CN116621949B - Method for increasing secretion expression of rabies virus G protein and application - Google Patents
Method for increasing secretion expression of rabies virus G protein and application Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for increasing secretion expression of rabies virus G protein and application thereof.
Background
Rabies (Rabies) is an acute infectious disease caused by Rabies virus (RABV) and is a zoonotic disease. Rabies virus is distributed worldwide and almost all mammals are susceptible to infection by this virus. The virus is highly neurotropic and, once it enters the human body, it reaches the brain along the nerve, resulting in fatal encephalitis. RABV is a minus-strand RNA virus containing a genome of about 12kb encoding five structural proteins: glycoprotein (G), RNA-dependent RNA polymerase (L), matrix protein (M), nucleoprotein (N) and phosphoprotein (P).
At present, no specific medicine is available for rabies virus infection, and vaccination is an effective means for preventing and controlling rabies. The total number of rabies vaccine injection per year in China is up to 1500 ten thousand injections. At present, rabies virus vaccines mainly comprise inactivated vaccine: purified Vero cell rabies virus inactivated vaccine, purified chicken embryo cultured inactivated virus vaccine and purified duck embryo vaccine cultured inactivated virus vaccine, and diploid cell cultured inactivated virus vaccine.
Glycoprotein (G) is a protein present on the surface of rabies virus particles, plays a key role in the process of infecting cells with rabies virus and stimulating an immune response in the body, and is the only structural protein capable of inducing the production of neutralizing antibodies (VNA) against RABV among all structural proteins. The G protein consists of 524 amino acids, and is divided into three functional areas, 1-459 is extracellular area, 460-480 is transmembrane area mainly composed of helix structure, 481-524 is intracellular area.
Early studies showed that eukaryotic cells expressed rabies G protein, with only a low dose required to induce mice to produce high levels of protective neutralizing antibodies.
Therefore, the mass preparation of high-purity G protein is particularly important for preventing and controlling rabies. However, the yield of the G protein produced by the current industrialized production is low, and the large-scale industrialized production cost is too high to produce and apply.
Disclosure of Invention
The invention aims to provide a method for increasing the secretory expression of rabies virus G protein, which is to design and express a G protein which keeps the original antigenicity and has a stable conformation on the basis of fully analyzing the conformation of rabies virus G protein. The engineered G-protein has stable conformation and is easy to express in eukaryotic expression systems.
The invention also aims to provide the G protein with stable conformation and application thereof, and the G protein has good antigenicity through a mouse immunoprotection experiment.
The aim and the technical problems of the invention are realized by adopting the following technical proposal. The method for increasing the secretory expression of the rabies virus G protein provided by the invention 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.
Further, in the step 1), the amino acid sequence of the G0 protein is shown as SEQ ID NO. 1; in the step 2), the amino acid sequence of the G1 protein is shown as SEQ ID NO. 2.
Further, in the step 1), the nucleotide sequence of the G0 protein is shown as SEQ ID NO. 3; in the step 2), the nucleotide sequence of the G1 protein is shown as SEQ ID NO. 4.
Further, in the step 3), the Nhe I restriction enzyme site and the initiation codon sequence are added to the 5 'end of the gene sequence, and the MLu I restriction enzyme site sequence is added to the 3' end.
Further, in the step 4), the expression vector is selected from pLV-eGFP vector, the competent cells are selected from DH5 alpha, and the host cells are selected from HEK293T cells.
The invention provides the G protein prepared by the method for increasing the secretory expression of the rabies virus G protein, and the nucleotide sequence of the G protein is shown as SEQ ID NO. 4.
The present invention provides a nucleotide sequence encoding a G protein as described above.
The present invention provides any one of the following applications of the G protein as described above:
1) The application of rabies virus vaccine preparation;
2) The application in preparing anti-rabies virus drugs;
3) The application in preparing rabies virus detection reagent or kit.
The invention provides a monoclonal strain prepared by the method for increasing the secretory expression of rabies virus G protein, and the nucleotide sequence of the G protein is shown as SEQ ID NO. 4.
By means of the technical scheme, the invention has the following advantages and beneficial technical effects:
1) The method for increasing the secretory expression of the rabies virus G protein improves the secretory expression quantity of the rabies virus G protein, the expression quantity of the mutated G protein is 26.4 times of the output before mutation, and the obtained purified protein has high stability and can be applied in large scale.
2) The rabies virus G1 protein vaccine provided by the invention can provide 100% protection rate, is safe, and has no influence on body weight and health.
Drawings
FIG. 1 shows a schematic diagram of the construction of G0/G1 proteins;
FIG. 2 shows a map of the cleavage assay of the pLV-G1-eGFP vector;
FIG. 3 shows the western blot validation of the expression of anti-his monoclonal antibody 1:7000 dilution;
figure 4 shows a steady line study;
FIG. 5 shows a graph of toxin-counteracting survival after immunization;
figure 6 shows a graph of the change in body weight of a vaccinated mouse against challenge.
Detailed Description
The invention discloses a method for increasing the secretion expression of rabies virus G protein and application thereof. The method for increasing the secretory expression of the rabies virus G protein 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.
The invention designs and expresses the G protein which keeps the original antigenicity and has stable conformation on the basis of fully analyzing the conformation of the rabies virus G protein. The engineered G-protein has stable conformation and is easy to express in eukaryotic expression systems. Through a mouse immunoprotection experiment, the vaccine has good antigenicity.
Examples
1 main experimental materials
HEK293 cells were purchased from ATCC CRL-3216;
cell culture media and serum were both purchased from Gibco company, usa;
BCA protein quantification kit was purchased from shanghai bi yun biotechnology limited.
2 vector construction
2.1 sequence selection and codon optimization
According to the invention, a standard strain CVS-11 glycoprotein (Genbank AAC 34683) of rabies virus is used as a template, 8 histidine sequences are added at the C end, and the full-length codon of the expressed G protein is optimally marked as the G0 protein. The mutated protein mutates 460-480 transmembrane amino acids into a flexible peptide sequence: GSGSGSGSGSGSGSGSGSGS, designated G1 protein. The construction schematic of the G0/G1 protein is shown in FIG. 1. The amino acid sequence of the G0 protein is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 3; the amino acid sequence of the G1 protein is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 4.
Nhe I restriction enzyme site and initial codon sequence are added to the 5 'end of the gene sequence of the sequence after the code optimization, and the sequence synthesis work is entrusted to be completed by Shanghai biological limited company at the 3' MLu I restriction enzyme site sequence.
Construction of 2.2pLV-G-eGFP vector
2.2.1 plasmid linearization
The pLV-eGFP vector was double digested with Nhe I and MLu I, and the fragment of the enzyme was electrophoresed as shown in FIG. 2.
2.2.2 connections
The target fragments recovered in the previous step were ligated with linearized pLV-eGFP vector using T4 DNA ligase, respectively, as follows.
2.2.3 conversion
Taking out competent cells DH5 alpha in a refrigerator, putting the cells into ice water to slowly melt, adding three groups of connection products into each tube, respectively, carrying out ice bath for 30min, carrying out heat shock for 45s at 42 ℃, quickly taking out the cells, putting the cells into ice for 3min, adding 500 mu L of liquid LB culture medium without any antibiotics, carrying out shake culture for 1h on a constant temperature shaking table at 37 ℃, centrifuging, reserving 100 mu L of supernatant resuspended thalli, coating the thalli on an LB plate containing Amp (100 mu g/mL), ensuring uniform coating, and carrying out constant temperature incubator culture at 37 ℃ for 12h.
2.2.4 plasmid extraction
Clones on plates were randomly picked, added to 2mL of ampicillin-resistant medium, and incubated overnight after repeated pipetting. Plasmids were extracted in small amounts using the tenna endotoxinfree plasmid extraction kit.
2.2.5 sequencing
The extracted plasmid is identified by enzyme digestion and sent to Shanghai biological limited company for sequencing, and after the sequence alignment is correct, the marker pLV-G0-eGFP or pLV-G1-eGFP is named.
3 transfection
1) One day prior to transfection, healthy log-phase HEK293T cells were digested with pancreatin and grown at 5X 10 5 Each was seeded onto 10cm cell culture dishes. Cells were incubated at 37℃with 5% CO 2 Culturing for 24 hours under the saturated humidity condition, and carrying out transfection operation when the cells reach 70% -80% density;
2) Transfection was performed using PEI transfection procedure. The lentiviral expression plasmid pLV-G0-eGFP or pLV-G1-eGFP, the lentiviral packaging plasmid psPAX2 and the lentiviral shuttle plasmid pMD2.G were diluted in a ratio of 10. Mu.g/well in 500. Mu.L of 1 XHBS and mixed well as follows;
the transfection system was as follows:
liquid A
PEI 48μL(N/P＝27.5)
1 XHBS was added to 500. Mu.L
Standing at room temperature for 10min
Liquid B
pLV-G1-eGFP 5μg
psPAX2 3.75μg
pMD2.G 1.25μg
1 XHBS was added to 500. Mu.L
Adding solution A into solution B, mixing, standing at room temperature for 20min, adding dropwise into cell culture supernatant of 10cm dish, mixing with shaking culture dish, and standing at 37deg.C with 5% CO 2 Culture supernatant was collected after 48 hours of continued culture in incubator.
Infection of HEK293T cells with lentiviruses
The virus liquid LV-G0-eGFP or LV-G1-eGFP obtained above is transfected into HEK293T cells, and the specific steps are as follows:
1) HEK293T cells in good growth state were digested and counted and diluted to 1X 10 with DMEM complete medium 5 Adding 24-well plate at a volume of 500 μl/well, preparing 2 multiple wells, placing into 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours;
2) 1.5mL EP tube procedure: polybrene is added into a DMEM complete medium to prepare a Polybrene-containing medium (the final concentration of Polybrene in the medium is 8 mug/mL);
adding 100 mu L of the prepared virus liquid LV-G0-eGFP or LV-G1-eGFP into the culture medium containing polybrene, and carrying out light blowing and mixing to obtain a culture medium containing slow viruses, wherein the final volume of the culture medium is 300 mu L;
3) The old culture medium of HEK293T cells cultured in 24-well plates for 24 hours is poured out, 300 mu L of culture medium containing slow virus is added into each well, and the mixture is put into 37 ℃ and 5% CO 2 Culturing in an incubator;
4) After 24h of cultivation, the lentivirus-containing medium in the 24-well plate was poured off, replaced with 500. Mu.L of DMEM complete medium, and placed in 37℃with 5% CO 2 Culturing in an incubator;
5 screening of monoclonal strains
1) The lentivirus-containing medium in the 24-well plate was poured out, and after 3-5 days of culture with 500. Mu.L of DMEM complete medium, the cells that had grown up the culture well were digested with pancreatin.
2) The HEK293T cells after the complete DMEM culture medium resuspension transfection are subcultured in a 96-well plate by a limiting dilution method, the proliferation condition of the 96-well plate cells is checked every day, and the clone number of each well of cells is observed under an inverted microscope after 10-15 d;
3) Wells containing 1 cell colony (i.e., 1 cell pellet) were selected from 96-well plates and monoclonal cells were cultured in 24-well plates.
Efficacy test example
6 secretory expression and stability assay
6.1Western blot sample processing
Monoclonal cells were selected from the wells cultured in 24-well plates, and after 3d of culture of the monoclonal cells in 24-well plates, the supernatant was collected. The supernatant was concentrated 10-fold and then added to a reduced 5 XSDS loading buffer.
6.2Western blot identification
1) 80. Mu.L of the concentrated supernatant was added to 20. Mu.L of a reduced 5 XSDS loading buffer. After centrifugation at 10,000rpm for 5min in a boiling water bath, 20. Mu.L of each supernatant was aspirated and subjected to 10% SDS-PAGE.
2) And (3) taking a separation gel after electrophoresis, transferring a membrane, and sealing for 2 hours by adopting 10% skimmed milk powder after transferring a PVDF membrane.
3) Incubation with anti-His polyclonal antibody overnight (1: 7000 dilution), TBST was washed 3 times for 10min each.
4) HRP-labeled rabbit anti-murine secondary antibody (1: 10000 dilution), incubation at 37℃for 1h, TBST wash 3 times for 10min each. The result of electrophoresis is shown in FIG. 3.
Protein 7 purification and quantification
7.1 protein purification
1) And continuously passaging a 293T cell recombinant cell strain HEK293T-G identified by Western blot and capable of stably secreting and expressing G protein, and expanding a cell working library. The collected cell culture supernatant was centrifuged to remove cells and debris, and then filtered through a 0.45 μm filter membrane.
2) After the Protein A purification column was properly connected to the BioLogic LP Protein purifier, 5 bed volumes of loading buffer (0.01M tris base) were continuously poured and the column equilibrated at 1mL/min to wash out the protection solution from the column. After the peak of the conductivity was stationary, the bed was washed 3 times more.
3) And (3) loading the culture medium supernatant sample processed in the first step at a speed of 1mL/min, wherein a blue peak (ultraviolet light-splitting peak) appears in a computer software real-time window, a red peak (conductivity peak) appears later, and the supernatant flowing through can be collected after 30 seconds after the red peak appears.
4) After the complete flow-through of the culture medium supernatant sample, the sample is washed by using a sampling buffer solution, the volume of a bed is 1mL/min and 5 times, and the non-target proteins are washed off. After the blue peak (ultraviolet light splitting peak) and the red peak (conductance peak) in the real-time window of the computer software are balanced, adding a loading buffer solution with the volume of 3-5 times of the bed.
5) The recombinant protein was then eluted with a protein eluent (150 mM imidazole, pH 8.5) while monitored with a BioLogic LP, and collection was started when a baseline rise was observed, i.e., an elution peak (blue line first peak, red line second peak).
6) And (3) after the eluting solution is collected until the eluting peak returns to the baseline, continuously balancing the volume of the column bed by 3-5 times by using the loading buffer solution, and regulating the flow rate to 1mL/min. The protein purifier was then washed by equilibrating 5 bed volumes with 20% ethanol.
7) The eluted proteins were dialyzed in PBS, the ionic components in the eluate were removed, and the dialysate was changed every 3 h.
7.2 determination of the purified protein concentration (BCA protein concentration determination method)
1) Preparing working solution, namely adding 1 volume BCA reagent B (50:1) into 50 volumes of BCA reagent A according to the number of standard substances and samples to prepare a proper amount of BCA working solution, and fully and uniformly mixing.
2) Dilution of standard: 10. Mu.L of the standard was diluted to 100. Mu.L with PBS (the standard was usually diluted with PBS) to a final concentration of 0.5mg/mL. The standard was added to the protein standard wells of the 96-well plate at 0,1,2,4,8,12,16,20 μl, and PBS was added to make up to 20 μl.
3) An appropriate volume of sample was added to the sample wells of the 96-well plate, and PBS was added to 20. Mu.L.
4) 200. Mu.L BCA working solution was added to each well, and the wells were left at 37℃for 30min.
5) Cooling to room temperature, measuring wavelength between 540-595nm with enzyme label instrument, and optimally calculating protein concentration according to standard curve.
The expression level of the G0 protein was determined to be 12.6. Mu.g/L, the expression level of the G1 protein was determined to be 332.5. Mu.g/L, and the yield per liter after mutation was 26.4 times that before mutation.
8 stability study
Purified protein (1 mg/mL), divided into 16 parts, each 0.5mL; placing 8 parts in a refrigerator at 4 ℃ and respectively sampling one part in 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks and 24 weeks and continuously sampling 7 times; placing 8 parts in a refrigerator at-80 ℃, and respectively sampling one part in 1 week, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks and 24 weeks and continuously sampling 7 times; the protein concentration was measured with BCA after each sampling, and the results are shown in table 1 and fig. 4, showing that the protein obtained after purification by the method of the present invention has good stability.
Table 1: stability detection
9 mice immunization experiments
Diluting the obtained antigen expression supernatant by using normal saline to ensure that the concentration of RV-G protein reaches 100 mug/mL, mixing the purified recombinant rabies virus G protein with 206 adjuvant according to the volume ratio of 1:1, emulsifying, and then storing at 4 ℃.
Female Bal b/c mice of 6 weeks of age were randomly divided into 3 groups (10 animals/group) and immunized by the intramuscular route (i.m.) with 100 μl of physiological saline, 100 μl of emulsified vaccine and commercial rabies inactivated vaccine, respectively. All mice were challenged six weeks after immunization with a 50LD50 dose of CVS-24 intra-brain route (i.c.), and after challenge, the mice were observed continuously for 21 days, weighed for weight, death, and analyzed for survival rate statistically.
As shown in fig. 5, rabies virus G1 protein vaccine can provide 100% protection rate of immunized mice, commercial vaccine can provide 90% protection rate, and the effect on body weight of mice is not significantly different from commercial vaccine (shown in fig. 6).
The present invention is not limited to the preferred embodiments, but can be modified, equivalent, and modified in any way without departing from the technical scope of the present invention.
Appendix:
1. pre-mutation G0 protein amino acid sequence: SEQ ID NO.1
MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVRTTKESLIIISPSVTDLDPYDKSLHSRVFPGGKCSGITVSSTYCSTNHDYTIWMPENPRPRTPCDIFTNSRGKRASKGNKTCGFVDERGLYKSLKGACRLKLCGVLGLRLMDGTWVAMQTSDETKWCPPDQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLKVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFKEGDEAEDFVEVHLPDVYKQISGVDLGLPNWGKYVLMTAGAMIGLVLIFSLMTWCRRANRPESKQRSFGGTGRNVSVTSQSGKVIPSWESYKSGGEIRLHHHHHHHH
2. The amino acid sequence of the mutated G1 protein SEQ ID NO.2
MVPQVLLFVPLLGFSLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVRTTKESLIIISPSVTDLDPYDKSLHSRVFPGGKCSGITVSSTYCSTNHDYTIWMPENPRPRTPCDIFTNSRGKRASKGNKTCGFVDERGLYKSLKGACRLKLCGVLGLRLMDGTWVAMQTSDETKWCPPDQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLKVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFKEGDEAEDFVEVHLPDVYKQISGVDLGLPNWGKYGSGSGSGSGSGSGSGSGSGSRRANRPESKQRSFGGTGRNVSVTSQSGKVIPSWESYKSGGEIRLHHHHHHHH
3. Nucleotide sequence after codon optimization of the G0 protein before mutation: SEQ ID NO.3
ATGGTGCCTCAAGTCCTCCTCTTCGTTCCCCTTTTGGGCTTTAGCCTGTGCTTCGGGAAGTTTCCTATTTACACCATCCCGGATAAGCTCGGCCCTTGGTCCCCAATTGACATCCACCATCTGTCTTGCCCCAATAATCTCGTCGTAGAAGATGAGGGATGTACCAACCTGAGCGAATTCTCATATATGGAACTGAAAGTTGGATACATCTCTGCCATAAAAGTGAATGGCTTCACGTGTACTGGCGTGGTCACCGAAGCGGAAACTTATACCAATTTCGTTGGATACGTCACAACCACCTTTAAAAGGAAGCACTTTAGACCGACCCCGGACGCCTGTAGAGCAGCATACAATTGGAAAATGGCCGGTGATCCCCGGTATGAAGAGAGCCTTCACAACCCATACCCAGATTACCACTGGCTGCGGACCGTGCGCACTACCAAGGAAAGTCTCATTATCATCTCCCCCAGCGTGACGGATCTGGATCCTTATGATAAGTCCCTGCACTCCCGCGTCTTTCCTGGAGGTAAATGTAGCGGCATTACCGTGAGCAGCACCTACTGCAGTACCAACCACGATTACACCATCTGGATGCCTGAAAACCCAAGGCCAAGGACTCCCTGCGACATCTTTACCAACTCTAGGGGAAAGCGGGCCTCAAAAGGGAACAAAACCTGTGGCTTTGTAGACGAGCGGGGGCTGTACAAGTCACTGAAGGGGGCTTGTAGGCTTAAGTTGTGCGGAGTTCTGGGACTGAGACTGATGGACGGTACATGGGTTGCGATGCAAACGTCCGACGAGACTAAATGGTGCCCCCCTGATCAGCTGGTGAATCTCCATGATTTTAGGTCCGACGAGATCGAGCATCTCGTGGTCGAGGAGTTGGTCAAGAAGCGGGAGGAGTGTCTCGATGCCCTGGAGAGTATCATGACCACCAAATCCGTCTCCTTTCGAAGGCTGAGCCACCTGAGGAAATTGGTTCCCGGGTTCGGAAAGGCTTATACTATTTTCAACAAAACTTTGATGGAGGCCGATGCCCATTACAAGTCCGTACGGACCTGGAACGAGATCATCCCATCTAAGGGTTGTTTGAAGGTCGGGGGGAGGTGTCACCCCCATGTGAATGGGGTATTTTTTAACGGGATCATCTTGGGCCCGGATGGACACGTGCTGATTCCAGAAATGCAGTCCAGCCTGCTGCAGCAACACATGGAGCTCCTCAAATCTAGTGTCATTCCCCTGATGCATCCCCTGGCAGATCCAAGTACGGTGTTCAAAGAGGGGGACGAAGCCGAGGATTTTGTGGAGGTTCACCTGCCAGACGTCTATAAACAGATATCCGGCGTGGACTTGGGCCTTCCTAATTGGGGCAAGTATGTGCTGATGACTGCCGGGGCTATGATCGGTCTGGTGCTGATCTTTTCACTGATGACATGGTGCAGACGGGCTAATAGACCCGAGAGTAAACAGCGGTCCTTCGGCGGAACCGGTCGGAATGTATCCGTCACTTCTCAGTCTGGCAAAGTCATCCCCAGCTGGGAATCCTATAAGTCAGGCGGCGAAATCCGACTGCACCACCACCACCACCACCA TCAT
4. Nucleotide sequence after codon optimization of the mutated G1 protein: SEQ ID NO.4
ATGGTGCCACAGGTCCTGCTTTTCGTCCCCCTGCTTGGATTTTCACTGTGTTTTGGCAAATTTCCAATCTACACAATTCCAGATAAGCTGGGTCCCTGGTCCCCCATCGATATCCACCACTTGTCTTGCCCCAATAATCTGGTGGTTGAGGACGAGGGCTGCACTAACCTGAGCGAATTCAGTTACATGGAGCTCAAGGTGGGATACATCTCTGCCATCAAAGTGAACGGTTTTACCTGTACCGGAGTTGTGACCGAGGCCGAAACCTACACTAACTTCGTCGGCTACGTCACCACAACGTTCAAGAGAAAACACTTCCGCCCTACACCTGACGCATGTAGGGCCGCATACAACTGGAAGATGGCCGGCGACCCACGCTATGAAGAAAGTCTCCACAACCCATACCCTGATTACCACTGGCTTCGGACCGTTAGGACGACCAAGGAGAGCCTCATCATCATCTCACCATCCGTGACCGACCTTGATCCTTACGACAAGAGCCTGCACTCAAGGGTTTTTCCCGGTGGGAAATGCTCTGGCATCACTGTTTCAAGTACGTATTGTTCCACAAACCATGATTACACGATCTGGATGCCCGAGAACCCGCGCCCACGCACGCCCTGTGATATCTTCACTAACTCACGCGGCAAGCGCGCCAGTAAGGGGAACAAAACATGCGGCTTTGTGGATGAGAGGGGTCTGTATAAGAGTCTGAAAGGCGCCTGTAGACTGAAGCTTTGCGGGGTGCTCGGTCTCCGGCTTATGGACGGAACCTGGGTAGCAATGCAGACCTCTGACGAGACCAAATGGTGTCCTCCAGATCAGTTGGTTAACTTGCACGACTTCAGATCTGACGAGATAGAACACCTCGTAGTGGAAGAGCTGGTGAAAAAAAGGGAGGAGTGTTTGGATGCCTTGGAAAGTATTATGACAACCAAGTCTGTGTCCTTCCGGCGACTCAGTCACCTGAGGAAGCTGGTGCCAGGCTTTGGAAAGGCCTACACGATTTTCAATAAGACGTTGATGGAAGCCGACGCACATTATAAGTCTGTGCGCACCTGGAATGAAATCATCCCTTCTAAAGGGTGCCTCAAAGTGGGTGGACGGTGCCATCCTCACGTGAACGGAGTGTTTTTCAATGGGATCATTCTCGGCCCCGATGGACATGTCCTTATCCCCGAAATGCAGAGTTCACTGCTTCAGCAGCACATGGAACTGCTCAAGTCCAGTGTCATCCCTTTGATGCACCCCTTGGCCGATCCGAGCACCGTCTTTAAGGAGGGGGATGAGGCAGAGGATTTCGTCGAAGTGCACCTGCCTGATGTGTATAAGCAGATAAGTGGAGTTGATCTCGGGCTGCCGAACTGGGGTAAATACGGCTCAGGCTCCGGCTCTGGGTCAGGAAGTGGCTCTGGATCCGGATCCGGCAGCGGATCTCGGCGCGCAAATAGACCCGAGAGCAAGCAGAGGTCATTTGGCGGGACTGGCCGGAATGTGTCCGTGACAAGCCAGAGCGGCAAGGTGATTCCGTCCTGGGAGAGCTACAAAAGCGGCGGGGAGATTCGGCTTCACCACCACCACCACCACCA TCAT
Claims (6)
1. A method for increasing secretory expression of rabies virus G protein, comprising the steps of:
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; the amino acid sequence of the G0 protein is shown as SEQ ID NO. 1; the nucleotide sequence of the G0 protein is shown as SEQ ID NO. 3;
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; the amino acid sequence of the G1 protein is shown as SEQ ID NO. 2; the nucleotide sequence of the G1 protein is shown as SEQ ID NO. 4;
3) Adding restriction enzyme sites and initial codon sequences into the gene sequence to synthesize a sequence; 4) Constructing an expression vector, transfecting host cells, and screening monoclonal strains to obtain monoclonal strains with increased secretion and expression of rabies virus G protein; the nucleotide sequence of the G protein is shown as SEQ ID NO. 4.
2. The method of increasing secreted expression of rabies virus G protein according to claim 1, wherein: in the step 3), nhe I restriction enzyme site and initial codon sequence are added to the 5 'end of the gene sequence, and MLu I restriction enzyme site sequence is added to the 3' end.
3. The method of increasing secreted expression of rabies virus G protein according to claim 1, wherein: in the step 4), the expression vector is selected from pLV-eGFP vector, the competent cells are selected from DH5 alpha, and the host cells are selected from HEK293T cells.
4. A nucleotide sequence encoding the G protein of claim 1.
5. Any one of the following applications of the G protein of claim 1:
1) The application of rabies virus vaccine preparation;
2) The application in preparing anti-rabies virus drugs;
3) The application in preparing rabies virus detection reagent or kit.
6. A monoclonal strain prepared by the method for increasing secretory expression of rabies virus G protein according to any one of claims 1-3, characterized in that: the nucleotide sequence of the G protein is shown as SEQ ID NO. 4.
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