe Ile Ile Ile Phe Ser Tyr Val Leu Ser Leu Gly Pro Tyr Cys

405 410 415

Phe Leu Ala Val Leu Ala Val Trp Val Asp Val Glu Thr Gln Val Pro

420 425 430

Gln Trp Val Ile Thr Ile Ile Ile Trp Leu Phe Phe Leu Gln Cys Cys

435 440 445

Ile His Pro Tyr Val Tyr Gly Tyr Met His Lys Thr Ile Lys Lys Glu

450 455 460

Ile Gln Asp Met Leu Lys Lys Phe Phe Cys Lys Glu Lys Pro Pro Lys

465 470 475 480

Glu Asp Ser His Pro Asp Leu Pro Gly Thr Glu Gly Gly Thr Glu Gly

485 490 495

Lys Ile Val Pro Ser Tyr Asp Ser Ala Thr Phe Pro Ala Ile Ser Ala

500 505 510

Glu Phe His His Thr Gly Leu Val Asp Pro Ser Ser Val Pro Ser Leu

515 520 525

Gly Cys Arg Ser Met Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gln

530 535 540

Arg Asn Glu Glu Lys Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu Lys

545 550 555 560

Gln Leu Gln Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg Leu Leu

565 570 575

Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met

580 585 590

Arg Ile Leu His Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp

595 600 605

Pro Gln Ala Ala Arg Ser Asn Ser Asp Gly Glu Lys Ala Thr Lys Val

610 615 620

Gln Asp Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala

625 630 635 640

Ala Met Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn

645 650 655

Gln Phe Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asn Phe

660 665 670

Asp Phe Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu Trp Glu Asp

675 680 685

Glu Gly Val Arg Ala Cys Tyr Glu Arg Ser Asn Glu Tyr Gln Leu Ile

690 695 700

Asp Cys Ala Gln Tyr Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala

705 710 715 720

Asp Tyr Val Pro Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr

725 730 735

Ser Gly Ile Phe Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His

740 745 750

Met Phe Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln

755 760 765

Cys Phe Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser

770 775 780

Tyr Asn Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu Gln Glu

785 790 795 800

Ala Leu Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg Trp Leu Arg Thr

805 810 815

Ile Ser Val Ile Leu Phe Leu Asn Lys Gln Asp Leu Leu Ala Glu Lys

820 825 830

Val Leu Ala Gly Lys Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala

835 840 845

Arg Tyr Thr Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro

850 855 860

Arg Val Thr Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile

865 870 875 880

Ser Thr Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr

885 890 895

Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg

900 905 910

Asp Ile Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu Leu

915 920 925

<210>105

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>105

catgtatgcc agcgtcctgc tcc 23

<210>106

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>106

gctatgcctg aagccagtct tgtg 24

<210>107

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>107

gcacctgctc ctgagcacct tctcc 25

<210>108

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>108

cacagcgctg cagccctgca gctggc 26

<210>109

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>109

ccagtgatga ctctgtccag cctg 24

<210>110

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>110

cagacacttg gcagggacga ggtg 24

<210>111

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>111

cttgtggtct actgcagcat gttccg 26

<210>112

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>112

catatccctc cgagtgtcca gcggc 25

<210>113

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>113

atggatcctt atcatggctt cctc 24

<210>114

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>114

caagaacagg tctcatctaa gagctcc 27

<210>115

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>115

ctctgatgcc atctgctgga ttcctg 26

<210>116

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>116

gtagtccact gaaagtccag tgatcc 26

<210>117

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>117

tggtggcgat ggccaacagc gctc 24

<210>118

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>118

gttgcgcctt agcgacagat gacc 24

<210>119

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>119

tcaacctgta tagcagcatc ctc 23

<210>120

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>120

aaggagtagc agaatggtta gcc 23

<210>121

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>121

gacacctgtc agcggtcgtg tgtg 24

<210>122

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>122

ctgatggaag tagaggctgt ccatctc 27

<210>123

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>123

gcgctgagcg cagaccagtg gctg 24

<210>124

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>124

cacggtgacg aagggcacga gctc 24

<210>125

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>125

agccatccct gccaggaagc atgg 24

<210>126

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>126

ccaggtaggt gtgcagcaca atggc 25

<210>127

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>127

ctgttcaaca gggctggttg gcaac 25

<210>128

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>128

atcatgtcta gactcatggt gatcc 25

<210>129

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>129

Thr Leu Glu Ser Ile Met

1 5

<210>130

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>130

Glu Tyr Asn Leu Val

1 5

<210>131

<211>5

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>131

Asp Cys Gly Leu Phe

1 5

<210>132

<211>36

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>132

Gly Ala Thr Cys Ala Ala Gly Cys Thr Thr Cys Cys Ala Thr Gly Gly

1 5 10 15

Cys Gly Thr Gly Cys Thr Gly Cys Cys Thr Gly Ala Gly Cys Gly Ala

20 25 30

Gly Gly Ala Gly

35

<210>133

<211>53

<212>PRT

＜213〉artificial sequence

<220>

＜223〉new sequence

<400>133

Gly Ala Thr Cys Gly Gly Ala Thr Cys Cys Thr Thr Ala Gly Ala Ala

1 5 10 15

Cys Ala Gly Gly Cys Cys Gly Cys Ala Gly Thr Cys Cys Thr Thr Cys

20 25 30

Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly Ala

35 40 45

Thr Gly Gly Thr Gly

50

Claims

1. the preparation method of recombinant host cell comprises the following steps:

(a) transfection expression carrier, described carrier comprise the polynucleotide of the g protein coupled receptor of encoding, and the aminoacid sequence that described g protein coupled receptor comprises is selected from:

SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQID NO:40;

(b) under the condition that allows expression vector expression g protein coupled receptor, cultivate host cell.