Canine distemper virus H protein monoclonal antibody 6G4H3 and application thereof

Abstract

The invention discloses a canine distemper virus H protein monoclonal antibody 6G4H3 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. Indirect immunofluorescence identification shows that the 9 obtained monoclonal antibodies can react with a low virulent vaccine strain (CDV-onderstoport), a neutralizing antibody detection result shows that six of the 9 obtained monoclonal antibodies react with giant panda / SX / 2014 (virulent isolated strain) and have neutralizing capacity, and 6G4H3 has the highest neutralizing titer (up to 27), can be used for preparing a therapeutic preparation for canine distemper virus, and can be used for preparing a therapeutic agent for canine distemper virus. And assistance is provided for research and development of therapeutic antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and particularly to a monoclonal antibody 6G4H3 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 a high incidence and high mortality rate. It can infect a variety of domestic and rare protected animals, as well as non-human primates. Since CDV was discovered, its host range has been continuously expanding. Now, its influence range has exceeded 8 orders and 20 families, and there are even rare wild protected animals such as the endangered Amur tiger and the national treasure giant panda. At the same time, some studies have shown that CDV may interact with human SLAM receptor (hSLAM) and human Nectin-4 receptor to infect human cell lines, which may mean that CDV virus may be transmitted from dogs to humans.

[0003] Currently, due to the differences caused by the continuous mutation of epidemic strains among wild animals, a large number of species cannot be effectively vaccinated against CDV; due to reasons such as gene recombination between vaccine strains and wild strains, the current vaccines cannot provide complete protection. The H protein is one of the glycoproteins on the surface of the CDV envelope, which can specifically bind to receptors (such as SLAM and nectin-4) on the surface of target cells, thereby mediating the invasion of the virus 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, developing specific therapeutic agents based on the H protein is of great significance for the treatment of canine distemper. Monoclonal antibodies have significant advantages in disease diagnosis and immunotherapy due to their strong specificity, high sensitivity, high titer, few cross-reactions, good repeatability, suitability for large-scale production, and low cost.

[0004] Currently, most domestic studies on monoclonal antibodies against CDV are used to establish diagnostic methods. Therapeutic antibodies with neutralizing ability are relatively rare, and most of them are obtained by immunizing mice with purified attenuated CDV strains, and it cannot be confirmed whether they only act on specific proteins. For example, Sun Weiwei (Sun Weiwei. Preparation of monoclonal antibodies against canine distemper virus H protein and establishment of a competitive ELISA detection method [D], 2020) immunized mice with purified Onderstepoort strain and prepared 7 hybridoma cell lines that stably secrete monoclonal antibodies against H protein. The virus neutralization test showed that the antibody neutralization titer of the supernatant of the hybridoma cell lines was 2 4 -2 8 , and the antibody neutralization titer of the ascites induced in BALB / c mice was 102 -10 6 , but since only the recombinant CDV H protein was used to verify the obtained monoclonal antibodies, it was impossible to determine whether they were specific only to the H protein. Shi Pengfei (Shi Pengfei. Preparation of Monoclonal Antibodies Against Canine Distemper Virus H Protein and Canine Parvovirus VP2 Protein and Screening of Antigenic Epitopes [D], 2021.) immunized mice with a truncated H protein expressed in prokaryotes, prepared two monoclonal antibodies against the CDV H protein and analyzed their antigenic epitopes. However, since neutralizing antibody detection was not performed, it was impossible to determine whether the obtained monoclonal antibodies had neutralizing ability. Bi Zhenwei et al. (BI Z, XIA X, WANG Y, et al. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein [J]. Microbiology and Immunology, 2015, 59(4): 202-8) evaluated the reactivity of 6 monoclonal antibodies against 5 CDV strains through indirect immunofluorescence and neutralization tests. The results showed that only 2 of the monoclonal antibodies had strong reactivity with the newly isolated canine distemper virus strain (CDV NJ01) in China, while they had no reactivity with the other 3 early isolated strains (CDV823, CDV851, and CDV846) and the vaccine strain Onderstepoort. The above studies indicate that certain specific antigenic sites of the H protein of currently prevalent strains in China have mutated, and the efficacy of existing monoclonal antibodies in treating prevalent strains of canine distemper has decreased.

[0005] Therefore, it is necessary to prepare monoclonal antibodies against the CDV H protein with relatively high neutralizing activity to facilitate the research and development of therapeutic antibodies. Summary of the Invention

[0006] One object of the present invention is to provide a monoclonal antibody 6G4H3 against the canine distemper virus H protein with relatively high neutralizing activity. The monoclonal antibody 6G4H3 against the 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] The monoclonal antibody 6G4H3 against the canine distemper virus H protein specifically reacts with both the attenuated vaccine strain and the virulent isolate of the canine distemper virus and has relatively high virus neutralizing activity. The highest neutralizing titer in the supernatant can reach 2 4 , and the highest neutralizing titer in the ascites can reach 2 7 .

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

[0009] A second object of the present invention is to provide the use of the above - mentioned monoclonal antibody 6G4H3 against canine distemper virus H protein in the preparation of a therapeutic agent for canine distemper virus.

[0010] A third object of the present invention is to provide a therapeutic agent for canine distemper virus, comprising the above - mentioned monoclonal antibody 6G4H3 against canine distemper virus H protein.

[0011] A fourth object of the present invention is to provide a nucleotide molecule encoding the above - mentioned monoclonal antibody 6G4H3 against canine distemper virus H protein.

[0012] A fifth object of the present invention is to provide an expression vector comprising the above - mentioned nucleotide molecule.

[0013] A sixth object 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:

[0015] In the present invention, the eukaryotically expressed CDV H protein was mixed with QuickAntibody - Mouse3W adjuvant and used to immunize BALB / c mice by intramuscular injection in the thigh. 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 through indirect ELISA, indirect immunofluorescence assay and neutralization assay. After expansion culture, ascites was prepared and purified, and they were identified. Nine monoclonal antibodies were successfully screened out, 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 the attenuated 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, among which the neutralizing titer of 6G4H3 was as high as 2 7 ; 6 out of them had the ability to neutralize the giant panda / SX / 2014 strain, among which the neutralizing titer of 6G4H3 was as high as 2 7 . In summary, the monoclonal antibody 6G4H3 against canine distemper virus H protein provided by the present invention can be used in the preparation of a therapeutic agent for canine distemper virus, providing assistance for the research and development of therapeutic antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other accompanying drawings can also be obtained based on these drawings.

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

[0018] Figure 2 is the neutralization titer against CDV in mouse serum;

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

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

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

[0022] Figure 6 is the indirect immunofluorescence assay of the supernatant of subcloned hybridoma cells; 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 is the neutralization effect diagram of the supernatant of hybridoma cells against CDV / R20 / 8-EGFP strain;

[0024] Figure 8 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 is the identification result of monoclonal antibody subclass; Figure 9Among them, A-I: Identification results of monoclonal antibody subtypes of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6.

[0026] Figure 10 To identify the specificity of monoclonal antibodies against CDV H protein by IFA; Figure 10 Among them, A-I: IFA test results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: IFA test result of CDV positive serum; K: Negative control;

[0027] Figure 11 To identify the specificity of monoclonal antibodies against CDV H 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 12 Neutralization fluorescence images of monoclonal antibodies against CDV / R20 / 8-EGFP strain; Figure 12 Among them, A-I: Neutralization fluorescence images of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: Fluorescence image of virus control;

[0029] Figure 13 Neutralization effect images of monoclonal antibodies against CDV-giantpanda / SX / 2014 strain; Figure 13 Among them, A-I: Neutralization effect images of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: Effect image of virus control. Specific embodiments

[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 making creative efforts fall within the scope of protection of the present invention.

[0031] The test materials used in the following examples were obtained from regular biochemical reagent stores without special instructions. The experimental methods in the following examples were 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 Epidemiology, 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 Epidemiology, 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 virus 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 Company in France (product number: PX-P6307) and is preserved 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, "Construction, Identification and Expression of Recombinant Baculoviruses of Canine Distemper Virus M, F, and H Genes", author: Yu Yicong.

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

[0037] African green monkey kidney cell (Vero) cell line expressing panda signaling lymphocytic activation molecule (SLAM) - GPSLAM-Vero 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 monoclonal antibodies against CDV H protein

[0039] For indirect immunofluorescence assay, CDV-Onderstepoort (attenuated vaccine strain, obtained by reverse genetic rescue) was used. For neutralization assay, recombinant CDV expressing green fluorescent protein (CDV / R20 / 8-EGFP) (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 were stored in this laboratory; BALB / c female mice were purchased from Beijing Speywood Biotechnology Co., Ltd.

[0040] 1 Animal immunization

[0041] BALB / c mice at 6 - 8 weeks of age were immunized, with a total of 5 mice. The immunogen was eukaryotic expression of CDV H protein, and the adjuvant was QuickAntibody-Mouse3W aqueous adjuvant from Bioworld. 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. The antibody titers were detected by indirect ELISA assay and neutralization assay. 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 mouse sera 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 is as follows: Dilute the protein to 2 μg / mL with the coating solution, add 100 μL to each well of the ELISA plate, and incubate overnight at 4 °C. Wash twice with PBST for 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 a gradient dilution downwards. The final dilution volume is 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 and wash three times with PBST for 5 min each time; Add 100 μL of HRP-labeled goat anti-mouse enzyme-linked 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 and wash five times with PBST for 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; Take the maximum dilution multiple when the OD 450 value of the antibody to be tested / OD 450 value of the negative control (S / N) ≥ 2.1 as the antibody titer.

[0045] The indirect ELISA method was used to detect the specific IgG against CDV in the serum of immunized mice. 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 performed. The test results showed (see Figure 1 ), and the antibody titers of the 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] Take the serum of mice that were positive in the indirect ELISA test for the CDV virus neutralization test to detect its neutralization titer against CDV / R20 / 8-EGFP (vaccine strain).

[0048] The specific method for detecting the neutralization titer is as follows: Inactivate the serum to be tested in a water bath at 56 °C for 30 min; Add 50 μL of DMEM incomplete medium to each well of a 96-well plate; Add 50 μL of the serum to be tested to the first well, mix well and then aspirate 50 μL and add it to the next well, and so on, make a 2-fold gradient dilution, make one repeat, and the final dilution volume is 50 μL; Add 50 μL of the virus solution with 100 TCID 50 to each well. At the same time, make 100 TCID 50 , 10 TCID 50 , 1 TCID50 Compared with the cell control, 100 μL per well, 6 wells for each gradient; place in a cell incubator at 37 °C and 5% CO2 for 1 h, and shake crosswise every 15 min in the middle; add 100 μL of Vero cell suspension to each well and continue to culture for 3 - 5 d; observe fluorescence under a fluorescence microscope; calculate the neutralization titer by the Reed - Muench method.

[0049] In order to select the mouse with the highest neutralization titer for the cell fusion experiment, the mouse serum detected as positive by the indirect ELISA test was used for the virus neutralization test to determine the neutralization titer of the mouse serum (see Figure 2 ). After detection, the highest serum neutralization titer reached 1:2048, and the mouse with the highest neutralization titer (number 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 to 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 best state is transparency.

[0053] Suspend the subcultured SP2 / 0 cells in 20 mL of RPMI 1640 incomplete medium in a 50 mL centrifuge tube, centrifuge at 1000 rpm for 7 min, discard the supernatant; add 30 mL of RPMI 1640 incomplete medium, centrifuge at 1000 rpm for 7 min, 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 cell number and store at room temperature for later use.

[0054] 4.2 Preparation of spleen cells

[0055] Select the mouse with the highest neutralization titer after the third immunization. After collecting blood from the eye socket, euthanize 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 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 at 1000 rpm for 5 min and discard the supernatant. Add 30 mL RPMI 1640 incomplete medium, centrifuge at room temperature 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 RPMI 1640 incomplete medium and calculate the volume of the cell suspension with a cell count of 1×10 8 cells and store at room temperature for later use.

[0056] 4.3 Cell fusion

[0057] Water - bath 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 drip 1 mL PEG into 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 RPMI 1640 incomplete medium to the fusion mixture and stir continuously for 4 min. Slowly add 10 mL RPMI 1640 incomplete medium to the fusion mixture. Incubate in a 37°C water - bath for 15 min. Slowly add 30 mL RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 min and discard the supernatant. Slowly add 40 mL RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 min and discard the supernatant. Slowly resuspend in 10 mL RPMI 1640 complete medium. Transfer the cell suspension to a cell culture flask containing 20 mL RPMI 1640 complete medium. Incubate the cell culture flask in a 37°C, 5% CO2 cell incubator for 16 - 24 h. Put 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 2Transfer the cells from the cell bottle 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; 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-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 a microscope every day after 7 days of cell fusion.

[0058] 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 a 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 incubate the 96-well plate in a 37 °C, 5% CO2 cell incubator for 3 - 4 days.

[0061] 5.2 Screening of Hybridoma Cells

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

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

[0064] The steps of the indirect immunofluorescence assay are as follows: Add 50 μL of DMEM incomplete medium per well to a 96-well plate (operate on ice, pre-cool DMEM in advance); add 50 μL of CDV-Onderstepoort (vaccine strain) virus solution per well (diluted to an MOI value of 0.1, operate on ice); add 100 μL of Vero cell suspension and incubate it in a cell culture 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 fix the cells per well, and fix at room temperature for 30 min; discard the cold acetone, add 300 μL of PBS per well, wash on a shaker at room temperature for 5 min, and repeat 3 times; add 100 μL of 2% BSA (prepared with PBS) per well for blocking, and incubate in a 37 °C incubator for 1 h; discard the liquid in the plate, add 50 μL of monoclonal antibody supernatant detected as positive by ELISA per well, add positive control wells and negative control wells (200-fold dilution of PBS), make three replicates, and incubate in a 37 °C incubator for 1 h; discard the liquid in the plate, add 300 μL of PBST per well, wash on a shaker at room temperature for 5 min, and 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 per well, and incubate in a 37 °C incubator for 1 h; discard the liquid in the plate, add 300 μL of PBST per well, wash on a shaker at room temperature for 5 min, and repeat 3 times (protected from light); discard the liquid in the plate, observe the results under an upright fluorescence microscope, and determine the wells showing green fluorescence signals as positive wells.

[0065] The fused hybridoma cells were screened by indirect ELISA (see Figure 3 ) and indirect immunofluorescence identification (see Figure 4 ), and 5 positive hybridoma cell strains were obtained, named 2C1, 2D1, 6A4, 6G4, and 6H4 respectively.

[0066] 6 Subcloning of hybridoma cell strains

[0067] Resuspend the 5 hybridoma cell strains detected as positive, after cell counting, take 100 cells, resuspend them in 1 mL of RPMI1640 complete medium, add them to a 15 mL centrifuge tube containing 10 mL of medium D, mix well by inverting up and down, let stand for 15 min, transfer to a 6-well plate, 2 mL per well, and at the same time add 2 mL of sterile PBS to the remaining 1 well, and incubate in a cell culture incubator at 37 °C and 5% CO2. After 10 - 14 days, pick and screen the subcloned cells.

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

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

[0070] Five hybridoma cell strains, 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 strains were obtained, which were 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 strains could all bind to the eukaryotic-expressed CDV H protein, and indirect immunofluorescence screening showed that the supernatants of these 9 hybridoma cell strains could all react with the CDV-Onderstepoort vaccine strain.

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

[0073] Table 1 Titers of Hybridoma Cell Supernatants

[0074]

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

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

[0077] 1 Ascites Preparation

[0078] Eighteen 8- to 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. 500 μL of adjuvant for ascites was intraperitoneally injected into each mouse; after 15 days, each mouse was injected with a hybridoma cell suspension containing approximately 8×10 5 cells (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 7 - 10 days later. The collected ascites was centrifuged at 3000 r / min for 10 min, the middle layer of the ascites was taken, and stored at -80 °C.

[0079] 2 Purification of ascites

[0080] The crude purification of mouse ascites was carried out by the caprylic 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, add 4 times the volume of acetate buffer, and adjust the pH value to 4.5; at room temperature, slowly add caprylic acid dropwise with a magnetic stirrer while stirring. The addition amount is 25 μL / mL, and the caprylic acid concentration is 33 μL / mL. Stir for 30 min; place at 4 °C and let stand overnight for sufficient precipitation; centrifuge at 4 °C, 10000 rpm for 30 min, collect the supernatant and filter through a funnel to remove impurities; after the supernatant is filtered through the funnel, measure the volume, add 10% 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, 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 280 of the effluent is close to 0; add the obtained dialysate 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 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 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 that the antibody preparation was successful.

[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: The kit was restored to room temperature, and the enzyme-linked immunosorbent assay (ELISA) plate was taken out. First, 50 μL of specimen diluent was added to each well of the ELISA plate, and then 50 μL of hybridoma cell supernatant was added. The supernatant of each hybridoma cell was added to 8 wells. 100 μL of negative and positive controls were added to 8 wells each. The plate was covered with a sealing film and incubated at 37 °C for 30 min. The liquid in the plate was discarded, and the plate was washed 5 times with the washing solution for 5 min each time. 100 μL of 8 kinds of enzyme-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, Lambda) were added to each well. The plate was covered with a sealing film and incubated at 37 °C for 30 min. The liquid in the plate was discarded, and the plate was washed 5 times with the washing solution for 5 min each time. 50 μL of chromogenic agent A and 50 μL of chromogenic agent B were added to each well, and the plate was developed at 37 °C for 20 min in the dark. 50 μL of stop solution was added to detect the OD 450 value. The subclass of the secondary antibody added to the well with the highest OD 450 value of the light chain and heavy chain subclasses was determined as the subclass of the monoclonal antibody.

[0084] After identification, except that the subclass of monoclonal antibody 2C1A1 was IgMκ, and the subclasses of monoclonal antibodies 6A4A5 and 6A4A6 were IgG2bκ, the subclasses of monoclonal antibodies secreted by the remaining cell lines were 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, GPSLAM-Vero cells (African green monkey kidney cell (Vero) cell line expressing giant panda signaling lymphocytic activation molecule (SLAM)) with a cell density of 70%-80% were infected with CDV-giant panda / SX / 2014 (a highly virulent isolate), and the uninfected cells were used as negative controls.

[0090] The 9 selected monoclonal antibodies, namely 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, were diluted and used as primary antibodies to identify the specificity of the 9 monoclonal antibodies 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 the giant panda / SX / 2014 strain (see Figure 10 A, B, E-J in the figure) and showed green positive fluorescence signals, while no fluorescence was observed for the monoclonal antibodies 6A4A5 and 6A4A6 (see Figure 10 C, D in the figure), indicating that the monoclonal antibodies 6A4A5 and 6A4A6 could not specifically recognize giant panda / SX / 2014 (virulent strain).

[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 10 μL / well for the Marker and 20 μL / well for the protein sample. Then perform Western Blot assay. 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 membranes 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 (200-fold dilution) with TBST containing 1% skim milk powder, 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 color developing 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 screened monoclonal antibodies, 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 CDV H protein as the coating antigen, the ELISA titer of the monoclonal antibody was determined by indirect ELISA assay. 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 ELISA titer of the monoclonal antibody was measured to reach 10 3 -10 8 , as shown in Table 3.

[0099] Table 3 ELISA titer table of monoclonal antibodies

[0100]

[0101]

[0102] 5.2 Identification of neutralizing titer of monoclonal antibody

[0103] The titer of the monoclonal antibody was identified by virus neutralization test. CDV / R20 / 8-EGFP (attenuated vaccine strain) and giant panda / SX / 2014 (virulent isolate) were respectively diluted to 100 TCID 50 / 50 μL for neutralizing antibody detection. The test method referred to Example 1. For giant panda / SX / 2014, after virus-induced cytopathic effect, the cytopathic effect was observed under a microscope to confirm its neutralizing titer, and the maximum dilution multiple of the cell wells without observed cytopathic effect was calibrated as its neutralizing titer.

[0104] 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 neutralizing 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 .

[0105] Table 4 Neutralizing titer table of monoclonal antibody

[0106]

[0107]

[0108] Example 3 Amino acid sequence analysis of monoclonal antibody 6G4H3 against canine distemper virus H protein

[0109] Through amino acid sequence analysis, the amino acid sequence of the heavy chain variable region of monoclonal antibody 6G4H3 against 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.

[0110] The above-described embodiments are only 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 6G4H3, characterized in that, The canine distemper virus H protein monoclonal antibody 6G4H3 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 canine distemper virus H protein monoclonal antibody 6G4H3 according to claim 1 in the preparation of a therapeutic agent for canine distemper virus.

3. A therapeutic preparation for canine distemper virus, characterized in that, Comprising the canine distemper virus H protein monoclonal antibody 6G4H3 according to any one of claims 1-3.

4. The therapeutic preparation for canine distemper virus according to claim 3, characterized in that, The canine distemper virus H protein monoclonal antibody 6G4H3 reacts specifically with both the attenuated vaccine strain and the virulent isolate of canine distemper virus, and has virus neutralizing activity.

5. The therapeutic preparation for canine distemper virus according to claim 4, characterized in that, The attenuated vaccine strain of canine distemper virus is CDV-Onderstepoort or CDV / R20 / 8-EGFP.

6. The therapeutic preparation for canine distemper virus according to claim 4, characterized in that The virulent isolate of canine distemper virus is CDV-giant panda / SX / 2014.

7. A nucleotide molecule encoding the canine distemper virus H protein monoclonal antibody 6G4H3 according to claim 1.

8. An expression vector comprising the nucleotide molecule according to claim 7.

9. A host cell, characterized in that, Containing the expression vector according to claim 8 or the nucleotide molecule according to claim 7 is integrated into the genome.

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