Hybridoma cell strain, canine distemper virus H protein monoclonal antibody and application

Abstract

The invention relates to the technical field of bioengineering, in particular to a hybridoma cell strain, a canine distemper virus H protein monoclonal antibody and application. The hybridoma cell strain is named as 8F5, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.46311. The canine distemper H protein immunogen is prepared, hybridoma cells capable of stably secreting antibodies are prepared through cell fusion and multiple subcloning, a large number of canine distemper virus H protein monoclonal antibodies are obtained through ascites preparation, the canine distemper virus H protein monoclonal antibodies can be used for canine distemper etiology and serology detection, and technical support is provided for canine distemper infection monitoring.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly to a hybridoma cell line, a monoclonal antibody against canine distemper virus H protein, and applications thereof. Background Art

[0002] Canine distemper virus, with the English name Canine Distemper Virus and the English abbreviation CDV, is a highly contagious virus belonging to the genus Morbillivirus of the Paramyxoviridae family. It mainly infects canids, but may also infect other animals. CDV is transmitted through the air, direct contact, and indirect contact. After infection, it can cause various symptoms, including respiratory symptoms, digestive system symptoms, nervous system symptoms, skin symptoms, and eye symptoms. The course of the disease can last from several weeks to several months, and severe cases may lead to death, especially in puppies and animals with a weakened immune system. Monoclonal antibodies have advantages such as strong specificity, high titer, good repeatability, low cost, and suitability for large-scale production, and thus have superiority in disease diagnosis and immunotherapy.

[0003] The main laboratory detection methods for diagnosing canine distemper virus at home and abroad mainly include: virus isolation and culture, electron microscopy observation, immunodiffusion test, indirect immunofluorescence test, virus neutralization test, PCR test, etc. However, these detection methods have the defect of low cross-reaction specificity. Therefore, developing a hybridoma cell line for producing relevant monoclonal antibodies is of great significance for improving the specificity of canine distemper detection methods. Summary of the Invention

[0004] To solve the above problems, the present invention provides a hybridoma cell line, a monoclonal antibody against canine distemper virus H protein, and applications thereof.

[0005] The present invention is achieved through the following technical solutions: A hybridoma cell line named 8F5, which is deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is: CGMCC NO. 46311.

[0006] The application of the above-mentioned hybridoma cell line in the preparation of a monoclonal antibody against canine distemper virus H protein.

[0007] A monoclonal antibody against canine distemper virus H protein, which is secreted by the above-mentioned hybridoma cell line or its subculture cell line.

[0008] Preferably, the monoclonal antibody against canine distemper virus H protein is prepared from the above-mentioned hybridoma cell line or its subculture cell line by the method of inducing ascites in vivo.

[0009] An immunogen of a monoclonal antibody against canine distemper virus H protein, wherein the monoclonal antibody against canine distemper virus H protein is the monoclonal antibody against canine distemper virus H protein described above; the immunogen of the monoclonal antibody against canine distemper virus H protein is the canine distemper virus H gene, and the nucleotide sequence of the canine distemper virus H gene is shown as SEQ ID NO. 1.

[0010] The application of the monoclonal antibody against canine distemper virus H protein in detecting canine distemper virus.

[0011] Preferably, the specific application method is to use the test sample as the coated antigen and the monoclonal antibody against canine distemper virus H protein as the primary antibody for indirect competitive ELISA detection.

[0012] Preferably, the monoclonal antibody against canine distemper virus H protein is used for preparing a canine distemper virus detection kit.

[0013] Preferably, the detection kit includes an ELISA detection component coated plate, an enzyme-labeled secondary antibody, and a chromogenic substrate.

[0014] Preferably, the monoclonal antibody is used for preparing a canine distemper virus diagnostic reagent.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a hybridoma cell line named 8F5, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is: CGMCC NO. 46311. The present invention prepares an immunogen of canine distemper H protein, and through cell fusion and multiple subclonings, a hybridoma cell that stably secretes antibodies is prepared. By preparing ascites, a large amount of monoclonal antibodies against canine distemper virus H protein can be obtained, which can be used for the etiology and serology detection of canine distemper, providing technical support for its infection monitoring. And it provides technical support for the scientific prevention and control of canine distemper.

[0016] Biological material deposit The hybridoma cell line provided by the present invention is named 8F5, and it was deposited in the China General Microbiological Culture Collection Center on December 26, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 46311. Brief description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Proliferation diagram of the CDV virus of the present invention; Figure 1 In it, A is normal vero cells; B is vero cells with lesions after inoculation with CDV.

[0019] Figure 2 Identification of 19T-H of the present invention; among them, M: DNA molecular weight Marker; 1: negative control; 2: amplified product of 19T-H bacterial liquid.

[0020] Figure 3 Identification diagram of the recombinant plasmid pCold TF-H of the present invention; among them, M: DNA molecular weight Marker; 1: negative control; 2: amplified product of CDV H gene.

[0021] Figure 4 Expression of the pCold TF-H recombinant protein of the present invention; among them, M: protein Marker; 1: supernatant of non-induced disruption; 2: precipitate of non-induced disruption; 3: supernatant of induced disruption; 4: precipitate of induced disruption; 5: supernatant of empty vector disruption; 6: precipitate of empty vector disruption.

[0022] Figure 5 Optimization diagram of the induction time of the pCold TF-H recombinant protein of the present invention; among them, M: protein Marker; 1-6: supernatants of disruption after induction for 14h, 16h, 18h, 20h, 22h, 24h.

[0023] Figure 6 Optimization diagram of the IPTG induction concentration of the pCold TF-H recombinant protein of the present invention; among them, M: protein Marker; 1-6: supernatants of disruption induced by 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM IPTG.

[0024] Figure 7 Optimization diagram of the IPTG induction concentration of the pCold TF-H recombinant protein of the present invention; among them, M: protein Marker; 1: protein flow-through solution; 2-8: elution solutions of 20mM, 40mM, 60mM, 100mM, 200mM, 500mM, 600mM imidazole.

[0025] Figure 8 Western Blot identification result of the pCold TF-H recombinant protein of the present invention; among them, M: protein Marker; 1: purified H protein.

[0026] Figure 9 Growth status of hybridoma cells after cell fusion of the present invention. Detailed implementation method

[0027] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] The beneficial effects of the present invention are illustrated by the following specific embodiments: Example 1 1. Cloning of H protein and construction of vector 1.1 Propagation of CDV virus Inoculate the CDV virus solution into Vero cells. After the cells show lesions such as vacuolar degeneration, coagulative necrosis, syncytium, and inclusion bodies, as Figure 1 shown, collect the infected supernatant, freeze-thaw it 3 times repeatedly, centrifuge at 8000 r / min for 30 min to remove cell debris, and collect the supernatant.

[0030] 1.2 Cloning of CDV H gene Use a kit to extract and reverse transcribe CDV RNA. Design primers based on the CDV H gene sequence registered in NCBI GenBank as shown in Table 1, perform PCR amplification, gel recovery, and subcloning of the gene. After mixing the CDV H gene and the pMD 19T vector, connect them in a water bath at 16°C for 3 h, and then transform the ligation product into competent Escherichia coli DH5ɑ. Use the 19T-H bacterial solution as a template and amplify the CDV H gene using the primers described in Table 1. As Figure 2 shown, a band with a size of approximately 1140 bp was amplified, which was consistent with the expected band size. The CDV H gene sequence is as shown in SEQ ID NO. 1.

[0031] SEQ ID NO. 1:

[0032] Table 1 Primer sequences for CDV H gene cloning

[0033] 1.3 Construction of the pCold TF prokaryotic expression vector Using the pMD 19T-H plasmid as a template, the CDV H gene was amplified with the primer sequences shown in Table 2. pCold TF was double digested with HindIII and XhoⅠ. After the PCR amplification of the CDV H gene and the enzymatic digestion reaction of the pCold TF vector, recovery and purification were carried out. The digested pCold TF vector was recombined with the amplified fragment of the CDV H gene. The ligation product was transformed into DH5α competent cells, and using the pCold TF-H bacterial liquid as a template, the CDV H gene was amplified with the primers described in Table 2. As Figure 3 shown, a band of approximately 1140 bp in size was amplified, which was consistent with the expected band size. The recombinant plasmid with a positive identification result was sent to a sequencing company for sequencing, and the recombinant plasmid with correct sequencing results was stored at -40 °C for future use.

[0034] Table 2 Primer sequences for CDV H gene homologous recombination

[0035] 1.4 Prokaryotic expression of CDV H protein and determination of the expression form The recombinant plasmid pCold TF-H was transformed into Escherichia coli BL21 competent cells, and SDS-PAGE analysis of the expression products, namely SDS-PAGE analysis, optimization of the induction time, purification, and ultrafiltration were carried out respectively.

[0036] (1) SDS-PAGE analysis of the expression products: Take 100 μL of the pCold TF-H recombinant bacterial liquid into 10 mL of LB liquid medium with ampicillin resistance, culture at 37 °C and 200 r / min until the OD 600 value of the bacterial liquid reaches 0.8, add isopropyl-β-D-thiogalactoside, that is, IPTG to a final concentration of 0.5 mM, induce at 16 °C and 180 r / min for 16 h, collect the bacteria by centrifugation at 4 °C, add 1 mL of 1× phosphate buffer, that is, 1× PBS to resuspend every 0.1 g of bacteria, after ultrasonic disruption, centrifuge at 10,000 rpm for 30 min at 4 °C, collect the supernatant and precipitate of the disrupted bacterial liquid, resuspend with 1× PBS and then add 5× loading buffer, that is, 5× Loading Buffer respectively, mix well and boil for 10 min. The SDS-PAGE electrophoresis results are as Figure 4The recombinant H protein was mainly expressed in the form of supernatant, and the target band appeared between 95 kDa and 100 kDa, which was consistent with the expected size of the expressed protein, 96 kDa.

[0037] (2)Optimization of the induction conditions for recombinant H protein: Take 1 mL of the induced bacterial liquid every 2 h after induction culture, and perform SDS-PAGE electrophoresis as in the previous step. The results are as Figure 5 shown. The expression level of the recombinant protein was optimal at 16 h of IPTG-induced expression. Add the recombinant bacterial liquid to the LB medium, and culture it on a shaker at 37 °C and 180 r / min. When the OD 600 value reaches 0.6, add IPTG to a final concentration of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM respectively, and transfer it to a shaker at 16 °C and 180 r / min for 16 h of oscillation culture. Perform SDS-PAGE electrophoresis as in the previous step. As Figure 6 shown, the recombinant protein was optimally induced at a concentration of 0.2 mM IPTG.

[0038] (3)Purification and ultrafiltration of H protein: After a large amount of the successfully expressed protein was expressed, it was purified by nickel column affinity chromatography. The specific operation steps are as follows: a. Add a white gasket at the bottom of the affinity chromatography column, add 3 mL of nickel column packing, resuspend the packing with 6 mL of wash buffer, and cover it with a white gasket on the top.

[0039] b. Add the successfully expressed protein to the affinity chromatography column and incubate overnight at 4 °C.

[0040] c. Collect the protein effluent.

[0041] d. Elute with 20 mM, 40 mM, 60 mM, 100 mM, 200 mM, 500 mM, 600 mM imidazole respectively and collect the eluate.

[0042] e. Add the above eluate to the SDS-PAGE 4×Loading Buffer, mix well, boil for 10 min, and then perform SDS-PAGE electrophoresis to observe the electrophoresis results.

[0043] f. Add the purified protein to a 50 KDa ultrafiltration tube, centrifuge horizontally at 4000×g for 10 min at 4 °C, collect the liquid and store it at -80 °C.

[0044] Purifying the CDV H protein by nickel column affinity chromatography can further obtain a protein with a higher purity requirement. The results are as Figure 7As shown, the purified CDV H protein can be eluted at an imidazole concentration of 500 mM, and a clear band appears at 96 kDa. There are no other protein bands in the purified protein sample at other positions.

[0045] 1.5 Protein immunoblotting, i.e., Western blot to identify CDV H protein Take the ultrafiltered H protein for detection, and the specific steps are as follows: a. Add SDS-PAGE 5×Loading Buffer to the purified protein, mix well, boil for 10 min, place on ice for 10 min, and then perform SDS-PAGE electrophoresis.

[0046] b. Electrophoresis: Perform electrophoresis at a voltage of 80 V.

[0047] c. Transfer membrane: Cut the target gel, and then cut a PVDF membrane, i.e., a polyvinylidene fluoride membrane, and four pieces of filter paper of the same size. Activate the PVDF membrane with methanol for 5 min, and then soak the PVDF membrane, filter paper, and gel in the transfer buffer for 20 min. After the time is up, place the above in the semi-dry transfer instrument in the order of two pieces of filter paper, PVDF membrane, gel, and two pieces of filter paper to transfer the membrane.

[0048] d. Blocking: Wash the PVDF membrane with Tris-buffered saline Tween buffer, i.e., TBST, 3 times, 10 min each time, and place it in the blocking solution to block overnight at 4°C.

[0049] e. Incubation: After blocking, add the primary antibody to the incubation solution at a ratio of 1:5000 and incubate overnight at 4°C. Then wash with TBST 3 times, 10 min each time.

[0050] f. Incubate with the secondary antibody for 1 h, wash with TBST 3 times, 10 min each time, and finally perform chemiluminescence.

[0051] g. Chemiluminescence: Keep in the dark, and the ratio of luminescent solution A:B = 1:1.

[0052] Perform Western Blot with His-tag antibody and the purified CDV H protein, and the results are as Figure 8 shown. A specific reaction occurs at 96 kDa, indicating that it can react specifically with the His-tag antibody.

[0053] 2. Animal immunization and cell fusion 2.1 Immunization of Balb / c mice Six-week-old female BALB / c mice were selected and immunized with purified CDV H protein as the antigen. The second immunization was carried out 7 days after the first immunization, and the third immunization was carried out 14 days after the second immunization. Seven days after two or three immunizations, the orbital blood of female BLAB / c mice was collected, and the serum was separated to detect the antibody level. The enzyme-linked immunosorbent assay (ELISA) results in Table 3 and Table 4 showed that the serum antibody level of the experimental mice after the second immunization was significantly lower than that after the third immunization. After the third immunization, all mice met the conditions for cell fusion.

[0054] Table 3 Detection of serum titer of mice after the second immunization

[0055] Table 4 Detection of serum titer of mice after the third immunization

[0056] 2.2 Preparation of feeder cells and resuscitation and activation of sp2 / 0 a. The non-immunized female Balb / c mice were sacrificed by cervical dislocation and immersed in 75% (v / v) alcohol for about 5 min.

[0057] b. The external skin of the mouse carcass was cut open under sterile conditions to expose the peritoneum, and a small incision was made in the center of the abdomen.

[0058] c. 3 mL of Dulbecco's modified Eagle's medium (DMEM basal medium) was aspirated with a syringe and injected into the peritoneal cavity of the mouse, and the washing and blowing were repeated 2 times. The extracted liquid contained peritoneal macrophages.

[0059] d. An appropriate amount of hypoxanthine-aminopterin-thymidine selection medium (HAT selection medium) was added and mixed evenly, and 100 μL was added to each well of a 96-well plate.

[0060] e. The cryopreserved sp2 / 0 cells were resuscitated one week before fusion.

[0061] f. When the cell density reached 80%, the cell size was uniform, and the cell state was good, the cells were resuspended and collected with the basal medium.

[0062] g. Centrifuge at 1200 r / min for 10 min, and pour off the supernatant.

[0063] 2.3 Preparation of immune spleen cells The preparation protocol of spleen cells is as follows: a. The immunized female Balb / c mice were bled from the orbital cavity to collect positive serum, and after being sacrificed by cervical dislocation, they were immersed in 75% (v / v) alcohol for about 5 min.

[0064] b. Tear the external skin of the mouse under sterile conditions, fix the mouse laterally and crosswise, take out the spleen, and homogenize it with 4 mL of basal medium.

[0065] c. Centrifuge at 1200 r / min for 10 min, and pour off the supernatant.

[0066] 2.4 Cell fusion Cell fusion is carried out according to the following steps: a. Pre-warm 40 mL of HAT culture medium, 15 mL of basal culture medium, and 0.8 mL of polyethylene glycol - 4000, and a beaker filled with water at 37 °C for later use.

[0067] b. Aspirate approximately 3×10 7 myeloma cells and 10 7 spleen lymphocytes, put the two into a 50 mL centrifuge tube, mix them well, and then supplement the basal culture medium to 40 mL.

[0068] c. Centrifuge at 1000 r / min for 10 min, pour off the supernatant, flick the bottom of the tube gently to mix the two cell precipitates into a paste.

[0069] d. Place the centrifuge tube in the pre-warmed beaker, aspirate 0.8 mL of polyethylene glycol - 4000, add it to the precipitate, and stir while adding for 1 min.

[0070] e. Let it stand in a water bath for 90 s, dilute polyethylene glycol - 4000 with 15 mL of pre-warmed basal culture medium, add 1 mL in the first 30 s, add 3 mL in the next 30 s, and then add the remaining 11 mL within 2 min.

[0071] f. Supplement the basal culture medium until it reaches 40 mL, centrifuge at 1000 r / min for 10 min, discard the supernatant, and keep the paste-like cell precipitate for later use.

[0072] g. Resuspend gently with 40 mL of HAT selection culture medium containing 20% (v / v) newborn bovine serum for later use.

[0073] h. Mix the feeder cells and the fused cells, add them to a 96-well plate with a multi-channel pipette, 200 μL per well, and culture them in a 37 °C, 5% (v / v) CO₂ incubator, and observe every day.

[0074] i. Detect when the cell colonies grow to one field of view.

[0075] 2.5 Indirect enzyme-linked immunosorbent assay (ELISA) to screen positive cell wells After coating with CDV H protein, 50 μL of the supernatant from each well of the 96-well plate was aspirated and added to the ELISA plate, and incubated in a 37 °C incubator for 1 h. Then it was washed three times with phosphate buffered saline-Tween 20, i.e., PBST. Then 100 μL of goat anti-mouse secondary antibody diluted 1:5000 was added to each well, incubated in a 37 °C incubator for 1 h, and washed three times with PBST. 100 μL of 3,3',5,5'-tetramethylbenzidine, i.e., TMB chromogenic solution, was added to each well for 15 min of color development. Finally, 100 μL of stop solution was added to each well to terminate the color development; OD was measured. 450 The OD value was recorded, and the wells with higher OD values and good cell growth were retained for subsequent screening of positive clones.

[0076] 2.6 Cloning of positive cells by limiting dilution The cloning method of positive cells is as follows: a. According to the detection results, wells with higher OD 450 values and good cell growth were selected for screening of positive wells.

[0077] b. The cells in the well to be cloned were dispersed with a micropipette, 100 μL was transferred into an EP tube, and an appropriate amount was aspirated for counting.

[0078] c. In the EP tube, it was diluted to 10 3 cells / mL with hypoxanthine-thymidine medium, i.e., HT medium.

[0079] d. 3.8 mL, 2.4 mL, and 2.0 mL of culture medium were respectively placed in three 4 mL centrifuge tubes for dilution.

[0080] e. 200 μL of cells diluted to 10 3 cells / mL were taken into the first centrifuge tube. After thorough mixing, 1.6 mL of the mixture was aspirated and added to the second centrifuge tube. After remixing evenly, 2 mL of the mixture was aspirated and added to the third centrifuge tube.

[0081] f. The cells in the centrifuge tubes were seeded at 100 μL / well into the feeder cell plate of a 96-well plate. The first centrifuge tube was used to seed one row, the second centrifuge tube was used to seed two rows, and the third centrifuge tube was used to seed five rows. The remaining cells in the EP tube were transferred to a 12-well plate.

[0082] g. Thereafter, the cell status of the cloning wells was observed daily.

[0083] 2.7 Secondary cloning and tertiary cloning of CDV H protein The screening method is the same as the above method until 80% of the entire plate are positive wells, which is regarded as the selection of monoclonal cells. Screening for positive cell lines requires experiments using the method of indirect ELISA to detect antibodies with purified and highly pure CDV H protein. Positive cell lines have a relatively high level of specific antibodies and need to undergo 3 rounds of continuous limited dilution to complete the screening. The changes in the antibody secretion levels of hybridomas are shown in Table 5. After final determination, 1 hybridoma cell was obtained. This 1 cell has a high level of specific antibodies and can secrete stably. It is named: 8F5. The growth status of hybridoma cells after cell fusion is as Figure 9 shown

[0084] Table 5 Changes in antibody secretion levels of hybridomas

[0085] 3. Ascites preparation and titer detection 3.1 Ascites preparation Inject 0.5 mL of liquid paraffin into the abdominal cavity of mice, inject 5×10 5 hybridoma cells, and perform intraperitoneal injection after 10 days. After about 7 days, the abdomen of the mice will show swelling of different sizes. Keep an eye on the condition of the mice at all times and extract the ascites in the body in a timely manner. Centrifuge the extracted ascites at 1000 r / min for 10 min. After all the ascites of the mice are taken out, mix them evenly and aliquot them for subsequent use.

[0086] 3.2 Ascites titer detection Dilute the purified H protein 1000, 3000, 9000, and 27000 times with the coating solution, coat the 96-well enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well at 4 °C overnight. After washing the plate three times with PBST, block it with 5% skim milk by volume for 1 h. Dilute the ascites 100, 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times with PBS, incubate at 100 μL / well at 37 °C for 0.5 h, wash the plate three times with PBST, add 100 μL of goat anti-mouse secondary antibody diluted 5000 times to each well, incubate at 37 °C for half an hour, wash the plate three times with PBST, add 100 μL of TMB chromogenic solution to each well for 15 min of color development, and add 100 μL of stop solution to each well to terminate the reaction and read the absorbance. The results are shown in Table 6. In the dilution ratio range of 1:1000 - 1:4000, the OD value corresponding to the 8F5 antibody is higher than 1. One hybridoma cell, 8F5, was obtained in this invention. This hybridoma cell can stably secrete monoclonal antibodies against CDV H protein.

[0087] Table 6 Results of indirect ELISA detection of 8F5 ascites

[0088] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0089] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line is named 8F5 and is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC NO. 46311.

2. Use of the hybridoma cell line according to claim 1 in the preparation of a monoclonal antibody against canine distemper virus H protein.

3. A monoclonal antibody against canine distemper virus H protein, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line according to claim 1 or its subculture cell line.

4. The monoclonal antibody against canine distemper virus H protein according to claim 3, characterized in that, The monoclonal antibody against canine distemper virus H protein is prepared from the hybridoma cell line according to claim 1 or its subculture cell line by the method of in vivo induction of ascites.

5. An immunogen of a canine distemper virus H protein monoclonal antibody, characterized in that, The monoclonal antibody against canine distemper virus H protein is the monoclonal antibody against canine distemper virus H protein according to claim 3; the immunogen of the monoclonal antibody against canine distemper virus H protein is the canine distemper virus H gene, and the nucleotide sequence of the canine distemper virus H gene is as shown in SEQ ID NO.

1.

6. Use of the monoclonal antibody against canine distemper virus H protein according to claim 3 in the detection of canine distemper virus.

7. The application according to claim 6, characterized in that, The specific application method is to use the sample to be tested as the coated antigen and the monoclonal antibody against canine distemper virus H protein as the primary antibody for indirect competitive ELISA detection.

8. The application according to claim 6, wherein, The monoclonal antibody against canine distemper virus H protein is used for the preparation of a canine distemper virus detection kit.

9. The application according to claim 8, characterized in that, The detection kit includes an ELISA detection component coated plate, an enzyme-labeled secondary antibody, and a chromogenic substrate.

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