CN108530521A - Recombinate the preparation and application of hepatitis C antigen - Google Patents

Recombinate the preparation and application of hepatitis C antigen Download PDF

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Publication number
CN108530521A
CN108530521A CN201810347268.0A CN201810347268A CN108530521A CN 108530521 A CN108530521 A CN 108530521A CN 201810347268 A CN201810347268 A CN 201810347268A CN 108530521 A CN108530521 A CN 108530521A
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hepatitis
antigen
seq
hcv
recombination
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李永刚
王琪
施苏洪
马超
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Nanjing Jingda Biological Technology Co Ltd
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Nanjing Jingda Biological Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61P31/12—Antivirals
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
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    • C—CHEMISTRY; METALLURGY
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    • C12N2770/24011—Flaviviridae
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    • G01—MEASURING; TESTING
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    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB

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Abstract

The invention discloses a kind of preparations and application of recombination hepatitis C antigen, recombinate the amino acid sequence such as SEQ ID NO of hepatitis C antigen：Shown in 1, protein structure is soluble protein.The nucleotide sequence of coding recombination hepatitis C antigen amino acid, such as SEQ ID NO：Shown in 2.The preparation method of the recombination hepatitis C antigen will include SEQ ID NO：The host cell of nucleotide sequence described in 2 is cultivated, the polypeptide obtained by isolation and purification culture or protein molecular antigen, obtains recombination hepatitis C antigen.The present invention contributes to the standardization and standardization that HCV core antigen detects, and can be used for preparing the kit of detection Hepatitis C Virus, is conducive to make up available reagent box detection HCV under-sensitive defects.

Description

Recombinate the preparation and application of hepatitis C antigen
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of preparation and application of recombination hepatitis C antigen.
Background technology
Hepatitis C is one of infectious disease caused by being infected by Hepatitis C Virus (HCV), worldwide popular, entirely There are about 1.7 hundred million people to have infected HCV for ball.After HCV infection human body, there is the people of nearly half that can become chronic hepatitis, it is also possible to cause Liver fibrosis, hepatic sclerosis, ascites and liver cancer, prognosis are poor.There are many ways to current diagnosis hepatitis c virus infection, most accurately Be exactly hepatitis C virus recombinant immune blotting (HCV-RIBA) and hepatitis C virus ribonucleic acid (HCV-RNA) detection, but this Two methods are complicated for operation, and experiment condition requires strictly, and of high cost, and hospital application is less.Laboratory traditional detection is mainly Anti-HCV enzyme-linked immunization, in recent years Hepatitis C virus core antigen (HCV-eAg) enzyme-linked immunization be also widely used in Clinic is mainly used for hepatitis c virus infection early diagnosis.HCV.Ab is detected and HCV-cAg detects easy to operate, common laboratory It can be operated, but may cause to fail to pinpoint a disease in diagnosis and mistaken diagnosis.
Viral hepatitis type C be can through blood, medical instrument, mother-to-baby transmission a kind of communicable disease, the whole world is annual There is three, 4,000,000 infection with hepatitis C virus.Hepatitis C can develop into liver fibrosis, hepatic sclerosis even liver cancer, Shire etc. mourns.One the study found that Hepatitis C Virus is the Major Risk Factors of liver cancer, the liver of some areas 73.3% Cancer patient is fallen ill due to HCV infection.Currently, effective prevention and treatment measure there is no to viral hepatitis type C, therefore, It timely and accurately detects hepatitis c virus infection, prevents HCV from propagating most important.For a long time, test in laboratory HCV infection, Generally use HCV.Ab enzyme-linked immunization.This method is simple and easy to operate, and experiment condition requirement is low, but may cause certain fail to pinpoint a disease in diagnosis And mistaken diagnosis.
Invention content
In view of the deficiencies in the prior art, the present invention is carried by gene recombination technology, cDNA clones HCV core antigen genes For a kind of recombination hepatitis C antigen and its preparation and application.
A kind of recombination hepatitis C antigen, amino acid sequence is as such as SEQ ID NO：Shown in 1, protein structure is can Dissolubility albumen.
The nucleotide sequence of coding recombination hepatitis C antigen amino acid, such as SEQ ID NO：Shown in 2.
A kind of host cell, which is characterized in that it includes SEQ ID NO：Nucleotide sequence described in 2.
The preparation method of the recombination hepatitis C antigen will include SEQ ID NO：Nucleotide sequence described in 2 Host cell is cultivated, the polypeptide obtained by isolation and purification culture or protein molecular antigen, obtains recombination hepatitis C antigen.
Recombination hepatitis C antigen disease caused by preparing hepatitis C virus vaccine or hepatitis C virus vaccine Application in the treatment preparation or preventing preparation of disease.
Application of the recombination hepatitis C antigen in preparing Hepatitis C Virus diagnostic preparation or diagnostic kit.
Advantageous effect：
The present invention overcomes Hepatitis C Virus detection sensitivity in the prior art is not high, it is susceptible to lacking for false negative result It falls into.Creatively it is found that the amino acid fragment of above-mentioned core antigen polymorphic site can be used as point of HCV core antigen detection Son label is for detecting Hepatitis C Virus, and the presence of the molecular labeling can indicate existing antigen quantitative detecting reagent Detection sensitivity reduces, the standardization and standardization for contributing to HCV core antigen to detect, and can be used for preparing detection hepatitis C virus The kit of poison is conducive to make up available reagent box detection HCV under-sensitive defects, further improve in the prior art Detection sensitivity and the accuracy for detecting the kit of Hepatitis C Virus, it is effective to be allowed to more wide spectrum.
Specific implementation mode
Embodiment 1
Using the plasmid pM-HCV_Genomeo containing HCV full-length genomes DNA as template, it is used in combination and is directed to core antigen gene Each segment design primer carries out PCR amplifications, and amplified production recycles corresponding specific amplified gene outcome through electrophoretic separation and glue, And be subcloned into pMD-18 (TaKaRa, Inc) carrier, respectively obtain each gene recombination plasmid.
P1: GGTCTGGAACAGATTATTCTCTCACCAT
P2: CTCGGAGGTGTTGCTGTCTCCCAAGAGCGTCCTT
By the restriction endonuclease sites of design, double digestion obtains corresponding genetic fragment from above-mentioned recombinant plasmid, using base Because Subcloned technology is by each gene pET41a connections of acquisition, corresponding HCV core antigens expression vector is built, is had The expression vector for having aforementioned Seq.2 coded sequences, after screening and identification, recombinant plasmid transformed Bacillus coli expression host BL21 bacterium, and induced expression analysis is carried out, the fusion protein antigen engineering bacteria of expression of HCV core antigen is established, is used to prepare Recombination fusion protein antigen.
Engineering bacteria is expressed through everfermentation, collection fermentation thalli, broken bacterium, separation, two step affinity chromatographys obtain purified fusion egg Bai Kangyuan obtains its amino acid sequence as such as SEQ ID NO through sequencing：Shown in 1, nucleotide sequence, such as SEQ ID NO：2 It is shown.
Embodiment 2 recombinates the isolation and purification of hepatitis C antigen
1, the chemical cracking of e. coli bl21 expression bacterium：
Thalline by induced expression uses chemical cleavage method, and 5-10ml bacterial lysates are added by every gram of wet bacterium（2% Triton-X100,10-50ug/ml lysozyme, 1mM DTT, 0.2M PBS, it is preferred to use the BugBuster of Merck ＆ Co., Inc. is molten Liquid）Abundant suspension thalline, 37 degree of slow shake incubate 1 hour;Divide in 4 degree of 12000 revs/min of high speed centrifugations 20, collects supernatant It is used for next step affinitive layer purification.
2, use two step affinity chromatography of gravitational method with purification of target recombinant protein, purity is more than 95%：
] A) by the above-mentioned supernatant after fully centrifuging, Ni2+-NTA affinity columns are through sample-loading buffer （100mMNaH2PO4,10mMTris,10mM2-ME,pH8.0）Fully after balance, the supernatant of cracking is added dropwise, collects efflux, takes 10ul samples are analyzed for SDS-PAGE.With eluent I（100mM NaH2PO4,10mM Tris,pH6.3）Column is fully washed to arrive OD280=0.01. Fraction collection eluents of efflux, sample when 10ul elutions being taken to start are analyzed for SDS-PAGE. With eluent II（100mM NaH2PO4,10mMTris,500mM Imidazole,pH8.0）Elute target protein：It collects every 1mL fractions take 10ul samples to be analyzed for SDS-PAGE respectively.Merge each pure component, to carry out GST affinity chromatography gels Column purifies in next step.
B) with the phosphate buffer of 10 times of bed volumes (4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4, 0.15MNaCl（pH7.3）) abundant balance columns, the sample after affinity chromatography is added, collection flows through component and is placed in On ice.With 10x volume 1xGST Bind/Wash Buffer (4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl（pH7.3）) column is washed, collection flows through component and is placed on ice.With 3x volumes 1xGSTWashBuffer （4.5mM Na2HPO4.12H2O, 1.5mM KH2PO4,0.15M NaCl, 2.7mM KCl10mM GST (reduced form) （pH7.3））Elute destination protein.Collection elution fraction is placed in waits for following protein electrophoretic analysis on ice, and merges identical albumen Component freezes spare in -20 degree.
The comparison of 3 difference HCV genotype sample core antigen levels of embodiment
1, case selection
In June, 2015 to September, collects 203 CHC patients serums, wherein male 130, women 73, and age 24-76 Sui puts down (± 8 .6 of 37 .3) year.HCV genotype using 2 .0 of Versant HCV Genotype (LiPA, Siemens Company, Marburg, Germany) detection, wherein genotype 1b is 41, and 2a is 43, and 3a is 31, and 3b is 43, and 6a is 45, substantially Cover Chinese Common genes type.HCV RNA use Roche Taqman HCV RNA kit quantifications, lowest detection to be limited to 15IU/ mL.Different HCV genotype patients are comparable in age, gender and HCV virus carrying capacity.
2, method
It is complete certainly in Abbott Laboratories Architect i2000 using Abbott Laboratories' Architect HCV Ag core antigen immue quantitative detection reagent boxes H C V antigens are carried out on dynamic Chemiluminescence Apparatus quantitatively to detect.The range of linearity quantitatively detected is 3 .0,0 f m o l/L- 20000fmol/L, lowest detection are limited to 3 .00fmol/L.One-way analysis of variance method and LSD methods is used to carry out respectively whole equal The Multiple range test of several comparisons and each group mean, P<0 .05 is the significant meaning of difference.
3, result
Different genotype patient group HCV antigen quantitative results show that the present invention recombinates hepatitis C antigen detection sensitivity height, has The standardization and standardization for helping HCV core antigen detection can be used for preparing the kit of detection Hepatitis C Virus, be conducive to Make up available reagent box detection HCV under-sensitive defects.
Sequence table
<110>Nanjing Jing Da Bioisystech Co., Ltd
<120>Recombinate the preparation and application of hepatitis C antigen
<141> 2018-04-18
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 314
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Leu Ser Asp Lys Asn Leu Val Ala Met Gly Cys Leu Ala Arg Asp Phe
1 5 10 15
Leu Pro Ser Thr Ile Ser Phe Thr Trp Asn Tyr Gln Asn Asn Thr Glu
20 25 30
Val Ile Gln Gly Ile Arg Thr Phe Pro Thr Leu Arg Thr Gly Gly Lys
35 40 45
Tyr Leu Ala Thr Ser Glu Val Leu Leu Ser Pro Lys Ser Val Leu Glu
50 55 60
Pro Ser Asp Glu Tyr Leu Val Cys Lys Val His Tyr Gly Gly Lys Asn
65 70 75 80
Arg Asp Leu His Val Pro Ile Pro Ala Val Ala Glu Met Asn Pro Asn
85 90 95
Val Asn Val Phe Val Pro Pro Arg Asp Gly Phe Ser Gly Arg Ala Pro
100 105 110
Arg Lys Ser Thr Leu Ile Cys Glu Ala Leu Asn Phe Thr Pro Lys Pro
115 120 125
Ile Thr Val Ser Trp Leu Lys Asp Gly Lys Leu Val Glu Ser Gly Phe
130 135 140
Thr Thr Asp Gly Val Thr Ile Glu Arg Lys Gly Leu Thr Pro Gln Thr
145 150 155 160
Tyr Lys His Ile Ser Thr Leu Thr Ile Ser Glu Ile Asp Trp Leu Asn
165 170 175
Leu Asn Val Tyr Thr Cys Arg Val Asp His Arg Ala Leu Thr Phe Leu
180 185 190
Lys Asn Val Ser Ser Thr Cys Ala Ala Ser Arg Pro Thr Asp Ile Leu
195 200 205
Thr Phe Thr Ile Pro Pro Ser Phe Ala Asp Ile Phe Leu Ser Lys Ser
210 215 220
Ala Asn Leu Thr Cys Leu Val Ser Asn Leu Thr Thr Tyr Glu Thr Leu
225 230 235 240
Asn Ile Ser Trp Ala Ser Gln Ser Gly Glu Pro Leu Glu Ala Lys Ile
245 250 255
Lys Ile Met Glu Ser His Ser Asn Gly Thr Phe Ser Ala Lys Pro Val
260 265 270
Ala Ser Val Cys Val Glu Gln Trp Asn Asn Arg Lys Glu Ser Val Cys
275 280 285
Thr Val Ser His Arg Asp Leu Met Ser Pro Gln Lys Lys Phe Ile Ser
290 295 300
Lys Pro Asn Glu Val Ser Lys His Pro Pro
305 310
<210> 2
<211> 942
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ccctgtctga taagaatctg gtggccatgg gctgcctggc ccgggacttc ctgcccagca 60
ccatttcctt cacctggaac taccagaaca acactgaagt catccagggt atcagaacct 120
tcccaacact gaggacaggg ggcaagtacc tagccacctc ggaggtgttg ctgtctccca 180
agagcgtcct tgaaccttca gatgaatacc tggtatgcaa agtacactac ggaggcaaaa 240
acagagatct gcatgtgccc attccagctg tcgcagagat gaaccccaat gtaaatgtgt 300
tcgtcccacc acgggatggc ttctctggcc gggcaccacg caagtctacg ctcatctgcg 360
aggccctaaa cttcactcca aaaccgatca cagtatcctg gctaaaggat gggaagctcg 420
tggaatctgg cttcaccaca gatggggtga ccatcgagcg gaaaggactg acaccccaaa 480
cctacaagca cataagcaca cttaccatct ctgaaatcga ctggctgaac ctgaatgtgt 540
acacctgccg tgtggatcac agggcactca ccttcttgaa gaacgtgtcc tccacatgtg 600
ctgccagtcg cccaacagac atcctaacct tcaccatccc cccctccttt gccgacatct 660
tcctcagcaa gtccgctaac ctgacctgtc tggtctcaaa cctgacgacc tatgaaaccc 720
tgaatatctc ctgggcttct cagagtggtg aaccactgga agccaaaatt aaaatcatgg 780
aaagccatag caatggcacc ttcagtgcta agccggtggc tagtgtttgt gtggaacagt 840
ggaataacag gaaggaaagt gtgtgtactg tgagccacag ggatctgatg tcaccacaga 900
agaaattcat ctcaaaaccc aatgaggtga gcaaacatcc ac 942
<210> 3
<211> 28
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ggtctggaac agattattct ctcaccat 28
<210> 4
<211> 34
<212> DNA/RNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ctcggaggtg ttgctgtctc ccaagagcgt cctt 34

Claims (6)

1. a kind of recombination hepatitis C antigen, which is characterized in that its amino acid sequence is as such as SEQ ID NO：Shown in 1, egg White structure is soluble protein.
2. the nucleotide sequence of claim 1 recombination hepatitis C antigen amino acid is encoded, such as SEQ ID NO：Shown in 2.
3. a kind of host cell, which is characterized in that it includes SEQ ID NO：Nucleotide sequence described in 2.
4. the preparation method of recombination hepatitis C antigen described in claim 1, which is characterized in that will include SEQ ID NO：2 The host cell of the nucleotide sequence is cultivated, and the polypeptide obtained by isolation and purification culture or protein molecular antigen obtain Recombinate hepatitis C antigen.
5. recombinating hepatitis C antigen described in claim 1 is preparing hepatitis C virus vaccine or hepatitis C virus vaccine draws Application in the treatment preparation or preventing preparation of the disease risen.
6. recombinating hepatitis C antigen described in claim 1 in preparing Hepatitis C Virus diagnostic preparation or diagnostic kit Using.
CN201810347268.0A 2018-04-18 2018-04-18 Recombinate the preparation and application of hepatitis C antigen Pending CN108530521A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113640519A (en) * 2021-08-25 2021-11-12 南通伊仕生物技术股份有限公司 Kit for specifically detecting hepatitis C and preparation method thereof
CN113667022A (en) * 2021-08-25 2021-11-19 南通伊仕生物技术股份有限公司 Fusion protein and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1462636A (en) * 2002-05-30 2003-12-24 北京迪威华宇生物技术有限公司 Vaccine of recombined albumen for preventing and treating infection of human C type hepatitis virus and its usage
CN103214561A (en) * 2013-03-12 2013-07-24 浙江家和制药有限公司 Human hepatitis c virus core antigen and preparation method and application thereof
CN105254724A (en) * 2015-12-01 2016-01-20 山东莱博生物科技有限公司 Truncated type hepatitis c virus HCV NS3 antigen and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1462636A (en) * 2002-05-30 2003-12-24 北京迪威华宇生物技术有限公司 Vaccine of recombined albumen for preventing and treating infection of human C type hepatitis virus and its usage
CN103214561A (en) * 2013-03-12 2013-07-24 浙江家和制药有限公司 Human hepatitis c virus core antigen and preparation method and application thereof
CN105254724A (en) * 2015-12-01 2016-01-20 山东莱博生物科技有限公司 Truncated type hepatitis c virus HCV NS3 antigen and preparation method and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113640519A (en) * 2021-08-25 2021-11-12 南通伊仕生物技术股份有限公司 Kit for specifically detecting hepatitis C and preparation method thereof
CN113667022A (en) * 2021-08-25 2021-11-19 南通伊仕生物技术股份有限公司 Fusion protein and application thereof
CN113667022B (en) * 2021-08-25 2024-03-22 南通伊仕生物技术股份有限公司 Fusion protein and application thereof
CN113640519B (en) * 2021-08-25 2024-05-31 南通伊仕生物技术股份有限公司 Kit for specifically detecting hepatitis C and preparation method thereof

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Application publication date: 20180914