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Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody generated by hybridoma cell strain and application

A hybridoma cell line, canine parvovirus technology, applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problem of unsatisfactory preventive effect in puppies

Active Publication Date: 2021-06-08
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the immune organs of puppies have not yet matured, the prevention effect of puppies through vaccine immunization is not ideal

Method used

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  • Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody generated by hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Canine Parvovirus Isolation

[0042] 1. Pretreatment of disease materials: Collect intestinal content samples of dogs suspected of being infected with canine parvovirus in a pet hospital in Lanzhou City, Gansu Province, add PBS solution at a volume ratio of 1:10, and centrifuge at 7000rpm for 15min. Take 500 μL of the supernatant and add an equal amount of chloroform, mix thoroughly, then centrifuge at 4000 rpm for 15 minutes, discard the precipitate, filter the supernatant through a 0.22 μm microporous membrane, and store it in a -20°C refrigerator for future use.

[0043] 2. Virus isolation: Inoculate F81 cells (purchased from Changchun Haiguang Biotechnology Co., Ltd., article number: HG-ATCCF81) with the above-mentioned treated disease samples by means of synchronous inoculation. 2 Cultivate in an incubator, and observe cytopathic changes (CPE) every day; continuous blind passage culture, CPE can be observed when it is passed to the 4th generation, and it is named C...

Embodiment 2

[0052] Establishment of hybridoma cell lines

[0053] 1. Experimental materials

[0054] 1. Antigen: the canine parvovirus VP2 protein was expressed as an antigen by the prokaryotic expression system in the above-mentioned embodiment 1;

[0055] 2. Culture medium: Hyclone DMEM medium was purchased from Thermo Company; HAT was purchased from SIGMA Company; HT was purchased from SIGMA Company;

[0056] 3. Experimental animals: female Balb / c mice, 8-12 weeks old, raised under SPF conditions;

[0057] 4. Other experimental materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from SIGMA Company; PEG4000 was purchased from MERCK Company; HRP-goat anti-mouse IgG antibody was purchased from abcam Company; the rest of the reagents were domestic analytically pure products .

[0058] 2. Method steps

[0059] 1. Animal immunity

[0060] (1) First immunization: mix equal volumes of VP2 antigen and complete Freund's adjuvant, and emulsify with a hand-he...

Embodiment 3

[0076] Preparation and Identification of Monoclonal Antibody Against Canine Parvovirus VP2 Protein

[0077] 1. Antibody preparation

[0078] Choose Balb / c mice aged 6-8 weeks, and inject 0.5 mL of liquid paraffin intraperitoneally, 0.5 mL per mouse. One week later, hybridoma cells were inoculated intraperitoneally, and the number of inoculated cells per mouse was 1×10 5 ~1×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.

[0079] The ascitic fluid was centrifuged (13000rpm, 30min) to remove cell components and other precipitates, and the supernatant was collected. Purify with Protein-Sepharose CL-4B, the upper column liquid is 20mM PBS buffer solution, the eluent of column chromatography is pH 2.7, 20mM glycine buffer solution, and the monoclonal antibody against canine parvovirus VP2 protein is obtained, which is tested by SDS -PAGE antibo...

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Abstract

The invention provides a hybridoma cell strain, a canine parvovirus VP2 protein monoclonal antibody generated by the hybridoma cell strain and application, and belongs to the technical field of monoclonal antibody preparation. The hybridoma cell strain 5A92B12 provided by the invention can be stably passaged and can continuously and stably secrete the canine parvovirus VP2 protein monoclonal antibody; after being cultured to 10 generations, the hybridoma cell strain 5A92B12 can still grow well and is stable in passage, and the supernatant titer of a culture solution can still reach 1x10<6> or above; the monoclonal antibody generated by the hybridoma cell strain and canine parvovirus VP2 protein can be specifically recognized; and the hybridoma cell strain has the advantages of high specificity and high sensitivity, has the characteristic of inhibiting canine parvovirus replication, can be applied to the detection and treatment of canine parvovirus diseases, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibody preparation, and in particular relates to a hybridoma cell line and the canine parvovirus VP2 protein monoclonal antibody produced by it and its application. Background technique [0002] Canine Parvovirus Disease (Canine Parvovirus Disease) is an infectious disease mainly characterized by hemorrhagic enteritis and non-suppurative myocarditis caused by Canine Parvovirus (CPV) infection. It mainly infects puppies. High, has become one of the most serious infectious diseases in the dog industry. [0003] Canine parvovirus is a single-stranded negative-strand DNA virus with a genome size of about 5kb, including two open reading frames (openreading frame, ORF). VP2; There is a hairpin structure at both ends of the genome, in which the 3' end is folded to form a "Y" structure, while the 5' end forms a "U" structure, which plays an important role in the replication and transcription of the ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/08G01N33/577G01N33/569A61K39/42A61P31/20C12R1/91
CPCC07K16/081G01N33/577G01N33/56983A61P31/20C07K2317/56A61K2039/505G01N2333/015Y02A50/30
Inventor 殷相平毛箬青王相伟周亚花殷娟斌孙跃峰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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