Monoclonal antibody 6A4A6 of H protein of canine distemper virus and application of monoclonal antibody 6A4A6

Abstract

The invention discloses a canine distemper virus H protein monoclonal antibody 6A4A6 and application thereof, and belongs to the technical field of monoclonal antibodies. According to the invention, nine monoclonal antibodies are screened through indirect ELISA (Enzyme-Linked Immunosorbent Assay) and indirect immunofluorescence identification, and the nine monoclonal antibodies are respectively named as 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6. According to indirect immunofluorescence identification, all the obtained nine monoclonal antibodies can react with a vaccine strain (CDV-onderstepoort), and all the obtained monoclonal antibodies can react with the vaccine strain (CDV-onderstepoort). A neutralizing antibody detection result shows that in the obtained nine monoclonal antibodies, 6A4A5 and 6A4A6 do not react with giant panda / SX / 2014 (virulent isolated strains) and only react with vaccine strains, and 6A4A6 has higher neutralizing titer (up to 26) compared with 6A4A5 and can be used for preparing a detection reagent and a treatment preparation for identifying canine distemper virus vaccine strains.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and particularly to a monoclonal antibody 6A4A6 against canine distemper virus H protein and its application. Background Art

[0002] Canine distemper (CD) is an acute, severe, and highly contagious infectious disease caused by canine distemper virus (CDV). It is widely distributed worldwide, with high morbidity and mortality rates. It 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] CDV virus particles are pleomorphic (usually round), belonging to the family Paramyxoviridae, genus Morbillivirus, 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 virus invasion into the host. 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 produce cellular immunity against CDV. Therefore, the development of specific therapeutic agents based on the H protein is of great significance for the treatment of canine distemper.

[0004] Currently, supportive therapy and antibiotic therapy are mostly used clinically as effective measures for treating canine distemper. Antiviral drugs such as ribavirin also have a certain effect on the treatment of canine distemper, but the effect is limited. Hyperimmune serum can neutralize the virus in animal blood, but the preparation cost is high, it is easy to transmit other viruses, and the antibody neutralization titers are uneven, making it difficult to standardize. Canine distemper virus vaccine is still the main preventive measure used clinically.

[0005] Currently, attenuated vaccine strains of canine distemper virus are used clinically. When evaluating the immune effect of canine distemper virus vaccine strains, it is necessary to avoid antibodies produced by natural infection with wild strains of canine distemper virus. However, currently, in addition to sequencing and analyzing the genes of virus strains for identification, there are no other necessary simple and rapid detection methods for differentiating and diagnosing wild strains and vaccine strains of canine distemper virus, which affects the detection of differentiating and diagnosing wild strain infections of canine distemper virus and antibodies produced by vaccine immunization, and is not conducive to the accurate diagnosis and treatment of canine distemper. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a monoclonal antibody 6A4A6 against canine distemper virus H protein. The subtype of the monoclonal antibody 6A4A6 against canine distemper virus H protein is IgG2bκ. The monoclonal antibody 6A4A6 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 shown as SEQ ID No:1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:2.

[0007] Another objective of the present invention is to provide the use of the above-mentioned monoclonal antibody 6A4A6 against canine distemper virus H protein in the preparation of a detection reagent for differentiating canine distemper virus vaccine strains.

[0008] Another objective of the present invention is to provide a detection reagent for differentiating canine distemper virus vaccine strains, which contains the above-mentioned monoclonal antibody 6A4A6 against canine distemper virus H protein.

[0009] Preferably, for the above-mentioned detection reagent for differentiating canine distemper virus vaccine strains, the canine distemper virus vaccine strain is CDV / R20 / 8-EGFP or Onderstepoort.

[0010] Preferably, for the above-mentioned detection reagent for differentiating canine distemper virus vaccine strains, the monoclonal antibody 6A4A6 against canine distemper virus H protein specifically reacts with the canine distemper virus vaccine strain, has virus neutralization activity, and does not react with the CDV virulent isolate.

[0011] Another objective of the present invention is to provide a nucleotide molecule encoding the above-mentioned monoclonal antibody 6A4A6 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] Another 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 this invention, BALB / c mice were immunized by intramuscular injection in the thigh with the eukaryotically expressed CDV H protein mixed with QuickAntibody-Mouse3W adjuvant. The spleen cells of the immunized mice were fused with myeloma cells using the semi-solid medium method. Hybridoma cell lines with neutralizing titers were screened out through indirect ELISA tests, indirect immunofluorescence tests, and neutralization tests. After expansion 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. Through indirect immunofluorescence identification, all the nine monoclonal antibodies obtained could react with Onderstepoort (vaccine strain). The neutralizing antibody detection results showed that 7 of the nine monoclonal antibodies obtained had the ability to neutralize the CDV / R20 / 8-EGFP strain, and 6 had the ability to neutralize the giant panda / SX / 2014 strain. Among them, 6A4A5 and 6A4A6 did not react with the giant panda / SX / 2014 strain (virulent isolate), but only reacted with the vaccine strain, and 6A4A6 had a higher neutralizing titer (2 6 ) than 6A4A5 and could be used to prepare detection reagents and therapeutic preparations for differentiating canine distemper virus vaccine strains. This invention is of great significance for the research and development of canine distemper virus diagnostic reagents, therapeutic preparations, and vaccines. 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 specific IgG titer of anti-CDV H protein in mouse serum;

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

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

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

[0021] Figure 5 It is the titer of specific IgG against the H protein of CDV in the supernatant of hybridoma cells after subcloning;

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

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

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

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

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

[0027] Figure 11 It is the specificity identification of CDV H monoclonal antibody by Western Blot; Figure 11 Among 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 12Neutralization fluorescence map of 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: Fluorescence map of virus control;

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

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

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

[0032] 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 operation 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-giantpanda / 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 for indirect immunofluorescence assay (attenuated vaccine strain, obtained by reverse genetic rescue), recombinant CDV expressing green fluorescent protein (CDV / R20 / 8 - EGFP) for neutralization assay (attenuated vaccine strain, obtained by reverse genetic rescue), African green monkey kidney (Vero) cells, myeloma (SP2 / 0) cells, and eukaryotic expression of CDV H protein are stored 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 of age 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 performed 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 buffer (3% skim milk powder, diluted with PBST) to each well and incubate at 37°C for 2 h; Discard the blocking buffer, 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 - fold with PBST) to the first well of each column and make serial 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 and 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, an indirect ELISA test was conducted. The test results showed (see Figure 1 ) that the antibody titers of mouse sera reached 1:640,000 - 1:5,120,000. Mice with ELISA titers were selected for neutralizing antibody detection.

[0046] 3 Neutralizing antibody detection

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

[0048] The specific method for detecting the neutralizing titer is as follows: The test serum 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 test serum 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 100 TCID 50 virus solution was added to each well. 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 days; 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 was used for the virus neutralization test to determine the neutralizing titer of mouse sera (see Figure 2 ). After detection, the highest serum neutralizing titer reached 1:2,048. 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. After discarding the supernatant, add 5 mL of RPMI 1640 complete medium to resuspend the cell pellet, and transfer it into a T25 cm 2 cell culture 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 transparent, which is 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 2×10 7 cells, 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, decapitate the mouse by cervical dislocation. Immerse it in 75% alcohol for 5 - 7 min and then transfer it into 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 to 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, and calculate the volume of the cell suspension with 1×10 8 cells, and store it at room temperature for later use.

[0056] 4.3 Cell fusion

[0057] Water bath the PEG and RPMI 1640 incomplete 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 RPMI 1640 incomplete medium to the fusion mixture and continuously stir for 4 min; slowly add 10 mL of RPMI 1640 incomplete medium to the fusion mixture. Incubate in a 37°C water bath for 15 min; slowly add 30 mL of RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 min and discard the supernatant; slowly add 40 mL of RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 min and discard the supernatant; resuspend slowly in 10 mL of RPMI 1640 complete medium; transfer the cell suspension to a cell flask containing 20 mL of RPMI 1640 complete medium; incubate the cell flask in a 37°C, 5% CO2 cell incubator for 16 - 24 hours; place medium D at room temperature one day in advance and shake medium D vigorously to mix well; transfer the fused cell suspension from the T75 cm 2 Transfer the cell suspension from the cell flask to a 50 mL conical tube, centrifuge at 1000 rpm for 7 min and discard the supernatant; resuspend the cells in RPMI 1640 complete medium to a total volume of 6 mL; transfer the cell suspension directly 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-ended 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% CO2 cell incubator for 10 - 14 days; observe the cell status under the microscope every day 7 days after cell fusion.

[0058] 5 Picking and screening of hybridoma cells

[0059] 5.1 Picking of hybridoma cells

[0060] Use a pipette and pipette tips to aspirate hybridoma cells from the 6-well plate under the microscope. Transfer single hybridoma cells to a 96-well cell culture plate containing HT medium, pipette the cells in each well to mix evenly, and then place 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 monoclonal antibodies with neutralizing ability, and the workload required for subsequent large-scale culture is extremely large. In the present invention, the fused cells are double-screened 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 (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, add 100 μL of 80% acetone solution pre-cooled at -20 °C to each well to fix the cells, and fix at room temperature for 30 min; discard the cold acetone, add 300 μL of PBS to each well, and wash on a shaker at room temperature for 5 min, repeat 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-fold), 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, repeat 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, repeat 3 times (protected from light); discard the liquid in the plate, and observe the results under an upright fluorescence microscope. The wells showing green fluorescence signals are determined as positive wells.

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

[0066] 6 Subcloning of hybridoma cell lines

[0067] The 5 hybridoma cell lines with positive detection results were resuspended. After cell counting, 100 cells were taken and resuspended in 1 mL of complete RPMI 1640 medium. Then they were added to a 15 mL centrifuge tube containing 10 mL of medium D. After mixing by inverting up and down, they were left standing for 15 min and 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. They were cultured in a cell incubator at 37 °C and 5% CO2 for 10 - 14 days, and then the subcloned cells were picked and screened.

[0068] 7 Detection of the titer of hybridoma cell supernatant

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

[0070] The 5 hybridoma cell lines 2C1, 2D1, 6A4, 6G4, and 6H4 were subcloned by the semi-solid medium method. After screening by indirect ELISA (see Figure 5 ) and indirect immunofluorescence identification (see Figure 6 ), 9 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 a neutralizing titer, 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] Example 2 Preparation and identification of monoclonal antibody against canine distemper virus H protein

[0076] The CDV / R20 / 8-EGFP (vaccine strain) for neutralization test, the CDV-giantpanda / SX / 2014 (virulent strain isolate) for indirect immunofluorescence and neutralization test, African green monkey kidney (Vero) cells, the African green monkey kidney cell line - GPSLAM-Vero expressing the signal lymphocytic activation molecule (SLAM) of giant panda, the eukaryotic-expressed H protein of CDV, and the prokaryotic-expressed H protein of CDV are all preserved in this laboratory; BALB / c female mice are purchased from Beijing Spey Foster Biotechnology Co., Ltd.

[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 are selected. Each mouse is intraperitoneally injected with 500 μL of ascites-specific adjuvant; after 15 days, each mouse is injected with a hybridoma cell suspension containing approximately 8×10 5 cells (suspended in PBS after being washed twice with PBS), and 3 mice are injected with each cell line. Ascites is collected when the abdomen of the mice becomes distended after 7 - 10 days. The collected ascites is centrifuged at 3000 r / min for 10 min, and the middle layer of the ascites is taken and stored at -80 °C.

[0079] 2 Ascites purification

[0080] The mouse ascites is roughly purified by the octanoic acid-ammonium sulfate method and then further purified through 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, and adjust the pH value to 4.5; at room temperature, slowly add octanoic acid dropwise while stirring with a magnetic stirrer, 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 10000 rpm for 30 min, collect the supernatant and filter it through a funnel to remove impurities; after the supernatant is filtered through the funnel, measure the volume, add 10% of the volume of 10×PBS, and adjust the pH value to 7.4; slowly add ammonium sulfate powder on ice within 30 min, 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 10000 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 OD of the effluent 280Close to 0; add the obtained dialysate to a purification column, 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 eluate 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 dye for analysis, the results showed that after ascites purification, there were two bands of heavy chain and light chain, with sizes of approximately 55 kDa and 20 kDa respectively (see Figure 8 ), indicating successful antibody preparation.

[0082] 3 Identification of monoclonal antibody subclasses

[0083] Use a mouse monoclonal antibody subclass identification kit (BF16001, Suzhou Bio-long Co., Ltd.) to identify the subclasses of the obtained monoclonal antibodies. The specific steps are as follows: Restore the kit to room temperature and take out the enzyme-linked immunosorbent assay (ELISA) plate; Add 50 μL of specimen diluent to each well of the ELISA plate first, then add 50 μL of hybridoma cell supernatant. Add the supernatant of each hybridoma cell to 8 wells, add 100 μL of negative and positive controls to each of 8 wells, stick on the sealing film and incubate at 37 °C for 30 min; Discard the liquid in the plate and wash it 5 times with the washing solution, 5 min each time; Add 100 μL of 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 it 5 times with the washing solution, 5 min each time; Add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B to each well, develop color at 37 °C in the dark for 20 min; Add 50 μL of stop solution and measure the OD450 value. Determine that the subclass of the secondary antibody added to the well with the highest OD 450 value is the monoclonal antibody subclass.

[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 24-well plates, GPSLAM-Vero cells (African green monkey kidney cell line (Vero) expressing giant panda signaling lymphocytic activation molecule (SLAM)) with a cell density of 70%-80% were infected with CDV-giant panda / SX / 2014 (strong strain), and the cells not infected with the virus were used as negative controls.

[0090] The 9 monoclonal antibodies 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6 screened out 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, 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 giant panda / SX / 2014 (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 amounts are: Marker: 10 μL / well, protein sample: 20 μL / well. Conduct Western Blot assay, 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, 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 (dilute 200 times), add to the NC membrane, incubate on a shaker at 4 °C overnight. After incubation, wash 4 times with TBST; (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 4 times with TBST; (6) Prepare the chromogenic solution: at a ratio of 1:1; (7) Expose, develop color and take pictures.

[0094] The results of Western Blot assay 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 among them, 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] Use the eukaryotic expression H protein of CDV as the coating antigen, and determine the ELISA titer of the monoclonal antibody through indirect ELISA assay. The specific operation refers to Example 1, and the primary antibody is the obtained monoclonal antibody (serial diluted 10-fold).

[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 antibody

[0102] The titer of the monoclonal antibody was identified by virus neutralization test. The CDV / R20 / 8-EGFP strain and the giant panda / SX / 2014 strain were diluted to 100 TCID 50 / 50 μL for neutralizing antibody detection. The test method referred to Example 1. After the giant panda / SX / 2014 strain caused cytopathic effect, the cytopathic situation was observed under the 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 the CDV / R20 / 8-EGFP strain, 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 antibody 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 antibody

[0105]

[0106] Example 3 Amino acid sequence analysis of monoclonal antibody 6A4A6 against 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 6A4A6 against canine distemper virus H protein 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. 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, which are used to illustrate the technical solutions of the present application, rather than to limit the same. 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 6A4A6, characterized in that, The subtype of the monoclonal antibody 6A4A6 against canine distemper virus H protein is IgG2bκ. The monoclonal antibody 6A4A6 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.

2. Use of the monoclonal antibody 6A4A6 against canine distemper virus H protein according to claim 1 in the preparation of a detection reagent for differentiating canine distemper virus vaccine strains.

3. A detection reagent for identifying canine distemper virus vaccine strains, characterized in that, Comprising the monoclonal antibody 6A4A6 against canine distemper virus H protein according to claim 1.

4. The detection reagent for identifying a canine distemper virus vaccine strain according to claim 3, characterized in that, The canine distemper virus vaccine strain is CDV / R20 / 8-EGFP or Onderstepoort.

5. The detection reagent for identifying canine distemper virus vaccine strains according to claim 3, characterized in that, The monoclonal antibody 6A4A6 against canine distemper virus H protein reacts specifically with the canine distemper virus vaccine strain, has virus neutralizing activity, and does not react with the CDV virulent isolate.

6. A nucleotide molecule encoding the monoclonal antibody 6A4A6 against canine distemper virus H protein according to claim 1.

7. An expression vector containing the nucleotide molecule according to claim 6.

8. A host cell containing the expression vector according to claim 7 or having the nucleotide molecule according to claim 6 integrated into its genome.

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