CN105085641A - Cytomembrane positioning signal peptide and coding sequence and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a cell membrane localization signal peptide and its coding sequence and application. The signal peptide is (a) or (b); (a) a polypeptide consisting of the amino acid sequence shown in Sequence 2 in the sequence listing; (b) the amino acid sequence of Sequence 2 is substituted by one or several amino acid residues and/or Or deletion and/or addition and derivative polypeptides with cell membrane localization signal peptide function. The signal peptide provided by the invention can target the target protein on the host cell membrane. The cell membrane localization signal peptide provided by the invention forms a fusion protein with a suitable target protein, which can be used to develop vaccines and drugs for treating virus, parasite, and bacterial infections, and realize efficient drug administration.

Description

A cell membrane localization signal peptide and its coding sequence and application

technical field

The invention relates to the technical field of genetic engineering, in particular to a cell membrane localization signal peptide and its coding sequence and application.

Background technique

Bacteria living in different environments respond to changes in the environment by secreting proteins with various biological functions to the cell surface or outside the cell to maintain their own survival and reproduction in the environment. Pathogens have a variety of protein secretion systems. Under different environmental conditions, they will use different protein secretion systems to interact with the host, secrete active proteins into host tissues or cells, and exert different physiological functions. Gram-negative bacteria have six protein secretion systems (I-VI). Different pathogenic bacteria have different protein secretion systems, and different protein secretion systems have different secretion mechanisms. According to the secretion pathway of the substrate protein, the type I, II, and V secretion systems secrete the protein around the bacterial cell but do not directly enter the host cell, and the type III, IV, and VI secretion systems directly transport the protein into the host cell. According to whether substrate protein secretion depends on signal peptide, the protein secretion system of bacteria is divided into Sec-dependent secretion system and Sec-independent secretion system, II and IV are Sec (Signalsequences)-dependent secretion system, I and III are Sec independent secretion system. Based on the characteristics of transporters in different protein secretion systems of bacteria, researchers have developed bacterial vectors for directional transporters for the construction of recombinant vaccines or anti-tumor vaccines for the prevention of diseases. For example, the signal peptide of the Salmonella type II secretion system substrate protein and the Streptococcus pneumoniae protein PspA are fused to establish a fusion expression vector, and the fusion protein is expressed in the attenuated Salmonella vaccine to construct a bivalent vaccine, which can prevent salmonellosis and pneumococcal disease at the same time; The type I or V protein secretion system of Salmonella presents tumor antigens or cytokines to host immune cells, and can be used to develop anti-tumor vaccines.

Bacterial type III secretion system (T3SS) consists of a group of proteins consisting of a transmembrane protein transport system that directly transports bacterially produced proteins from the bacterial cytoplasm to the host cell. Aiming at the mechanism by which T3SS transports proteins into host cells, researchers have carried out relevant research, using T3SS to transport heterologous antigens into animals for the treatment of diseases caused by viruses, intracellular parasites, and parasites, as well as for the presentation of Cytokine therapy for colon cancer, melanoma, breast cancer, etc. In order to realize T3SS transporting the target protein, it is necessary to clarify the signal peptide sequence of the substrate protein transported by T3SS, and use the signal peptide to localize the target protein into the host cell. According to the current research progress, the substrate protein secreted through the T3SS pathway does not have a typical Sec-dependent signal sequence, and its secretion signal peptide may be located at different positions of the substrate protein, or it may be the 5' mRNA encoding the substrate protein. Therefore, identifying the transport signal peptide of T3SS substrate protein is the key to the development of targeted vaccines and drugs.

Marine fish farming is developing rapidly in my country and is an important part of aquaculture in my country. Due to the expansion of farming scale, deterioration of farming environment, and germplasm degradation, diseases caused by bacteria, viruses, and parasites have seriously affected the fish farming industry. The development of disease prevention and control methods relying on antibiotics and disinfectants is likely to cause antibiotic residues in aquatic products and environmental microbial resistance, affecting food safety and the sustainable development of aquaculture. The method of relying on vaccines to prevent and control diseases can reduce the use of chemical agents such as antibiotics, and has been widely recognized and promoted internationally. A variety of inactivated vaccines and genetically engineered vaccines developed by Chinese researchers have good immune protection effects. However, the current vaccines have the following problems: one is that most of them are vaccines that stimulate humoral immunity, and these vaccines are difficult to treat diseases caused by viruses, parasites and bacteria parasitic in cells; the other is that most of the existing vaccines are single or monovalent Vaccines, while there are many types of pathogens in fish farming in my country, single or monovalent vaccines for diseases caused by one pathogen have been difficult to meet the needs of aquaculture development. In view of these circumstances, the development of targeted delivery of multiple/polyvalent vaccines is one approach to address the above-mentioned problems.

Edwardsiella tarda is one of the important pathogenic bacteria of fish. It is an intracellular parasite, which can invade fish epithelial cells and phagocytic cells, and grow and multiply in the cells, eventually leading to hemorrhagic sepsis in fish. The bacterium has a variety of pathogenic factors, among which T3SS is an important pathogenic factor, which has the functions of assisting bacteria to parasitize in host cells and resisting phagocytic immune killing. The T3SS of Edwardsiella tarda consists of 35 genes, which encode device proteins, effector proteins, molecular chaperones and regulatory proteins, and four substrate proteins (EseG, FliC, EseJ, and Orf13) are known to pass through the T3SS of Edwardsiella tarda. The T3SS is transported into the host cell, which lays the foundation for the identification of the Edwardsiella tarda T3SS substrate protein transport signal peptide in the present invention.

Contents of the invention

The purpose of the present invention is to provide a cell membrane localization signal peptide and its coding sequence and application.

To achieve the above object, the technical solution adopted in the present invention is:

A cell membrane localization signal peptide, the signal peptide is (a) or (b);

(a) a polypeptide consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing;

(b) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence of Sequence 2 and a derivative polypeptide having the function of a cell membrane localization signal peptide.

A gene for a cell membrane localization signal peptide, the gene for a cell membrane localization signal peptide is the DNA molecule described in any one of 1)-3):

1) The DNA molecule shown in sequence 1 in the sequence listing;

2) Hybridizing with the DNA sequence defined in 1) under stringent conditions and encoding a DNA molecule with a cell membrane localization signal peptide functional protein;

3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) and encoding a protein with a cell membrane localization signal peptide function.

Recombinant expression vector, expression cassette, recombinant bacterium or transgenic cell line containing said DNA molecule.

The recombinant plasmid obtained by inserting the DNA molecular sequence into the multiple cloning site of the plasmid pCX340.

The application of a cell membrane localization signal peptide in positioning a target protein on a cell membrane.

The target protein is specifically β-lactamase protein (TEM1).

The cell membrane localization signal peptide provided by the present invention is formed into a fusion protein with a suitable target protein, which can be used to develop vaccines and drugs for treating virus, parasite, and bacterial infections, and achieve high-efficiency drug delivery.

A method for localizing a target protein in a cell, using the polypeptide shown in Sequence 2 in the sequence listing or its derivative peptide as a cell membrane localization signal peptide to localize the target protein on the cell membrane.

The cells are specifically HeLa cells or flounder kidney cells. The method is as follows: introducing the fusion gene into Edwardsiella tarda LSE40, infecting the cells and expressing it, so as to localize the target protein on the cell membrane of the cells; The gene sequence of the β-lactamase protein.

The present invention has advantages:

The cell membrane localization signal peptide provided by the invention can target the target protein to the cell membrane. The cell membrane positioning signal peptide provided by the present invention is formed into a fusion protein with a suitable target protein, wherein the T3SS pathway of Edwardsiella tarda is used to construct a vector system for directional transport of the target protein, aiming at presenting the foreign protein into the host cell, It provides tools for the development of highly efficient multivalent vaccines with Edwardsiella lentus as the carrier, which can be used to develop vaccines and drugs for the treatment of viruses, parasites, and bacterial infections, and to achieve efficient drug delivery.

Description of drawings

Fig. 1 is the vector map of the recombinant plasmid pCX108 of the present invention; it is the product obtained by linking the sequence 1 of the sequence listing with NdeI and EcoRI restriction sites and the pCX341 plasmid.

Fig. 2 is a verification result diagram of the construction of the recombinant plasmid pCX108; it is obtained by double-digesting the plasmid pCX108 with NdeI and EcoRI to obtain the pCX341 plasmid and a 324bp insert DNA fragment. Lanes 1 and 2 are recombinant plasmids.

Fig. 3 is the detection diagram of plasmid pCX341 and recombinant plasmid pCX108 expressing fusion protein in Edwardsiella tarda LSE40; It is that pCX341 and pCX108 are transformed into Edwardsiella tarda LSE40 respectively, obtain recombinant bacteria LSE40pCX341 and LSE40pCX108, after DMEM culture, extract LSE40pCX341 and The supernatant extracellular protein (ECP) and total cell protein (TCP) of LSE40pCX108 culture were detected by westernblot to detect the expression of fusion protein. The bacterial cytoplasmic protein DnaK was used as a quality control protein to detect whether the ECP component was contaminated by the TCP component.

Figure 4 is a diagram showing the detection of fusion protein expression after recombinant bacteria LSE40pCX341 and LSE40pCX108 infect HeLa cells; it is to infect HeLa cells with recombinant bacteria LSE40pCX341 and LSE40pCX108, extract HeLa cell protein, and use western blot to detect the expression of fusion protein. β-actin is a cytoplasmic protein, which is used to detect whether the cell membrane phase of HeLa is contaminated by cytoplasmic phase proteins.

Figure 5 is a diagram showing the detection of fusion protein expression in flounder kidney tissue infected with recombinant bacteria LSE40pCX341 and LSE40pCX108; after the recombinant bacteria LSE40pCX341 and LSE40pCX108 were injected intramuscularly into flounder, the protein of flounder kidney cells was extracted, and the expression of fusion protein was detected by western blot Condition.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The test methods in the subordinate examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Plasmid pCX341: donated by the Institute of Hydrobiology, Chinese Academy of Sciences. The pCX341 plasmid carries the β-lactamase (β-TEM1) gene as the target protein gene.

Edwardsiella tarda (Edwardsiella tarda) LSE40: It was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on January 23, 2013. The preservation number is: CGMCCNo.7199, and the address of the preservation unit is: No. 1, Beichen West Road, Chaoyang District, Beijing Court No. 3.

HeLa cells: purchased from ATCC, catalog number CCL-2 ^™ .

Example 1 Identification of Transit Signal Peptide

1. Construction of recombinant plasmids

1. DNA template preparation: Genomic DNA of Edwardsiella tarda LSE40 was extracted.

2. PCR amplification: with the DNA in step 1 as a template, 108For (AT CATATG CCTTGGATGCCACGATTGC, the underline is the NdeI restriction site) and 108Rev ( GAATTC TCGGCCTTGGCCTTTTGCTCGGCCT, the underline is the EcoRI restriction site) for PCR Amplified to obtain a PCR product with a size of 324bp. The PCR amplification system (50μl) is: 10×buffer5μl, MgCl ₂ (25mM) 5μl, dNTPmixture (10mMeach) 1μl, PfuTaqpolymerase (5U/μl) 0.5μl, 108For (10μM) 1μl, 108Rev (10μM) 1μl, DNA template 1μl , add ddH ₂ O to the rest. The PCR amplification conditions were: 94°C for 5 minutes; 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s; 72°C for 10 minutes.

3. Digestion: the PCR product in step 2 was double-digested with restriction endonucleases NdeI and EcoRI (37° C. for 4 hours) to obtain the digested product. At the same time, plasmid pCX341 was double digested with restriction endonucleases NdeI and EcoRI, and a linear plasmid DNA fragment of about 5600 bp was recovered.

4. Ligation: Ligate the PCR digestion product in step 3 with the pCX341 linear DNA to obtain the recombinant plasmid pCX108. The map of the pCX108 plasmid is shown in Figure 1. When the pCX108 plasmid was double-digested with NdeI and EcoRI, the 324bp DNA fragment shown in SEQ ID No.1 was obtained (Figure 2). According to the sequencing results, the structural characteristics of the recombinant plasmid pCX108 are described as follows: Insert sequence 1 of the sequence listing between the NdeI and EcoRI restriction sites of the plasmid pCX341 as shown in the 1st to 324th nucleotides from the 5' end DNA molecule (a fragment consisting of the first to 108 amino acid residues from the N-terminal of SEQIDNo.2 of the expression sequence table, referred to as 108aa), the DNA molecule forms a fusion DNA molecule with the β-lactamase gene on the pCX341 plasmid, expressing The fusion protein of 108aa and β-lactamase, referred to as 108aa-TEM1.

SEQ ID No.1

ATGCCTTGGATGCCACGATTGCGCTGTGCTATGGCCAGGCCGACGGCAACCCAGGCTATACACTGCTGCGCCAGCGGGCACAGGATCTGCTGGATACGCTTAATCAACCCTAAGGAGCCTATGATGATCAACGGCGTTTTCACCGATGCACCGCTGCCACCCACCGCGGCGCCGGGGCCTTGCCCCGCGCCAAGCCGCAGGGAGATCGGCACCCAGTGCGACCTGCTGGGCATAGAGGCGCCGCGCAGCCGAGCGCCGGGGGCCTCAGCGCCCCTGGACCAGCTCAGGGCACAGGCGCAGGAGGCCGAGCAAAAGGCCAAGGCC

Sequence features:

Length: 324 base pairs

Type: nucleic acid

Chain type: double chain

Topology: Linear

Molecule type: DNA

Specific name: EseGCDS (1-324bp)

Original source: Edwardsiella tarda LSE40

SEQ ID No.2

Sequence features:

Length: 108 amino acids

Type: amino acid sequence

Chain type: single chain

Topology: Linear

Molecule Type: Protein Primary Structure

Specific name: EseG1-108 amino acid residue sequence

Original source: Edwardsiella tarda LSE40

2. Expression and secretion detection of fusion protein

1. Construction of Edwardsiella tarda expressing fusion protein

1.1 Preparation of Competent Bacteria: Edwardsiella tarda pathogenic strain LSE40 was cultivated with TSB broth (purchased from Sigma Company), cultivated at 25°C for 24-36 hours, centrifuged at 4°C for 10 minutes at 4000g, and collected the thalline , washed three times with glycerol saline (30ml glycerol, 100ml distilled water) at a temperature of 4-8°C and resuspended to a concentration of 1×10 ⁸ cells/mL, the resulting cell suspension was the competent bacteria.

1.2 Transformation: The constructed recombinant plasmid pCX108 was electroporated to transform the competent bacteria obtained in 1.1, and the transformed bacteria were cultivated in TSA medium (purchased from Sigma) containing 10 μg/mL tetracycline for 36-48 hours, and the cells containing the recombinant plasmid were screened. Edwardsiella tarda colonies yielded LSE40pCX108. Edwardsiella tarda containing plasmid pCX341 was screened by the same method and used as a blank control, named LSE40pCX341.

2. Extraction of bacterial extracellular protein (ECP) and total cellular protein (TCP)

LSE40pCX108 and LSE40pCX341 were respectively inoculated in DMEM (purchased from Gibco) medium, cultivated at 25°C and 5% _CO for 24-36 hours, centrifuged a part of the bacterial culture at 4000g for 10 minutes at 4°C, and the supernatant Sterilize by filtering through a 0.22 μm microporous membrane, and the filtrate is the extracellular protein (ECP) component; resuspend the corresponding bacterial pellet in PBS (pH 7.4) and break it with ultrasonic waves, and centrifuge at 12,000g at 4°C for 5 minutes. The supernatant was taken to obtain the total cell protein (TCP) fraction. Add the above two components into frozen trichloroacetic acid to a final concentration of 10%, place on ice for 1-2 hours, centrifuge at 12,000g at 4°C for 20 minutes, wash the precipitate three times with ice acetone and air-dry, wash with an appropriate amount of PBS (pH7. 4) Dissolving the precipitate to obtain ECP protein solution and TCP protein solution respectively.

3. Detection of fusion proteins

After SDSPage electrophoresis, the ECP and TCP obtained in step 2 were transferred to a 0.45 μm PVDF membrane (purchased from Roche), using mouse anti-TEM1 monoclonal antibody as the primary antibody (purchased from QEBBioscience), and horseradish peroxidase The labeled goat anti-mouse IgG was used as the secondary antibody (purchased from Boshide Biotechnology), and Western Blot detection was carried out. The results are shown in Figure 3. The positive hybridization band of the fusion protein can be detected in the ECP and TCP of LSE40pCX108, indicating that the 108aa-TEM1 fusion protein is in Edwardsi Expressed in the bacterial cell and secreted outside the bacterial cell; a protein-positive hybridization band was detected in the TCP of LSE40pCX341, but not detected in the ECP, indicating that TEM1 was expressed in the Edwardsiella cell, but could not be secreted outside the bacterial cell .

3. Localization of fusion protein in Hela cells

Hela cells were infected with LSE40pCX108, and the localization of the fusion protein in Hela cells was detected. The specific method is:

1. Culture of Hella cells: Digest the cultured HeLa cells into single cells with 0.25% trypsin, resuspend to a cell concentration of 2×10 ⁵ cells/ml, inoculate into cell culture plates, and incubate at 35°C, 5% Cultured for 24 hours under CO ₂ until the cells adhered to the wall.

2. Cultivation of recombinant bacteria: Inoculate LSE40pCX341 and LSE40pCX108 in TSB medium respectively, culture on a shaking table at 25°C for 18 hours, transfer the bacterial solution to fresh TSB medium at a ratio of 1:20 (v:v) After culturing for 5 hours, take the bacterial solution and centrifuge at 4°C for 10 minutes to collect the bacteria, resuspend in DMEM medium, wash by centrifugation for 3 times, resuspend in DMEM medium, and adjust the bacterial concentration to 10 ⁸ cells/ml.

3. Recombinant bacteria infect HeLa cells: Add the bacterial suspension obtained in step 2 to the HeLa cells prepared in step 1 at a ratio of MOI (Multiplicity of infection) of 10:1 (v:v), at 35°C, 5% After 5 hours of cultivation in CO ₂ , replace with fresh DMEM medium (containing 200 μg/ml gentamycin), and after 1 hour of cultivation, replace with fresh DMEM medium (containing 16 μg/ml gentamycin), and cultivate for 18 hours.

4. Extraction and detection of different components of HeLa cells: Gently discard the HeLa cell culture medium in step 3, wash the cells with PBS 3 times, collect HeLa cells, add 5ml HB buffer (1M sucrose, 14.8ml double distilled water , 0.2ml of 300mM imidazole, 200μl of 100mMPMSF), washed 3 times at 1500g, centrifuged at 4°C for 15 minutes, collected the cell pellet, resuspended in 1ml of HB buffer, repeatedly blown the cells with a 1ml syringe until they were completely broken, and the cell lysate was obtained at 1500g , Centrifuge at 4°C for 20 minutes, transfer the supernatant to an ultracentrifuge tube, and centrifuge at 40,000g at 4°C for 60 minutes, the supernatant is the mass phase of HeLa cells; wash the precipitate twice with HB buffer and add to the supernatant, etc. Volume of HB buffer resuspended, as the membrane phase of HeLa cells. The phase-splitting protein of HeLa cells was detected by western blot, and the results are shown in Figure 4. In HeLa cells infected with LSE40pCX108, a positive band of fusion protein 108aa-TEM1 was detected in the membrane phase, but 108aa-TEM1 was not detected in the mass phase. band; while the HeLa cells infected with LSE40pCX341, no positive band of fusion protein 108aa-TEM1 was detected in the membrane phase and plasma phase. These results indicated that the recombinant plasmid pCX108 localized TEM1 on the Hela cell membrane.

Implementation 2 Localization detection of fusion protein in flounder cells

1. Cultivation of recombinant bacteria: Inoculate LSE40pCX341 and LSE40pCX108 in TSB medium respectively, culture on a shaking table at 25°C for 18 hours, transfer the bacterial solution to fresh TSB medium at a ratio of 1:20 (v:v) After culturing for 5 hours, the bacterial solution was collected by centrifugation at 5000g at 4°C for 10 minutes to collect the bacterial cells, washed three times with PBS and resuspended, and the bacterial concentration was adjusted to 10 ⁷ cells/ml.

2. Infected flounder: buy flounder (Paralichthysolivaceus) from a farm and raise them temporarily in the aquarium for a week, then take 3 fishes, take internal organs for bacterial isolation, and use them for experiments after testing that there is no bacterial infection. Divide the flounder into two groups, 20 tails/group, respectively take the LSE40pCX341 and LSE40pCX108 bacterial solutions in step 1 and inject flounder intramuscularly with 0.1ml/tail, and after 48 hours of infection, take 10 head kidneys of flounder from each group for analyze.

3. Extraction and detection of protein in the kidney tissue cells of flounder: cut the kidney of flounder in step 2 with scissors, soak it in PBS (containing 10 Units/ml sodium heparin), and filter it with a cell strainer with a pore size of 100 μm (purchased from BDFalcon) , the filtrate was centrifuged at 1500g for 15 minutes, and the cells were collected. The cells were washed twice with PBS and then resuspended in HB buffer. The cells were homogenized by adding TritonX100, and the flounder kidney cells were phase-separated as described above, and the membrane phase was extracted. The mass phase protein was detected by western blot. The results are shown in Figure 5. In the flounder kidney cells infected with LSE40pCX108, a positive band of 108aa-TEM1 fusion protein could be detected in the membrane phase, but no positive band of 108aa-TEM1 was detected in the plasma phase; In flounder kidney cells, no positive band of fusion protein 108aa-TEM1 was detected in both membrane phase and plasma phase. These results indicated that the recombinant plasmid pCX108 localized TEM1 on the kidney cell membrane of flounder.

Claims (7)

1. A cell membrane localization signal peptide, characterized in that: the signal peptide is (a) or (b); (a) a polypeptide consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing; (b) Substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence of Sequence 2 and a derivative polypeptide having the function of a cell membrane localization signal peptide. 2. A gene of the cell membrane localization signal peptide according to claim 1, characterized in that: the gene of the cell membrane localization signal peptide is any DNA molecule in 1)-3): 1) The DNA molecule shown in sequence 1 in the sequence listing; 2) Hybridizing with the DNA sequence defined in 1) under stringent conditions and encoding a DNA molecule with a cell membrane localization signal peptide functional protein; 3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) and encoding a protein with a cell membrane localization signal peptide function. 3. The gene of the cell membrane localization signal peptide according to claim 2, characterized in that: a recombinant expression vector, an expression cassette, a recombinant bacterium or a transgenic cell line containing the DNA molecule. 4. The gene of the cell membrane localization signal peptide according to claim 3, characterized in that: the recombinant plasmid obtained by inserting the DNA molecular sequence into the multiple cloning site of the plasmid pCX340. 5. The application of the cell membrane localization signal peptide according to claim 1 in positioning the target protein on the cell membrane. 6. The application of the cell membrane localization signal peptide of claim 1 according to claim 6, characterized in that: the target protein is specifically β-lactamase protein (TEM1). 7. A method for localizing a target protein in a cell, characterized in that: the polypeptide shown in Sequence 2 in the Sequence Listing or its derivative peptide is used as a cell membrane localization signal peptide to localize the target protein on the cell membrane.