Monoclonal antibody 2D1B1 of H protein of canine distemper virus and application of monoclonal antibody 2D1B1

Abstract

The invention discloses a canine distemper virus H protein monoclonal antibody 2D1B1 and application thereof, and belongs to the technical field of monoclonal antibodies. Nine monoclonal antibodies are screened out through indirect ELISA (enzyme-linked immunosorbent assay) and indirect immunofluorescence identification. Indirect immunofluorescence identification shows that the obtained nine monoclonal antibodies can react with a vaccine strain (CDV-onderstoport), a Western Blot test result shows that the screened nine monoclonal antibodies can be subjected to specific reaction with eukaryotic expression CDV H protein, and 2D1B1 can also be subjected to specific reaction with prokaryotic expression CDV H protein, that is, 2D1B1 can not only react with an attenuated vaccine strain and a virulent isolated strain, but also react with a prokaryotic expression CDV H protein, and can also react with a prokaryotic expression CDV H protein. The compound can react with the CDV H protein expressed in eukaryotic and prokaryotic modes, has high neutralization titer (26), and can be used for preparing a broad-spectrum detection reagent and a treatment preparation for identifying the canine distemper virus H protein.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to a monoclonal antibody 2D1B1 against canine distemper virus H protein and its application. Background Art

[0002] Canine distemper (CD) is an acute, severe, highly contagious infectious disease caused by canine distemper virus (CDV). It is widely distributed worldwide, with high morbidity and mortality rates, and can infect a variety of domestic and rare protected animals, as well as non-human primates. In recent years, the host range of CDV has been continuously expanding, and giant pandas have also become one of the hosts that can be infected by CDV. Since the first report of CDV infection in giant pandas in 1997, 7 giant pandas have died due to CDV infection.

[0003] The CDV virus particles are pleomorphic (usually round), belonging to the Paramyxoviridae family, Morbillivirus genus, with a diameter between 100 nm and 250 nm. It is an enveloped single-stranded negative-sense non-segmented RNA virus. The H protein is one of the glycoproteins on the surface of the CDV envelope, which can specifically bind to receptors on the surface of target cells (such as SLAM and nectin-4), thereby mediating the invasion of the virus into the host. The full length of the H protein of CDV consists of 1947 nucleotides, encoding 607 amino acids, and the size of the H protein is about 78 kDa. Its structure is a tetramer, which binds to the host-specific receptor, resulting in the infection of the virus to cells. The H protein belongs to type II glycoprotein and contains three regions: the N-terminal cytoplasmic tail region, the transmembrane region, and the C-terminal extracellular domain. The cytoplasmic tail region contains a transmembrane signal region and an anchor site, and the C-terminal forms the "head" of the H protein that can bind to the receptor. The H protein can activate the F protein to mediate plasma membrane fusion, so the H gene is crucial for the pathogenicity of CDV. There are many neutralizing antigenic epitopes on the H protein, which is the main antigen that induces the body to produce neutralizing antibodies and can effectively guide animals to generate cellular immunity against CDV. Therefore, developing specific therapeutic preparations based on the H protein is of great significance for the treatment of canine distemper.

[0004] Monoclonal antibodies have advantages such as strong specificity, high sensitivity, high titer, less cross-reactivity, good repeatability, low cost, and suitability for large-scale production. They have superiority in disease diagnosis and immunotherapy and have currently been widely used in the diagnosis and treatment of diseases.

[0005] However, in existing research, the functions of monoclonal antibodies against the H protein of canine distemper virus are relatively single. For example, Cao Zhigang et al. (Cao Zhigang, Yi Li, Cheng Yuening, et al. Preparation of monoclonal antibodies against canine distemper virus nucleoprotein and identification of its linear antigenic epitopes [J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(07): 578-82.) immunized mice with the truncated N protein of CDV expressed by adenovirus and prepared 2 monoclonal antibodies against the CDV N protein. One of them reacted with both the strong strain and the vaccine strain of CDV, and the other reacted with the weak strain of CDV, but neither reacted with Vero cells. However, no virus neutralization test was performed, so it was impossible to determine whether they had neutralization ability. Liu Yuxiu et al. (LIU Y, HAO L, LI X, et al. Development and Characterization of Canine Distemper Virus Monoclonal Antibodies [J]. Monoclon Antib Immunodiagn Immunother, 2017, 36(3): 119-23) prepared 5 monoclonal antibodies by the limiting dilution method. Four of them could recognize both the wild strain and the vaccine strain of CDV, but they also had no neutralization ability. Mu Yong et al. (Mu Yong, Hou Xiaoxuan, He Shuaijie, et al. Screening of monoclonal antibodies against the H protein of canine distemper virus from whole canine sources based on single B cell antibody technology [J]. Chinese Veterinary Science: 1-11) screened four monoclonal antibodies against the CDV H protein based on the single B cell antibody screening technology. After identification, all 4 monoclonal antibodies could specifically recognize the native conformation of the H protein on the surface of CDV, but still had no neutralization ability. Shi Pengfei (Shi Pengfei. Preparation of monoclonal antibodies against the H protein of canine distemper virus and the screening of antigenic epitopes of the VP2 protein of canine parvovirus [D], 2021) immunized mice with the prokaryotically expressed CDV H protein, prepared two monoclonal antibodies, and performed antigenic epitope analysis, but did not detect neutralizing antibodies and could not determine whether they had neutralization ability. Bi Zhenwei et al. (Bi Zhenwei, Xu Libo, Xia Xingxia, et al. Preparation of monoclonal antibodies for differentiating and neutralizing strong and weak strains of canine distemper virus [J]. Jiangsu Agricultural Sciences, 2020, 48(20): 178-82) immunized mice with the purified weak strain CDV 851 and prepared a monoclonal antibody with neutralization ability. The neutralization titer of the supernatant of the hybridoma cell line reached 1:2 8 , and the neutralization titer of the ascites reached 1:10 6 . However, Western Blot identification, indirect immunofluorescence identification, and virus neutralization identification all showed that this monoclonal antibody could only react with the CDV vaccine strain and not with the CDV strong strain, and could not be used as a therapeutic monoclonal antibody. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a multifunctional monoclonal antibody 2D1B1 against canine distemper virus H protein. The monoclonal antibody 2D1B1 against canine distemper virus H protein comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:2.

[0007] Among them, the monoclonal antibody 2D1B1 against canine distemper virus H protein specifically reacts with both the attenuated vaccine strain and the virulent isolate of canine distemper virus, and specifically reacts with CDV H protein expressed in eukaryotes and prokaryotes, and simultaneously has virus neutralizing activity.

[0008] In a preferred embodiment, the attenuated vaccine strain of canine distemper virus is CDV-Onderstepoort strain or CDV / R20 / 8-EGFP strain. The virulent isolate of canine distemper virus is CDV-giant panda / SX / 2014 strain.

[0009] Another objective of the present invention is to provide the application of the above-mentioned monoclonal antibody 2D1B1 against canine distemper virus H protein in the preparation of a detection reagent or a therapeutic preparation for canine distemper virus vaccine strains.

[0010] A further objective of the present invention is to provide a detection reagent or a therapeutic preparation for canine distemper virus vaccine strains, which contains the above-mentioned monoclonal antibody 2D1B1 against canine distemper virus H protein.

[0011] Yet another objective of the present invention is to provide a nucleotide molecule encoding the above-mentioned monoclonal antibody 2D1B1 against canine distemper virus H protein.

[0012] Another objective of the present invention is to provide an expression vector containing the above-mentioned nucleotide molecule.

[0013] A further objective of the present invention is to provide a host cell containing the above-mentioned expression vector or having the above-mentioned nucleotide molecule integrated into its genome.

[0014] Compared with the prior art, the technical effects of the present invention are as follows:

[0015] In the present invention, BALB / c mice were immunized by intramuscular injection in the thigh with eukaryotically expressed CDV H protein mixed with QuickAntibody-Mouse3W adjuvant. The spleen cells of the immunized mice were fused with myeloma cells by the semi-solid medium method. Hybridoma cell lines with neutralizing titers were screened out through indirect ELISA, indirect immunofluorescence assay and neutralization assay. After expanded culture, ascites were prepared and purified, and they were identified. Nine monoclonal antibodies were successfully screened out and named 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6 respectively. Identified by indirect immunofluorescence, all the nine monoclonal antibodies obtained could react with the vaccine strain (CDV-Onderstepoort); the results of neutralizing antibody detection showed that 7 out of the 9 monoclonal antibodies obtained had the ability to neutralize the CDV / R20 / 8-EGFP strain, and 6 monoclonal antibodies could react with the virulent strain (CDV-giant panda / SX / 2014); the results of Western Blot assay indicated that all the 9 monoclonal antibodies screened out could specifically react with eukaryotically expressed CDV H protein, and the protein size was 75 kDa. Among them, 2D1B1 could also specifically react with prokaryotically expressed CDV H protein, that is, 2D1B1 could not only react with the attenuated vaccine strain and virulent isolated strain, but also react with eukaryotically and prokaryotically expressed CDV H protein, and at the same time had a high neutralizing titer (2 6 ), and it is a multifunctional monoclonal antibody against CDV H protein, which can be used to prepare detection reagents and therapeutic preparations for CDV H protein. The present invention is of great significance for the development of diagnostic reagents and the research and development of therapeutic preparations for canine distemper virus. Brief Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0017] Figure 1 is the titer of specific IgG against CDV H protein in mouse serum;

[0018] Figure 2 is the neutralizing titer of anti-CDV in mouse serum;

[0019] Figure 3 is the titer of specific IgG against CDV H protein in the supernatant of hybridoma cells;

[0020] Figure 4 is the indirect immunofluorescence assay of the supernatant of hybridoma cells;Figure 4 In which, A-E: Results of indirect immunofluorescence assay for 2C1, 2D1, 6A4, 6G4, 6H4; F: Results of indirect immunofluorescence assay for mouse positive serum; G: Negative control;

[0021] Figure 5 Is the specific IgG titer of anti-CDV H protein in the supernatant of hybridoma cells after subcloning;

[0022] Figure 6 Is the indirect immunofluorescence assay for the supernatant of hybridoma cells after subcloning; Figure 6 In which, A-I: Results of indirect immunofluorescence assay for 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: Results of indirect immunofluorescence assay for mouse positive serum; K: Negative control;

[0023] Figure 7 Is the neutralization effect diagram of hybridoma cell supernatant against CDV / R20 / 8-EGFP strain;

[0024] Figure 8 Is the SDS electrophoresis analysis of purified CDV H monoclonal antibody; Figure 8 In which, A-I: Results of SDS-PAGE analysis for 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; M: Protein Marker; 1: Unpurified ascites; 2: Purified ascites;

[0025] Figure 9 Is the identification result of monoclonal antibody subclass; Figure 9 In which, A-I: Identification results of monoclonal antibody subtypes for 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6.

[0026] Figure 10 Is the specificity identification of CDV H protein monoclonal antibody by IFA; Figure 10 In which, A-I: IFA detection results for 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: IFA detection results for CDV positive serum; K: Negative control;

[0027] Figure 11 Is the specificity identification of CDV H monoclonal antibody by Western Blot; Figure 11Among them, A-I: Western blot results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; M: Protein Marker; 1: Eukaryotic expressed H protein; 2: Prokaryotic expressed H protein;

[0028] Figure 12 Is the neutralization fluorescence map of the monoclonal antibody against CDV / R20 / 8-EGFP strain; Figure 12 Among them, A-I: Neutralization fluorescence maps of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: Virus control fluorescence map;

[0029] Figure 13 Is the neutralization effect map of the monoclonal antibody against CDV-giant panda / SX / 2014 strain; Figure 13 Among them, A-I: Neutralization effect maps of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: Virus control effect map. Specific implementation manners

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in combination with embodiments and drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0031] The test materials used in the following embodiments are all obtained from regular biochemical reagent stores without special instructions. The experimental methods in the following embodiments are all conventional methods without special instructions.

[0032] The recombinant CDV expressing green fluorescent protein (CDV / R20 / 8-EGFP) was constructed and preserved by the Laboratory of Animal Virology and Special Animal Epidemic Disease of the Military Veterinary Research Institute (see the reverse genetic operating system and its application of the canine distemper virus CDV / R-20 / 8 vaccine strain disclosed in the patent publication number CN102329809B). The parental virus CDV / R20 / 8 of CDV / R20 / 8-EGFP is a live attenuated vaccine strain widely used in China's production practice and is preserved in the Laboratory of Animal Virology and Special Animal Epidemic Disease of the Military Veterinary Research Institute (see the construction of the recombinant canine distemper virus CDV / R-20 / 8 vaccine strain expressing rabies virus G protein disclosed in the patent publication number CN102344913B).

[0033] The wild strain / strong virulent isolate CDV-giant panda / SX / 2014 was isolated and preserved by the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute (for the method, see the article Feng N, Yu Y, Wang T, Wilker P, Wang J, Li Y, Sun Z, Gao Y, Xia X. Fatal canine distemper virus infection of giant pandas in China. Sci Rep. 2016 Jun 16;6:27518.).

[0034] The eukaryotic expression recombinant CDV H protein was purchased from ProteoGenix, France (product number: PX-P6307) and stored in the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute.

[0035] The prokaryotic H protein was expressed and purified by the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute. For the method, see the graduation thesis of Jilin Agricultural University Canine Distemper Virus M, F, H Gene Recombinant Baculovirus Construction, Identification and Expression Research, author: Yu Yicong.

[0036] The CDV-Onderstepoort strain is a standard vaccine strain (attenuated vaccine strain) (product of Fort Dodge Animal Health, USA: Genbank: AF305419).

[0037] The African green monkey kidney cell (Vero) cell line-GPSLAM-Vero expressing the giant panda signaling lymphocytic activation molecule (SLAM) was constructed by the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute (for the method, see: Feng N, Liu Y, Wang J, Xu W, Li T, Wang T, Wang L, Yu Y, Wang H, Zhao Y, Yang S, Gao Y, Hu G, Xia X. Canine distemper virus isolated from a monkey efficiently replicates on Vero cells expressing non-human primate SLAM receptors but not human SLAM receptor. BMC Vet Res. 2016 Aug 2;12(1):160).

[0038] Example 1 Preparation and Screening of Hybridoma Cell Lines Stably Secreting Anti-CDV H Protein Monoclonal Antibodies

[0039] CDV - Onderstepoort (attenuated vaccine strain, obtained by reverse genetic rescue) for indirect immunofluorescence assay, recombinant CDV expressing green fluorescent protein (CDV / R20 / 8 - EGFP) (attenuated vaccine strain, obtained by reverse genetic rescue) for neutralization assay, African green monkey kidney (Vero) cells, myeloma (SP2 / 0) cells, and eukaryotic expression of CDV H protein are preserved in our laboratory; BALB / c female mice are purchased from Beijing Speyford Biotechnology Co., Ltd.

[0040] 1 Animal Immunization

[0041] BALB / c mice at 6 - 8 weeks old were immunized, a total of 5 mice. The immunogen was eukaryotic expression of CDV H protein, and the adjuvant was QuickAntibody - Mouse3W aqueous adjuvant from Bioron. The immunization interval was 2 weeks, and a total of 4 immunizations were carried out. The immunization dose was 50 μL adjuvant + 50 μL H protein (0.5 mg / mL) per mouse. Blood was collected from the mice 7 days after the 2nd, 3rd, and 4th immunizations and serum was separated. Indirect ELISA assay and neutralization assay were used to detect the antibody titer. Cell fusion was carried out 7 days after the 4th immunization, blood was collected and serum was separated to detect the titer.

[0042] 2 Indirect ELISA Detection

[0043] The enzyme - linked immunosorbent assay (ELISA) plate was coated with eukaryotic expression of CDV H protein. The sera of mice 7 days after the 2nd, 3rd, and 4th immunizations were used as the primary antibody, and HRP - labeled goat anti - mouse IgG was used as the secondary antibody for indirect ELISA detection.

[0044] The specific operation of indirect ELISA detection is as follows: Dilute the protein to 2 μg / mL with coating buffer, add 100 μL to each well of the ELISA plate, and incubate overnight at 4°C. Wash twice with PBST, 5 min each time; Add 300 μL of blocking solution (3% skim milk powder, diluted with PBST) to each well and incubate at 37°C for 2 h; Discard the blocking solution, add 100 μL of PBST containing 1% skim milk powder to each well. Add 100 μL of the serum dilution to be tested (1% skim milk powder, diluted 40,000 times with PBST) to the first well of each column, and make gradient dilutions downward, with a final dilution volume of 100 μL. At the same time, set positive and negative controls and incubate at 37°C for 1 h; Discard the liquid in the plate, wash three times with PBST, 5 min each time; Add 100 μL of HRP - labeled goat anti - mouse enzyme - labeled secondary antibody (BS12478) diluted 1:20,000 to each well of the ELISA plate, place it in an incubator at 37°C, and incubate for 1 h; Discard the liquid in the plate, wash five times with PBST, 5 min each time; Add 100 μL of TMB chromogenic solution to each well, develop color in the dark for 10 min; Add 50 μL of stop solution to each well; Measure the OD 450 value; Use the OD 450 value of the antibody to be tested / OD 450The maximum dilution multiple with a value (S / N) ≥ 2.1 is taken as the antibody titer.

[0045] After immunization, the specific IgG against CDV in the serum of mice was detected by the indirect ELISA method. Five immunized mice (numbered 413 - 417) were bled 7 days after the 2nd, 3rd, and 4th immunizations respectively. After separating the serum, the indirect ELISA test was carried out. The test results showed (see Figure 1 ) that the antibody titer of mouse serum reached 1:640000 - 1:5120000. Mice with ELISA titer were selected for neutralizing antibody detection.

[0046] 3 Neutralizing antibody detection

[0047] The serum of mice detected as positive by indirect ELISA was used for the CDV virus neutralization experiment to detect the neutralizing titer produced against the CDV / R20 / 8 - EGFP vaccine strain.

[0048] The specific method for detecting the neutralizing titer is as follows: The serum to be tested was inactivated in a 56°C water bath for 30 min; 50 μL of DMEM incomplete medium was added to each well of a 96 - well plate; 50 μL of the serum to be tested was added to the first well. After mixing, 50 μL was aspirated and added to the next well, and so on, for a 2 - fold serial dilution with one repeat, and the final dilution volume was 50 μL; 50 μL of the virus solution with 100 TCID 50 was added to each well, and at the same time, 100 TCID 50 , 10 TCID 50 , 1 TCID 50 and cell controls were set, 100 μL for each well, with 6 wells for each gradient; it was placed in a 37°C, 5% CO2 cell incubator for 1 h, and shaken crosswise every 15 min in the middle; 100 μL of Vero cell suspension was added to each well and continued to be cultured for 3 - 5 d; fluorescence was observed under a fluorescence microscope; the neutralizing titer was calculated by the Reed - Muench method.

[0049] In order to select mice with the highest neutralizing titer for cell fusion experiments, the serum of mice detected as positive by the indirect ELISA test was used for the virus neutralization test to determine the neutralizing titer of mouse serum (see Figure 2 ). After detection, the highest serum neutralizing titer reached 1:2048. The mouse with the highest neutralizing titer (numbered 414) was selected for cell fusion.

[0050] 4 Cell fusion experiment

[0051] 4.1 Resuscitation and culture of SP2 / 0 cells

[0052] Take out the cryopreserved SP2 / 0 cells from the liquid nitrogen tank, quickly shake them in a 37°C water bath to melt them. Take 5 mL of RPMI 1640 complete medium to resuspend the cells, centrifuge at 1000 rpm for 7 min, discard the supernatant, then add 5 mL of RPMI 1640 complete medium to resuspend the cell pellet, and transfer it into a T25 cm 2 cell flask, and incubate overnight in a cell culture incubator at 37°C and 5% CO2. Observe the cells after 12 h. The cell state is round, without aggregated clumps, and the transparency is in the best state.

[0053] Suspend the passaged SP2 / 0 cells in 20 mL of RPMI 1640 incomplete medium in a 50 mL centrifuge tube, centrifuge at 1000 rpm for 7 min, and discard the supernatant; add 30 mL of RPMI 1640 incomplete medium, centrifuge at 1000 rpm for 7 min, and discard the supernatant; repeat 2 times for a total of 3 washes; resuspend the cells in 25 mL of RPMI 1640 incomplete medium; calculate the volume of the cell suspension with a cell count of 2×10 7 and store it at room temperature for later use.

[0054] 4.2 Preparation of splenocytes

[0055] Take the mouse with the highest neutralization titer after the third immunization. After collecting blood from the eye socket, sacrifice the mouse by cervical dislocation. Immerse it in 75% alcohol for 5 - 7 min and then transfer it to a laminar flow hood. Fix it on a foam board with a 1 mL syringe needle, cut open the abdominal skin, open the abdominal cavity, remove the excess fat and connective tissue on the surface of the spleen, rinse the surface of the spleen with RPMI 1640 incomplete medium, place a 200-mesh cell sieve on the mouth of a 50 mL centrifuge tube and moisten it with 10 mL of RPMI 1640 incomplete medium, press the spleen with a 5 mL syringe plunger and grind it clockwise, collect the cells into a 50 mL centrifuge tube, centrifuge at room temperature and 1000 rpm for 5 min, and discard the supernatant; add 30 mL of RPMI 1640 incomplete medium, centrifuge at room temperature and 1000 rpm for 7 min, and discard the supernatant. Repeat 2 times for a total of 3 washes; resuspend the cells in 25 mL of RPMI 1640 incomplete medium, calculate the volume of the cell suspension with a cell count of 1×10 8 and store it at room temperature for later use.

[0056] 4.3 Cell fusion

[0057] Water bath the PEG and incomplete RPMI 1640 medium to 37°C; put the prepared SP2 / 0 cells and spleen cells into a 50 mL conical tube; centrifuge at 1000 rpm for 7 min and discard the supernatant; slowly add 1 mL of PEG to the cells with a 1 mL pipette over 1 min, and slowly stir the cells with the pipette tip for 1 min; add 4 mL of incomplete RPMI 1640 medium to the fusion mixture and continuously stir for 4 min; slowly add 10 mL of incomplete RPMI 1640 medium to the fusion mixture. Incubate in a 37°C water bath for 15 min; slowly add 30 mL of complete RPMI 1640 medium, centrifuge at 1000 rpm for 7 min and discard the supernatant; slowly add 40 mL of complete RPMI 1640 medium, centrifuge at 1000 rpm for 7 min and discard the supernatant; slowly resuspend in 10 mL of complete RPMI 1640 medium; transfer the cell suspension to a cell culture flask containing 20 mL of complete RPMI 1640 medium; incubate the cell culture flask in a 37°C, 5% CO2 cell incubator for 16 - 24 h; place medium D at room temperature one day in advance and shake it vigorously to mix well; transfer the fused cell suspension from the T75 cm 2 Transfer the cell culture flask to a 50 mL conical tube, centrifuge at 1000 rpm for 7 min and discard the supernatant; resuspend the cells in complete RPMI 1640 medium to a total volume of 6 mL; directly transfer the cell suspension to a bottle containing 60 mL of medium D. Invert the bottle gently several times to mix thoroughly. Incubate at room temperature for 15 min to allow the bubbles to rise to the top; use a 20 mL syringe and a blunt-tipped needle to slowly add 1 mL of the cell suspension in medium D to the 6-well plate, taking care to avoid generating bubbles during the process. Tilt each plate so that the medium is evenly distributed at the bottom of the plate; incubate in a 37°C, 5% CO 2 Cell incubator for 10 - 14 d; observe the cell status under a microscope every day after 7 d of cell fusion.

[0058] 5 Picking and screening of hybridoma cells

[0059] 5.1 Picking of hybridoma cells

[0060] Use a pipette and pipette tip to aspirate hybridoma cells from the 6-well plate under a microscope. Transfer single hybridoma cells to a 96-well cell culture plate containing HT medium, pipette to mix the cells in each well, and then incubate the 96-well plate in a 37°C, 5% CO2 cell culture incubator for 3 - 4 days.

[0061] 5.2 Screening of hybridoma cells

[0062] Currently, most monoclonal screening is carried out using indirect ELISA tests, which can screen out a large number of hybridoma cells secreting antibodies. However, the disadvantages are that false positives are prone to occur, it is difficult to screen and obtain monoclonal antibodies with neutralizing ability, and the workload required for subsequent large-scale culture is extremely large. In the present invention, double screening of the fused cells is carried out by indirect ELISA and indirect immunofluorescence, which largely avoids the phenomenon of false positives in the results of indirect ELISA, reduces the workload in the subsequent culture of hybridoma cell lines, and makes it easier to obtain monoclonal antibodies with stable expression and high titer.

[0063] Specifically, first, an indirect ELISA test is used for preliminary screening. The primary antibody is the supernatant of hybridoma cells without dilution, and the positive serum of immunized mice is diluted to an OD 450 close to 1 as a positive control. Then, an indirect immunofluorescence test is used to further screen the supernatant of hybridoma cells detected as positive by the indirect ELISA test.

[0064] The steps of the indirect immunofluorescence experiment are as follows: Add 50 μL / well of DMEM incomplete medium to a 96-well plate (operate on ice, and pre-cool DMEM in advance); Add 50 μL / well of CDV-Onderstepoort (vaccine strain) virus solution to the 96-well plate (diluted to an MOI value of 0.1, operate on ice); Add 100 μL of Vero cell suspension and place it in a cell incubator at 37°C and 5% CO2 for 48 h; Take out the 96-well plate, discard the medium in the plate, and add 100 μL of 80% acetone solution pre-cooled at -20°C to each well to fix the cells for 30 min at room temperature; Discard the cold acetone, add 300 μL of PBS to each well, and wash on a shaker at room temperature for 5 min, repeating 3 times; Add 100 μL of 2% BSA (prepared with PBS) to each well for blocking and incubate in a 37°C incubator for 1 h; Discard the liquid in the plate, add 50 μL of the monoclonal antibody supernatant detected as positive by ELISA to each well, add positive control wells and negative control wells (PBS diluted 200 times), make three replicates, and incubate in a 37°C incubator for 1 h; Discard the liquid in the plate, add 300 μL of PBST to each well, and wash on a shaker at room temperature for 5 min, repeating 3 times; Discard the liquid in the plate, add 50 μL of Alexa 488-labeled goat anti-mouse fluorescent secondary antibody (A0428) diluted 1:500 and Evans blue dye solution diluted 1:500 to each well, and incubate in a 37°C incubator for 1 h; Discard the liquid in the plate, add 300 μL of PBST to each well, and wash on a shaker at room temperature for 5 min, repeating 3 times (protected from light); Discard the liquid in the plate and observe the results under an upright fluorescence microscope. Determine the wells showing green fluorescence signals as positive wells.

[0065] The fused hybridoma cells are identified by indirect ELISA (see Figure 3 ) and indirect immunofluorescence (seeFigure 4 ) Screening was performed to obtain 5 positive hybridoma cell lines, named 2C1, 2D1, 6A4, 6G4, and 6H4 respectively.

[0066] Subcloning of 6 hybridoma cell lines

[0067] The 5 detected positive hybridoma cell lines were resuspended. After cell counting, 100 cells were taken and resuspended in 1 mL of complete RPMI1640 medium, added to a 15 mL centrifuge tube containing 10 mL of medium D. After mixing by inverting up and down, it was allowed to stand for 15 min, then transferred to a 6-well plate, 2 mL per well. At the same time, 2 mL of sterile PBS was added to the remaining 1 well. It was cultured in a 37 °C, 5% CO2 cell incubator for 10 - 14 days, and then the subcloned cells were picked and screened.

[0068] Detection of the titer of hybridoma cell supernatant

[0069] According to the above indirect ELSIA method, the ELISA titer of the hybridoma cell supernatant was detected, and the primary antibody was the hybridoma cell supernatant. After expanding the culture of the subcloned hybridoma cell lines, the hybridoma cell supernatant was taken for neutralizing antibody detection.

[0070] The 5 hybridoma cell lines of 2C1, 2D1, 6A4, 6G4, and 6H4 were subcloned by the semi-solid medium method, and screened by indirect ELISA (see Figure 5 ) and indirect immunofluorescence identification (see Figure 6 ). Nine positive hybridoma cell lines were obtained, named 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6 respectively.

[0071] Indirect ELISA screening showed that the supernatants of these 9 hybridoma cell lines could all bind to the eukaryotic-expressed CDV H protein, and indirect immunofluorescence screening showed that the supernatants of these 9 hybridoma cell lines could all react with the CDV-Onderstepoort vaccine strain.

[0072] The supernatants of the 9 obtained hybridoma cell lines were subjected to a virus neutralization test to detect whether they had neutralizing ability. The results showed that 7 of them had neutralizing titers, namely 2D1B1, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6, and the highest neutralizing titer reached 1:16 (see Table 1 and Figure 7 ).

[0073] Table 1 Titer table of hybridoma cell supernatant

[0074]

[0075] Preparation and Identification of Monoclonal Antibody Against Canine Distemper Virus H Protein in Example 2

[0076] CDV / R20 / 8-EGFP (attenuated vaccine strain) for neutralization test, CDV-giant panda / SX / 2014 strain (virulent isolate) for indirect immunofluorescence and neutralization test, African green monkey kidney (Vero) cells, African green monkey kidney cell line - GPSLAM-Vero expressing panda signaling lymphocytic activation molecule (SLAM), eukaryotic-expressed H protein of CDV, and prokaryotic-expressed H protein were all preserved in our laboratory; BALB / c female mice were purchased from Beijing Sino Biological Inc.

[0077] 1 Ascites Preparation

[0078] Eighteen 8 - 10-week-old SPF-grade female BALB / c mice and 9 hybridoma cell lines that stably secrete CDV H protein antibodies screened by the present invention were selected. Each mouse was intraperitoneally injected with 500 μL of ascites-specific adjuvant; after 15 days, each mouse was injected with approximately 8×10 5 hybridoma cell suspension (suspended in PBS after washing twice with PBS), and 3 mice were injected with each cell line. Ascites was collected when the mouse abdomen was distended after 7 - 10 days. The collected ascites was centrifuged at 3000 r / min for 10 min, and the middle layer of the ascites was taken and stored at -80 °C.

[0079] 2 Ascites Purification

[0080] The crude purification of mouse ascites was carried out by the octanoic acid-ammonium sulfate method, and then further purified by a Protein A purification column (all liquids need to be filtered through a 0.22 μm filter). The specific experimental steps are as follows: Take 5 mL of ascites and add 4 times the volume of acetate buffer, adjust the pH value to 4.5; Slowly add octanoic acid dropwise with magnetic stirring at room temperature, the addition amount is 25 μL / mL, the octanoic acid concentration is 33 μL / mL, and stir for 30 min; Place it at 4 °C and let it stand overnight to fully precipitate; Centrifuge at 4 °C and 10,000 rpm for 30 min, collect the supernatant and filter it through a funnel to remove impurities; After the supernatant is filtered through a funnel, measure the volume, add 10% volume of 10×PBS, and adjust the pH value to 7.4; Slowly add ammonium sulfate powder within 30 min on ice, the addition amount is 0.706 mg / mL, and keep stirring. Stir with a magnetic stirrer at 4 °C for 2 h; Centrifuge at 4 °C and 10,000 rpm for 30 min, discard the supernatant, suspend the precipitate with 2 mL of PBS, load it into a dialysis bag, place it in PBS, and dialyze overnight at 4 °C at 200 rpm; Add PBS to the Protein A purification column until the OD280 of the effluent is close to 0; Add the obtained dialysis solution to the purification column and load the sample repeatedly 3 times to ensure that the protein binds fully to the packing material; Wash the purification column with 10 mL of PBS; Elute with 5 mL of eluent (4.5 mL of 0.1 M glycine solution with pH 2.7 + 0.5 mL of Tris-HCl buffer with pH 9); Collect the eluent with 1.5 mL centrifuge tubes, 1 mL per tube, and add 100 μL of Tris-HCl buffer with pH 9.0; After the purified antibody is analyzed correctly by SDS-PAGE, store it at -80 °C.

[0081] After SDS-PAGE electrophoresis and staining with Coomassie Brilliant Blue staining solution for analysis, the results showed that there were two bands of heavy chain and light chain after ascites purification, and the sizes were approximately 55 kDa and 20 kDa respectively (see Figure 8 ), indicating the successful preparation of the antibody.

[0082] 3 Identification of monoclonal antibody subclasses

[0083] The subclass of the obtained monoclonal antibody was identified using a mouse monoclonal antibody subclass identification kit (BF16001, Suzhou Bioron Co., Ltd.). The specific steps were as follows: Restore the kit to room temperature and take out the enzyme-linked immunosorbent assay (ELISA) plate. First, add 50 μL of specimen diluent to each well of the ELISA plate, then add 50 μL of hybridoma cell supernatant. Add the supernatant of each hybridoma cell to 8 wells, and add 100 μL each of negative and positive controls to 8 wells each. Stick on the sealing film and incubate at 37 °C for 30 min. Discard the liquid in the plate and wash 5 times with the washing solution, 5 min each time. Add 100 μL each of 8 enzyme-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, Lambda) to each well, stick on the sealing film and incubate at 37 °C for 30 min. Discard the liquid in the plate and wash 5 times with the washing solution, 5 min each time. Add 50 μL each of chromogenic agent A and chromogenic agent B to each well, and develop color at 37 °C for 20 min in the dark. Add 50 μL of stop solution and detect the OD 450 value. Determine that the subclass of the secondary antibody added to the well with the highest OD 450 value of the light chain and heavy chain subclasses is the subclass of the monoclonal antibody.

[0084] After identification, except that the subclass of monoclonal antibody 2C1A1 is IgMκ and the subclasses of monoclonal antibodies 6A4A5 and 6A4A6 are IgG2bκ, the subclasses of monoclonal antibodies secreted by the remaining cell lines are all IgG1κ (see Figure 9 and Table 2)

[0085] Table 2 Identification Table of Monoclonal Antibody Subclasses

[0086]

[0087] 4 Identification of Monoclonal Antibody Characteristics

[0088] 4.1 Indirect Immunofluorescence Assay

[0089] In a 24-well plate, infect GPSLAM-Vero cells (an African green monkey kidney cell (Vero) cell line expressing the giant panda signaling lymphocytic activation molecule (SLAM)) with a cell density of 70%-80% with CDV-giant panda / SX / 2014 (a virulent isolate), and use the non-infected cells as a negative control.

[0090] The 9 selected monoclonal antibodies, 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, were diluted and used as primary antibodies, and the specificity of the 9 monoclonal antibodies was identified by indirect immunofluorescence assay. The specific method was as follows: After observing the lesions (usually 48 h), the cells were fixed with pre-cooled 4% paraformaldehyde for 20 min at 100 μL / well; 100 μL / well of 2% BSA (diluted with PBS) was added and blocked at 37 °C for 1 h; 100 μL / well of the diluted monoclonal antibody (diluted 500-fold) was added and incubated at 37 °C for 1 h; washed 3 times with PBST; 100 μL / well of Alexa 488-labeled goat anti-mouse IgG diluted 1:500 was added to each well and incubated at 37 °C for 1 h; washed 3 times with PBST; the results were observed with a fluorescence microscope.

[0091] The results showed that the monoclonal antibodies 2C1A1, 2D1B1, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6 and mouse positive serum could specifically recognize the giant panda / SX / 2014 strain (see Figure 10 A, B, E-J in Figure 10 showed green positive fluorescence signals, while no fluorescence was observed for the monoclonal antibodies 6A4A5 and 6A4A6 (see

[0092] 4.2 Western Blot Assay

[0093] Add the eukaryotic expression CDV H protein sample and 6× protein loading buffer into a centrifuge tube at a ratio of 5:1, mix well, boil in boiling water for 10 min. The loading amount is 10 μL / well for Marker and 20 μL / well for the protein sample. Perform Western Blot test, and the specific steps are as follows: (1) Electrophoresis: 80 V, 1 h; (2) Transfer: 300 mA, 70 min; A. Take out the gel, cut the target band, and cut NC membrane and fiber pads with the same size as the gel; B. Place the transfer template with the white side down (arrange in the order of white board → sponge pad → fiber pad → NC membrane → gel → fiber pad → sponge → black board); C. Fill with transfer buffer, add ice bags, and place the transfer tank in an ice basin; (3) Blocking: Immerse the NC membrane in 5% skim milk powder diluted with TBST, and block on a shaker at room temperature for 2 h; (4) Primary antibody incubation: Dilute 9 monoclonal antibodies with TBST containing 1% skim milk powder (200-fold dilution), add to the NC membrane, and incubate on a shaker at 4 °C overnight. After incubation, wash with TBST 4 times; (5) Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse IgG (H+L) 1:20000 with TBST containing 1% skim milk powder, incubate on a shaker at room temperature for 1 h. After incubation, wash with TBST 4 times; (6) Prepare the chromogenic solution: at a ratio of 1:1; (7) Expose, develop color and take pictures.

[0094] The results of Western Blot test showed that the 9 monoclonal antibodies screened out, namely 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, could all specifically react with the eukaryotic expression CDV H protein. The protein size was 75 kDa, which was consistent with the expected result (see Figure 11 ), and 2D1B1 could also specifically react with the prokaryotic expression H protein.

[0095] Identification of the titer of monoclonal antibodies

[0096] 5.1 Identification of the ELISA titer of monoclonal antibodies

[0097] Using the eukaryotic expression H protein of CDV as the coating antigen, the ELISA titer of the monoclonal antibody was determined by indirect ELISA test. The specific operation referred to Example 1, and the primary antibody was the obtained monoclonal antibody (10-fold serial dilution).

[0098] After indirect ELISA identification, the measured ELISA titer of the monoclonal antibody could reach 10 3 -10 8 , as shown in Table 3.

[0099] Table 3 ELISA titer table of monoclonal antibodies

[0100]

[0101] 5.2 Identification of neutralization titer of monoclonal antibodies

[0102] The titer of the monoclonal antibody was identified by virus neutralization test. CDV / R20 / 8-EGFP (vaccine strain) and giant panda / SX / 2014 (virulent strain) were respectively diluted to 100 TCID 50 / 50 μL for neutralizing antibody detection, and the test method referred to Example 1. After the giant panda / SX / 2014 strain produced lesions, the lesion conditions were observed under a microscope to confirm its neutralization titer, and the maximum dilution multiple of the cell wells without observed cytopathic effect was calibrated as its neutralization titer.

[0103] The results of the CDV / R20 / 8-EGFP neutralization test showed that 7 out of the 9 obtained monoclonal antibodies had the ability to neutralize CDV / R20 / 8-EGFP, namely 2D1B1, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, and the highest neutralization titer reached 2 7 ; The results of the CDV-giant panda / SX / 2014 neutralization test showed that 6 out of the 9 obtained monoclonal antibodies had the ability to neutralize CDV-giant panda / SX / 2014, namely 2D1B1, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, and the highest titer reached 2 7 (see Table 4). The neutralization effect diagrams of the monoclonal antibodies against CDV / R20 / 8-EGFP and giant panda / SX / 2014 are shown in Figure 12 and Figure 13 .

[0104] Table 4 Neutralization titer table of monoclonal antibodies

[0105]

[0106] Example 3 Amino acid sequence analysis of monoclonal antibody 2D1B1 of canine distemper virus H protein

[0107] Through amino acid sequence analysis, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D1B1 of canine distemper virus H protein is shown in SEQ ID No:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No:2. Among them, the heavy chain variable region belongs to the IGH type, and the light chain variable region belongs to the IGK type.

[0108] The above-described embodiments are merely specific implementation manners of the present application, used to illustrate the technical solutions of the present application, rather than limiting it. The protection scope of the present application is not limited thereto. Although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: Any person skilled in the art within the technical scope disclosed by the present application can still modify the technical solutions recorded in the foregoing embodiments, or can easily think of changes, or perform equivalent replacements on some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. All should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A canine distemper virus H protein monoclonal antibody 2D1B1, characterized in that The canine distemper virus H protein monoclonal antibody 2D1B1 comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No:

2.

2. The monoclonal antibody 2D1B1 against canine distemper virus H protein according to claim 1, characterized in that, The canine distemper virus H protein monoclonal antibody 2D1B1 specifically reacts with both the attenuated vaccine strain and the virulent isolate of canine distemper virus, and specifically reacts with the CDV H protein expressed in eukaryotes and prokaryotes, and simultaneously has virus neutralizing activity.

3. The monoclonal antibody 2D1B1 against canine distemper virus H protein according to claim 2, characterized in that, The attenuated vaccine strain of canine distemper virus is the CDV-Onderstepoort strain or the CDV / R20 / 8-EGFP strain.

4. The monoclonal antibody 2D1B1 against canine distemper virus H protein according to claim 2, characterized in that, The virulent isolate of canine distemper virus is the CDV-giant panda / SX / 2014 strain.

5. Use of the canine distemper virus H protein monoclonal antibody 2D1B1 as claimed in claim 1 in the preparation of a detection reagent for canine distemper virus H protein.

6. Use of the canine distemper virus H protein monoclonal antibody 2D1B1 as claimed in claim 1 in the preparation of a therapeutic preparation for canine distemper virus H protein.

7. A detection reagent for canine distemper virus H protein, characterized in that, Comprising the canine distemper virus H protein monoclonal antibody 2D1B1 as claimed in claim 1.

8. A therapeutic preparation of canine distemper virus H protein, characterized in that, Comprising the canine distemper virus H protein monoclonal antibody 2D1B1 as claimed in claim 1.

9. A nucleotide molecule encoding the canine distemper virus H protein monoclonal antibody 2D1B1 as claimed in claim 1.

10. An expression vector, characterized in that, Comprising the nucleotide molecule as claimed in claim 9.

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