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Preparation method and application of anti-PCV2 monoclonal antibody

A monoclonal antibody and sequence technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of high technical and equipment requirements, failure to popularize and apply, and time-consuming and labor-intensive operations, and achieve a single Anti-purity, extensive research application value and commercial use value, strong specific effect

Active Publication Date: 2020-06-26
河南中泽生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

With the continuous development of molecular biology, PCR has been widely used in the laboratory to detect disease pathogens due to its advantages of rapidity and high sensitivity. Promotion and application in production and breeding

Method used

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  • Preparation method and application of anti-PCV2 monoclonal antibody
  • Preparation method and application of anti-PCV2 monoclonal antibody
  • Preparation method and application of anti-PCV2 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046]Example 1: Preparation and identification of hybridoma cell lines secreting anti-PCV2 monoclonal antibody

[0047] 1. Main material

[0048] PCV2a Cap protein subunit vaccine is commercially available, BALB / c mice were purchased from Zhengzhou University School of Medicine, myeloma cell SP2 / 0 was purchased from the Laboratory of Molecular Immunology, School of Life Sciences, Zhengzhou University, porcine circovirus type 2 Cap protein was purchased from From the Laboratory of Molecular Immunology, School of Life Sciences, Zhengzhou University, PCV2 KF strain, porcine circovirus type 1 PCV1, PCV3, CSFV Shimen strain, porcine reproductive and respiratory syndrome virus PRRSV BJ-4 strain, porcine pseudorabies virus PRV HN-JZ-16 and porcine epidemic diarrhea virus PEDV were purchased from the Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences.

[0049] 2. Main Reagents

[0050] Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, PEG-1...

Embodiment 2

[0103] Example 2: Preparation of anti-PCV2 monoclonal antibody ascites

[0104] The monoclonal hybridoma cell line 3B6 in Example 1 was expanded and cultured, and the titer of the culture supernatant was measured by ELISA to ensure the stability of the monoclonal cell line, and the cells were collected for large-scale preparation of monoclonal antibodies.

[0105] 1. Preparation and Purification of Monoclonal Antibody from Ascites

[0106] (1) Select multiparous female BALB / c mice and inject 500 μl sterilized paraffin intraperitoneally to stimulate immune cells to promote the proliferation of hybridoma cells;

[0107] (2) Observe the state of the mice, and after 7 to 10 days, the 7 Inject an amount of 3 cells into the monoclonal positive cells prepared in advance, and observe the status of the mice in time. After about 10 days, extract the ascites, centrifuge at 8000 r / min, and centrifuge at 4°C for 20 minutes to remove grease and cell sediment. Collect the ascites supernatan...

Embodiment 3

[0125] Example three: Identification of anti-PCV2 monoclonal antibodies

[0126] 1. Isotype identification

[0127] The identification of monoclonal antibody subtype and type was performed according to the instructions of the Mouse Monoclonal Antibody Isotyping Kit.

[0128] The identification results of monoclonal antibody subclass and subtype showed that the subtype of monoclonal antibody 3B6 was IgG2a, as shown in Table 2, and its light chain type was Kappa type.

[0129] Table 2 Identification of monoclonal antibody subtypes

[0130] .

[0131] 2. Affinity identification

[0132] The PCV2 Cap protein was diluted with CBS solution to a concentration of 0.5 µg / mL and 1 µg / mL coating solution to coat the microplate respectively, and the ascites titer of monoclonal antibody was determined by indirect ELISA method, with monoclonal antibody concentration as the abscissa, OD 450 The value is the ordinate, and the corresponding two indirect ELISA reaction curves are drawn. ...

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Abstract

The invention discloses a preparation method and application of an anti-PCV2 monoclonal antibody. The anti-PCV2 monoclonal antibody, and DNA sequences and amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are disclosed. The preparation method of the antibody is designed. The antibody is applied in an antigen / antibody detection kit, antigen / antibody immunochromatography test paper, IFA, IPMA and a PCV2 or PCV2 Cap immunoaffinity column. The antibody can simultaneously recognize two major epidemic subtypes which are PCV2a and PCV2b, and has good reactogenicity with PCV2 totivirus and PCV2 Cap protein, but is free of cross reaction with PCV1 and PCV3 subtypes and porcine viruses, and lays a foundation for the study of etiology and pathogenesis of PCV2 and clinical detection of PCV2 pathogens.

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to a preparation method and application of an anti-PCV2 monoclonal antibody. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to the Circoviridae family (Circoviridae), a member of the genus Circovirus (Circovirus). It exists widely and is internationally recognized as an important immunosuppressive pathogen that endangers the large-scale pig farming in all countries in the world. [0003] Among them, PCV2 is the main pathogen that causes PMWS, porcine dermatitis and nephropathy syndrome (Porcine dermatitis and nephropathy syndrome, PDNS), porcine respiratory disease complex (Porcine respiratory disease complex, PRDC) and other diseases, causing serious losses to the pig industry. At present, the main prevailing strain in my country is still PCV2. PCV2 infection leads to the reduction of pig herd immunity, and the subsequent mixed or se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N15/06G01N33/577
CPCC07K16/081C12N15/02G01N33/577
Inventor 张改平王爱萍陈玉梅蒋敏刘东民刘运超周景明刘燕凯
Owner 河南中泽生物工程有限公司
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