Anti-rabies virus neutralizing antibody as well as preparation and use thereof

Abstract

An anti-rabies virus neutralizing antibody and preparation and a use thereof. Provided is an anti-rabies virus neutralizing antibody, comprising a heavy chain variable region SEQ ID NO. 1-SEQ ID NO. 3, a light chain variable region SEQ ID NO. 4-SEQ ID NO. 5, and GAS. The anti-rabies virus neutralizing antibody has good neutralizing activity; a high isoelectric point provides advantages in the stability of antibody formulations; a high Tm value indicates good thermal stability; strong affinity with recombinant rabies virus glycoproteins shows promise for the mass production of rabies virus neutralizing antibodies, providing technical support for the preparation of low-cost, safe, and effective rabies passive immune formulations.

Description

Anti-rabies virus neutralizing antibody and its preparation and application

[0001] This invention claims priority to the Chinese patent application filed with the China Patent Office on December 7, 2023, with application number 2023116860416 and invention name “A neutralizing antibody against rabies virus and its preparation and application”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-rabies virus neutralizing antibody and its preparation and application. Background Art

[0003] Rabies virus belongs to the Rhabdoviridae family and the genus Lyssavirus. The virus is bullet-shaped, 75-80 nm in diameter and 175-200 nm long. It consists of an inner nucleocapsid containing a 40 nm core and an outer dense envelope with numerous filamentous projections on its surface, each with a hammer-shaped distal end. The entire viral surface exhibits a honeycomb-like, hexagonal structure. The viral genome consists of a negative-stranded, single-stranded RNA with a molecular weight of 4.6106. The viral genome is 11,932 nucleotides long, of which approximately 91% encode five known structural proteins: the glycoprotein (GP), the envelope matrix protein (M2P), the capsid matrix protein (M1P), the nucleoprotein (NP), and the transcriptase protein (LP). The genomic RNA is bound to 180 NP molecules to form a ribonucleoprotein (RNP), which protects the RNA from degradation and provides a suitable structural foundation for genome replication and transcription. M2P is the smallest structural protein of rabies virus (molecular weight of only 25103). It connects the viral outer membrane and the GP and nucleocapsid on the membrane. GP is a typical transmembrane glycoprotein that binds to acetylcholine receptors, rendering the virus neurotoxic. It also induces the production of neutralizing antibodies and stimulates cellular immunity in the host, providing protection against rabies virus attack. NP is a group-specific antigen of rabies virus that can induce the body to produce complement-fixing antibodies. NP-induced protection against rabies virus is generated by the interaction of various cytokines (such as antibodies, monokines, and lymphocytes); it also promotes neutralizing antibodies. Rabies virus GP and NP may also induce the body to produce interferon.

[0004] Rabies is an acute zoonotic disease caused by the rabies virus, primarily affecting the central nervous system. Rabies virus is typically transmitted to humans through saliva or bites from infected animals. Currently available anti-rabies immunoglobulins on the market have certain issues or limitations, such as the difficulty in mass production due to blood source issues, the potential risk of disease transmission, and serum sickness.

[0005] scFvs are composed of the variable regions of the heavy and light chains of an antibody, linked by a short peptide of 15-20 amino acids. They do not contain an Fc fragment and are classified as small molecule genetically engineered antibodies. A complete antibody consists of two heavy chains (H) and two light chains (L). Through artificial modification, only the variable regions can be expressed. The heavy chain variable region (VH) and light chain variable region (Vu) are linked to form a recombinant gene using a synthetic linker gene. The antibody expressed from this recombinant gene is called a single-chain antibody (scFv). Structurally, the N-terminus of the heavy chain can be linked to the C-terminus of the light chain, or the N-terminus of the light chain can be linked to the C-terminus of the heavy chain. Linkers are typically 15-25 amino acids long and are typically composed of glycine (Gly) and serine (Ser). They possess a certain degree of flexibility and protease resistance. Linkers connect the VH and VL proteins, maintaining a certain degree of flexibility so that the functional regions of the VH and Vi proteins can fold and still pair, forming a monovalent antigen-binding site.

[0006] The specificity and low immunogenicity of scFvs make them a promising alternative to traditional therapeutic modalities, improving the accuracy of targeting specific molecules while avoiding adverse side effects. scFvs can be used for rabies virus detection. In the case of rabies infection, treatment is only available shortly after exposure, and accurate diagnosis is crucial for patient survival. scFvs are also less expensive than traditional monoclonal antibodies and are widely available.

[0007] Summary of the Invention

[0008] In order to solve the above problems, the present invention provides an anti-rabies virus neutralizing antibody, which includes a heavy chain variable region, a light chain variable region and a linker. The heavy chain variable region and light chain variable region of the anti-rabies virus neutralizing antibody have specific CDR sequences and can specifically neutralize rabies virus.

[0009] In one aspect, the present invention provides an anti-rabies virus neutralizing antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:

[0010] (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO. 1;

[0011] (2) The amino acid sequence of HCDR2 is shown in SEQ ID NO. 2;

[0012] (3) The amino acid sequence of HCDR3 is shown in SEQ ID NO. 3;

[0013] (4) The amino acid sequence of LCDR1 is shown in SEQ ID NO. 4;

[0014] (5) The amino acid sequence of LCDR2 is GAS;

[0015] (6) The amino acid sequence of LCDR3 is shown in SEQ ID NO.5.

[0016] Specifically, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 or a sequence having more than 80% sequence similarity to SEQ ID NO.6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a sequence having more than 80% sequence similarity to SEQ ID NO.7.

[0017] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 or a sequence having a sequence similarity of more than 85% to SEQ ID NO.6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a sequence having a sequence similarity of more than 85% to SEQ ID NO.7.

[0018] Further preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 or a sequence having more than 90% sequence similarity to SEQ ID NO.6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a sequence having more than 90% sequence similarity to SEQ ID NO.7.

[0019] More preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 or a sequence having more than 95% sequence similarity to SEQ ID NO.6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a sequence having more than 95% sequence similarity to SEQ ID NO.7.

[0020] Most preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 or a sequence having a sequence similarity of more than 98% to SEQ ID NO.6; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a sequence having a sequence similarity of more than 98% to SEQ ID NO.7.

[0021] Specifically, the anti-rabies virus neutralizing antibody also includes a linker.

[0022] More specifically, the amino acid sequence of the linker is as shown in SEQ ID NO.8.

[0023] In another aspect, the present invention provides a nucleic acid of the aforementioned anti-rabies virus neutralizing antibody.

[0024] Specifically, the sequence of the nucleic acid is shown in SEQ ID NO.9.

[0025] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0026] Specifically, the expression vector can be a plasmid, a phage, or a virus.

[0027] In another aspect, the present invention provides a cell comprising the aforementioned anti-rabies virus neutralizing antibody or nucleic acid or expression vector.

[0028] Specifically, the cell is a eukaryotic cell or a prokaryotic cell.

[0029] In another aspect, the present invention provides a method for detecting rabies virus, wherein the detection method is performed by using the aforementioned anti-rabies virus neutralizing antibodies, and the method is not a disease diagnosis or treatment method.

[0030] In another aspect, the present invention provides a rabies virus detection kit, comprising the aforementioned anti-rabies virus neutralizing antibody or nucleic acid or expression vector or cell.

[0031] In another aspect, the present invention provides the use of the aforementioned anti-rabies virus neutralizing antibody or nucleic acid or expression vector or cell in the preparation of a rabies passive immunization preparation.

[0032] In another aspect, the present invention provides a rabies passive immunization preparation comprising the aforementioned anti-rabies virus neutralizing antibody.

[0033] Specifically, pharmaceutically acceptable excipients are also included.

[0034] Preferably, the pharmaceutically acceptable excipient is selected from polysorbate, histidine, sucrose, arginine, sodium chloride, methionine, acetate, trehalose, proline, sorbitol, sodium phosphate, poloxamer 188, ethylenediaminetetraacetic acid, citric acid, mannitol, glutamate, glycine, sodium citrate, sodium succinate and / or lactic acid.

[0035] The technical effects achieved by the present invention are:

[0036] (1) The in vitro neutralizing activity of C07 antibody against CVS-11 strain was determined by RFFIT and was approximately 1500 IU / mg.

[0037] (2) Non-reducing capillary electrophoresis analysis showed that the immunoglobulin monomer content of C07 antibody was higher than 95%.

[0038] (3) The isoelectric point test results showed that the isoelectric point of the antibody was generally high, with the main isoelectric point being about 8.95.

[0039] (4) The affinity of C07 antibody to recombinant rabies virus glycoprotein is 2.393E-10M.

[0040] (5) The C07 antibody has good thermal stability, with the lowest Tm value of approximately 72°C.

[0041] (6) The C07 antibody has 100% neutralization ability against 13 virus strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the purity of antibodies by non-reducing CE-SDS.

[0043] Figure 2 shows the isoelectric point analysis of C07 antibody.

[0044] Figure 3 is a graph showing the affinity determination between the C07 antibody and the recombinant rabies virus glycoprotein.

[0045] FIG4 is a graph showing Tm values ​​determined by differential scanning calorimetry. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0047] Example 1

[0048] 1.1 Main experimental materials

[0049] (1) Cells, viruses, and vectors

[0050] Baby hamster kidney (BSR) cells and the rabies virus challenge virus standard (CVS) were obtained from the National Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention; HEK293 EBNA1 cells were purchased from the Peking Union Medical College Cell Resource Center. The human anti-rabies virus phage library was constructed by the Fifth Research Laboratory of the Lanzhou Institute of Biological Products.

[0051] (2) Bacterial strains

[0052] The Escherichia coli (E. coli) TG1 strain was preserved and provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products.

[0053] (3) Phage antibody library

[0054] The screened rabies virus phage library was provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products. This library was obtained after three rounds of screening using purified and inactivated CVS-11 as the antigen. The construction of this library is described in: Bi Siying, Mao Xiaoyan, Chen Jijun, et al. Construction and screening of a human anti-rabies virus scFv phage antibody library [J]. Chinese Journal of Biological Products, 2012, 25(12): 1578-1582. DOI: 10.13200 / j.cjb.2012.12.19.bisy.004.

[0055] (4) CHO-K1 cells

[0056] The whole-molecule antibody was expressed in CHO-K1 cells, which were obtained from ATCC.

[0057] 1.2 Main reagents and equipment

[0058] Vent DNA polymerase was purchased from New Brunswick, Wisconsin-Madison; restriction endonucleases and T4 DNA ligase were purchased from Promega (Cat. No. M1801); plasmid extraction kits were purchased from Qiagen (Cat. Nos. 12163 and 12125); national standard for human anti-rabies virus immunoglobulin was purchased from the China Food and Drug Administration (national standard for human anti-rabies virus immunoglobulin); fluorescein isothiocyanate (FITC)-labeled anti-rabies virus nucleoprotein antibody was purchased from Beijing Kangsiertai Medical Research Center (Cat. No. CAR100); OPM CD07, OPM CD18, and OPM CD20 culture medium was purchased from Shanghai Aopuma Biotechnology Co., Ltd. (catalog numbers: P091307 and F091718, respectively); 2YT bacterial culture medium was purchased from BD; ampicillin and kanamycin were purchased from Shanghai Sangon Biotechnology Co., Ltd.; glucose was purchased from Amresco (catalog number 0188); helper phage M13K07 was provided by Lanzhou Institute of Biological Products Co., Ltd.; Mabselect SuRe affinity purification medium (catalog number 17543801) was purchased from Sterfan. A fluorescence microscope was purchased from Olympus IX71; a DNA gel recovery kit was purchased from Qigen (catalog number 28704); and PrimeScript TM The First Strand cDNA Synthesis Kit was purchased from Bio-Ray Biotechnology Co., Ltd. (Cat. No. 6210A). Human rabies immune globulin (HRIG) was produced by Sinopharm Wuhan Blood Products Co., Ltd. The SPR analysis CM5 chip His capture kit was purchased from Cytiva (Cat. Nos. BR100012 and 28995056, respectively).

[0059] 1.3 Analysis of anti-rabies virus neutralization activity using immunofluorescence focus inhibition assay (RFFIT)

[0060] 1.3.1 Neutralization Virus Dilution Determination

[0061] The rabies virus CVS-11 (challenge virus standard) to be tested was diluted three-fold using DMEM medium containing 10% calf serum. 50 μL of the diluted virus was added to a 96-well plate. Two wells of each dilution were used as parallels. 1.0×10 6Add 50 μL of BSR cell suspension at a concentration of 1 cell / mL. Add 50 μL of culture medium to each test well, incubate at 37°C with 5% CO2 for 24 hours. Discard the supernatant. Wash the cells once with PBS and fix them with 200 μL of pre-cooled 80% acetone for 10 minutes; discard the acetone and let them stand at room temperature for 15 minutes. Dilute the FITC-labeled anti-rabies virus nucleoprotein antibody 200 times with PBS, add 100 μL to each well, incubate at 37°C for 1 hour, discard the liquid, and wash three times with PBS. Observe the infection ratio of cells at different dilutions under a fluorescence microscope. The virus dilution at which 80%-95% of the cells are infected is the virus dilution used for neutralization.

[0062] 1.3.2 RFFIT determination of in vitro neutralization activity

[0063] The phage or transiently expressed antibody to be analyzed, and the national standard of rabies immunoglobulin were diluted 1:3 starting from 10 times, for a total of 8 dilutions; DMEM medium containing 10% newborn calf serum was used as a negative control. 50 μL of each of the sample to be tested, standard, and negative control were added to a 96-well cell culture plate, and 50 μL of neutralizing virus was added to each well. After neutralization at 37°C for 1 hour, 1.0×10 6 Incubate 50 μL of a BSR cell suspension (100 μg / mL) at 37°C in 5% CO2 for 24 hours. Discard the supernatant. Wash the cells once with PBS and fix them with 200 μL of pre-chilled 80% acetone at -30°C for 10 minutes. Discard the acetone and let the cells stand at room temperature for 15 minutes. Dilute FITC-labeled anti-rabies virus nucleoprotein antibody 200-fold with PBS, add 100 μL to each well, incubate at 37°C for 1 hour, discard the liquid, and wash three times with PBS. Observe and record the infection rate of each well under a fluorescence microscope. Calculate the antibody activity according to the formula for the rabies immunoglobulin titer assay in the Pharmacopoeia of the People's Republic of China (Volume III).

[0064] The results of preliminary screening of the neutralizing activity of monoclonal phage antibodies by RFFIT analysis showed that the phage neutralizing activity was 3.67 IU / mL.

[0065] 1.4 Preliminary screening of monoclonal phage antibody particles by RFFIT analysis

[0066] 1.4.1 Preparation of monoclonal phage antibody particles

[0067] Take 100 μL of phage after three rounds of screening and add it to 1 mL of E. coli TG1 culture medium (A600nm = 0.5-0.7), mix well, and infect at room temperature for 30 minutes. Spread the culture medium onto four 150 mm 2YT-A plates and incubate at 30°C overnight. Select clones for culture and add each clone to a 96-well plate containing 100 μL of 2YT medium containing ampicillin and incubate at 37°C at 220 rpm overnight. Take 10 μL of the culture and add it to 140 μL of fresh 2YT medium containing ampicillin and incubate at 37°C at 220 rpm for 1.5 hours. Add 15 μL of M13K07 helper phage, mix well, and infect at room temperature for 30 minutes. Centrifuge at 4°C, 8 cm radius, 6000 rpm for 10 min, resuspend the cells in 150 μL of 2YT medium containing ampicillin and kanamycin, and culture overnight at 30°C, 220 rpm; centrifuge at 4°C, 8 cm radius, 6000 rpm for 20 min, and collect the supernatant.

[0068] 1.4.2 RFFIT analysis of monoclonal phage antibody particles

[0069] Take 3.3 μL of culture supernatant, add 46.7 μL of DMEM medium and 50 μL of diluted neutralizing virus, neutralize at 37℃ for 1 hour, and then add 1.0×10 6 50 μL of a BSR cell suspension (100 μg / mL) was incubated at 37°C with 5% CO2 for 24 hours. The supernatant was discarded. The cells were washed once with PBS and fixed with 200 μL of pre-cooled 80% acetone for 10 minutes. The acetone was discarded and the cells were allowed to stand at room temperature for 15 minutes. 100 μL of FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200-fold in PBS and added to each well. The cells were incubated at 37°C for 1 hour, the solution was discarded, and the cells were washed three times with PBS. The infection rate of each well was observed and recorded under a fluorescence microscope. Wells showing significant inhibition of viral growth were designated as positive. 50 μL of bacterial suspension was removed from the corresponding 96-well plate and added to 1 mL of 2YT-A medium. The cells were incubated overnight at 37°C at 220 rpm. 300 μL of the bacterial suspension was added to 300 μL of autoclaved glycerol and stored at -70°C. Another 300 μL of the bacterial suspension was sent for sequencing. Sequencing results were compared online using vbase2 to confirm the correct scFv sequence. Correct sequences were then aligned using DNASTAR 7.1, and clones with non-duplicate sequences were selected for the next step. The phage sequencing primer sequence was CGA AGGAGACAGTCATAATG.

[0070] Phage particle sequencing showed that the light chain of the C07 antibody belongs to the human KV1 family, and the heavy chain belongs to the human HV3 family. The ScFv nucleotide sequence is as follows:

[0071] C07 antibody ScFv nucleotide sequence SEQ ID NO.9:

[0072] The amino acid sequence of the heavy chain variable region of the C07 antibody ScFv is SEQ ID NO.6:

[0073] The amino acid sequence of the light chain variable region of the C07 antibody ScFv is SEQ ID NO.7:

[0074] 1.4.3 Purification and activity determination of monoclonal phage antibody particles

[0075] Add 15 μL of the preserved bacterial suspension to 1.5 mL of 2YT medium containing ampicillin and glucose. Prepare phage particles according to 1.4.1. Add 1 / 5 volume of 20% PEG-8000 + 2.5 mol / L NaCl solution to the resulting supernatant, mix thoroughly, and incubate on ice for 1 hour. Centrifuge at 12,000 rpm, 8 cm radius, for 20 minutes to precipitate the phage antibody particles. Resuspend the pellet in 1.0 mL of PBS-1% BSA and centrifuge at 12,000 rpm, 8 cm radius, for 5 minutes. Discard the pellet and freeze at -70°C.

[0076] 1.5 Whole-molecule antibody expression and purification

[0077] 1.5.1 Whole-molecule antibody expression

[0078] Take a piece of recombinant engineered cells stored in liquid nitrogen, resuscitate, add OPM CD07 medium, and culture at 37°C, 5% CO2; after the cells grow to an appropriate density, use OPM CD07 to adjust the cell density to 0.5-2.0 cells / ml, and culture at 37°C, 5% CO2. Feed the cells on days 3, 5, 7, 9, and 11 of culture. The feed ingredients are OPM-CHO CDF18 and OPM-CHO CDF26, and the amount recommended by the culture medium manufacturer is added.

[0079] 1.5.2 Whole-molecule antibody purification

[0080] After incubation, the cell suspension was centrifuged at 2000 g for 10 minutes, and the supernatant was clarified and filtered using a 0.45 μm capsule filter. Chromatography was performed using a Stovepane Mabselect SuRe affinity chromatography medium. The medium was equilibrated with PBS, and after loading, intermediate washes were performed with PBS. The target protein was eluted with 10 mmol / L sodium acetate solution (pH 3.5) to collect the target protein.

[0081] Example 2 Analysis of whole-molecule antibody activity

[0082] 2.1 Activity assay

[0083] The protein concentration of the purified antibody was determined using the A280 assay. The purified antibody was then used to determine its in vitro neutralizing activity according to 1.3.2.

[0084] The in vitro neutralizing activity of C07 antibody against CVS-11 strain was determined by RFFIT and was approximately 1500 IU / mg.

[0085] 2.2 Antibody purity analysis

[0086] Antibody purity was analyzed using non-reducing CE-SDS. Purified monoclonal antibodies were assayed using CE-SDS. The non-reducing CE-SDS sample was diluted to 1 mg / mL, and 5% by volume of 0.8 mol / L iodoacetamide was added. The sample was heated at 70°C for 15 minutes. After injection, the concentration was run at 15 kV for 15 minutes, and the A214 nm value was measured. After analysis, the spectrum was integrated to calculate the antibody monomer purity.

[0087] Non-reducing capillary electrophoresis analysis showed that the immunoglobulin monomer content of C07 antibody was higher than 95%. The detection spectrum is shown in Figure 1.

[0088] 2.3 Isoelectric point determination

[0089] Isoelectric point analysis was performed using the CIEF method. The sample was diluted with deionized water to a working concentration of 10 mg / mL. 200 μL of 3 mmol / L urea-CIEF Gel, 12 μL of ampholyte, 20 μL of cathode stabilizing solution, 2 μL of anode stabilizing solution, 2 μL of PI marker A, 2 μL of PI marker B, 2 μL of PI marker C, and 10 μL of the antibody to be detected were mixed. The main detection parameters of capillary electrophoresis are as follows: capillary length 21 cm, capillary temperature 20°C, sample temperature 10°C, focusing voltage 25 kV, separation voltage 30 kV, and UV detector wavelength 280 nm.

[0090] The isoelectric point test results showed that the antibody has a high isoelectric point overall, with a main isoelectric point of approximately 8.95. The isoelectric point test spectrum is shown in Figure 2.

[0091] 2.4 Affinity determination

[0092] A capture assay was used to determine the equilibrium dissociation constant (KD) between a recombinant human anti-rabies virus monoclonal antibody and the rabies virus glycoprotein. The capture molecule, Anti-His Antibody, was coated on a CM5 chip, and the glycoprotein was captured as the ligand. The antibody was then measured as the analyte. The Anti-His Antibody was diluted to 1-10 μg / mL with sodium acetate and mixed thoroughly. The immobilization program was selected. The chip surface was first activated with a mixture of NHS / EDC. The Anti-His Antibody was then coupled to the selected channel at a flow rate of 10 μL / min for 420 seconds. Finally, a blank channel was blocked with ethanolamine. HBS-EP+Buffer was diluted 10-fold as the sample diluent and running buffer, and the single-cycle knetics program was run. To capture the ligand, the glycoprotein was first diluted 1200-fold with running buffer. The flow rate was 10 μL / min, and the capture time was 100 seconds. The test sample was then serially diluted (5 concentration points), using running buffer as a blank control. The analyte binding time was 120 seconds, the dissociation time was 600 seconds, and the flow rate was 30 μL / min. The regeneration solution was glycine, with a binding time of 30 seconds and a flow rate of 30 μL / min. The analysis software was opened and the equation was fitted using a 1:1 binding model to automatically determine the KD value.

[0093] The affinity map determined by Biacore is shown in Figure 3. After calculation, the affinity of C07 antibody to recombinant rabies virus glycoprotein is 2.393E-10M.

[0094] 2.5 Thermal stability analysis

[0095] Thermal stability analysis was performed using a microcal differential scanning calorimeter (Microcal PEAQ-DSC). The test sample was treated with buffer to a protein concentration of approximately 1 g / L and then tested on the instrument.

[0096] Tm is an important indicator of protein thermal stability; the higher the Tm, the better the protein's stability. The LZR07 antibody exhibited three distinct peaks, as shown in Figure 4 and Table 1. Its lowest Tm value was approximately 72°C, indicating good thermal stability.

[0097] Table 1 C07 antibody thermal stability test results

[0098] 2.6 Mouse neutralization assay to determine protection against street strains

[0099] (1) Domestic rabies virus strains

[0100] There are 15 rabies virus strains isolated from different regions and at different times in China, namely: JX13-417, ZJ13-431, JX13-189, JX12-234, JX2017-45, JX2017-28, GN07, Hubei, Shaanxi-HZ, SC-HZ, BD06, JX08-45, ZJ-QZ, ZJ12-03, and DRV.

[0101] (2) Rabies virus preparation and LD50 determination

[0102] Rabies virus seed (mouse brain toxic homogenate) was diluted 100-fold with DMEM and injected intracerebrally into 15-18g mice, with 25 μL per mouse. The mice were observed after injection and the onset of dying mice was observed after 4 days. The brain tissues of infected mice were homogenized by adding 5 mL / g DMEM culture medium containing antibiotics to prepare a 20% brain tissue homogenate, which was frozen at -80°C. When determining the LD50, the frozen homogenate was taken, melted and mixed, and then diluted 10-fold. Three gradients of 10-3, 10-4, and 10-5 were injected intracerebrally into mice, with 25 μL per mouse. Ten mice were injected per group. The mice were observed for 14 days after injection and the LD50 of different strains was calculated based on the mortality of the mice.

[0103] (3) Antibody neutralization test for rabies virus

[0104] Rabies virus C07 antibodies were diluted to the designated titer (40 IU / mL) and mixed with an equal volume of virus solution containing 100 LD50. The mixture was neutralized by incubation at 37°C for 60 minutes and then placed on ice. The neutralized mixture was injected intracerebrally into 15-18 g Kunming mice, with 25 μL injected per mouse, and 10 mice per group. For each strain, a control group without antibody (replaced with an equal volume of PBS) and a control group receiving rabies human immunoglobulin (HRIG) diluted to 40 IU / mL were also established. Mice were kept and observed for 28 days, and mortality was recorded. The neutralization capacity of the antibodies against prevalent rabies strains was analyzed. The results are shown in Table 2.

[0105] Table 2 20IU / mL antibody to 50LD 50 Death of mice after rabies virus neutralization

[0106] As can be seen from Table 2, with 28 days as the data endpoint, all mice in the control groups of each virus strain became ill and died, with an incubation period ranging from 6 to 15 days; the control HRIG antibody at 20 IU / mL had complete neutralization ability against all 15 virus strains; the antibody had 100% neutralization ability against 13 of the viruses; in the experimental groups that were not completely neutralized, the mortality rate of mice was 20%-30%, but the incubation period of mice was extended by 4 to 8 days compared with the virus-challenged control group.

Claims

1. An anti-rabies virus neutralizing antibody, characterized in that: It comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO.1; (2) The amino acid sequence of HCDR2 is shown in SEQ ID NO.2; (3) The amino acid sequence of HCDR3 is shown in SEQ ID NO. 3; (4) The amino acid sequence of LCDR1 is shown in SEQ ID NO. 4; (5) The amino acid sequence of LCDR2 is GAS; (6) The amino acid sequence of LCDR3 is shown in SEQ ID NO.

5.

2. The anti-rabies virus neutralizing antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6 or a sequence having more than 80% sequence similarity to SEQ ID NO.6; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.7 or a sequence having more than 80% sequence similarity to SEQ ID NO.

7.

3. The anti-rabies virus neutralizing antibody according to any one of claims 1-2, characterized in that: The anti-rabies virus neutralizing antibody also includes a linker.

4. The anti-rabies virus neutralizing antibody according to claim 3, characterized in that: The amino acid sequence of the linker is shown in SEQ ID NO.

8.

5. A nucleic acid encoding the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4.

6. The nucleic acid according to claim 5, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.

9.

7. An expression vector comprising the nucleic acid of claim 5 or 6.

8. A cell comprising the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 6, or the expression vector according to claim 7.

9. The cell according to claim 8, characterized in that The cells are eukaryotic cells or prokaryotic cells.

10. A method for detecting rabies virus, characterized in that: The detection is carried out by using the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4, wherein the method is a non-disease diagnosis or treatment method.

11. A rabies virus detection kit, characterized in that: It comprises the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 6, the expression vector according to claim 7, or the cell according to any one of claims 8 to 9.

12. Use of the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 6, the expression vector according to claim 7, or the cell according to any one of claims 8 to 9 in the preparation of a rabies passive immunization preparation.

13. A rabies passive immunization preparation, characterized in that: Comprising the anti-rabies virus neutralizing antibody according to any one of claims 1-4.

14. The rabies passive immunization preparation according to claim 13, characterized in that: Pharmaceutically acceptable excipients are also included.

15. The rabies passive immunization preparation according to claim 14, characterized in that: The pharmaceutically acceptable excipient is selected from polysorbate, histidine, sucrose, arginine, sodium chloride, methionine, acetate, trehalose, proline, sorbitol, sodium phosphate, poloxamer 188, ethylenediaminetetraacetic acid, citric acid, mannitol, glutamate, glycine, sodium citrate, sodium succinate and / or lactic acid.

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