CN116621949B - A method and application for increasing the secretion and expression of rabies virus G protein - Google Patents

Abstract

The invention discloses a method for increasing the secretion expression of rabies virus G protein and application thereof. The method comprises the following steps: 1) Taking the glycoprotein amino acid sequence of a standard strain of rabies virus as a template, adding 8 histidine sequences at the C end, and optimally marking the full-length codon of the expressed glycoprotein as G0 protein; 2) The amino acids of the transmembrane region of the G0 protein 460-480 are mutated into a flexible peptide sequence: GSGSGSGSGSGSGSGSGSGS, obtaining mutant G1 protein after optimizing the password; 3) Adding restriction enzyme sites and initial codon sequences into the gene sequence to synthesize a sequence; 4) And constructing an expression vector, transfecting host cells, and screening monoclonal strains to obtain the monoclonal strains with increased secretion and expression of rabies virus G protein. On the basis of rabies virus G protein conformation, the invention designs and expresses a G protein which keeps the original antigenicity and has stable conformation, and is easy to express in a eukaryotic expression system; through a mouse immunoprotection experiment, the vaccine has good antigenicity.

Description

A method and application for increasing the secretion and expression of rabies virus G protein

Technical field

The invention belongs to the field of biotechnology, and in particular relates to a method and application for increasing the secretion and expression of rabies virus G protein.

Background technique

Rabies is a zoonotic acute infectious disease mainly caused by rabies virus (RABV). Rabies virus is distributed globally and nearly all mammals are susceptible to infection. The virus is highly neurotropic and once it enters the body, it travels along the nerves to the brain, causing fatal encephalitis. RABV is a negative-strand RNA virus containing a genome of approximately 12 kb, encoding five structural proteins: glycoprotein (G), RNA-dependent RNA polymerase (L), matrix protein (M), nucleoprotein (N), and phosphoprotein (P ).

There is currently no specific medicine for rabies virus infection, and vaccination is an effective means to prevent and control rabies. The total number of people vaccinated against rabies in my country every year is as high as 15 million. At present, rabies virus vaccines are mainly inactivated vaccines: purified Vero cell rabies virus inactivated vaccine, purified chicken embryo cultured inactivated virus vaccine, purified duck embryo cultured inactivated virus vaccine, and diploid cell cultured inactivated virus vaccine. of inactivated virus vaccines.

Glycoprotein (G) is a protein present on the surface of rabies virus particles. It plays a key role in the process of rabies virus infecting cells and stimulating the body's immune response. It is the only structural protein among all structural proteins that can induce the production of neutralizing antibodies against RABV ( VNA) structural protein. G protein consists of 524 amino acids and is divided into three functional regions, 1-459 is the extracellular region, 460-480 is the transmembrane region mainly composed of helical structure, and 481-524 is the intracellular region.

Previous studies have shown that rabies G protein expressed in eukaryotic cells can induce mice to produce high levels of protective neutralizing antibodies at only a low dose.

Therefore, the large-scale preparation of high-purity G protein is particularly important for preventing and controlling the occurrence of rabies. However, the current industrial production of G protein has a low yield, and the cost of large-scale industrial production is too high to allow production and application.

Contents of the invention

The purpose of the present invention is to provide a method for increasing the secretion and expression of rabies virus G protein, which is to design and express a G protein that retains its original antigenicity and has a stable conformation on the basis of fully analyzing the conformation of rabies virus G protein. The modified G protein has a stable conformation and is easily expressed in eukaryotic expression systems.

Another object of the present invention is to provide the above-mentioned G protein with a stable conformation and its application, and verify that it has good antigenicity through mouse immune protection experiments.

The purpose of the present invention and solving its technical problems are achieved by adopting the following technical solutions. A method for increasing the secretion and expression of rabies virus G protein proposed according to the present invention includes the following steps:

1) Use the glycoprotein amino acid sequence of the standard strain of rabies virus as a template, add 8 histidine sequences to the C-terminus, and optimize the codons of the full-length expressed glycoprotein to label it as G0 protein;

2) Mute the amino acids 460-480 of the transmembrane region of the G0 protein into a flexible peptide sequence: GSGSGSGSGSGSGSGSGSGS to obtain the code-optimized mutant G1 protein;

3) After adding restriction enzyme sites and start codon sequences to the gene sequence, the sequence is synthesized;

4) Construct an expression vector, and after transfecting host cells, screen monoclonal strains to obtain monoclonal strains with increased secretion and expression of rabies virus G protein.

Further, in step 1), the amino acid sequence of the G0 protein is as shown in SEQ ID NO.1; in step 2), the amino acid sequence of the G1 protein is as shown in SEQ ID NO.2.

Further, in step 1), the nucleotide sequence of the G0 protein is shown in SEQ ID NO.3; in step 2), the nucleotide sequence of the G1 protein is shown in SEQ ID NO.4.

Further, in step 3), the Nhe I restriction endonuclease site and start codon sequence are added to the 5' end of the gene sequence, and the MLu I restriction endonuclease site sequence is added to the 3' end.

Further, in step 4), the expression vector is pLV-eGFP vector, the competent cell is DH5α, and the host cell is HEK293T cells.

The present invention provides G protein prepared by the method for increasing the secretion and expression of rabies virus G protein as described above, and the nucleotide sequence of the G protein is shown in SEQ ID NO. 4.

The present invention provides nucleotide sequences encoding G proteins as described above.

The present invention provides any of the following applications of G protein as described above:

1) Application in preparing rabies virus vaccine;

2) Application in the preparation of anti-rabies virus drugs;

3) Application in preparing rabies virus detection reagents or kits.

The present invention provides monoclonal strains prepared by the above-mentioned method of increasing the secretion and expression of rabies virus G protein. The nucleotide sequence of the G protein is shown in SEQ ID NO. 4.

Through the above technical solutions, the present invention has the following advantages and beneficial technical effects:

1) The method for increasing the secretion and expression of rabies virus G protein provided by the present invention increases the secretion and expression of rabies virus G protein. The expression amount of G protein after mutation and the output per liter are 26.4 times that before mutation, and the obtained purified protein is stable. It has high performance and can be applied on a large scale.

2) The rabies virus G1 protein vaccine provided by the present invention can provide 100% protection rate, is safe, and has no impact on body weight and health.

Description of the drawings

Figure 1 shows a schematic diagram of G0/G1 protein construction;

Figure 2 shows the enzyme digestion identification diagram of pLV-G1-eGFP vector;

Figure 3 shows westernblot verification of expression of anti-his monoclonal antibody at a 1:7000 dilution;

Figure 4 shows the stable row study;

Figure 5 shows the challenge survival curve after immunization;

Figure 6 shows the changes in body weight of mice after immunization with the vaccine.

Detailed ways

The invention discloses a method and application for increasing the secretion and expression of rabies virus G protein. The method for increasing the secretion and expression of rabies virus G protein includes the following steps:

1) Use the glycoprotein amino acid sequence of the standard strain of rabies virus as a template, add 8 histidine sequences to the C-terminus, and optimize the codons of the full-length expressed glycoprotein to label it as G0 protein;

2) Mute the amino acids 460-480 of the transmembrane region of the G0 protein into a flexible peptide sequence: GSGSGSGSGSGSGSGSGSGS to obtain the code-optimized mutant G1 protein;

3) After adding restriction enzyme sites and start codon sequences to the gene sequence, the sequence is synthesized;

4) Construct an expression vector, and after transfecting host cells, screen monoclonal strains to obtain monoclonal strains with increased secretion and expression of rabies virus G protein.

On the basis of fully analyzing the conformation of rabies virus G protein, the present invention designs and expresses a G protein that retains its original antigenicity and has a stable conformation. The modified G protein has a stable conformation and is easily expressed in eukaryotic expression systems. Through mouse immune protection experiments, it has been verified that it has good antigenicity.

Example

1 Main experimental materials

HEK293 cells were purchased from ATCC CRL-3216;

Cell culture medium and serum were purchased from Gibco, USA;

BCA protein quantification kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

2 vector construction

2.1 Sequence selection and codon optimization

The present invention uses the standard strain CVS-11 glycoprotein of rabies virus (GenbankAAC34683) as a template, adds 8 histidine sequences to the C-terminus, and optimizes the expression of the full-length G protein codons and labels it as G0 protein. The mutated protein mutates 460-480 amino acids in the transmembrane region into a flexible peptide sequence: GSGSGSGSGSGSGSGSGS, named G1 protein. The schematic diagram of G0/G1 protein construction is shown in Figure 1. The amino acid sequence of the G0 protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3; the amino acid sequence of the G1 protein is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4 .

The Nhe I restriction endonuclease site and start codon sequence were added to the 5' end of the gene sequence of the code-optimized sequence, and the 3' MLu I restriction endonuclease site sequence was added. The synthesis of this sequence was entrusted to Shanghai Sangon. BIOLOGICAL LIMITED COMPLETE.

2.2 Construction of pLV-G-eGFP vector

2.2.1 Plasmid linearization

The pLV-eGFP vector was double digested with Nhe I and MLu I, and the electrophoresis pattern of the digested fragments is shown in Figure 2.

2.2.2 Connection

Use T4 DNA ligase to ligate the target fragments recovered in the previous step with the linearized pLV-eGFP vector. The system is as follows.

2.2.3 Conversion

Take out the competent cells DH5α from the refrigerator and place them in ice water to slowly melt. Add the three sets of ligation products to each tube respectively, bathe in ice for 30 minutes, heat shock at 42°C for 45 seconds, quickly take them out and place them in ice for 3 minutes, and add the solution without any antibiotics. 500 μL of liquid LB culture medium was cultured on a constant temperature shaker at 37°C with shaking at 150 r/min for 1 hour, then centrifuged, leaving 100 μL of supernatant to resuspend the bacterial cells, and spread it on an LB plate containing Amp (100 μg/mL) to ensure even coating. Incubate in a constant temperature incubator at 37°C for 12 hours.

2.2.4 Plasmid extraction

Randomly pick the clones on the plate, add 2 mL of ampicillin-resistant medium, pipet repeatedly and then culture overnight. The plasmid was extracted in a small amount using Tiangen endotoxin-free plasmid extraction kit.

2.2.5 Sequencing

The extracted plasmid was digested and identified and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After the sequence alignment was correct, the plasmid was named pLV-G0-eGFP or pLV-G1-eGFP.

3 Transfection

1) One day before transfection, trypsinize HEK293T cells in logarithmic phase that are growing well and seed them in a 10cm cell culture dish at 5 × 10 ⁵ cells. The cells were cultured for 24 hours at 37°C, 5% CO ₂ and saturated humidity. Transfection was performed when the cells reached a density of 70%-80%;

2) Transfection is performed using PEI transfection procedures. Dilute lentiviral expression plasmid pLV-G0-eGFP or pLV-G1-eGFP, lentiviral packaging plasmid psPAX2 and lentiviral shuttle plasmid pMD2.G at a ratio of 10 μg/well 4:3:1 in 500 μL 1×HBS and mix Mix well, as follows;

The transfection system is as follows:

A liquid

PEI 48μL(N/P＝27.5)

1×HBS Make up to 500μL

Leave at room temperature for 10 minutes

B liquid

pLV-G1-eGFP 5μg

psPAX2 3.75μg

pMD2.G 1.25μg

1×HBS Make up to 500μL

Add solution A to solution B, mix thoroughly, let stand at room temperature for 20 minutes, then gently add dropwise to the cell culture supernatant in a 10cm dish, shake the dish gently to mix, and place in a 37°C, 5% CO ₂ incubator. After continuing to culture for 48 hours, the culture supernatant was collected.

4 Lentivirus infection of HEK293T cells

Transfect HEK293T cells with the viral liquid LV-G0-eGFP or LV-G1-eGFP obtained above. The specific steps are as follows:

1) Digest and count HEK293T cells that are growing well and dilute them to 1×10 ⁵ cells/mL with DMEM complete medium. Add 500 μL/well to a 24-well plate. Prepare 2 duplicate wells and place them in 37°C, 5% CO ₂ Cultivate in the incubator for 24 hours;

2) Take a 1.5mL EP tube and operate: add polybrene to DMEM complete medium to prepare a medium containing Polybrene (the final concentration of Polybrene in the medium is 8 μg/mL);

Add 100 μL of the prepared virus liquid LV-G0-eGFP or LV-G1-eGFP to the above polybrene-containing culture medium to make the final volume 300 μL and mix gently by blowing to prepare a lentivirus-containing culture medium;

3) Pour out the old medium of the HEK293T cells cultured in the 24-well plate for 24 hours, add 300 μL of lentivirus-containing medium to each well, and place it in a 37°C, 5% CO ₂ incubator;

4) After 24 hours of culture, pour out the lentivirus-containing culture medium in the 24-well plate, replace it with 500 μL DMEM complete culture medium, and place it in a 37°C, 5% CO ₂ incubator;

5 Monoclonal strain screening

1) Pour out the lentivirus-containing culture medium in the 24-well plate and replace it with 500 μL DMEM complete culture medium. After culturing for 3-5 days, trypsinize the cells that cover the culture wells.

2) Resuspend the transfected HEK293T cells in DMEM complete medium, passage them in a 96-well plate using the limiting dilution method, and continue to culture them. Check the cell proliferation in the 96-well plate every day, and observe the cells in each well under an inverted microscope after 10-15 days. number of clones;

3) Select the well containing 1 cell colony (i.e. 1 cell clump) in the 96-well plate and culture the monoclonal cells in the 24-well plate.

Efficacy test example

6. Secretion expression and stability testing

6.1 Western blot sample processing

Select the monoclonal cell wells cultured in the 24-well plate. When the monoclonal cells are cultured in the 24-well plate for 3 days, collect the supernatant. The supernatant was concentrated 10 times and reduced 5×SDS loading buffer was added.

6.2 Western blot identification

1) Add 80 μL of concentrated supernatant to 20 μL of reduced 5×SDS loading buffer. Place in a boiling water bath for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, and then draw 20 μL of each supernatant for 10% SDS-PAGE electrophoresis.

2) After electrophoresis, take the separation gel and transfer it to the membrane. After transferring to the PVDF membrane, use 10% skim milk powder to block for 2 hours.

3) Incubate overnight with anti-His polyclonal antibody (1:7000 dilution), wash 3 times with TBST, 10 min each time.

4) Incubate HRP-labeled rabbit anti-mouse secondary antibody (1:10000 dilution), incubate at 37°C for 1 hour, and wash 3 times with TBST, 10 minutes each time. The electrophoresis results are shown in Figure 3.

7Protein purification and quantification

7.1 Protein purification

1) Continuous passage of the recombinant cell line HEK293T-G, a 293T cell line identified by Western blot that can stably secrete and express G protein, to expand the cell library. The collected cell culture supernatant was centrifuged to remove cells and debris, and then filtered with a 0.45 μm filter membrane.

2) After correctly connecting the Protein A purification column to the BioLogic LP protein purifier, continuously perfuse 5 times the column bed volume of loading buffer (0.01M tris base) and equilibrate the column at 1mL/min to wash away the protective solution in the column. . After the electroconductive peak is stable, wash 3 times the column bed volume.

3) Load the culture medium supernatant sample processed in the first step at a speed of 1mL/min. A blue peak (UV spectroscopy peak) will appear first in the real-time window of the computer software, and then a red peak (conductance peak) will appear. The red peak will appear. After flowing for another 30 seconds, the supernatant can be collected.

4) After the culture supernatant sample has completely flowed through, use loading buffer to flow wash at 1 mL/min, 5 times the column bed volume, to wash away non-target proteins. After the blue peak (UV spectroscopy peak) and red peak (conductivity peak) in the real-time window of the computer software have become balanced, add 3-5 times the column bed volume of the loading buffer.

5) Then use protein eluent (150mM imidazole, pH 8.5) to elute the recombinant protein, and monitor it with BioLogic LP. When an increase in the baseline is observed, an elution peak appears (the blue line peaks first, and the red line peaks later). Start collecting.

6) Collect the eluate until the elution peak returns to the baseline, continue to balance 3 to 5 times the column bed volume with the loading buffer, and adjust the flow rate to 1 mL/min. Then use 20% ethanol to balance 5 times the column bed volume and clean the protein purifier.

7) The eluted protein is dialyzed in PBS to remove ionic components in the eluate, and the dialysate is replaced every 3 hours.

7.2 Determination of purified protein concentration (BCA protein concentration determination method)

1) Prepare working solution: According to the number of standards and samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix thoroughly.

2) Dilute the standard: Take 10 μL of the standard and dilute it to 100 μL with PBS (the standard can generally be diluted with PBS) to make the final concentration 0.5 mg/mL. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard into the protein standard wells of the 96-well plate, and add PBS to make up to 20 μL.

3) Add appropriate volume of sample to the sample well of the 96-well plate, and add PBS to 20 μL.

4) Add 200 μL BCA working solution to each well and place at 37°C for 30 minutes.

5) Cool to room temperature, use a microplate reader to measure the wavelength between 540-595nm, 562nm is the best, and calculate the protein concentration based on the standard curve.

The G0 protein expression level was determined to be 12.6 μg/L, and the G1 protein expression level was 332.5 μg/L. The output per liter after the mutation was 26.4 times that before the mutation.

8Stability studies

Purified protein (1 mg/mL) was divided into 16 parts, each part was 0.5 mL; 8 parts were placed in a 4°C refrigerator for 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks. Take one sample each for 7 consecutive times; place 8 samples in a -80°C refrigerator and take one sample each for 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks for 7 consecutive samples. times; BCA was used to detect the protein concentration after each sampling, and the results are shown in Table 1 and Figure 4, indicating that the protein obtained after purification by the method of the present invention has good stability.

Table 1: Stability testing

9 Mouse Immunity Experiment

Dilute the harvested expressed antigen supernatant with physiological saline so that the concentration of RV-G protein reaches 100 μg/mL. Then mix the purified recombinant rabies virus G protein and 206 adjuvant at a volume ratio of 1:1, emulsify and place at 4 Store at ℃.

Six-week-old female Bal b/c mice were randomly divided into 3 groups (10 mice/group), and were immunized with 100 μL of normal saline, 100 μL of emulsified vaccine and commercial inactivated rabies vaccine through intramuscular route (i.m.). . Six weeks after immunization, all mice were challenged with 50LD50 dose of CVS-24 via intracerebral route (i.c.). After the challenge, observation was continued for 21 days. The body weight and death status of the mice were weighed, and the survival rate of the mice was statistically analyzed.

The results are shown in Figure 5. The rabies virus G1 protein vaccine can provide 100% protection rate for immunized mice, and the commercial vaccine can provide 90% protection rate. The impact on mouse body weight is not significantly different from that of commercial vaccines ( As shown in Figure 6).

The above are only preferred embodiments of the present invention and do not limit the present invention in any form. Therefore, any simple modifications to the above embodiments may be made based on the technical essence of the present invention without departing from the technical content of the present invention. , equivalent changes and modifications, all still fall within the scope of the technical solution of the present invention.

appendix:

1. Amino acid sequence of G0 protein before mutation: SEQ ID NO.1

MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVRTTKESLIIISPSVTDLDPYDKSLHSRVFPGGKCSGITVSSTYCSTNHDYTIWMPENPRPRPRTPCDIFTNS RGKRASKGNKTCGFVDERGLYKSLKGACRLKLCGVLGLRLMDGTWVAMQTSDETKWCPPDQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLKVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFHLKEGDEAEDFVEV PDVYKQISGVDLGLPNWGKYVLMTAGAMIGLVLIFSLMTWCRRANRPESKQRSFGGTGRNVSVTSQSGKVIPSWESYKSGGEIRLHHHHHHHH

2. Amino acid sequence of G1 protein after mutation SEQ ID NO.2

MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVRTTKESLIIISPSVTDLDPYDKSLHSRVFPGGKCSGITVSSTYCSTNHDYTIWMPENPRPRPRTPCDIFTNS RGKRASKGNKTCGFVDERGLYKSLKGACRLKLCGVLGLRLMDGTWVAMQTSDETKWCPPDQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLKVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFHLKEGDEAEDFVEV PDVYKQISGVDLGLPNWGKYGSGSGSGSGSGSGSGSSRRANRPESKQRSFGGGTGRNVSVTSQSGKVIPSWESYKSGGEIRLHHHHHHHH

3. Nucleotide sequence after codon optimization of G0 protein before mutation: SEQ ID NO.3

ATGGGTGCCTCAAGTCCTCCTTCGTTCCCCTTTTGGGCTTTAGCCTGTGCTTCGGGAAGTTTCCTATTTACACCATCCCGGATAAGCTCCGGCCCTTGGTCCCCAATTGACATCCACCATCTGTCTTGCCCCAATAATCTCGTCGTAGAAGATGAGGGATGTACCAACCTGAGCGAATTCTCATATATGGAACTGAAAGTTGGATACATCTCTGCCATAAAAGTGAATGGCTTCACGTGTACTGGGCTGGTCACCGAAGCGGA AACTTATACCAATTTCGTTGGATACGTCACAACCACCTTTAAAAGGAAGCACTTTAGACCGACCCCGGACGCCTGTAGAGCAGCATACAATTGGAAAATGGCCGGTGATCCCCGGTATGAAGAGAGCCTTCACAACCCATACCCAGATTACCACTGGCTGCGGACCGTGCGCACTACCAAGGAAAGTCTCATTATCATCTCCCCCAGCGTGACGGATCTGGATCCTTATGATAAGTCCCTGCACTCCCGCGTCTTTCCTGGAGGTAAATGTAGCG GCATTACCGTGAGCAGCACCTACTGCAGTACCAACCACGATTACACCATCTGGATGCCTGAAAACCCAAGGCCAAGGACTCCCTGCGACATCTTTACCAACTCTAGGGGGAAAGCGGGCCTCAAAAGGGAACAAAACCTGTGGCTTTGTAGACGAGCGGGGGCTGTACAAGTCACTGAAGGGGGCTTGTAGGCTTAAGTTGTGCGGAGTTCTGGGACTGAGACTGATGGACGGTACATGGGTTGCGATGCAAACGTCCGACGAGACT AAATGGTGCCCCCCTGATCAGCTGGTGAATCTCCATGATTTTAGGTCCGACGAGATCGAGCATCTCGTGGTCGAGGAGTTGGTCAAGAAGCGGGAGGAGTGTCTCGATGCCCTGGAGAGTATCATGACCACCAAATCCGTCTCCTTTCGAAGGCTGAGCCACCTGAGGAAATTGGTTCCCGGGTTCGGAAAGGCTTATACTATTTTCAACAAAACTTTGATGGAGGCCGATGCCCATTACAAGTCCGTACGGACCTGGAACGAGA TCATCCCATCTAAGGGTTGTTTGAAGGTCGGGGGGAGGTGTCACCCCATGTGAATGGGGGTATTTTTTAACGGGATCATCTTGGGCCCGGATGGACACGTGCTGATTCCAGAAATGCAGTCCAGCCTGCTGCAGCAACACATGGAGCTCCTCAAATCTAGTGTCATTCCCTGATGCATCCCCTGGCAGATCCAAGTACGGTGTTCAAAGAGGGGGACGAAGCCGAGGATTTTGTGGAGGTTCACCTGCCAGACGTCTATAAACAG ATATCCGGCGTGGACTTGGGCCTTCCTAATTGGGGCAAGTATGTGCTGATGACTGCCGGGGCTATGATCGGTCTGGTGCTGATCTTTTCACTGATGACATGGTGCAGACGGGCTAATAGACCCGAGAGTAAACAGCGGTCCTTCGGCGGAACCGGTCGGAATGTATCCGTCACTTCTCAGTCTGGCAAAGTCATCCCCAGCTGGGAATCCTATAAGTCAGGCGGCGAAATCCGACTGCACCACCACCACCACCACCACCA TCAT

4. Codon-optimized nucleotide sequence of G1 protein after mutation: SEQ ID NO.4

AtggccacagGGCTGCTGCTTTTTCCCCCCCCCTGCTGGGGGGGATTTTGTGCAAATCAATCCATCCAGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCA is AtaatctgggttttttttttttttttttttttttttttttttttttttggggggCTGCTGCTACTGAGAATTCAGGGGGGGGGGGGGGGGGGGGGGGGGCCCAAACGGGGGGGGCGCGCGAAAAAAAAAAAAC CCTACTACTACTACTTTTCGGGGGCTACCACACACACACAACAAGAGAGAACACACACCCCCCCTGCACTGCACCCCCAACAACAAGGCCCCCCCCCCCCCTAAGAAGTCCCCCCCC AcACCCACCCCCCCTTACCCCTGGGGGGGGACCGCGACCCAGAGCCACACACCCCCCCCCCCCCCGACCGACGACGCCCTCAGGCAGGGGGGGGGGGGGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCACCCCACAGCAC GGAAAATGCTGCTGCTCTGTGTTCAAGTACGTACCAAACCACACACACGACCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCGCGCGCGCGCGCCCCAGT AAGGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGTGTAGTGCGCGCGCGCGCGCTGTGAGCTGAGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGCAATGCAGCAGA CctctCTGACGAGACAAAAATGTGTGTCCTCAGAGAGGTGTTGCACACGACAGACGACACACACCTCTCTCTGGGGGGGGGGGGGGGCTGCTGCTTGA is AAGTATTTTACAACAACAAGTCTGTGTCCCGCGCGCGACAGTCTCTGAGGGCCCCCCCCCCCCCCCCCCCCTACACACAATTTCACCCCACCACGACCACACGACCACACACACACGACACACCACACCACACCACCCACCACCCACCCCACCCCCCACCCCCCACGCGCACGCGCACGCACTCACGCACTCACGCACTCATCAC GCGCACTGGAATGAAATCATCCCCCTCTCTCTGCTCTCTCAAGGGGGGGGGGGGCCCCCTCTCTCTCTCTCTCTCGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCAAGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCC AGTTCTCTGCACAGCACACACACTGAACTGCTCCAGTCCAGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGAgGAgGAGAGAGCACCACCCCCCC TGATGTGTGTGTAGCAGATAGGGGGAGAGAGAGGGGGGGCTGCGGGGGGGGGGGGCTCCTCCTCTCTCTCTGGGGGGGGGGGGGGGGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCCCTCAT CCGAGAGCAGCAGGGTCATTTTGGGGGGGGGGGGGGGGAATGTGTGCCCCAGCGGGGGGTGTCCTCTCTCTAGGGGGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCC can Accaccatcat

Claims (6)

1. A method for increasing the secretion and expression of rabies virus G protein, characterized by comprising the following steps: 1) Use the glycoprotein amino acid sequence of the standard strain of rabies virus as a template, add 8 histidine sequences at the C-terminus, and optimize the full-length codons of the expressed glycoprotein to label it as G0 protein; the G0 protein amino acid sequence is such as SEQ ID NO.1 is shown; the G0 protein nucleotide sequence is shown as SEQ ID NO.3; 2) Mute the amino acids in the transmembrane region 460-480 of the G0 protein into a flexible peptide sequence: GSGSGSGSGSGSGSGSGSGS to obtain a code-optimized mutant G1 protein; the amino acid sequence of the G1 protein is as shown in SEQ ID NO. 2; the G1 The protein nucleotide sequence is shown in SEQ ID NO.4; 3) After adding restriction endonuclease sites and start codon sequences to the gene sequence, the sequence is synthesized; 4) Constructing an expression vector, transfecting host cells, and screening single clones to obtain increased secretion and expression of rabies virus G protein The monoclonal strain; the nucleotide sequence of the G protein is shown in SEQ ID NO. 4. 2. The method for increasing the secretion and expression of rabies virus G protein as claimed in claim 1, characterized in that: in step 3), an Nhe I restriction endonuclease site and a start codon are added to the 5' end of the gene sequence. sequence, add the MLu I restriction endonuclease site sequence at the 3' end. 3. The method for increasing the secretion and expression of rabies virus G protein as claimed in claim 1, characterized in that: in step 4), the expression vector selects pLV-eGFP vector, the competent cell selects DH5α, and the host cell selects HEK293T. cell. 4. A nucleotide sequence encoding the G protein of claim 1. 5. Any of the following applications of the G protein according to claim 1: 1) Application in preparing rabies virus vaccine; 2) Application in the preparation of anti-rabies virus drugs; 3) Application in preparing rabies virus detection reagents or kits. 6. The monoclonal strain prepared by the method of increasing the secretion and expression of rabies virus G protein according to any one of claims 1 to 3, characterized in that: the nucleotide sequence of the G protein is as shown in SEQ ID NO.4 .