CN109111507B

CN109111507B - Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof

Info

Publication number: CN109111507B
Application number: CN201810963128.6A
Authority: CN
Inventors: 孙乐; 张翠娟
Original assignee: Zhejiang Pedichang Science And Technology Development Co ltd
Current assignee: Zhejiang Pedichang Science And Technology Development Co ltd
Priority date: 2017-08-22
Filing date: 2018-08-22
Publication date: 2021-12-24
Anticipated expiration: 2038-08-22
Also published as: CN109111507A

Abstract

The invention provides a virus recombinant glycoprotein, which is formed by replacing a signal peptide of a target virus glycoprotein with a signal peptide of a human or animal secretory protein. The invention also provides a method for efficiently expressing the recombinant glycoprotein by using the eukaryotic cell. The invention solves the world technical problems of low expression quantity and poor secretion of virus recombinant glycoprotein in eukaryotic cells, lays a solid foundation for developing vaccines and virus protective antibody detection reagents, and has good application prospect.

Description

Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a virus recombinant glycoprotein, a eukaryotic cell high-efficiency expression method and application thereof.

Background

Rabies is a type B infectious disease which is reported and managed by the law of China and is one of the highest acute infectious diseases of human mortality so far, and the pathogeny of Rabies is Rabies virus (Rabies virus), and the virus is susceptible to human and all warm-blooded animals. The fatality rate of rabies is close to 100%, although humanized anti-rabies virus antibody drugs have been greatly developed in recent years, no very effective therapeutic drug is developed at present, and therefore, rabies vaccines are still the most effective means for preventing and treating rabies at present.

The rabies virus genome is nonsegmented single-strand negative-strand RNA, has a total length of about 12kb, and respectively encodes five main structural proteins, namely an enzyme protein (L), a glycoprotein (G), a matrix protein (M), a phosphorylated protein (P) and a nucleoprotein (N). Wherein, the G protein contains a plurality of protective antigen sites and can induce and generate protective antibody against rabies virus.

The prevention of rabies after exposure recommended by the World Health Organization (WHO) is mainly a method of combining rabies vaccine injection with anti-rabies virus immunoglobulin. In order to improve the safety of the vaccine, recombinant hepatitis B vaccines and recombinant human papilloma virus vaccines are on the market at a time. However, the eukaryotic expression amount of the rabies virus recombinant glycoprotein is too low, the prokaryotic expression protein conformation is not right, and the research on the recombinant rabies vaccine has not been progressed. The commonly used rabies vaccine for human is still inactivated vaccine, and has certain safety risk.

In addition, different virus strains are used by different manufacturers, different virus expression hosts are used, the production process is different, the quality is different, and the control of rabies is directly influenced. At present, the experiment for inhibiting virus infection cells is generally adopted to evaluate the effectiveness of the vaccine, which wastes time and labor. The kit for detecting the rabies protective antibody titer by using the ELISA indirect method developed by the recombinant rabies G protein coated plate is simple, convenient, accurate and high in repeatability, and has great social benefit for improving the rabies vaccine quality in China.

Disclosure of Invention

The invention aims to provide a virus recombinant glycoprotein and a method for efficiently expressing the virus recombinant glycoprotein in eukaryotic cells.

It is another object of the present invention to provide various uses of the viral recombinant glycoproteins.

In order to achieve the object, in a first aspect, the present invention provides a viral recombinant glycoprotein which is a recombinant protein formed by replacing a signal peptide of a target viral glycoprotein with a signal peptide of a human or animal secretory protein.

Optionally, a His tag and a flexible Linker can be connected to the N terminal and/or the C terminal of the virus recombinant glycoprotein.

The signal peptide of the human or animal secretory protein comprises but is not limited to a signal peptide of a human antibody IgG heavy chain and/or human albumin.

The target virus glycoprotein of the invention includes, but is not limited to, rabies virus G protein and Ebola virus GP1 protein.

In a second aspect, the invention provides a rabies virus recombinant glycoprotein, which is formed by replacing a signal peptide of a rabies virus G protein with a signal peptide of a human IgG protein heavy chain, and the amino acid sequence of the recombinant glycoprotein is shown as SEQ ID NO. 1.

In a third aspect, the invention provides an Ebola virus recombinant glycoprotein, which is a recombinant glycoprotein formed by replacing a signal peptide of an Ebola virus GP1 protein with a signal peptide of human albumin, wherein an amino acid sequence of the recombinant glycoprotein is shown as SEQ ID NO. 3.

In a fourth aspect, the invention provides a nucleic acid encoding the recombinant glycoprotein.

In a fifth aspect, the invention provides a biological material containing the nucleic acid, wherein the biological material is an expression cassette, an expression vector, a cloning vector, an engineering bacterium or a host cell and the like.

In a sixth aspect, the present invention provides a method for efficiently expressing the recombinant glycoprotein using a eukaryotic cell, comprising the steps of:

1) obtaining and optimizing a nucleic acid encoding the recombinant glycoprotein according to any one of claims 1 to 4 by a PCR method, a DNA recombination method or an artificial synthesis method based on a known protein sequence through codon correspondence and host expression frequency;

2) cloning the nucleic acid into a eukaryotic expression vector, transforming or transfecting host cells, propagating the host cells to achieve expression of the viral recombinant glycoprotein in the eukaryotic cells, and collecting the target protein from the cell culture.

Wherein, the eukaryotic expression vector includes but is not limited to pcDNA 3. The host cell includes but is not limited to 293 cell, CHO.

In a seventh aspect, the present invention provides a monoclonal or polyclonal antibody developed based on the recombinant glycoprotein.

In an eighth aspect, the present invention provides a vaccine, wherein the effective component is the virus recombinant glycoprotein, and optionally comprises pharmaceutical excipients.

In a ninth aspect, the present invention provides a colloidal gold test strip, wherein the test strip is coated with the recombinant glycoprotein, or a target virus antibody detection reagent or a target virus infection diagnosis reagent prepared from the recombinant glycoprotein.

In a tenth aspect, the present invention provides any one of the following uses of the recombinant glycoprotein:

1) the application in preparing vaccine and antiviral drug;

2) the application in preparing a target virus antibody detection reagent or a target virus infection diagnosis reagent;

3) the application in the preparation of colloidal gold test paper strips;

4) the application in preparing monoclonal antibody or polyclonal antibody;

5) the application in ELISA detection of target virus antibodies.

In one embodiment of the invention, the method for efficiently expressing the rabies virus recombinant glycoprotein by using the eukaryotic cell comprises the following steps:

1) obtaining and optimizing nucleic acid for coding the recombinant glycoprotein by a PCR method, a DNA recombination method or an artificial synthesis method according to the amino acid sequence of the rabies virus recombinant glycoprotein through the codon correspondence and host expression frequency;

2) cloning the nucleic acid into eukaryotic expression vector pcDNA3, transforming or transfecting 293 cell, propagating with host cell to realize the expression of virus recombinant glycoprotein in eukaryotic cell, and collecting target protein from cell culture.

The invention further provides application of the rabies virus recombinant glycoprotein in vaccine preparation.

The invention further provides the rabies virus recombinant glycoprotein for immunizing animals or human to produce antibodies.

The invention further provides application of the rabies virus recombinant glycoprotein in rabies virus antibody detection.

The invention further provides application of the rabies virus recombinant glycoprotein in rabies virus antibody diagnosis.

The invention further provides application of the rabies virus recombinant glycoprotein in a rabies virus antibody ELISA detection method.

The invention further provides application of the rabies virus recombinant glycoprotein in a rabies virus antibody gold-labeled test strip.

The rabies virus recombinant glycoprotein provided by the invention can be used for rabies vaccine development because the antibody generated after immunizing a mouse can recognize different rabies virus strains. The purified rabies virus recombinant glycoprotein has cross reaction with serum of animals or human immunized by different rabies vaccines, and can be used for developing animal or human anti-rabies virus antibody diagnostic reagents.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention successfully solves the worldwide technical problems of low expression level and poor secretion (difficult to pass through cell membranes) of the virus recombinant glycoprotein in eukaryotic cells, lays a solid foundation for developing vaccines and virus protective antibody detection reagents, and has good application prospect.

Secondly, the eukaryotic cells are utilized to efficiently express the rabies virus recombinant glycoprotein, the expression quantity of the rabies virus recombinant glycoprotein in the supernatant of the transient 293 cell is up to 10mg/L, and the commercial mass production can be carried out.

Thirdly, the antibody generated after the mouse is immunized by the rabies virus recombinant glycoprotein can identify different rabies virus strains, can be used for rabies vaccine development, and has huge market prospect; the purified rabies virus recombinant glycoprotein has cross reaction with serum of animals or human immunized by different rabies vaccines, can be used for developing animal or human anti-rabies virus antibody diagnostic reagents, and has good application prospect.

Drawings

FIG. 1 is a schematic diagram showing the structure of plasmid pcDNA3 in example 2 of the present invention.

FIG. 2 shows the SDS-PAGE detection of the recombinant rabies G glycoprotein according to example 2 of the present invention.

FIG. 3 shows the results of Westernblot assay for rabies G glycoprotein (no signal peptide added) in example 2 of the present invention. Wherein lanes 1-4 are: cell supernatant, empty vector expression cell supernatant, cell lysate and empty vector cell lysate.

FIG. 4 shows that the purified rabies virus recombinant G glycoprotein is coated with different concentrations in example 2 of the invention, and is detected by using an anti-glycoprotein antibody, and the antibody presents a gradient signal for different coating amounts of glycoprotein.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 Synthesis of rabies Virus recombinant glycoprotein and nucleic acid thereof and construction of expression vector

The signal peptide of the heavy chain of the human IgG protein replaces the signal peptide of the rabies virus glycoprotein, and the obtained recombinant protein is the rabies virus recombinant glycoprotein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1. The Rabies virus is from Rabes virus strain CTN-1.

According to the protein sequence shown in SEQ ID NO. 1, the nucleic acid for coding the rabies virus recombinant glycoprotein is obtained and optimized by utilizing an artificial synthesis method through the codon correspondence and host expression frequency.

Example 2 expression of rabies Virus recombinant glycoprotein Using eukaryotic cells

(I) construction of recombinant plasmid

The optimized rabies virus recombinant glycoprotein nucleic acid sequence is cut by restriction enzyme and inserted into an expression vector pcDNA3 (figure 1), and escherichia coli DH5 alpha competent cells are transformed. And (5) after the successful construction of the vector is verified, preparing the recombinant plasmid. The partial sequence of the constructed recombinant plasmid is shown in SEQ ID NO. 2.

(II) plasmid extraction (using Tiangen plasmid extraction kit)

Shaking the bacteria: 4ml of culture medium containing corresponding antibiotics is added into a 14ml lock cap tube, the sterilized toothpick is dipped with the gene synthesis puncture bacteria and is put into an LB culture medium for overnight culture.

1. Column balancing: 500. mu.L of the equilibrium solution BL was added to the adsorption column CP4 (the adsorption column was placed in the collection tube), and the tube was centrifuged at 12000rpm (about 13400g) for 1min to remove the waste liquid from the collection tube, and the adsorption column was replaced in the collection tube.

2. 5-15ml of overnight-cultured bacterial suspension was added to a centrifuge tube, and centrifuged at 12000rpm (about 13400g) for 1min to remove the supernatant as much as possible. Note that: when the bacterial liquid is more, the bacterial precipitates can be collected into a centrifugal tube through centrifugation for several times, the bacterial amount is better to be fully cracked, and the extraction efficiency of plasmids is reduced due to insufficient cracking of excessive bacteria.

3. To the centrifuge tube where the pellet of the cells remained, 500. mu.L of solution Pl (RNaseA was added in advance) was added, and the pellet of the bacterial cells was thoroughly suspended using a pipette or a vortex shaker.

4. The cells were lysed by adding 500. mu.L of solution P2 to the tube and gently inverting the tube 6 to 8 times.

5. Adding 700 mu L of the solution P3 into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing, wherein white flocculent precipitates appear. Centrifuge at 12000rpm (about 13400g) for 10min, at which time a pellet forms at the bottom of the tube.

6. Adding the supernatant collected in the previous step into filtering column Cs (the filtering column is put into the collecting pipe), centrifuging at 12000rpm (about 13400g) for 2min, and carefully adding the solution obtained in the collecting pipe after centrifugation into adsorption column CP4 (the adsorption column is put into the collecting pipe) in portions.

7.12000rpm (about 13400g) for l min, the waste liquid in the collection tube was discarded, and the adsorption column CP4 was placed in the collection tube.

8. Adding 500 μ L deproteinized solution P4 into adsorption column CP4, centrifuging at 12000rpm (about 13400g) for L min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 in the collection tube.

9. Adding 600 μ L rinsing solution PW (to check whether anhydrous ethanol has been added) into adsorption column CP4, centrifuging at 12000rpm (about 13400g) for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube.

10. 600. mu.L of the rinsing solution PW 4 was added to the adsorption column CP4, and centrifuged at 12000rpm (13400g) for L min to remove the waste liquid from the collection tube.

11. The adsorption column CP4 was replaced in the collection tube and centrifuged at 12000rpm (about 13400g) for 2min to remove the residual rinse from the adsorption column. To ensure that downstream experiments were not affected by residual ethanol, adsorption column CP4 was uncapped and left at room temperature for several minutes to allow complete drying of the residual rinse solution from the adsorption material.

12. The adsorption column was placed in a clean centrifuge tube, and then 300. mu.L of elution buffer TB was dropped into the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12000rpm (about 13400g) for L min to collect the plasmid solution in the centrifuge tube.

(III) detection of plasmid DNA concentration and purity

The concentration and purity of the obtained plasmid DNA were measured by a microspectrophotometer (OD 260/280).

(IV) ethanol precipitation

Plasmid was precipitated overnight by adding 3 volumes of absolute ethanol and 0.1 volumes of 3M sodium acetate. 12000rpm, 4 ℃, centrifuging for 10min, drying in a biological safety cabinet, precipitating, dissolving with pyrogen-free water, and detecting the concentration by a micro spectrophotometer to ensure that the concentration is about 1 mug/mul.

POLODELIverer^TM3000 transient transfection of 293 cells transfection conditions

1. Cells were plated into 6-well plates one day before transfection, 3X 10 cells per well⁵～3.5×10⁵The cells were cultured in 1.5ml complete medium at 37 ℃ in 5% CO₂The cells were incubated overnight under conditions to achieve a cell density of 60% -80% before transfection.

2. Take 4. mu.g of DNA and 8. mu.l of POLODELECTRER^TM3000 diluted in 250ml of Opti-MEM medium, mixed well and left at room temperature for 5 min.

3. Polodeiverer^TM3000 solution is dropped into the DNA solution, blown by a gun or evenly mixed by vortex, and then placed for 15min at room temperature.

4. The mixture is dropped into the culture medium evenly, the culture dish is shaken up lightly, and the liquid does not need to be changed before transfection.

5. Transfected cells were incubated at 37 ℃ with 5% CO₂Culturing for 48-72h under the condition, and changing the solution CD293 after 24h of transfection.

6. Supernatants were collected 48-72h after transfection for transient expression analysis by ELISA assay.

POLODELIverer^TM3000 Mass transfection

1. Large transient transfections of 100ml cells were performed using established transfection conditions and supernatants collected for testing.

2. The supernatant was purified by a nickel column and the purification was checked by ELISA.

3. Protein purity was verified by SDS-PAGE and protein concentration was quantified by BCA.

(VII) results of the experiment

1. The result of SDS-PAGE detection of the recombinant rabies G glycoprotein is shown in FIG. 2.

Western blot analysis of rabies G glycoprotein (control) without signal peptide is shown in FIG. 3. As can be seen from fig. 3, G protein was only present in the cell lysate and was not detected in the cell supernatant.

2. The expression amount and purification yield of recombinant rabies G glycoprotein in eukaryotic cell supernatants are shown in table 1 and fig. 4.

Gradient coating of enzyme label plate with cell supernatant at 1:10, 1:30, 1:100, 1:300, 1:1000, 1:3000, 1:10000, adding 100 μ L per well, reacting at 4 deg.C overnight; after overnight incubation, washing the plate with PBS 3 times, and blocking with 5% milk-containing PBS at room temperature for 1 hr; after blocking, washing the plate 1 time with PBS, adding 2 μ g/ml primary anti-NM 57 diluted with 5% milk-containing PBS, and reacting at room temperature for 1 hr; washing the plate with PBS for 3 times, drying, adding HRP-labeled goat anti-mouse secondary antibody diluted at 1:2000 with PBS containing 5% milk, and reacting at room temperature for 1 hr; after the reaction is finished, washing the plate for 5 times by using PBS solution, drying by beating, adding 100 mu l of substrate (substrate A solution and substrate B solution are uniformly mixed in equal volume) into each hole, and reacting for 20min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (0.1M H) was added to each well₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained.

The actual protein obtained by Ni column purification was 32 mg/L.

TABLE 1

3. Coating the purified rabies virus recombinant G glycoprotein with different concentrations, and detecting by using an anti-glycoprotein antibody, wherein the antibody presents a gradient signal aiming at the glycoprotein with different coating amounts. The ELISA results of the rabies glycoprotein specific monoclonal antibody for identifying and purifying the rabies recombinant G protein are shown in Table 2. Wherein, the monoclonal antibody 7G3 recognizes G protein linear epitope, and the monoclonal antibody NM57 recognizes conformation epitope. The concentration of coating NM57 was 2. mu.g/ml, and the sensitivity of NM57 to recognize G protein was 10 ng/well. The monoclonal antibody NM57 is purchased from New drug research and development Limited liability company of North China pharmaceutical group, and 7G3 is provided by Kyoto Biotechnology (Beijing) Limited company.

TABLE 2

4. The results of the titer of human sera after vaccine immunization was detected after recombinant rabies glycoprotein are shown in table 3. The sensitivity reaches 100 ng/ml.

TABLE 3

Coating the enzyme label plate with purified rabies G protein, wherein the concentration of the enzyme label plate is 1 mu G/ml, 100 mu L of the enzyme label plate is added into each hole, and the enzyme label plate reacts overnight at 4 ℃; after overnight incubation, the plates were washed 3 times with PBS solution and blocked with 3% BSA-PBS at 37 ℃ for 1 hr; after blocking was complete, the plates were washed 3 times with PBS solution. Diluting NM57 with blank matrix human serum, diluting with 3% BSA 20 times and 100 times respectively, adding sample, adding 100 μ L per well, and reacting at 37 deg.C for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, and drying by beating; adding HRP-labeled goat anti-human secondary antibody diluted by PBS containing 3% BSA at a ratio of 1:5000, and reacting at room temperature for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, drying by beating, adding 100 mu l of substrate (substrate A solution and substrate B solution are uniformly mixed in equal volume) into each hole, and reacting for 5min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (0.1M H) was added to each well₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained.

5. The results of the titer of the sera from dogs immunized with the vaccine after recombinant rabies glycoprotein are shown in tables 4-5. The 50-fold dilution mean was 0.291, and Nc +3 × SD was 0.482.

TABLE 4

Coating the enzyme label plate with purified rabies G protein, wherein the concentration of the enzyme label plate is 1 mu G/ml, 100 mu L of the enzyme label plate is added into each hole, and the enzyme label plate reacts overnight at 4 ℃; after overnight incubation, the plates were washed 3 times with PBS solution and blocked overnight at 4 ℃ with 3% BSA-PBS; after blocking was complete, the plates were washed 3 times with PBS solution. Diluting 11 parts of canine blank matrix serum with 20% CS-PBS 20 times, 50 times, 100 times and 200 times respectively, adding sample, adding 100 μ L per well, and reacting at 37 deg.C for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, and drying by beating; adding HRP-labeled rabbit anti-canine secondary antibody diluted by PBS containing 3% BSA at a ratio of 1:10000, and reacting at room temperature for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, drying by beating, adding 100 mu l of substrate (substrate A solution and substrate B solution are uniformly mixed in equal volume) into each hole, and reacting for 6min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (0.1M H) was added to each well₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained.

TABLE 5

Three dog blood background information: 432621# >10.26,43263# -1.5,202145 # -5.92.

Coating the enzyme label plate with purified rabies G protein, wherein the concentration of the enzyme label plate is 1 mu G/ml, 100 mu L of the enzyme label plate is added into each hole, and the enzyme label plate reacts overnight at 4 ℃; after overnight incubation, the plates were washed 3 times with PBS solution and blocked overnight at 4 ℃ using PBS solution containing 3% BSA; after blocking was complete, the plates were washed 3 times with PBS solution. Diluting 4 parts of dog positive serum with 20% CS-PBS respectively by 0 times, 10 times, 25 times and 50 times, directly loading, and diluting 100 times with blank matrix dog serumDiluting 2 times, diluting with 20% CS-containing PBS 50 times, adding 100 μ L per well, and reacting at 37 deg.C for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, and drying by beating; adding HRP-labeled rabbit anti-canine secondary antibody diluted by 3% BSA-PBS solution at a ratio of 1:10000, and reacting at room temperature for 1 hr; after the reaction is finished, washing the plate for 3 times by using PBS solution, drying by beating, adding 100 mu l of substrate (substrate A solution and substrate B solution are uniformly mixed in equal volume) into each hole, and reacting for 6min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (0.1M H) was added to each well₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained.

6. The purified antibody 7G3 recognized the rabies virus recombinant G glycoprotein at a titer of about 10. mu.g/ml (Table 6).

TABLE 6

7. The results of cross-reaction ELISA of tail blood of mice and rabies viruses of different strains after the mice are immunized by the recombinant rabies glycoprotein are shown in Table 7.

TABLE 7

As can be seen from Table 7, the PDC001 mouse tail blood recognizes the strongest signal of Jilin Meifeng, recognizes that Liaoning is large, and the Norcheng organism has a signal of 0.8-1.0, recognizes that Henan far is large and OD is about 0.4, and recognizes that other vaccine signals are weak.

The rabies G protein coating concentration is 1 mug/ml, the vaccine of each manufacturer is coated after being diluted by 1:100, each hole is coated with 100 mug of enzyme label plate respectively, and the reaction is carried out overnight at 4 ℃; after overnight incubation, washing the plate with PBS 3 times, and blocking with 5% milk-containing PBS at room temperature for 1 hr; after blocking, washing the plate 1 time with PBS, adding mouse tail blood diluted 1:500 with 5% milk-containing PBS, and cooling at room temperatureThe time should be 1 hr; washing the plate with PBS for 3 times, drying, adding HRP-labeled goat anti-mouse secondary antibody diluted at 1:2000 with PBS containing 5% milk, and reacting at room temperature for 1 hr; after the reaction is finished, washing the plate for 5 times by using PBS solution, drying by beating, adding 100 mu l of substrate (substrate A solution and substrate B solution are uniformly mixed in equal volume) into each hole, and reacting for 20min under the conditions of light shielding and room temperature; then 50. mu.L of stop solution (0.1M H) was added to each well₂SO₄) After mixing, the OD is read on a microplate reader₄₅₀The value is obtained.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Arg Ser Gly Val Pro Pro Lys Val Val Asn Tyr Glu Ala Gly Glu Trp

85 90 95

Ala Glu Asn Cys Tyr Asn Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu

100 105 110

Cys Leu Pro Ala Ala Pro Asp Gly Ile Arg Gly Phe Pro Arg Cys Arg

115 120 125

Tyr Val His Lys Val Ser Gly Thr Gly Pro Cys Ala Gly Asp Phe Ala

130 135 140

Phe His Lys Glu Gly Ala Phe Phe Leu Tyr Asp Arg Leu Ala Ser Thr

145 150 155 160

Val Ile Tyr Arg Gly Thr Thr Phe Ala Glu Gly Val Val Ala Phe Leu

165 170 175

Ile Leu Pro Gln Ala Lys Lys Asp Phe Phe Ser Ser His Pro Leu Arg

180 185 190

Glu Pro Val Asn Ala Thr Glu Asp Pro Ser Ser Gly Tyr Tyr Ser Thr

195 200 205

Thr Ile Arg Tyr Gln Ala Thr Gly Phe Gly Thr Asn Glu Thr Glu Tyr

210 215 220

Leu Phe Glu Val Asp Asn Leu Thr Tyr Val Gln Leu Glu Ser Arg Phe

225 230 235 240

Thr Pro Gln Phe Leu Leu Gln Leu Asn Glu Thr Ile Tyr Thr Ser Gly

245 250 255

Lys Arg Ser Asn Thr Thr Gly Lys Leu Ile Trp Lys Val Asn Pro Glu

260 265 270

Ile Asp Thr Thr Ile Gly Glu Trp Ala Phe Trp Glu Thr Lys Lys Asn

275 280 285

Leu Thr Arg Lys Ile Arg Ser Glu Glu Leu Ser Phe Thr Val Val Ser

290 295 300

Asn Gly Ala Lys Asn Ile Ser Gly Gln Ser Pro Ala Arg Thr Ser Ser

305 310 315 320

Asp Pro Gly Thr Asn Thr Thr Thr Glu Asp His Lys Ile Met Ala Ser

325 330 335

Glu Asn Ser Ser Ala Met Val Gln Val His Ser Gln Gly Arg Glu Ala

340 345 350

Ala Val Ser His Leu Thr Thr Leu Ala Thr Ile Ser Thr Ser Pro Gln

355 360 365

Ser Leu Thr Thr Lys Pro Gly Pro Asp Asn Ser Thr His Asn Thr Pro

370 375 380

Val Tyr Lys Leu Asp Ile Ser Glu Ala Thr Gln Val Glu Gln His His

385 390 395 400

Arg Arg Thr Asp Asn Asp Ser Thr Ala Ser Asp Thr Pro Ser Ala Thr

405 410 415

Thr Ala Ala Gly Pro Pro Lys Ala Glu Asn Thr Asn Thr Ser Lys Ser

420 425 430

Thr Asp Phe Leu Asp Pro Ala Thr Thr Thr Ser Pro Gln Asn His Ser

435 440 445

Glu Thr Ala Gly Asn Asn Asn Thr His His Gln Asp Thr Gly Glu Glu

450 455 460

Ser Ala Ser Ser Gly Lys Leu Gly Leu Ile Thr Asn Thr Ile Ala Gly

465 470 475 480

Val Ala Gly Leu Ile Thr Gly Gly Arg Arg Thr Arg Arg Glu Ala Ile

485 490 495

Val Asn Ala Gln Pro Lys Cys Asn Pro Asn Leu His Tyr Trp Thr Thr

500 505 510

Gln Asp Glu Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly

515 520 525

Pro Ala Ala Glu Gly Ile Tyr Ile Glu Gly Leu Met His Asn Gln Asp

530 535 540

Gly Leu Ile Cys Gly Leu Arg Gln Leu Ala Asn Glu Thr Thr Gln Ala

545 550 555 560

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

565 570 575

Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr

580 585 590

Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu Pro His Asp Trp Thr

595 600 605

Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp Phe Val Asp

610 615 620

Lys Thr Leu Pro Asp Gln Gly Asp Asn Asp Asn Trp Trp Thr Gly Trp

625 630 635 640

Arg Gln Trp Ile Pro Ala Gly Ile Gly Val Thr Gly Val Ile Ile Ala

645 650 655

Val Ile Ala Leu Phe Cys Ile Cys Lys Phe Val Phe

660 665

Claims

1. The method for efficiently expressing the rabies virus recombinant glycoprotein by utilizing the eukaryotic cells is characterized in that a signal peptide of a human antibody heavy chain is used for replacing a signal peptide of target virus glycoprotein; the amino acid sequence of the rabies virus recombinant glycoprotein is shown as SEQ ID NO. 1, the DNA sequence for coding the recombinant glycoprotein is shown as SEQ ID NO.2, and host cells for expressing the recombinant glycoprotein comprise 293 cells and CHO.