Preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV

A prrsv-n-igy, antibody technology, applied in the biological field, achieves the effect of stable physical properties and reducing the occurrence rate of false positives

Inactive Publication Date: 2010-06-02
ZHEJIANG UNIV
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Problems solved by technology

According to antigenic differences, PRRSV isolates from various regions are divided into European type (prototype strain is LV) and North America

Method used

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  • Preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV
  • Preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV
  • Preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV

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Embodiment Construction

[0025] The preparation technology and application of the antibody agent of the present invention will be further described in conjunction with the implementation below.

[0026] 1 Materials and methods

[0027] 1.1 Antigen preparation

[0028]Referring to the "Molecular Cloning Experiment Guide" and the PRRSV N protein gene sequence published on GenBank (GenBank accession number: FJ175688), PCR amplified the N gene, introduced EcoR I / HindIII restriction sites, and cloned the amplified fragment into prokaryotic expression In the vector pET-30a(+), transform the competent E.coli BL21(DE3)pLysS strain, pick a single colony and identify it by PCR and sequencing; -1 After induction with IPTG for 4 hours, the expression product was extracted and detected by SDS-PAGE; the recombinant protein was purified and expressed by Ni-NTAAgarose affinity chromatography, immunogenicity was detected by Western blot and protein concentration was determined by Bradford method, and the purified pro...

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Abstract

The invention relates to the research for virus antibody and provides a preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV. The preparation for antibody comprises the steps of preparing antigen by amplifying N genes on protein gene sequence PCR of PRRSV-N and extracting animal immunity and specific yolk immunity globulin. By using the recombined PRRSV-N protein of an antigen immunity layer, the specific PRRSV-N-IgY antibody is prepared. The prepared specific PRRSV-N-IgY antibody can be applied to detecting IFA of PRRSV. Compared with the antibody of animals such as rabbit, rat, and the like, IgY has stable physical property and can obviously decrease the occurrence rate of false positive without activating mammal complement, without combining with mammal Fc receptor or germ and without nonspecific reaction with RF (rheumatoid factors).

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an anti-porcine reproductive and respiratory syndrome virus (porcine productive and respiratory syndrome virus, PRRSV) nucleocapsid protein (N) protein-specific yolk immunoglobulin (anti-PRRSV-N-IgY antibody) The preparation of and its application in the detection of PRRSV by indirect immunofluorescence detection (IFA) method. Background technique [0002] The main pathogen of porcine reproductive and respiratory syndrome (porcine productive and respiratory syndrome, PRRS) is porcine reproductive and respiratory syndrome virus (porcine productive and respiratory syndrome virus, PRRSV), which was first discovered in the United States in 1987. It mainly manifests clinically as abortion, Premature births, stillbirths, mummies, a gradual decline in the survival rate of piglets, and respiratory symptoms in bred pigs, etc. Bring catastrophic economic loss to pig industry. In 1991, Wensvo...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K16/02G01N33/569
Inventor 李肖梁方维焕
Owner ZHEJIANG UNIV
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