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A kind of PRRS virus murine monoclonal antibody and its preparation method and application

A monoclonal antibody and blue ear virus technology, applied in the biological field, can solve problems such as inability to achieve large-scale on-site detection

Active Publication Date: 2021-03-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the more common detection methods are mainly molecular biological detection and serological detection. Because molecular biological detection requires instruments and related technical personnel, large-scale on-site detection cannot be achieved.

Method used

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  • A kind of PRRS virus murine monoclonal antibody and its preparation method and application
  • A kind of PRRS virus murine monoclonal antibody and its preparation method and application
  • A kind of PRRS virus murine monoclonal antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Antigen Preparation

[0061] Inactivated highly pathogenic PRRSV TP strain (that is, HP-PRRSV TP strain, obtained from reference: Yang, H., et al., CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus.Antiviral Res , 2018.151: p.63-70.).

Embodiment 2

[0062] The preparation of embodiment 2 monoclonal antibody

[0063] 1. The preparation method of PRRS mouse-derived monoclonal antibody is as follows:

[0064] Animal immunization: 10-week-old female Balb / c mice (purchased from the Animal Center of Southern Medical University, Guangdong Province) were selected.

[0065] (1) Initial immunization: 300 μg of antigen (prepared in Example 1) and complete Freund's adjuvant were emulsified at a ratio of 1:1 by volume and injected subcutaneously at multiple points;

[0066] (2) Secondary immunization: 2 weeks later, the same amount of antigen as the first immunization plus Freund's incomplete adjuvant was emulsified at a ratio of 1:1 by volume and injected subcutaneously at multiple points;

[0067] (3) Three times of immunization: 2 weeks later, the same amount of antigen as the first immunization plus Freund's incomplete adjuvant was injected subcutaneously at multiple points (blood was collected 10 days later to measure its potenc...

Embodiment 3

[0089] Embodiment 3 Monoclonal antibody of the present invention distinguishes highly pathogenic blue ear virus and traditional blue ear virus

[0090] 1. ELISA experiment verified monoclonal antibody to distinguish highly pathogenic PRRS virus from traditional PRRS virus

[0091] (1) coating: dilute respectively PRRSV TP strain (the highly pathogenic PRRSV TP strain inactivated in embodiment 1, be called for short PRRSV TP strain) with carbonate coating buffer, PRRSV CH-1a strain (purchased from Shandong Lvdu Biotechnology Co., Ltd., referred to as PRRS CH-1A strain, PRRS JN strain, PRRS QT strain, PRRS LQ strain, PRRS TY strain (the virus strains were all purchased in Shandong Ludu Biotechnology Co., Ltd.), MARC-145 (Marc145 cells, purchased from Guangzhou Alexander Biotechnology Co., Ltd.). Add 0.1ml of coating solution containing PRRSV TP strain to the reaction wells of different polystyrene plates and the coating solution containing PRRSVCH-1a strain overnight at 4 ° C. ...

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Abstract

The invention discloses a PRRSV murine monoclonal antibody and preparation method and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequences are respectively as shown in SEQ IDNO. 4-6; and the light chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequences are respectively as shown in SEQ IDNO. 10-12. The murine monoclonal antibody can be specifically combined with highly pathogenic form PRRSV instead of being combined withconventional PRRSV, so as to effectively diagnose whether the swine is affected with the highly pathogenic form PRRSV, and treat the swines affected with different PRRSV in a distinguished manner; the murine monoclonal antibody can also be used for developing antibody diagnostic kits and test stripes for distinguishing highly pathogenic form PRRSV and conventional PRRSV and used for preparing drugs for diagnosing PRRSV.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mouse-derived monoclonal antibody against PRRS virus and its preparation method and application. Background technique [0002] PRRS virus is an important single-stranded RNA virus that endangers the development of the pig industry. Its RNA is about 15KB long and contains 10 open reading frames, namely ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6 , ORF7; where ORF1 encodes two large multimeric proteins, which can be cleaved into 14 non-structural proteins. ORF2a, ORF2b, ORF3-7and ORF5a encode seven structural proteins, namely glycoprotein GP2, envelope protein E protein, GP3, GP4, GP5, M, N and ORF5a protein. Among them, GP5 and M proteins can form dimers, while GP2, GP3, and GP4 can form trimers and assist the virus to enter the host cell. According to nucleotide sequence analysis, PRRS virus can be divided into two genotypes, European type and North Amer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/06G01N33/577G01N33/569
CPCC07K16/10C07K2317/56C12N15/02G01N33/56983G01N33/577G01N2333/08
Inventor 唐勇严俊杰王宏
Owner JINAN UNIVERSITY
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