A rabies virus G protein mutant and its application

Abstract

The present invention discloses a rabies virus G protein mutant and its application. The present invention first discloses that the rabies virus G protein mutant is 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; or, a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the protein obtained by mutating the 177th amino acid of the rabies virus G protein shown in SEQ ID NO.1 from asparagine to lysine. Further disclosed is the application of the above-mentioned rabies virus G protein mutant in the preparation of human rabies virus antibody detection kits and reagents. The rabies virus mutant of the present invention can bind to rabies virus IgG antibodies in human serum / plasma with high affinity and can be used to quantitatively detect the rabies virus antibody titer level in human serum / plasma, thereby facilitating dynamic monitoring of the immune effect of the vaccine.

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 its application. Background Art

[0002] Rabies is a zoonotic disease 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 appearance, with a helical symmetric nucleocapsid, an envelope on the surface, and single-stranded RNA inside. The rabies virus genome encodes five structural proteins: 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- and post-exposure care, with rabies vaccination being a key measure. After vaccination, the body produces rabies virus-neutralizing antibodies, the concentration of which determines the protective effect of the vaccine. According to WHO standards, a serum titer of neutralizing antibodies effectively binding to rabies virus must reach or exceed 0.5 international units (IU / mL) to be considered adequately protective. If the antibody titer falls below this standard, booster immunizations are necessary until the required level is reached. Therefore, dynamic monitoring of rabies antibody levels after vaccination is crucial for rabies prevention. Currently, the main rabies vaccines on the Chinese market include: a primary hamster kidney cell rabies vaccine prepared by adapting the aGV strain to primary hamster kidney cells; a purified Vero cell rabies vaccine prepared by adapting the aGV, CTN-1, and PV strains to Vero cells; and an adult diploid rabies vaccine prepared by adapting the PM-1503 strain to human diploid cells. Vero cell rabies vaccine production accounts for approximately 90% of the national rabies vaccine production.

[0004] Chemiluminescence (CL) refers to the emission of light that accompanies a chemical reaction. Chemiluminescent immunoassays (CLIA) combine CL with antigen-antibody immune reactions, offering advantages such as high specificity, high sensitivity, a wide detection range, and a high degree of automation. CLIA can quantitatively measure antibody levels in serum with far greater 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 changes 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. 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 development of rabies vaccines and diagnostic reagents, the impact of antigen glycosylation needs to be fully considered.

[0006] Most of the test 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 represented by A1) or A2):

[0012] A1) a protein obtained by mutating 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 C-terminus of the resulting 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 resulting fusion protein is named mutant GM protein, and its amino acid sequence is shown in SEQ ID NO.4.

[0016] To facilitate 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. The His-tagged sequence of the rabies virus G protein shown in SEQ ID NO. 1 is named GC protein, and the amino acid sequence of the GC protein can be shown in SEQ ID NO. 3. A mutant GM protein is obtained by mutating amino acid position 177 of the rabies virus G protein shown in SEQ ID NO. 1 and adding a His tag, 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 amino acids 177-179 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 amino acids 177-179 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 FIG). Figure 4 ) makes the 177-179 glycosylation modification site NCS (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 antigen epitopes I and IV blocked 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] Recombinant plasmids and / or recombinant cells comprising the above nucleotide sequence are also within the scope of protection 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 chemiluminescence kit further comprises one or more of a mouse anti-human IgG secondary antibody with a label, a sample diluent, a rabies immunoglobulin calibrator, 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 0.01 M Tris, 0.2 M NaCl, 1% casein, 0.1% Tween-20, 1% Triton-100, and 0.1% ProClin 300;

[0026] The rabies immunoglobulin calibrator is prepared by diluting the rabies immunoglobulin national standard (37 IU / ml) with a calibrator diluent (0.01 M Tris, 0.2 M NaCl, 2% BSA, 0.1% ProClin 300) 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-iodinephenol;

[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. The lack of glycosylation at the site improves the detection rate of the rabies virus G protein mutant for rabies virus IgG antibodies in serum / plasma, while also providing better consistency for rabies virus neutralizing antibody titers. The rabies virus G protein mutant is coupled to magnetic particles to prepare a chemiluminescent reagent for detecting rabies virus IgG antibody levels in serum / plasma. Compared to the unmutated protein, the luminescent reagent prepared using the rabies virus G protein mutant as an antigen has better sensitivity and specificity for detecting rabies virus IgG antibodies and is highly correlated with neutralization test results. Therefore, the rabies virus mutant of the present invention can bind to rabies virus IgG antibodies in human serum / plasma with high affinity and can be used to quantitatively detect rabies virus antibody titer levels in human serum / plasma, facilitating dynamic monitoring of the immune effect of vaccines. It can also be used to prepare reagents and kits for detecting rabies virus antibody titer levels with higher detection sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The specific embodiments of the present invention will be further described in detail below with reference to 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 shows the SDS-PAGE of the purification of GC protein and mutant GM protein.

[0035] Figure 3 This is the polymorphism diagram and proportion of the main glycosylated 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 chemiluminescence kit detection results and the neutralization test results for 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 following detailed description 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, compared to the other three vaccine strains, only the PV strain has an N-glycosylation site in the NCS sequence at positions 177-179. The present invention uses the PV strain G protein as an antigenic substrate to analyze whether glycosylation at amino acid N 177 affects 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 sequence of the G protein of the existing Chinese vaccine strain PV (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 (GenBank: M13215.1) was obtained and a His tag was added (the resulting 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 was codon-optimized and the sequence was synthesized (Shanghai Bioengineering Co., Ltd.) and cloned into the pCMV3 vector, named pCMV3-GC.

[0044] Remove the DH-5α competent cells and immediately place 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 30 minutes; place in a 42℃ water bath for 45 seconds, then place on ice for 2 minutes; add 900ul SOC medium, mix, shake and culture at 37℃ 200rpm for 1 hour, centrifuge, remove the supernatant, take an appropriate amount of bacterial liquid to apply to LB plates containing ampicillin, and culture at 37℃ for 19 hours to screen for blue and 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 16 hours, collect the bacterial liquid for plasmid microextraction, and collect the amplified recombinant pCMV3-GC plasmid for later use.

[0045] The pCMV3-GC plasmid was subjected to site-directed mutagenesis to prepare the pCMV3-GM plasmid. The 177th amino acid of the rabies virus GC protein was mutated from asparagine (N) to lysine (K) (i.e., the mutant GM protein). The 531st position of the nucleotide sequence shown in SEQ ID NO.5 needed to be 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 prepared 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 to 50μL, and mixed. PCR reaction procedure: pre-denaturation (1 cycle): 95°C, 3 min; PCR reaction (18 cycles): 95°C, 30 sec; 60°C, 1 min; 68°C, 2.5 min; extension (1 cycle): 68°C, 10 min. After the PCR reaction, add 1 μl of DPn I enzyme to the PCR product, mix thoroughly, and digest at 37°C for 1 h to digest the plasmid. Thaw DH-5α competent cells, add 5 μl of the DPn I digestion product, and incubate on ice for 30 minutes. Incubate in a 42°C water bath for 45 seconds, then incubate on ice for 2 minutes. Add 400 μl of LB medium, mix thoroughly, and incubate at 150 rpm for 1 hour. After incubation, spread the culture onto an LB plate containing ampicillin and incubate inverted at 37°C for 16 hours. Bacteria containing the mutant plasmid will appear blue. Select five blue-spotted colonies for colony PCR. The PCR product is then sent for sequencing, confirming that the T at position 531 of the nucleotide sequence of SEQ ID NO. 5 has been mutated to the G at position 531 of the nucleotide sequence of SEQ ID NO. 6, indicating that the pCMV3-GC plasmid has been mutated into the pCMV3-GM plasmid. Amplify the recombinant pCMV3-GM plasmid for future use.

[0046] The recombinant plasmids pCMV3-GM and pCMV3-GC were transfected into HEK-293 cells, cultured in suspension at 37°C, 5% CO2, and 130 rpm for 3 days, and the supernatant was collected by centrifugation at 5000 rpm for 20 min for purification. The protein was first crudely purified using a nickel column. 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 through a 0.22 μm filter membrane and mixed with an equal volume of binding buffer. The sample was then loaded at a flow rate of 1 mL / min and eluted with different concentrations of elution buffer (5, 25, and 500 mmol / L imidazole) at a flow rate of 2 mL / 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 against PBS overnight. The dialyzed eluate was purified by ion chromatography column. The column was first rinsed with 5 times the column volume of ultrapure water, and then balanced with 5 times the column volume of binding buffer. The dialyzed eluate was filtered with a 0.22 μm filter membrane and 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-balanced with 10 times the column volume 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 FIG. 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. The results showed that the 177th amino acid of GC protein was asparagine (N), and the 177-179 amino acids NCS had glycosylation modifications (main types such as Figure 3 The mutant GM protein has a lysine (K) at position 177 and no glycosylation modification at positions 177-179 (KCS). 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 the figure below. 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 blocked by sugar chains more exposed, which is conducive to 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 chemiluminescence 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: 0.1 M 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 bead storage solution: 0.01M PBS, pH 7.4; 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, mark them as GC and GM, add 20 μl of magnetic particles to each tube, add 300 μl of magnetic bead washing solution, place on a shaker at 300 rpm, wash for 3 minutes; place on a magnetic stand, discard the supernatant, and wash twice more.

[0060] Activate the magnetic microparticles by adding 20 μl of 10 mg / ml EDC and NHS respectively, place on a shaker at 300 rpm and activate at 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 store 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 with calibrator diluent (0.01 M Tris, 0.2 M NaCl, 2% BSA, 0.1% ProClin 300) 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 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] 2) Rabies virus antibody chemiluminescence immunoassay process

[0070] A fully automatic chemiluminescence analyzer was used as the detection tool, and the methodology was an indirect method, that is, 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) were added to the instrument in sequence, and the cells were incubated at 37°C for 20 minutes, followed by magnetic separation and washing 5 times. 50 μl of HRP-mouse anti-human IgG secondary antibody was then added, and the cells were incubated at 37°C for 25 minutes, followed by magnetic separation and washing 5 times. 50 μl of luminescent solution A and 50 μl of luminescent solution 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 Detection of 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 appropriate amount of NaHCO3, and adjust the pH to 7.6.

[0075] 3. 80% cold acetone: Measure 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 viral suspension: Take CVS-11 virus seed and make appropriate dilutions. Inoculate well-grown BSR cells at an infection dose of 0.1. Incubate at 37°C, 5% CO2 for 1 day, then transfer to 34°C for further incubation. After 2 days, collect the culture supernatant and centrifuge at 4000 rpm at 4°C for 10 minutes to remove cell debris. Take the supernatant and add 10% fetal bovine serum. After mixing, the obtained viral suspension is divided into small tubes and frozen below -80°C for later use.

[0079] 2. Pre-titration of virus solution: Take one 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 100 μl of virus solution and add it to 400 μl of DMEM culture medium containing 10% fetal bovine serum, mix thoroughly, and transfer 50 μl of each dilution to a 96-well culture plate. Make two replicates for each dilution, and add 5x10 6 50 μl of a BSR cell suspension (100 μg / ml) was added and cultured at 37°C, 5% CO2 for 24 hours. After the incubation period, the supernatant was discarded, the cells were washed once with PBS, and 50 μl of 80% cold acetone was added to each well. The cells were fixed at 4°C for 30 minutes. The acetone was removed by aspiration, and after evaporation and drying, 50 μl of a 1:50 diluted FITC-labeled rabies virus nucleoprotein antibody was added to each well for staining. The cells were incubated at 37°C for 30 minutes, washed three times with PBS, and dried. 50 μl of 80% glycerol was added to each well. The cells were observed under a fluorescence microscope, and the number of fluorescent foci in each well was counted.

[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 for the neutralization test. The virus with this dilution is selected as the neutralization virus.

[0081] 4. Serially dilute the rabies immunoglobulin standard and serum sample (inactivated at 56°C for 30 minutes) three-fold using DMEM culture medium containing 10% fetal bovine serum. That is, pre-add 100 μl of culture medium to each well of a 96-well culture plate, add 50 μl of the standard or serum sample to it, dilute it 1:3, mix thoroughly, and then pipette 50 μl into 100 μl of culture medium in the next well to make a 1:9 dilution. Repeat this serial dilution until a dilution of 1:729 is reached to obtain the diluted standard and serum sample. Add 50 μl of neutralizing virus to each well of the diluted standard and serum sample. At the same time, set up normal cell control wells (add only 100 μl of DMEM to the well) and neutralizing virus control wells (add 50 μl of neutralizing virus to 100 μl of DMEM culture medium containing 5% fetal bovine serum). Mix well and neutralize at 37°C for 1 hour. Add 50 μl of 1x10 6 A BSR cell suspension at a concentration of 100 μl / ml was incubated at 37°C in 5% CO2 for 24 hours. After the incubation period, the culture medium was aspirated, and 100 μl of PBS was added to each well for washing and aspiration. 50 μl of 80% acetone, precooled to 4°C, was added to each well and fixed at 4°C for 30 minutes. The acetone was aspirated, and after evaporation and drying, 50 μl of a 1:50 fluorescently labeled rabies virus nucleoprotein antibody was added. The plate was incubated at 37°C for 30 minutes. The liquid was aspirated, and the plate was washed two to three times with PBS. The plate was then spin-dried, and 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 sample 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. Among them, 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 for rabies virus neutralizing antibodies (results are shown in Table 1). 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 overall coincidence 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 results of 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 had 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 of antibodies and RFFIT detection of neutralizing antibodies for different antigens

[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 scope of protection of the present invention.

Claims

1. Use of a rabies virus G protein mutant in the preparation of a rabies virus antibody detection kit or reagent, characterized in that: The rabies virus G protein mutant is shown as A1) or A2): A1) a protein obtained by mutating amino acid 177 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 C-terminus of the protein.

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

3. The chemiluminescence kit according to claim 2, characterized in that The chemiluminescence kit further comprises one or more of a mouse anti-human IgG secondary antibody with a label, a sample diluent, a rabies immunoglobulin calibrator, a luminescent liquid A, and a luminescent liquid B.

4. The chemiluminescence kit according to claim 3, characterized in that The marker in the mouse anti-human IgG secondary antibody with a marker is acridinium ester, horseradish peroxidase or alkaline phosphatase.

5. The use according to claim 1, or the chemiluminescence kit according to any one of claims 2 to 4, characterized in that: The rabies virus antibody is a human rabies virus antibody.

6. The use according to claim 5, characterized in that The rabies virus antibody is a human rabies virus IgG antibody.

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