Rabies virus G protein mutant and application thereof

Abstract

The invention discloses a rabies virus G protein mutant and application thereof. The invention firstly discloses a rabies virus G protein mutant which is a protein obtained by mutating the 177th amino acid of the rabies virus G protein as shown in SEQ ID NO.1 from asparagine to lysine; or the fusion protein is obtained by connecting the N terminal or / and the C terminal of the protein obtained by mutating asparagine into lysine in the 177th amino acid of the rabies virus G protein as shown in SEQ ID NO.1 to a label. The invention further discloses application of the rabies virus G protein mutant in preparation of a human rabies virus antibody detection kit and a reagent. The rabies virus mutant disclosed by the invention can be combined with a rabies virus IgG antibody in human serum / plasma with high affinity, and can be used for quantitatively detecting the titer level of the rabies virus antibody in the human serum / plasma, so that the immune effect of a vaccine can be dynamically monitored.

Description

Technical Field

[0001] The present invention relates to the field of biological immunology, and more specifically, to a rabies virus G protein mutant and application thereof. Background Art

[0002] Rabies is a zoonosis caused by a virus found in the saliva of infected animals. It is transmitted to pets and humans through bites or open wounds. Rabies virus (RV) is a single-stranded negative-sense RNA virus belonging to the genus Lyssavirus of the family Rhabdoviridae. Its virus particles are bullet-shaped in shape, with a helical symmetric nucleocapsid, an envelope on the surface, and single-stranded RNA inside. The rabies virus genome encodes five structural proteins, namely glycoprotein (G), nucleoprotein (N), dimerase (L), phosphoprotein (NS) and matrix (M). Glycoprotein (G) is the main protective antigen that can induce the production of neutralizing antibodies. It is an important target protein for the development of rabies vaccines, antiviral drugs and antibody diagnostic reagents.

[0003] The key to preventing rabies lies in standardized pre-exposure and post-exposure treatment, among which rabies vaccination is an important means. After vaccination, the body produces rabies virus neutralizing antibodies, and their concentration level determines the protectiveness of vaccination. According to WHO standards, the neutralizing antibody titer that effectively binds to rabies virus in serum must reach or exceed 0.5 international units (IU / mL) to be sufficiently protective. If the antibody titer is lower than this standard, booster immunization is required until the antibody reaches the required level. Therefore, dynamic monitoring of rabies antibody levels after vaccination plays an important role in preventing rabies. At present, the main rabies vaccines on the Chinese market include: primary hamster kidney cell rabies vaccine prepared by adapting aGV strain to primary hamster kidney cells, aGV strain, CTN-1 strain, and PV strain to Vero cells to prepare purified Vero cell rabies vaccine, and PM-1503 strain adapted to human diploid cells to prepare adult diploid rabies vaccine; the production of Vero cell rabies vaccine accounts for about 90% of the national production of rabies vaccines.

[0004] Chemiluminescence (CL) refers to the emission of light that accompanies a chemical reaction. Chemiluminescent Immunoassay (CLIA) combines chemiluminescence with antigen-antibody immune reactions and has the advantages of high specificity, high sensitivity, wide detection range, and high degree of automation. CLIA can quantitatively detect antibody levels in serum with a much higher sensitivity than traditional methods and can dynamically monitor changes in antibody levels.

[0005] Glycosylation is a common modification of viral proteins and has an important impact on the biological functions of the virus. For pathogens such as rabies virus, glycosylation not only helps the folding, transport and assembly of viral proteins, but also may evade recognition and attack by the host immune system by masking antigenic epitopes and misleading immune responses. Antigen glycosylation modification will change the spatial structure and exposure mode of antigenic epitopes, thereby affecting the binding of antibodies to antigens. N-linked glycosylation is a common type of glycosylation modification of proteins, and its glycosylation modification mainly occurs on the specific amino acid motif of NXS / T (asparagine-X-serine / threonine, X can be any amino acid except proline). In the research and development of rabies vaccines and diagnostic reagents, the impact of antigen glycosylation needs to be fully considered.

[0006] Most of the kits on the market for quantitative detection of human rabies virus IgG antibodies are ELISA kits, which have many manual operation steps, many influencing factors, and lower sensitivity than chemiluminescence, and cannot quickly and dynamically monitor antibody levels before and after vaccination.

[0007] Therefore, it is necessary to develop a new human rabies virus IgG antibody detection kit to solve the above problems. Summary of the invention

[0008] One object of the present invention is to provide a rabies virus G protein mutant, which can bind to rabies virus IgG antibodies in human serum / plasma with high affinity.

[0009] Another object of the present invention is to provide a use of the above-mentioned rabies virus G protein mutant in the preparation of rabies virus antibody detection kits and reagents.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The present invention first provides a rabies virus G protein mutant, wherein the rabies virus G protein mutant is as shown in A1) or A2):

[0012] A1) a protein comprising a mutation of amino acid 177 of the rabies virus G protein shown in SEQ ID NO.1 from asparagine (N) to lysine (K);

[0013] A2) A fusion protein obtained by mutating the 177th amino acid of the rabies virus G protein shown in SEQ ID NO.1 from asparagine (N) to lysine (K) and connecting a tag to the N-terminus or / and the C-terminus of the protein.

[0014] Furthermore, the protein obtained in A1) is named mutant G1 protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0015] Furthermore, the tag in A2) can be a His tag, and the obtained fusion protein is named mutant GM protein, and its amino acid sequence is shown in SEQ ID NO.4.

[0016] In order to facilitate the subsequent protein purification of the rabies virus G protein shown in SEQ ID NO.1, a purification tag, such as a His tag, is added, and the sequence of the rabies virus G protein shown in SEQ ID NO.1 with the His tag added is named GC protein, and the amino acid sequence of the GC protein can be shown in SEQ ID NO.3. The 177th amino acid site of the rabies virus G protein shown in SEQ ID NO.1 is mutated and a His tag is added to obtain a mutant GM protein, and the amino acid sequence is shown in SEQ ID NO.4.

[0017] In the present invention, since the 177th amino acid of the rabies virus G protein or GC protein is asparagine (N), the 177-179 amino acids on the G or GC protein have a glycosylation modification site NCS; the 177th amino acid of the mutant G1 or GM protein is lysine (K), and the 177-179 amino acids on the mutant G1 or GM protein are non-glycosylation modification sites KCS, that is, the 177th amino acid of the mutant G1 or GM protein is mutated from asparagine (N) to lysine (K) (the spatial three-dimensional structure is shown in Figure 4 ) makes the glycosylation modification sites NCS at positions 177-179 (containing the main types of N-glycosylation such as Figure 3 The glycosylation modification site of G or GC protein will affect the binding of the antibody to antigenic site I and antigenic site IV, while the non-glycosylation modification site KCS of mutant G1 or GM protein reduces the steric hindrance of the original sugar chain of amino acids 177-179 of mutant G1 or GM protein, making the antigenic epitopes I and IV shielded by sugar chains more exposed, which is beneficial to the binding of mutant G1 or GM protein to rabies virus antibody.

[0018] The nucleotide sequence encoding the above-mentioned rabies virus G protein mutant is also within the protection scope of the present invention.

[0019] Furthermore, the nucleotide sequence encoding the mutant G1 protein is shown in SEQ ID NO.6, positions 1 to 1572; the nucleotide sequence encoding the mutant GM protein is shown in SEQ ID NO.6.

[0020] The recombinant plasmid and / or recombinant cell comprising the above nucleotide sequence is also within the protection scope of the present invention.

[0021] The present invention further provides the use of the rabies virus G protein mutant in the preparation of rabies virus antibody detection kits and reagents.

[0022] The present invention further provides a chemiluminescent kit for detecting rabies virus antibodies, wherein the chemiluminescent kit comprises magnetic particles coated with the rabies virus G protein mutant.

[0023] Furthermore, the chemiluminescent kit also includes one or more of a mouse anti-human IgG secondary antibody with a marker, a sample diluent, a rabies immunoglobulin calibrator, and a luminescent liquid A and a luminescent liquid B.

[0024] Furthermore, the label in the mouse anti-human IgG secondary antibody with a label can be acridinium ester, horseradish peroxidase (HRP), alkaline phosphatase, preferably horseradish peroxidase.

[0025] In a specific embodiment of the present invention, the sample diluent contains 0.01M Tris, 0.2M NaCl, 1% casein, 0.1% Tween-20, 1% Triton-100, 0.1% ProClin 300;

[0026] The rabies immunoglobulin calibrator is diluted with a calibrator diluent (0.01M Tris, 0.2M NaCl, 2% BSA, 0.1% ProClin 300) to sequentially dilute the rabies immunoglobulin national standard (37 IU / ml) to concentrations of 0 IU / ml, 0.5 IU / ml, 2 IU / ml, 4 IU / ml, 10 IU / ml, and 16 IU / ml to obtain a diluted standard;

[0027] The luminescent solution A contains 0.1M Tris, 0.18mM luminol and 0.1M p-iodophenol;

[0028] The luminescent liquid B is 3 mM hydrogen peroxide.

[0029] In a specific embodiment of the present invention, the rabies virus antibody is a human rabies virus antibody, preferably, a human rabies virus IgG antibody.

[0030] The beneficial effects of the present invention are as follows:

[0031] The rabies virus G protein mutant of the present invention contains a non-glycosylated modification site KCS, and the lack of site glycosylation improves the detection rate of the rabies virus G protein mutant for rabies virus IgG antibodies in serum / plasma, and at the same time has better consistency for the rabies virus neutralizing antibody titer. The rabies virus G protein mutant is coupled to magnetic particles to prepare a chemiluminescent reagent to detect the rabies virus IgG antibody level in serum / plasma. Compared with the unmutated protein, the rabies virus G protein mutant is used as the antigen to prepare a luminescent reagent to detect the rabies virus IgG antibody with better sensitivity and specificity, and has a high correlation with the neutralization test results. Therefore, the rabies virus mutant of the present invention can bind to the rabies virus IgG antibody in human serum / plasma with high affinity, can be used for quantitative detection of the rabies virus antibody titer level in human serum / plasma, is helpful for dynamically monitoring the immune effect of the vaccine, and can be used to prepare reagents and kits for detecting the rabies virus antibody titer level with higher detection sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0033] Figure 1 This is a comparison of the 177th amino acid sequence of different rabies virus vaccine strains.

[0034] Figure 2 The figure is the SDS-PAGE picture of the purification of GC protein and mutant GM protein.

[0035] Figure 3 It is the polymorphism diagram and proportion of the main glycosylation-modified sugar chains at amino acids 177-179 of G protein or GC protein.

[0036] Figure 4 The spatial structure diagram and main antigenic sites of G protein (A) and mutant G1 protein (B).

[0037] Figure 5 Correlation between the results of the chemiluminescence kit and the neutralization test results for the preparation of GC protein (A) and mutant GM protein (B). DETAILED DESCRIPTION

[0038] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] The present invention analyzes the amino acid sequences of the existing rabies vaccine strains (PM1503, CTN-1, 4aGV and PV) in China, and the comparison results of the 177th amino acid sequence are as follows: Figure 1 As shown, it can be seen that compared with the other three vaccine strains, only the N in the NCS sequence of the PV strain at positions 177-179 is an N-glycosylation modification site. The present invention uses the G protein of the PV strain as an antigen substrate to analyze whether the glycosylation of the amino acid N at position 177 will affect the recognition and binding of rabies virus IgG antibodies and neutralizing antibodies in serum.

[0041] Example 1 Preparation and purification of rabies virus GC protein and its mutant GM protein

[0042] The amino acid sequence of the rabies virus mutant protein was modified based on the amino acid of the G protein of the existing vaccine strain PV strain in China (GenBank: AAA47218.1, i.e., SEQ ID NO.1). The specific process is as follows:

[0043] The nucleotide sequence of the G protein of the vaccine strain PV strain (GenBank: M13215.1) was obtained and a His tag was added (the obtained nucleotide sequence is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.3, named GC protein), the HEK293 expression system codon was optimized and the sequence was synthesized (Shanghai Biotechnology Co., Ltd.), and cloned into the pCMV3 vector, named pCMV3-GC.

[0044] Take out the DH-5α competent cells and immediately put them on ice for 5 minutes to thaw; take 1μl pCMV3-GC plasmid and add it to 100μl DH-5α competent cells, stir gently to mix, and place on ice for 30min; place in a 42℃ water bath for 45s, and then place on ice for 2min; add 900ul SOC culture medium, mix, and culture at 37℃ 200rpm for 1h, centrifuge, remove the supernatant, take an appropriate amount of bacterial liquid to apply to the LB plate containing ampicillin, and culture at 37℃ for 19h to screen for blue-white spots; pick 5 white-spot colonies for colony PCR, and agarose gel electrophoresis shows that they are all positive clones containing the target gene. Pick one of the positive pCMV3-GC clone colonies for LB liquid amplification, culture at 37℃ 250rpm for 16h, collect the bacterial liquid for plasmid microextraction, and collect the amplified recombinant pCMV3-GC plasmid for use.

[0045] The pCMV3-GC plasmid was subjected to site-directed mutagenesis to prepare the pCMV3-GM plasmid. The amino acid at position 177 of the GC protein of the rabies virus was mutated from asparagine (N) to lysine (K) (i.e., the mutant GM protein), and the nucleotide sequence at position 531 shown in SEQ ID NO.5 was mutated from T to G. PrimerX was used to design forward and reverse mutation primers (nucleotide sequences such as SEQ ID NO.7 and SEQ ID NO.8). The mutation reaction system was configured according to the instructions of the site-directed mutagenesis kit: 10ng pCMV3-GC plasmid DNA, 1μl 10μM forward / reverse mutation primers, 5μl 5 10xPfu Buffer, 1μl 10mM dNTP Mix, 1.2μl 2.5U / μlPfu DNA Polymerase, and sterile water was added to 50μL and mixed. PCR reaction program: pre-denaturation (1 cycle): 95℃, 3min; PCR reaction (18 cycles): 95℃, 30sec; 60℃, 1min; 68℃, 2.5min; extension (1cylce): 68℃, 10min. After the PCR reaction, add 1μl DPn I enzyme to the PCR product, mix well and digest at 37℃ for 1h to digest the plasmid. Thaw DH-5α competent cells, add 5μl DPn I enzyme digestion product, ice bath for 30min; 42℃ water bath for 45s, then ice bath for 2min; add 400μl LB medium, mix well, and culture at 150 rpm for 1h. After the incubation, spread the bacterial solution on the LB plate containing ampicillin, and culture at 37℃ for 16h. The bacteria containing the mutant plasmid will appear blue. Pick 5 blue-spotted colonies for colony PCR, and send the PCR product for sequencing. It is confirmed that the 531st T of the nucleotide sequence described in SEQ ID NO.5 is mutated to the 531st G of the nucleotide sequence of SEQ ID NO.6, that is, the pCMV3-GC plasmid is mutated to the pCMV3-GM plasmid. Amplify the recombinant pCMV3-GM plasmid for use.

[0046] The recombinant plasmids pCMV3-GM and pCMV3-GC were transfected into HEK-293 cells, respectively, and suspended and cultured at 37°C, 5% CO2, 130rpm for 3 days. The supernatant was collected for purification by centrifugation at 5000rpm for 20min. The protein was first crudely purified with a nickel column, and the column was first rinsed with 5 column volumes of ultrapure water, and then equilibrated with 5 column volumes of binding buffer. The collected cell supernatant was filtered with a 0.22μm filter membrane and mixed with an equal volume of binding buffer, then loaded, with a flow rate of 1mL / min, eluted with different concentrations of elution buffer (5, 25, 500mmol / L imidazole), a flow rate of 2mL / min, and the eluate was collected. Immediately after elution, the column was re-equilibrated with 10 column volumes of binding buffer. The purified protein eluate was dialyzed with PBS overnight. The dialyzed eluate was purified by an ion chromatography column. The column was first rinsed with 5 column volumes of ultrapure water, and then equilibrated with 5 column volumes of binding buffer. The dialyzed eluate was filtered with a 0.22 μm filter membrane and then loaded with the sample at a flow rate of 1 mL / min. The eluate was linearly eluted with elution buffer (1 mol / L Nacl) at a flow rate of 1 mL / min. The eluate was collected and immediately re-equilibrated with 10 column volumes of binding buffer after elution. The eluate was concentrated with an ultrafiltration tube and the concentration was adjusted to 1.5 mg / mL. The purity of the protein was verified by SDS-PAGE (the results are shown in Figure 2 ), to obtain GC protein (whose amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.5) and mutant GM protein (whose amino acid sequence is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.6).

[0047] The prepared GC protein and mutant GM protein were subjected to N-glycosylation detection, and the results showed that the 177th amino acid of the GC protein was asparagine (N), and the 177-179 amino acids NCS had glycosylation modifications (the main types were as follows Figure 3 The mutant GM protein has a lysine (K) at position 177, and the amino acids KCS at positions 177-179 are not glycosylated. Based on literature research, the spatial structure diagram of the G protein and mutant G1 protein and the main antigenic sites were constructed using PyMOL software. The results are shown in Figure 4 As shown, the N-glycosylation site of amino acids 177-179 of the G protein will affect the binding of the antibody to antigenic site I and antigenic site IV, while the amino acids 177-179 of the mutant G1 protein are non-glycosylated sites, which makes the antigenic epitopes I and IV shielded by sugar chains more exposed, which is beneficial for binding to rabies virus antibodies.

[0048] Example 2 Detection of rabies virus antibodies

[0049] 1. Chemiluminescent immunoassay for rabies virus antibodies

[0050] 1) Preparation of rabies virus antibody chemiluminescent kit

[0051] 1. The buffer components used in the rabies virus antibody chemiluminescence kit are as follows:

[0052] Magnetic bead washing solution: 0.01M PBS solution, pH 7.4

[0053] Magnetic bead activation buffer: 0.1M MES solution, pH 5.0

[0054] Magnetic bead blocking solution: 0.01M PBS, pH 7.4; 0.5% BSA; 0.1% ProClin 300

[0055] Magnetic beads storage solution: 0.01M PBS, pH7.4; 0.5% BSA; 0.1% ProClin 300; 3% glycerol

[0056] Sample diluent: 0.01M Tris, 0.2M NaCl, 1% casein, 0.1% Tween-20, 1% Triton-100, 0.1% ProClin 300

[0057] Enzyme-labeled secondary antibody diluent: 0.01M PBS, pH 7.4; 1% casein; 0.1% Tween-20; 1mg / mL Bronidox; 0.1% ProClin 300

[0058] 2. Preparation of magnetic microparticles coated with GC protein and mutant GM protein

[0059] Take two EP tubes respectively, mark them as GC and GM, add 20 μl magnetic microparticles respectively, add 300 μl magnetic bead washing solution, place on a shaker at 300 rpm, wash for 3 min; place on a magnetic rack, discard the supernatant, and wash twice more.

[0060] Activate magnetic microparticles by adding 20 μl of 10 mg / ml EDC and NHS respectively, activate on a shaker at 300 rpm and room temperature for 30 min; place on a magnetic rack and discard the supernatant; add 300 μL of magnetic bead activation buffer, wash for 3 min, place on a magnetic rack, discard the supernatant, and wash twice more.

[0061] Add 30 μL of the prepared GC protein and mutant GM protein to the magnetic particles in the GC and GM tubes, respectively, mix well, and coat at room temperature for 1 hour; after coating, place on a magnetic stand, discard the supernatant, add 20 μL of magnetic bead blocking solution and block at room temperature for 30 minutes, place on a magnetic stand, and discard the supernatant.

[0062] Add 2 ml of magnetic bead preservation solution to resuspend the protein-coated magnetic microparticles, mix well to obtain magnetic microparticles coated with GC protein or mutant GM protein, and place at 4°C for use.

[0063] 3. Preparation of enzyme-labeled secondary antibodies

[0064] Take 2 μl of commercial horseradish peroxidase (HRP)-labeled mouse anti-human IgG secondary antibody and add it to 12 ml of enzyme-labeled secondary antibody diluent, dilute it 1:6000, and obtain HRP-mouse anti-human IgG secondary antibody for use.

[0065] 4. Preparation of rabies immunoglobulin calibrator

[0066] The rabies immunoglobulin national standard (37 IU / ml) was diluted in sequence to concentrations of 0 IU / ml, 0.5 IU / ml, 2 IU / ml, 4 IU / ml, 10 IU / ml, and 16 IU / ml using calibration diluent (0.01 M Tris, 0.2 M NaCl, 2% BSA, 0.1% ProClin 300) to obtain the diluted standard, i.e., the rabies immunoglobulin calibrator.

[0067] 5. Assembly of rabies virus antibody chemiluminescence kit

[0068] The rabies virus antibody chemiluminescent kit includes magnetic particles coated with GC protein or mutant GM protein, HRP-mouse anti-human IgG secondary antibody, sample diluent, rabies immunoglobulin calibrator, luminescent solution A (0.1M Tris, 0.18mM luminol and 0.1M p-iodinephenol) and luminescent solution B (3mM hydrogen peroxide).

[0069] II) Rabies virus antibody chemiluminescent immunoassay process

[0070] A fully automatic chemiluminescence analyzer was used as the detection tool, and the methodology was an indirect method, that is, the instrument sequentially added 10 μl of sample (serum sample or calibrator), 90 μl of sample diluent, and 20 μl of magnetic particles (magnetic particles coated with GC protein or mutant GM protein), incubated at 37°C for 20 min, and then magnetically separated and washed 5 times. Then, 50 μl of HRP-mouse anti-human IgG secondary antibody was added, and after incubation at 37°C for 25 min, magnetically separated and washed 5 times, 50 μl of luminescent liquid A and 50 μl of luminescent liquid B were added for luminescent reaction, the light signal was collected, and the antibody test concentration was recorded.

[0071] 2. Rapid fluorescent focus inhibition test (RFFIT) for rabies virus neutralizing antibodies

[0072] 1) Reagent configuration

[0073] 1. DMEM culture medium containing 5% fetal bovine serum: Take DMEM cell culture medium containing 5% fetal bovine serum, add antibiotics to a final concentration of 100U / ml antibiotics, and add glutamine to a final concentration of 0.03%, add appropriate amount of NaHCO3, and adjust the pH to 7.6.

[0074] 2. DMEM culture medium containing 10% fetal bovine serum: Take DMEM cell culture medium containing 10% fetal bovine serum, add antibiotics to a final concentration of 100U / ml antibiotics, and add glutamine to a final concentration of 0.03%, add an appropriate amount of NaHCO3, and adjust the pH to 7.6.

[0075] 3. 80% cold acetone: Take 80 ml of acetone, add 20 ml of 0.1 mol / L PBS (pH 7.6), mix well, seal, and store at 4°C.

[0076] 4. FITC-labeled rabies virus nucleoprotein antibody: Take 100 μl of commercial FITC-labeled rabies virus nucleoprotein antibody and add it to 4.9 ml of PBS, dilute it 1:50, mix well and set aside.

[0077] 2) Neutralization test

[0078] 1. Preparation of virus suspension: Take CVS-11 virus seed and make appropriate dilution, inoculate well-growing BSR cells with an infection dose of 0.1 MOI, culture at 37°C, 5% CO2 for 1 day, then transfer to 34°C for further culture. Collect the culture supernatant after 2 days, centrifuge at 4°C 4000 rpm for 10 minutes to remove cell debris, take the supernatant and add 10% fetal bovine serum, mix well to obtain the virus suspension, divide into small tubes, and freeze below -80°C for later use.

[0079] 2. Pre-titration of virus solution: Take 1 tube of frozen virus suspension, thaw it in running water, and make 5-fold serial dilutions starting from 1:5 on a 24-well culture plate to obtain virus solution; take 100ul of virus solution and add it to 400μl of DMEM culture medium containing 10% fetal bovine serum, mix it thoroughly, and transfer 50μl of each dilution to a 96-well culture plate. Make 2 copies of each dilution in parallel, and add 5x10 6 / ml BSR cell suspension 50μl, cultured at 37℃, 5% CO2 for 24 hours. After the culture, discard the supernatant, wash once with PBS, add 80% cold acetone, 50μl per well, fix at 4℃ for 30 minutes, remove the acetone, add 50μl 1:50 diluted FITC-labeled rabies virus nucleoprotein antibody to each well after evaporation and drying, incubate at 37℃ for 30 minutes, wash 3 times with PBS, spin dry, add 80% glycerol 50μl per well, observe under a fluorescence microscope, and count the number of fluorescent foci in each well.

[0080] 3. Determination of the optimal dilution of the virus for neutralization test: Count the proportion of fluorescent foci in each well under a fluorescence microscope. The virus dilution at which 80% to 95% of the cells are infected by the virus is the optimal dilution of the virus for neutralization test. The virus with this dilution is selected as the neutralization virus.

[0081] 4. Use DMEM culture medium containing 10% fetal bovine serum to perform a 3-fold serial dilution of the rabies immunoglobulin standard and serum sample (56°C, 30 minutes inactivation), that is, pre-add 100 μl of culture medium to each well of the 96-well culture plate, take 50 μl of the standard or serum sample and add it, dilute 1:3, mix thoroughly, and then take 50 μl and add it to the next well of 100 μl culture medium to make a 1:9 dilution. In this way, the serial dilution reaches a dilution of 1:729 to obtain the diluted standard and serum sample. Add 50 μl of neutralization virus to each well of the diluted standard and serum sample, and set up a normal cell control well (only add 100 μl of DMEM to the well) and a neutralization virus control well (100 μl of DMEM culture medium containing 5% fetal bovine serum, add 50 μl of neutralization virus), mix well and neutralize at 37°C for 1 hour, add 50ul 1x10 6 BSR cell suspension of 100 μl / ml was cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the culture medium was aspirated, 100 μl PBS was added to each well for washing and aspirated, 50 μl of 80% acetone precooled to 4°C was added to each well, fixed at 4°C for 30 minutes, the acetone was aspirated, and 50 μl of 1:50 fluorescently labeled rabies virus nucleoprotein antibody was added after evaporation and drying, incubated at 37°C for 30 minutes, the liquid was aspirated, the plate was washed 2 to 3 times with PBS, and dried, 50 μl of 80% glycerol was added to each well, the number of fluorescent foci was observed under a fluorescence microscope, and the titer of rabies virus neutralizing antibodies in the serum samples was calculated.

[0082] 5. The RFFIT neutralization test was used to detect serum samples from 24 clinical volunteers. The neutralizing antibody concentrations of the samples are shown in Table 1, where No.1-No.4 are sera from volunteers who were not vaccinated with rabies vaccine, and No.5-No.24 are sera from volunteers who were vaccinated with rabies vaccine.

[0083] 3. Correlation between chemiluminescent immunoassay and rapid fluorescent focus inhibition test (RFFIT)

[0084] The same 24 clinical serum samples with confirmed concentrations were tested using the chemiluminescent immunoassay in step one and the rapid fluorescent focus inhibition test (RFFIT) in step two to detect rabies virus neutralizing antibodies (results are shown in Table 1). The analysis of the results showed that compared with the rapid fluorescent focus inhibition test (RFFIT), the sensitivity of the chemiluminescent immunoassay of clinical serum samples with mutant GM protein and GC protein as antigens was 85.71% and 80.95%, respectively, the specificity was 100%, and the total compliance rates were 88% and 84%, respectively. The results of the chemiluminescent immunoassay with mutant GM protein as antigen and the rapid fluorescent focus inhibition test (RFFIT) were highly correlated (r=0.959), while the results of the chemiluminescent immunoassay with GC protein as antigen were less correlated with the rapid fluorescent focus inhibition test (RFFIT) than the chemiluminescent immunoassay with mutant GM protein as antigen (r=0.831); the results after logarithmic transformation of the data are as follows Figure 5 As shown in the figure, compared with the chemiluminescent immunoassay results using GC protein as the antigen, the chemiluminescent immunoassay results using mutant GM protein as the antigen have a better linear relationship with the rapid fluorescent focus inhibition test (RFFIT) test results (represented by "neutralization test" in the figure) (Y=1.213*X+0.1259, R 2 =0.9386).

[0085] Table 1 Comparison of the results of chemiluminescent immunoassay for different antigens and neutralizing antibodies detected by RFFIT

[0086] Sample No. Neutralizing antibody concentration (IU / ml) GM (IU / ml) GC (IU / ml) No.1 0.1 0.2 0.2 No.2 0.1 0.2 0.2 No.3 0.1 0.2 0.2 No.4 0.1 0.2 0.2 No.5 2 1.538 0.794 No.6 10 7.938 4.972 No.7 3.42 1.722 0.641 No.8 1.97 2.482 0.224 No.9 0.5 0.399 0.2 No.10 1.5 0.884 0.2 No.11 10.26 6.657 7.357 No.12 4.5 3.129 2.655 No.13 10.26 5.031 5.747 No.14 17.77 4.414 1.974 No.15 23.38 7.853 0.667 No.16 30.77 6.972 0.627 No.17 1.14 1.092 0.63 No.18 2.60 0.464 2.217 No.19 1.97 1.504 0.58 No.20 30.77 12.807 20.751 No.21 40.50 12.65 8.444 No.22 0.5 0.392 0.2 No.23 7.79 3.841 2.101 No.24 4.5 3.423 1.218

[0087] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A rabies virus G protein mutant, characterized in that: The rabies virus G protein mutant is shown in A1) or A2): A1) a protein obtained by mutating the 177th amino acid of the rabies virus G protein shown in SEQ ID NO.1 from asparagine to lysine; A2) A fusion protein obtained by mutating the 177th amino acid of the rabies virus G protein shown in SEQ ID NO.1 from asparagine to lysine and connecting a tag to the N-terminus or / and the C-terminus of the protein.

2. The rabies virus G protein mutant according to claim 1, characterized in that The amino acid sequence of the rabies virus G protein mutant is shown in SEQ ID NO.2 or SEQ ID NO.

4.

3. A nucleotide sequence encoding the rabies virus G protein mutant according to claim 1 or 2.

4. The nucleotide sequence according to claim 3, characterized in that The nucleotide sequence is shown in positions 1 to 1572 of SEQ ID NO.6 or in SEQ ID NO.

6.

5. A recombinant plasmid or recombinant cell comprising the nucleotide sequence of claim 3 or 4.

6. Use of the rabies virus G protein mutant according to claim 1 or 2 in the preparation of rabies virus antibody detection kits and reagents.

7. A chemiluminescent kit for detecting rabies virus antibodies, characterized in that: The chemiluminescent kit comprises magnetic particles coated with the rabies virus G protein mutant according to claim 1 or 2.

8. The chemiluminescent kit according to claim 7, characterized in that: The chemiluminescent kit also includes one or more of mouse anti-human IgG secondary antibody with a marker, a sample diluent, a rabies immunoglobulin calibrator, a luminescent liquid A and a luminescent liquid B.

9. The chemiluminescent kit according to claim 8, characterized in that: The marker in the mouse anti-human IgG secondary antibody with a marker is acridinium ester, horseradish peroxidase or alkaline phosphatase.

10. The use according to claim 6, or the chemiluminescence kit according to any one of claims 7 to 9, characterized in that: The rabies virus antibody is a human rabies virus antibody, preferably, a human rabies virus IgG antibody.

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