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

Abstract

The present invention discloses a canine distemper virus H protein monoclonal antibody 6G4H3 and its application, belonging to the technical field of monoclonal antibodies. The present invention screened out 9 monoclonal antibodies through indirect ELISA and indirect immunofluorescence identification, and named them 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6 respectively. After indirect immunofluorescence identification, the 9 monoclonal antibodies obtained were all able to react with the attenuated vaccine strain (CDV-Onderstepoort), and the neutralizing antibody test results showed that 6 of the 9 monoclonal antibodies obtained reacted with giant panda / SX / 2014 (a highly virulent isolate) and had neutralizing ability, among which 6G4H3 had the highest neutralizing titer (up to 2 7 ), which can be used to prepare therapeutic preparations for canine distemper virus and provide support for the development of therapeutic antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to a monoclonal antibody 6G4H3 based on the H protein of canine distemper virus and applications thereof. Background Art

[0002] Canine distemper (CD) is an acute, severe, and highly contagious disease caused by the canine distemper virus (CDV). It is widely distributed worldwide, with high morbidity and mortality rates, affecting a wide range of domestic and endangered species, including non-human primates. Since its discovery, CDV has continuously expanded its host range, now affecting over eight orders and 20 families, including endangered Amur tigers and giant pandas. Furthermore, studies have shown that CDV can interact with the human SLAM receptor (hSLAM) and the human nectin-4 receptor to infect human cell lines, potentially suggesting the potential for CDV transmission from dogs to humans.

[0003] Currently, due to the differences caused by the continuous mutation of prevalent strains among wild animals, a large number of species cannot be effectively vaccinated against CDV; and the current vaccines cannot provide complete protection due to factors such as genetic recombination between vaccine strains and wild strains. 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 H protein contains many neutralizing antigenic epitopes and is the main antigen that induces the body to produce neutralizing antibodies, which can effectively guide animals to produce cellular immunity against CDV. Therefore, the development of specific therapeutic preparations 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, low cross-reactivity, good reproducibility, suitability for large-scale production and low cost.

[0004] At present, most of the research on CDV monoclonal antibodies in China is used to establish diagnostic methods. Therapeutic antibodies with neutralizing ability are relatively rare, and most of them are purified CDV attenuated strains used to immunize mice. It is impossible to confirm whether they only act on specific proteins. For example, Sun Weiwei (Sun Weiwei. Preparation of monoclonal antibodies to canine distemper virus H protein and establishment of competitive ELISA detection method [D], 2020) used purified Onderstepoort strains to immunize mice and prepared 7 hybridoma cell lines that stably secreted anti-H protein monoclonal antibodies. The virus neutralization test showed that the antibody neutralization titer of the hybridoma cell line supernatant was 2 4 -2 8 The neutralizing titer of the antibody induced in BALB / c mouse ascites was 102 -10 6 However, since only recombinant CDV H protein was used to verify the monoclonal antibodies obtained, it is impossible to determine whether they are only targeted at 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.) used prokaryotically expressed truncated H protein to immunize mice, prepared two CDV H protein monoclonal antibodies and analyzed their antigenic epitopes, but since no neutralizing antibody test was performed, it is impossible to determine whether the monoclonal antibodies obtained have 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 by indirect immunofluorescence and neutralization tests. The results showed that only two of the monoclonal antibodies reacted strongly with the newly isolated canine distemper virus strain in China (CDV NJ01), while they showed no reactivity with the other three earlier isolated strains (CDV823, CDV851, and CDV846) as well as the vaccine strain Onderstepoort. These studies suggest that mutations at specific antigenic sites in the H protein of the strain currently circulating in China have reduced the effectiveness of existing monoclonal antibodies against the circulating canine distemper strain.

[0005] Therefore, it is necessary to prepare monoclonal antibodies with high neutralizing activity against CDV H protein to provide support for the development of therapeutic antibodies. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a canine distemper virus H protein monoclonal antibody 6G4H3 with high neutralizing activity, wherein 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 shown in SEQ ID No: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0007] The canine distemper virus H protein monoclonal antibody 6G4H3 reacts specifically with both the attenuated vaccine strain and the virulent isolated strain of canine distemper virus and has high virus neutralizing activity. The highest neutralizing titer of the supernatant can reach 2 4 , the neutralization titer in ascites can reach up to 2 7 .

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

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

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

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

[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 the above-mentioned nucleotide molecule integrated into its genome.

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

[0015] The present invention utilizes eukaryotic expression of CDV H protein mixed with QuickAntibody-Mouse3W adjuvant to immunize BALB / c mice by thigh muscle injection, and the spleen cells of the immunized mice are fused with myeloma cells using a semi-solid culture medium method. Hybridoma cell lines with neutralizing titers are screened out by indirect ELISA test, indirect immunofluorescence test and neutralization test, and ascites is prepared and purified after expanded culture, and identified, and 9 monoclonal antibodies are successfully screened out, which are named 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6 respectively. After indirect immunofluorescence identification, the 9 monoclonal antibodies obtained can react with the attenuated vaccine strain (CDV-Onderstepoort). The neutralizing antibody test results show that 7 of the 9 monoclonal antibodies obtained have neutralizing ability against CDV / R20 / 8-EGFP strain, among which 6G4H3 has the highest neutralizing titer of 2 7 ; Six strains showed neutralizing ability against giant panda / SX / 2014 strain, among which 6G4H3 had the highest neutralizing potency of 2 7 In summary, the canine distemper virus H protein monoclonal antibody 6G4H3 provided by the present invention can be used to prepare therapeutic preparations for canine distemper virus, providing support for the research and development of therapeutic antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

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

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

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

[0020] Figure 4 Indirect immunofluorescence assay for hybridoma cell supernatant; Figure 4 Middle, AE: results of indirect immunofluorescence assay of 2C1, 2D1, 6A4, 6G4, and 6H4; F: results of indirect immunofluorescence assay of mouse positive serum; G: negative control;

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

[0022] Figure 6 Indirect immunofluorescence assay of hybridoma cell supernatant after subcloning; Figure 6 Indirect immunofluorescence assay results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6; J: indirect immunofluorescence assay results of mouse positive serum; K: negative control;

[0023] Figure 7 The figure shows the neutralization effect of hybridoma cell supernatant on CDV / R20 / 8-EGFP strain;

[0024] Figure 8 The purified CDV H monoclonal antibody was analyzed by SDS electrophoresis; Figure 8 In the figure, AI: SDS-PAGE analysis results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6; M: protein marker; 1: unpurified ascites; 2: purified ascites;

[0025] Figure 9 This is the result of monoclonal antibody subclass identification; Figure 9In the figure, the subtype identification results of monoclonal antibodies AI: 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6.

[0026] Figure 10 To identify the specificity of CDV H protein monoclonal antibodies for IFA; Figure 10 Middle, AI: IFA test results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6; J: IFA test results of CDV-positive serum; K: negative control;

[0027] Figure 11 To identify the specificity of CDV H monoclonal antibody for Western Blot; Figure 11 In the figure, AI: Western Blot results of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6; M: Protein Marker; 1: Eukaryotic expression of H protein; 2: Prokaryotic expression of H protein;

[0028] Figure 12 It is the fluorescence graph of neutralization of CDV / R20 / 8-EGFP strain by monoclonal antibody; Figure 12 Middle, AI: neutralization fluorescence images of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6; J: virus control fluorescence image;

[0029] Figure 13 This is a graph showing the neutralization effect of monoclonal antibodies against CDV-giant panda / SX / 2014 strain; Figure 13 In the figure, AI: neutralization effect diagram of 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6; J: virus control effect diagram; K: negative control. DETAILED DESCRIPTION

[0030] To help those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0031] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores. The experimental methods in the following examples were conventional methods unless otherwise specified.

[0032] The recombinant CDV expressing green fluorescent protein (CDV / R20 / 8-EGFP) was constructed and preserved by the Animal Virology and Special Animal Epidemiology Laboratory 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 patent publication No. CN102329809B). The parental virus CDV / R20 / 8 of CDV / R20 / 8-EGFP is an attenuated vaccine strain widely used in my country's production practice and is preserved in the Animal Virology and Special Animal Epidemiology Laboratory 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 patent publication No. CN102344913B).

[0033] The wild-type / virulent isolate CDV-giant panda / SX / 2014 was isolated and stored in the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute (for methods, see 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: 275-18).

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

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

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

[0037] GPSLAM-Vero, an African green monkey kidney cell (Vero) cell line expressing giant panda signaling lymphoid activator molecule (SLAM), was constructed by the Laboratory of Animal Virology and Special Animal Epidemiology, Military Veterinary Research Institute (for methods, 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 (an attenuated vaccine strain obtained by reverse genetic rescue) was used for indirect immunofluorescence assays, and recombinant CDV expressing green fluorescent protein (CDV / R20 / 8-EGFP) (an attenuated vaccine strain obtained by reverse genetic rescue) was used for neutralization experiments. African green monkey kidney (Vero) cells, myeloma (SP2 / 0) cells, and eukaryotic expression of CDV H protein were all maintained in our laboratory. BALB / c female mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0040] 1Animal immunization

[0041] Five BALB / c mice, 6 to 8 weeks old, were immunized with eukaryotically expressed CDV H protein and Biolon's QuickAntibody-Mouse3W aqueous adjuvant. Immunizations were repeated 2 weeks apart for a total of four times, with a dose of 50 μL adjuvant plus 50 μL H protein (0.5 mg / mL) per mouse. Seven days after the second, third, and fourth immunizations, blood was collected and serum was isolated. Antibody titers were measured using indirect ELISA and neutralization assays. Seven days after the fourth immunization, cell fusion was performed, and blood and serum were collected and titers were measured.

[0042] 2. Indirect ELISA test

[0043] Indirect ELISA was performed by coating an ELISA plate with eukaryotic CDV H protein, using mouse serum 7 days after the second, third, and fourth immunizations as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody.

[0044] The specific operation of indirect ELISA detection is as follows: dilute the protein to 2 μg / mL with coating solution, add 100 μL to each well of the ELISA plate, incubate at 4°C overnight, wash twice with PBST, 5 min / time; add 300 μL of blocking solution (3% skim milk powder, diluted in PBST) to each well, incubate at 37°C for 2 h; discard the blocking solution, add 100 μL of PBST containing 1% skim milk powder to each well, and add 100 μL of the serum dilution solution to be tested (1% skim milk powder, PBST) to the first well of each column. 40000 dilution), and then make gradient dilution downward to a final volume of 100μL. At the same time, make positive and negative controls and incubate at 37℃ for 1h. Discard the liquid in the plate and wash three times with PBST, 5min / time. Add 100μL of 1:20000 diluted HRP-labeled goat anti-mouse enzyme-labeled secondary antibody (BS12478) to each well of the enzyme-labeled plate, place in a 37℃ incubator and incubate for 1h. Discard the liquid in the plate and wash five times with PBST, 5min / time. Add 100μL of TMB colorimetric solution to each well and color for 10min in the dark. Add 50μL of stop solution to each well. Measure OD 450 Value; OD of the antibody to be tested 450 Value / negative control OD 450 The maximum dilution factor with a value (S / N) ≥ 2.1 was taken as the antibody titer.

[0045] The indirect ELISA method was used to detect the specific IgG of anti-CDV in the serum of the immunized mice. Blood was collected from the 5 immunized mice (numbers 413-417) 7 days after the 2nd, 3rd and 4th immunizations, and the serum was separated and subjected to indirect ELISA test. The test results showed that (see Figure 1 ), the mouse serum antibody titer reached 1:640000-1:5120000, and mice with ELISA titer were selected for neutralizing antibody detection.

[0046] 3 Neutralizing antibody detection

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

[0048] The specific method for neutralization titer detection is as follows: the serum to be tested is inactivated in a 56°C water bath for 30 minutes; 50 μL of DMEM incomplete medium is added to each well of a 96-well plate; 50 μL of the serum to be tested is added to the first well, mixed, and then 50 μL is aspirated and added to the next well, and so on, making a 2-fold serial dilution, repeating one time, and the final dilution volume is 50 μL; 50 μL of 100 TCID is added to each well 50 The virus solution was prepared at 100 TCID 50 、10TCID 50, 1TCID 50 For cell control, add 100 μL per well, and each gradient has 6 wells; place in a 37°C, 5% CO2 cell incubator for 1 hour, and shake crosswise every 15 minutes; add 100 μL of Vero cell suspension to each well and continue to culture for 3-5 days; observe fluorescence under a fluorescence microscope; calculate neutralization titer by the Reed-Muench method.

[0049] In order to select mice with the highest neutralization titer for cell fusion experiments, virus neutralization tests were performed using mouse serum that was positive in indirect ELISA tests to determine the neutralization titer of mouse serum (see Figure 2 After testing, the serum neutralization titer was as high as 1:2048, and the mouse with the highest neutralization titer (No. 414) was selected for cell fusion

[0050] 4. Cell fusion experiment

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

[0052] Remove the frozen SP2 / 0 cells from the liquid nitrogen tank, shake them quickly in a 37°C water bath to thaw them, take 5 mL of RPMI 1640 complete medium to resuspend the cells, centrifuge at 1000 rpm for 7 minutes, discard the supernatant, add 5 mL of RPMI 1640 complete medium to resuspend the cell pellet, and transfer it to a T25 cm 2 The cells were placed in a flask and incubated overnight at 37°C in a cell culture incubator with 5% CO2. After 12 hours, the cells were observed and the optimal state was when they were round, free of aggregates, and permeable.

[0053] The passaged SP2 / 0 cells were suspended in 20 mL of RPMI 1640 incomplete medium in a 50 mL centrifuge tube, centrifuged at 1000 rpm for 7 min, and the supernatant was discarded. 30 mL of RPMI 1640 incomplete medium was added, and the cells were centrifuged at 1000 rpm for 7 min, and the supernatant was discarded. This was repeated twice, for a total of three washes. The cells were resuspended in 25 mL of RPMI 1640 incomplete medium. Calculate the number of cells per 2 × 10 7 The volume of cell suspension corresponding to the number of cells was stored at room temperature for future use.

[0054] 4.2 Preparation of splenocytes

[0055] The mouse with the highest neutralization titer after the third immunization was selected. After eyeball blood collection, the mouse was sacrificed by cervical dislocation and immersed in 75% alcohol for 5-7 minutes. After transfer to a clean bench, the mouse was fixed to a foam board with a 1mL syringe needle. The abdominal skin was cut open, the abdominal cavity was opened, and excess fat and connective tissue on the surface of the spleen were removed. The spleen surface was rinsed with RPMI 1640 incomplete medium. A 200-mesh cell sieve was placed in the mouth of a 50mL centrifuge tube and moistened with 10mL RPMI 1640 incomplete medium. The spleen was pressed with a 5mL syringe plunger and triturated clockwise. The cells were collected into a 50mL centrifuge tube and centrifuged at 1000rpm for 5 minutes at room temperature. The supernatant was discarded. 30mL RPMI 1640 incomplete medium was added and the cells were centrifuged at 1000rpm for 7 minutes at room temperature. The supernatant was discarded. Repeat two times for a total of three washes. The cells were resuspended in 25mL RPMI 1640 incomplete medium and 1×10 8 The volume of cell suspension corresponding to the number of cells was stored at room temperature for future use.

[0056] 4.3 Cell fusion

[0057] Heat PEG and RPMI 1640 incomplete medium in a water bath to 37°C; place the prepared SP2 / 0 cells and spleen cells in a 50 mL conical tube; centrifuge at 1000 rpm for 7 minutes and discard the supernatant; use a 1 mL pipette to slowly add 1 mL of PEG to the cells dropwise for 1 minute, and slowly stir the cells with the pipette tip for 1 minute; add 4 mL of RPMI 1640 incomplete medium to the fusion mixture and stir continuously for 4 minutes; slowly add 10 mL of RPMI 1640 incomplete medium to the fusion mixture. Incubate in a 37°C water bath for 15 minutes; slowly add 30 mL of RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 minutes, and discard the supernatant; slowly add 40 mL of RPMI 1640 complete medium, centrifuge at 1000 rpm for 7 minutes, and discard the supernatant; slowly resuspend 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 incubator for 16-24 hours; bring medium D to room temperature one day in advance and shake the medium D vigorously to mix it thoroughly; transfer the fused cell suspension from the T75 cm 2Transfer the cells from the flask to a 50mL conical tube, centrifuge at 1000rpm for 7 minutes, and discard the supernatant. Resuspend the cells in complete RPMI 1640 medium to a total volume of 6mL. Transfer the cell suspension directly to a bottle containing 60mL of Medium D. Gently invert the bottle several times to mix thoroughly. Incubate at room temperature for 15 minutes, allowing air bubbles to rise to the top. Using a 20mL syringe and a blunt-end needle, slowly add 1mL of the cell suspension in Medium D to each 6-well plate, taking care to avoid introducing air bubbles. Tilt each plate to evenly distribute the medium across the bottom of the plate. Incubate in a 37°C, 5% CO2 incubator for 10-14 days. Observe the cells daily under a microscope after 7 days of confluency.

[0058] 5. Hybridoma cell selection and screening

[0059] 5.1 Hybridoma cell selection

[0060] Use a pipette and pipette tip to aspirate hybridoma cells from a 6-well plate under a microscope. Transfer a single hybridoma cell to a 96-well cell culture plate containing HT medium. Mix the cells in each well by pipetting up and down, 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 cell lines are screened using indirect ELISA, which can screen a large number of antibody-secreting hybridoma cells. However, the disadvantages are that false positives are prone to occur, it is difficult to screen for neutralizing monoclonal antibodies, and the workload required for subsequent expanded culture is extremely high. The present invention uses indirect ELISA and indirect immunofluorescence to perform dual screening on the fused cells, which largely avoids the false positive phenomenon of indirect ELISA results, reduces the workload of subsequent hybridoma cell line culture, and makes it easier to obtain monoclonal antibodies with stable expression and high titer.

[0063] Specifically, the indirect ELISA test was first used for preliminary screening. The primary antibody was the hybridoma cell supernatant, which was not diluted. The positive serum of the immunized mice was diluted to OD 450 The indirect ELISA test was used to further screen the hybridoma cell supernatants that tested positive.

[0064] The indirect immunofluorescence experiment steps are as follows: add 50 μL / well of DMEM incomplete medium to a 96-well plate (operate on ice, DMEM is pre-cooled 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 culture it in a 37°C, 5% CO2 cell culture incubator for 48 hours; remove the 96-well plate, discard the culture 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 them at room temperature for 30 minutes; discard the cold acetone, and add PBS to each well. 300 μL, wash on a shaker at room temperature for 5 minutes, repeat 3 times; add 100 μL of 2% BSA (in PBS) to each well for blocking, and incubate at 37°C incubator for 1 hour; discard the liquid in the plate, add 50 μL of the monoclonal antibody supernatant tested positive by ELISA to each well, add positive control wells and negative control wells (200-fold dilution of PBS), do three replicates, and incubate at 37°C incubator for 1 hour; discard the liquid in the plate, add 300 μL of PBST to each well, wash on a shaker at room temperature for 5 minutes, repeat 3 times; discard the liquid in the plate, add 1:500 diluted Alexa 488-labeled goat anti-mouse fluorescent secondary antibody (A0428) and 1:500 diluted Evans blue staining solution 50 μL to each well, and incubate at 37°C incubator for 1 hour; discard the liquid in the plate, add PBST to each well 300 μL, wash on a shaker at room temperature for 5 minutes, repeat 3 times (protect from light); discard the liquid in the plate, observe the results under an upright fluorescence microscope, and determine that the wells showing green fluorescent signals are positive wells.

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

[0066] 6 Subcloning of hybridoma cell lines

[0067] Resuspend the five hybridoma cell lines that tested positive, count the cells, 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 them by inversion, let them stand for 15 minutes, transfer them to a 6-well plate, 2 mL per well, and add 2 mL of sterile PBS to the remaining well. Place the plate in a 37°C, 5% CO2 cell culture incubator and culture for 10-14 days. After that, pick and screen the subcloned cells.

[0068] 7. Hybridoma cell supernatant titer detection

[0069] The ELISA titer of the hybridoma cell supernatant was tested according to the above-mentioned indirect ELISA method, with the primary antibody being the hybridoma cell supernatant. After the subcloned hybridoma cell line was expanded and cultured, the hybridoma cell supernatant was collected for neutralizing antibody detection.

[0070] The five hybridoma cell lines 2C1, 2D1, 6A4, 6G4, and 6H4 were subcloned by the semi-solid culture method and analyzed by indirect ELISA (see Figure 5 ) and indirect immunofluorescence identification (see Figure 6 ) were screened and 9 positive hybridoma cell lines were obtained, which were named 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6.

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

[0072] The supernatants of the 9 hybridoma cells were subjected to virus neutralization tests to test their neutralization ability. The results showed that 7 strains had neutralization titers, namely 2D1B1, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, with the highest neutralization titer reaching 1:16 (see Table 1 and Figure 7 ).

[0073] Table 1 Hybridoma cell supernatant titer

[0074]

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

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

[0077] 1. Ascites Preparation

[0078] Eighteen 8-10 week old SPF female BALB / c mice were selected and nine hybridoma cells that stably secreted CDV H protein antibodies screened by the present invention were injected into the peritoneal cavity of each mouse with 500 μL of ascites special adjuvant. After 15 days, each mouse was injected with about 8×10 5 Hybridoma cell suspension of 100 cells (washed twice with PBS and then suspended in PBS) was injected into 3 mice per strain. When abdominal distension was observed in the mice on day 7-10, ascites was collected and centrifuged at 3000 rpm for 10 min. The middle layer of ascites was collected and frozen at -80°C.

[0079] 2 Ascites purification

[0080] The mouse ascites was crudely purified by the octanoic acid-ammonium sulfate method and then further purified by a Protein A purification column (all liquids needed to be filtered with a 0.22 μm filter). The specific experimental steps were as follows: 5 mL of ascites was added to 4 times the volume of acetate buffer and the pH value was adjusted to 4.5; octanoic acid was slowly added dropwise with stirring using an electromagnetic stirrer at room temperature, the amount added was 25 μL / mL, the octanoic acid concentration was 33 μL / mL, and stirring was performed for 30 minutes; it was placed at 4°C and allowed to stand overnight for sufficient precipitation; it was centrifuged at 4°C and 10,000 rpm for 30 minutes, the supernatant was collected and filtered with a funnel to remove impurities; after filtering the supernatant with a funnel, the volume was measured and 10% volume of 10× PBS was added to adjust the pH value to 7.4; ammonium sulfate powder was slowly added within 30 minutes on ice, the amount added was 0.706 mg / mL, and stirring was continued, and stirring was performed with an electromagnetic stirrer at 4°C for 2 hours; it was centrifuged at 4°C and 10,000 rpm for 30 minutes, the supernatant was discarded, and 2 mL Suspend the precipitate in PBS, place it in a dialysis bag, place it in PBS, and dialyze it overnight at 200 rpm at 4°C; add PBS to the Protein A purification column until the OD value of the effluent is 280 Close to 0; add the obtained dialysate to the purification column and repeat the sample loading 3 times to ensure that the protein is fully bound to the filler; wash the purification column with 10 mL PBS; elute with 5 mL eluent (4.5 mL 0.1 M glycine solution with pH 2.7 + 0.5 mL Tris-HCl buffer with pH 9); collect the eluate in a 1.5 mL centrifuge tube, 1 mL per tube, and add 100 μL Tris-HCl buffer with a pH value of 9.0; the purified antibody is stored at -80°C after SDS-PAGE analysis.

[0081] After SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, the results showed that the purified ascites fluid had two bands, heavy chain and light chain, with sizes of 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 subclasses of the monoclonal antibodies were identified using a mouse monoclonal antibody subclass identification kit (BF16001, Suzhou Biolong Company). The specific steps are as follows: the kit was restored to room temperature and the ELISA plate was removed; 50 μL of the specimen diluent was first added to each well of the ELISA plate, followed by 50 μL of hybridoma cell supernatant. 8 wells of each hybridoma cell supernatant were added, and 100 μL of each negative and positive control were added to each well. The plate was sealed with film and incubated at 37°C for 30 min. The liquid in the plate was discarded. The plate was washed five times with cleaning solution, each time for 5 minutes; 100 μL of each of 8 enzyme-labeled secondary antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, and Lambda) was added to each well, and the plate was sealed with film and incubated at 37°C for 30 minutes; the liquid in the plate was discarded, and the plate was washed five times with cleaning solution, each time for 5 minutes; 50 μL of color developer A and color developer B were added to each well, and the plate was developed at 37°C in the dark for 20 minutes; 50 μL of stop solution was added to detect OD 450 Determine the OD value of light chain and heavy chain subclass 450 The secondary antibody subclass added to the well with the highest value is the monoclonal antibody subclass.

[0084] After identification, except for the monoclonal antibody 2C1A1 which was IgMκ and the monoclonal antibodies 6A4A5 and 6A4A6 which were IgG2bκ, the subclass of the monoclonal antibodies secreted by the other cell lines were IgG1κ (see Figure 9 and Table 2)

[0085] Table 2 Monoclonal antibody subclass identification table

[0086]

[0087] 4 Characterization of monoclonal antibodies

[0088] 4.1 Indirect immunofluorescence assay

[0089] GPSLAM-Vero cells (African green monkey kidney (Vero) cell line expressing giant panda signaling lymphoid activation molecule (SLAM)) at a cell density of 70%-80% were infected with CDV-giant panda / SX / 2014 (a highly virulent isolate) in 24-well plates, and cells not infected with the virus were used as negative controls.

[0090] The nine selected monoclonal antibodies, 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5, and 6H4H6, were diluted and used as primary antibodies. The specificity of the nine monoclonal antibodies was identified by indirect immunofluorescence. The specific method was as follows: after lesions were observed (generally 48 hours), cells were fixed with pre-chilled 4% paraformaldehyde (100 μL / well) for 20 minutes; 2% BSA (diluted in PBS) (100 μL / well) was added for blocking at 37°C for 1 hour; 100 μL / well of the diluted monoclonal antibody (500-fold dilution) was added and incubated at 37°C for 1 hour; the cells were washed three times with PBST; 100 μL / well of Alexa 488-conjugated goat anti-mouse IgG (1:500 dilution) was added to each well and incubated at 37°C for 1 hour; the cells were washed three times with PBST; and the results were observed using a fluorescence microscope.

[0091] The results showed that monoclonal antibodies 2C1A1, 2D1B1, 6G4H1, 6G4H3, 6H4F4, 6H4H5, 6H4H6 and mouse positive serum could specifically recognize giant panda / SX / 2014 strain (see Figure 10 A, B, EJ) showed green positive fluorescent signals, while monoclonal antibodies 6A4A5 and 6A4A6 (see Figure 10 No fluorescence was observed in (C, D), indicating that monoclonal antibodies 6A4A5 and 6A4A6 could not specifically recognize giant panda / SX / 2014 (a virulent strain).

[0092] 4.2 Western Blot Assay

[0093] The eukaryotic expression CDV H protein sample and 6× protein loading buffer were added to a centrifuge tube at a ratio of 5:1, mixed evenly, and boiled in boiling water for 10 minutes. The sample loading volume was 10 μL / well for marker and 20 μL / well for protein sample. Western Blot test was performed. The specific steps were as follows: (1) electrophoresis: 80V, 1h; (2) transfer: 300mA, 70min; A. Remove the gel, cut the target band, and cut the NC membrane and fiber pad of the same size as the gel; B. Turn the transfer template white side down (arrange them in the order of white board → sponge pad → fiber pad → NC membrane → gel → fiber pad → sponge → black board); C. Fill the transfer buffer, add ice packs, and place the transfer tank in an ice basin; (3) Blocking: Soak the NC membrane with 5% skim milk powder diluted with TBST and shake at room temperature. Block for 2 hours; (4) Primary antibody incubation: dilute 9 monoclonal antibodies (200-fold dilution) with TBST containing 1% skim milk powder, add them to the NC membrane and incubate overnight on a shaker at 4°C. After incubation, wash 4 times with TBST; (5) Secondary antibody incubation: dilute HRP-labeled goat anti-mouse IgG (H+L) 1:20,000 with TBST containing 1% skim milk powder, incubate on a shaker at room temperature for 1 hour. After incubation, wash 4 times with TBST; (6) Prepare color development solution: 1:1 ratio; (7) Expose to color and take pictures.

[0094] Western Blot results showed that the nine monoclonal antibodies selected, 2C1A1, 2D1B1, 6A4A5, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, could react specifically with the eukaryotic CDV H protein, with a protein size of 75 kDa, which was consistent with the expected results (see Figure 11 ), among which 2D1B1 can also react specifically with prokaryotically expressed H protein.

[0095] 5. Identification of Monoclonal Antibody Titer

[0096] 5.1 Identification of Monoclonal Antibody ELISA Titer

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

[0098] After indirect ELISA identification, the monoclonal antibody ELISA titer can reach 10 3 -10 8 , see Table 3.

[0099] Table 3 Monoclonal Antibody ELISA Titer

[0100]

[0101]

[0102] 5.2 Identification of neutralizing titer of monoclonal antibodies

[0103] The titer of the monoclonal antibody was identified by virus neutralization test, using CDV / R20 / 8-EGFP (attenuated vaccine strain) and giant panda / SX / 2014 (virulent isolate) diluted to 100 TCID 50 Neutralizing antibody detection was performed using 50 μL of the solution. The test method was similar to that in Example 1. After the virus produced lesions in giant panda / SX / 2014, the lesions were observed under a microscope to confirm the neutralization titer. The maximum dilution factor of the wells in which no cytopathic effect was observed was designated as the neutralization titer.

[0104] The results of CDV / R20 / 8-EGFP neutralization test showed that 7 of the 9 monoclonal antibodies obtained had the ability to neutralize CDV / R20 / 8-EGFP, namely 2D1B1, 6A4A6, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, with the highest neutralization titer reaching 2 7 The results of the CDV-giant panda / SX / 2014 neutralization test showed that 6 of the 9 monoclonal antibodies obtained had neutralizing ability against CDV-giant panda / SX / 2014, namely 2D1B1, 6G4H1, 6G4H3, 6H4F4, 6H4H5 and 6H4H6, with the highest titer reaching 2 7 (See Table 4). The neutralization effect of monoclonal antibodies on CDV / R20 / 8-EGFP and giant panda / SX / 2014 is shown in the figure. Figure 12 and Figure 13 .

[0105] Table 4 Monoclonal Antibody Neutralization Titer

[0106]

[0107]

[0108] Example 3 Amino Acid Sequence Analysis of Canine Distemper Virus H Protein Monoclonal Antibody 6G4H3

[0109] Amino acid sequence analysis revealed that the amino acid sequence of the heavy chain variable region of the canine distemper virus H protein monoclonal antibody 6G4H3 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. The heavy chain variable region is of the IGH type, and the light chain variable region is of the IGK type.

[0110] The above-described embodiments are only specific implementation methods of the present application, which are used to illustrate the technical solutions of the present application, rather than to limit them. The scope of protection of the present application is not limited thereto. Although the present application has been described in detail with reference to the above-mentioned embodiments, those skilled in the art should understand that any person skilled in the art can modify or easily conceive of changes to the technical solutions described in the above-mentioned embodiments within the technical scope disclosed in the present application, or perform equivalent replacements for some of the technical features thereof; and these modifications, changes or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application. They should all be included in the scope of protection of the present application. Therefore, the scope of protection of the present application shall be subject to the scope of protection 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 includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region 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.

2. Use of the canine distemper virus H protein monoclonal antibody 6G4H3 as claimed in claim 1 in the preparation of a therapeutic preparation for canine distemper virus.

3. A therapeutic preparation for canine distemper virus, characterized in that, The invention comprises the canine distemper virus H protein monoclonal antibody 6G4H3 according to any one of claims 1 to 2.

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

5. The therapeutic preparation for canine distemper virus according to claim 4, characterized in that The attenuated canine distemper virus vaccine strain 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 canine distemper virus isolate was CDV-giant panda / SX / 2014.

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

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

Images