A kind of anti-rabies virus neutralizing antibody and its preparation and application

Abstract

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. The present invention provides an anti-rabies virus neutralizing antibody, including 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, and has advantages in the stability of antibody preparations; a high Tm value and good thermal stability; a strong affinity with recombinant rabies virus glycoprotein, and can neutralize a variety of street virus strains well. It is expected to achieve mass production of rabies virus neutralizing antibodies, providing technical support for the preparation of low-cost, safe and effective rabies passive immunization preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-rabies virus neutralizing antibody and a preparation and application thereof. Background Art

[0002] Rabies virus belongs to the family Rhabdoviridae and the genus Lyssavirus. The virus is bullet-shaped, with a diameter of 75-80nm and a length of 175-200nm. The inner layer is a nucleocapsid containing a 40nm core, and the outer layer is a dense envelope with many filamentous protrusions on the surface, the distal end of which is hammer-shaped. The entire surface of the virus presents a honeycomb hexagonal structure. The genome of the virus is a negative-stranded single-stranded RNA with a molecular weight of 4.6106. The viral genome is 11,932 nucleotides long, of which about 91% of the nucleotides are involved in encoding five known structural proteins, namely glycoprotein (GP), envelope matrix protein (M2P), capsid matrix protein (M1P), nucleoprotein (NP) and transcriptase protein (LP). The genomic RNA is combined with 180 NP molecules to form a ribose nucleoprotein (RNP), which protects the RNA from degradation and provides a suitable structural basis for the replication and transcription of the genome. M2P is the smallest structural protein of rabies virus (molecular weight is only 25103), which can connect the outer membrane of the virus and the GP and nucleocapsid on the membrane. GP is a typical transmembrane glycoprotein that can bind to acetylcholine receptors to make the virus neurotoxic. It can also induce the production of neutralizing antibodies and stimulate cellular immunity in the host, and has a protective effect against the attack of rabies virus. NP is a group-specific antigen of rabies virus, which can cause the body to produce complement-fixing antibodies. The anti-rabies virus protection induced by NP is produced by the interaction of various cytokines (such as antibodies, monokines and lymphocytes); it can also promote neutralizing antibodies. Rabies virus GP and NP may also induce the body to produce interferon.

[0003] Rabies is an acute zoonotic infectious disease caused by the rabies virus, which mainly invades the central nervous system. The rabies virus is usually transmitted to humans by infected animals through saliva or bites. Currently, the anti-rabies virus immunoglobulins on the market all have certain problems or limitations, such as the inability to mass produce due to blood source problems, potential risks of disease transmission or serum sickness, etc.

[0004] scFv is composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected by a short peptide of 15-20 amino acids. It does not contain Fc fragments and is a small molecule genetic engineering antibody. The complete antibody is composed of two heavy chains (heavy chain, H) and two light chains (light chain, L). Through artificial modification, it can only express the variable region. The antibody heavy chain variable region (VH) and the light chain variable region (Vu) are connected into a recombinant gene through an artificially synthesized linker gene. The antibody expressed by the recombinant gene is called a single-chain antibody (scFv). Structurally, the N-terminus of the heavy chain can be connected to the C-terminus of the light chain, or the N-terminus of the light chain can be connected to the C-terminus of the heavy chain. The length of the linker is usually 15-25 amino acids, usually composed of glycine (Gly) and serine (Ser), with a certain elasticity and protease resistance. The role of the linker is to connect VH and VL, and maintain a certain elasticity so that the functional regions of VH and Vi can still be paired after folding to form a monovalent antigen binding site.

[0005] The specificity and low immunogenicity of scFvs make them a good alternative to traditional forms of treatment, improving the accuracy of targeting specific molecules and avoiding adverse side effects. scFvs can be applied to the detection of rabies virus. In the case of rabies infection, which is only treatable shortly after exposure, accurate diagnosis is crucial for the survival of patients. scFvs are cheaper than traditional monoclonal antibodies and can be widely popularized. Summary of the invention

[0006] 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 the light chain variable region of the anti-rabies virus neutralizing antibody have specific CDR sequences and can specifically neutralize rabies virus.

[0007] 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:

[0008] (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO.1;

[0009] (2) The amino acid sequence of HCDR2 is shown in SEQ ID NO.2;

[0010] (3) The amino acid sequence of HCDR3 is shown in SEQ ID NO. 3;

[0011] (4) The amino acid sequence of LCDR1 is shown in SEQ ID NO. 4;

[0012] (5) The amino acid sequence of LCDR2 is GAS;

[0013] (6) The amino acid sequence of LCDR3 is shown in SEQ ID NO.5.

[0014] 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.

[0015] 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 85% 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 85% sequence similarity to SEQ ID NO.7.

[0016] 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.

[0017] 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.

[0018] Most 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 98% 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 98% sequence similarity to SEQ ID NO.7.

[0019] Specifically, the anti-rabies virus neutralizing antibody also includes a linker.

[0020] More specifically, the amino acid sequence of the linker is as shown in SEQ ID NO.8.

[0021] In yet another aspect, the present invention provides a nucleic acid of the aforementioned anti-rabies virus neutralizing antibody.

[0022] Specifically, the sequence of the nucleic acid is shown in SEQ ID NO.9.

[0023] In yet another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0024] Specifically, the expression vector can be a plasmid, a phage, or a virus.

[0025] In yet another aspect, the present invention provides a cell comprising the aforementioned anti-rabies virus neutralizing antibody or nucleic acid or expression vector.

[0026] Specifically, the cell is a eukaryotic cell or a prokaryotic cell.

[0027] 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 antibody, and the method is a non-disease diagnosis or treatment method.

[0028] In yet 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.

[0029] In yet 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.

[0030] In yet another aspect, the present invention provides a rabies passive immunization preparation, comprising the aforementioned anti-rabies virus neutralizing antibody.

[0031] Specifically, pharmaceutically acceptable excipients are also included.

[0032] 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.

[0033] The technical effects achieved by the present invention are:

[0034] (1) The in vitro neutralizing activity of C07 antibody against CVS-11 strain was determined using RFFIT and was approximately 1500 IU / mg.

[0035] (2) Non-reducing capillary electrophoresis analysis showed that the immunoglobulin monomer content of C07 antibody was higher than 95%.

[0036] (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.

[0037] (4) The affinity of C07 antibody to recombinant rabies virus glycoprotein is 2.393E-10M.

[0038] (5) The C07 antibody has good thermal stability, with the lowest Tm value of approximately 72°C.

[0039] (6) The C07 antibody has 100% neutralization ability against 13 virus strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The purity of the antibody was determined by non-reducing CE-SDS.

[0041] Figure 2 This is the isoelectric point analysis of C07 antibody.

[0042] Figure 3 This is the affinity determination graph of C07 antibody and recombinant rabies virus glycoprotein.

[0043] Figure 4 The Tm value spectrum is determined by differential scanning calorimetry. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0045] Example 1

[0046] 1.1 Main experimental materials

[0047] (1) Cells, viruses and vectors

[0048] Baby hamster kidney (BSR) cells and rabies virus challenge virus standard (CVS) were obtained from the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention; HEK293 EBNA1 cells were purchased from Peking Union Medical College Cell Resource Center. The human anti-rabies virus phage library was constructed by the Fifth Research Laboratory of Lanzhou Institute of Biological Products.

[0049] (2) Bacterial species

[0050] The Escherichia coli (E. coli) TG1 strain was preserved and provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products.

[0051] (3) Phage antibody library

[0052] The screened rabies virus phage library was provided by the Fifth Research Laboratory of Lanzhou Institute of Biological Products. The library used purified and inactivated CVS-11 as antigen and was obtained after three rounds of screening. The construction of the library can be found in: Bi Siying, Mao Xiaoyan, Chen Jijun, et al. Construction and screening of 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.

[0053] (4) CHO-K1 cells

[0054] The whole-molecule antibody was expressed in CHO-K1 cells obtained from ATCC.

[0055] 1.2 Main reagents and equipment

[0056] Vent DNA polymerase was purchased from NEB; restriction endonucleases and T4 DNA ligase were purchased from Promega (Cat. No. M1801); plasmid extraction kits were purchased from Qiagen (Cat. No. 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 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. (item numbers: P091307, F091718); 2YT bacterial culture medium was purchased from BD; ampicillin and kanamycin were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; glucose was purchased from Amresco (item number 0188); helper phage M13K07 was provided by Lanzhou Institute of Biological Products Co., Ltd.; MabselectSuRe affinity purification medium (item number 17543801) was purchased from Stovan. Fluorescence microscope was purchased from Olympus IX71; DNA gel recovery kit was purchased from Qigen (item number 28704); PrimeScript TM1st 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. SPR analysis CM5 chip His capture kit was purchased from Cytiva, with catalog numbers BR100012 and 28995056, respectively.

[0057] 1.3 Analysis of anti-rabies virus neutralization activity using immunofluorescence focus inhibition assay (RFFIT)

[0058] 1.3.1 Neutralization virus dilution determination

[0059] The rabies virus CVS-11 (challenge virus standard) to be tested was diluted three times in 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 6 50 μL of BSR cell suspension with a concentration of cells / mL. Add 50 μL of culture medium to each test well, 5% CO2, and culture at 37°C for 24 hours. Discard the supernatant. Wash the cells once with PBS, add 200 μL of pre-cooled 80% acetone to fix for 10 minutes; discard the acetone and let stand at room temperature for 15 minutes. FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200 times with PBS, 100 μL was added to each well, incubated at 37°C for 1 hour, the liquid was discarded, and washed 3 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 with the virus is the virus dilution for neutralization.

[0060] 1.3.2 RFFIT determination of in vitro neutralization activity

[0061] The phage or transiently expressed antibody to be analyzed, and the national standard of rabies immunoglobulin were diluted 1:3 times from 10 times, with a total of 8 dilutions; DMEM medium containing 10% newborn calf serum was used as a negative control. 50 μL of each 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, 1.0×10 650 μL of BSR cell suspension with a concentration of cells / mL, 5% CO2, and culture at 37°C for 24 hours. Discard the supernatant. Wash the cells once with PBS, add 200 μL of pre-cooled 80% acetone at -30°C to fix for 10 minutes; discard the acetone and let stand at room temperature for 15 minutes. FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200 times with PBS, 100 μL was added to each well, incubated at 37°C for 1 hour, the liquid was discarded, and washed 3 times with PBS. Observe and record the infection ratio of each well under a fluorescence microscope. Calculate the antibody activity according to the formula in the rabies immunoglobulin titer assay in the Pharmacopoeia of the People's Republic of China (Volume 3).

[0062] The results of preliminary screening of monoclonal phage antibody neutralization activity by RFFIT analysis showed that the phage neutralization activity was 3.67 IU / mL.

[0063] 1.4 Preliminary screening of monoclonal phage antibody particles by RFFIT analysis

[0064] 1.4.1 Preparation of monoclonal phage antibody particles

[0065] Take 100 μL of phage after 3 rounds of screening, add to 1 mL of E. coli TG1 bacterial solution (A600nm = 0.5-0.7), mix well, and infect at room temperature for 30 minutes. Spread the bacterial solution on 4 150mm 2YT-A plates and incubate at 30°C overnight. Select clones for culture, add each clone to a 96-well plate containing 100 μL of 2YT medium containing ampicillin, and incubate at 37°C, 220 r / min overnight. Take 10 μL of culture and add 140 μL of fresh 2YT medium containing ampicillin, incubate at 37°C for 1.5 hours at 220 r / min, 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 r / min, for 10 min, resuspend the cells in 150 μL of 2YT medium containing ampicillin and kanamycin, and culture overnight at 30°C, 220 r / min; centrifuge at 4°C, 8 cm radius, 6 000 r / min, for 20 min, collect the supernatant.

[0066] 1.4.2 RFFIT analysis of monoclonal phage antibody particles

[0067] 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 1.0×10 650 μL of BSR cell suspension with a concentration of 100 μg / mL was added, and cultured at 37°C for 24 h with 5% CO2. The supernatant was discarded. The cells were washed once with PBS, and 200 μL of pre-cooled 80% acetone was added to fix for 10 min; the acetone was discarded and the cells were allowed to stand at room temperature for 15 min. FITC-labeled anti-rabies virus nucleoprotein antibody was diluted 200 times with PBS, and 100 μL was added to each well, incubated at 37°C for 1 h, the liquid was discarded, and the cells were washed 3 times with PBS. The infection ratio of each well was observed and recorded under a fluorescence microscope, and the wells with obvious inhibition of virus growth were marked as positive wells. 50 μL of bacterial solution was taken from the 96-well plate corresponding to the positive wells, 1 mL of 2YT-A medium was added, and the cells were cultured overnight at 37°C and 220 r / min. 300 μL of bacterial solution was taken, 300 μL of autoclaved glycerol was added, and stored at -70°C; another 300 μL of bacterial solution was sent for sequencing. The sequencing results were compared online using vbase2 to determine whether they were the correct scFv sequences; the correct sequences were compared using DNASTAR 7.1, and clones with non-repetitive sequences were selected for the next step of the experiment. The phage sequencing primer sequence was CGAAGGAGACAGTCATAATG.

[0068] Phage particle sequencing showed that the light chain of C07 antibody belongs to the human KV1 family, the heavy chain belongs to the human HV3 family, and the ScFv nucleotide sequence is as follows:

[0069] C07 antibody ScFv nucleotide sequence SEQ ID NO.9:

[0070] ATGGCCCAGGTACAGCTGCAGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAGGCTCTCCTGTGCTGCCTCTGGATTCATTTTCAGCAACTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTTGAGTGGGTGGCAGTTATCTCATATGAGGGAAGAATTCCTGACTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAGGAACACACTTTACATGCAATTGAACCGCCTGACACCTGAGGACACGGCAATGTATTACTGTGCGAAAATAATGACAGATGGAAGCGGTTTTGATCTCTGGGGCCAGGGGACAATGGTCACTGTCTCCTCAGGTGGCGGCGGTTCCGGAGGTGGTGGTTCTGGCGGTGGTGGCAGCGACATCGTGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGGTTAATAGTTACCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0071] Amino acid sequence of the heavy chain variable region of the ScFv of C07 antibody, SEQ ID NO.6:

[0072] MAQVQLQQSGGGVVQPGGSLRLSCAASGFIFSNFGMHWVRQAPGKGLEWVAVISYEGRIPDYSDSVKGRFTISRDNSRNTLYMQLNRLTPEDTAMYYCAKIMTDGSGFDLWGQGTMVTVSS

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

[0074] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYGASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQVNSYPITFGQGTRLEIK

[0075] 1.4.3 Purification and activity determination of monoclonal phage antibody particles

[0076] Take 15 μL of the preserved bacterial solution and add it to 1.5 mL of 2YT medium containing ampicillin and glucose, and prepare phage particles according to 1.4.1. Add 1 / 5 volume of 20% PEG-8000 + 2.5 mol / L NaCl solution to the obtained supernatant, mix well, keep it in an ice bath for 1 hour, centrifuge it at a centrifugal radius of 8 cm and 12 000 r / min for 20 minutes, and the precipitate is the phage antibody particles. Resuspend the precipitate in 1.0 mL PBS-1% BSA, centrifuge it at a centrifugal radius of 8 cm and 12 000 r / min for 5 minutes, discard the precipitate, and freeze it at -70℃.

[0077] 1.5 Whole-molecule antibody expression and purification

[0078] 1.5.1 Whole-molecule antibody expression

[0079] Take a recombinant engineered cell preserved in liquid nitrogen, add OPM CD07 medium after recovery, 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 is added on the 3rd, 5th, 7th, 9th and 11th days of cell culture. The feed ingredients are OPM-CHO CDF18 and OPM-CHOCDF26, and they are added according to the amount recommended by the culture medium manufacturer.

[0080] 1.5.2 Whole-molecule antibody purification

[0081] After the culture, the cell suspension was centrifuged at 2000g for 10 min, and the supernatant was clarified and filtered using a 0.45μm capsule filter. The chromatography medium was equilibrated with PBS, and after loading, PBS was used for intermediate washing, and 10mmol / L sodium acetate solution at pH 3.5 was used for elution to collect the target protein.

[0082] Example 2 Analysis of whole molecule antibody activity

[0083] 2.1 Activity assay

[0084] The protein concentration of the purified antibody was determined using the A280 method. The purified antibody was used to determine the in vitro neutralization activity according to 1.3.2.

[0085] The in vitro neutralizing activity of C07 antibody against CVS-11 strain was determined by RFFIT to be approximately 1500 IU / mg.

[0086] 2.2 Antibody purity analysis

[0087] The purity of the antibody was analyzed using the non-reducing CE-SDS method. The purity of the purified monoclonal antibody was detected using CE-SDS. The non-reducing CE-SDS sample was diluted to 1 mg / mL, 5% volume of 0.8 mol / L iodoacetamide was added, heated at 70°C for 15 min, and the sample was injected and run at 15 kV for 15 min to detect the A214 nm value. After the detection, the spectrum was integrated to calculate the purity of the antibody monomer.

[0088] Non-reducing capillary electrophoresis analysis showed that the immunoglobulin monomer content of C07 antibody was higher than 95%. Figure 1 .

[0089] 2.3 Isoelectric point determination

[0090] Isoelectric point analysis was performed using the cief method. The sample was diluted with deionized water to a working concentration (10 mg / mL). Mix 200 μL of 3 mmol / L urea-cIEF Gel, 12 μL of amphoteric electrolytes, 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. 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, UV detector wavelength 280 nm.

[0091] The isoelectric point test results show that the isoelectric point of the antibody is generally high, with the main isoelectric point being about 8.95. Figure 2 .

[0092] 2.4 Affinity determination

[0093] The equilibrium dissociation constant KD of recombinant human anti-rabies virus monoclonal antibody and rabies virus glycoprotein was detected by capture method. The capture molecule Anti-His Antibody was coated on the CM5 chip, the glycoprotein was captured as the ligand, and then the antibody was measured as the analyte. Anti-His Antibody was diluted to 1-10μg / mL with sodium acetate and mixed evenly. Program operation: Select the immobilization program, first activate the chip surface with a mixture of NHS / EDC, then couple Anti-His Antibody to the selected channel, the flow rate is 10μL / min, the time is 420sec, and finally the blank channel is blocked with ethanolamine. HBS-EP+Buffer was diluted 10 times as the test sample diluent and running buffer, and the singlecycle knetics program was run. When capturing the ligand, the glycoprotein was first diluted 1200 times with the running buffer, the flow rate was 10μL / min, and the capture time was 100sec. Then the test sample was diluted in gradient (5 concentration points), with running buffer as blank control, analyte binding time 120sec, dissociation time 600sec, flow rate 30μL / min, regeneration solution was glycine, binding time 30sec, flow rate 30μL / min. The analysis software was opened, and the 1:1Binding model was used for equation fitting to automatically obtain the KD value.

[0094] Biacore affinity profiles are shown in Figure 3 As shown, after calculation, the affinity of C07 antibody to recombinant rabies virus glycoprotein is 2.393E-10M.

[0095] 2.5 Thermal stability analysis

[0096] Thermal stability analysis was performed using a microcal differential scanning calorimeter (Microcal PEAQ-DSC). The test sample was treated with a buffer solution to a protein concentration of about 1 g / L and then tested on the instrument.

[0097] Tm is an important indicator of protein thermal stability. The higher the Tm, the better the stability of the protein. LZR07 antibody showed three obvious peaks. Figure 4 As shown in Table 1, the lowest Tm value is about 72°C, indicating that the antibody has good thermal stability.

[0098] Table 1 C07 antibody thermal stability test results

[0099] Tm1(℃) Tm2(℃) Tm3(℃) 72.02 78.98 84.94

[0100] 2.6 Mouse neutralization test to determine protection against street strains

[0101] (1) Domestic rabies virus strains

[0102] There are 15 rabies virus strains isolated from different regions and 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.

[0103] (2) Preparation of rabies virus and determination of LD50

[0104] Take the rabies virus strain (mouse brain toxin homogenate), dilute it 100 times with DMEM, and inject 15-18g mice into the brain, 25μL per mouse, observe after injection, and take the brain tissue of the dying mice at -20℃ for centralized storage. Add the DMEM culture medium containing antibiotics to the brain tissue of the infected mice at a ratio of 5mL / g for homogenization, prepare 20% brain tissue homogenate, and freeze it at -80℃. When determining LD50, take the above-mentioned frozen homogenate, melt and mix it, and then dilute it 10 times. Take 3 gradients of 10-3, 10-4, and 10-5 for intracerebral injection of mice, 25μL per mouse, 10 mice per group, observe for 14 days after injection, and calculate the LD50 of different strains according to the death of mice.

[0105] (3) Antibody neutralization test for rabies virus

[0106] Rabies virus C07 antibodies were taken separately, diluted to the specified titer (40 IU / mL), mixed with an equal volume of virus solution containing 100 LD50, incubated at 37°C for 60 minutes, and placed on ice. The neutralized mixture was injected intracerebrally into 15-18g Kunming mice, 25 μL per mouse, and 10 mice were injected per group; each strain was also set up with a control group without antibody (replaced with an equal volume of PBS) and a rabies human immunoglobulin (HRIG) control group (diluted to 40 IU / mL). The mice were kept and observed for 28 days, and the death of mice was recorded. The neutralization ability of the antibodies against the epidemic strains of rabies was analyzed. The results are shown in Table 2.

[0107] Table 2 20IU / mL antibody to 50LD 50 Death of mice after rabies virus neutralization

[0108]

[0109]

[0110] As can be seen from Table 2, with 28 days as the data endpoint, all 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 of 20 IU / mL has complete neutralization ability for all 15 virus strains; the antibody has 100% neutralization ability for 13 of the viruses; in the experimental group that was 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-infected 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 is a single-chain antibody, which 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: Comprising the anti-rabies virus neutralizing antibody according to any one of claims 1-4.

12. Use of the anti-rabies virus neutralizing antibody according to any one of claims 1 to 4 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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