CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof

A technology for respiratory syndrome and pig reproduction, applied in the field of bioengineering, can solve the problems of long duration of immunity, weakened antigenicity, safety concerns, etc., and achieve the effect of being beneficial to prevention and control

Inactive Publication Date: 2014-04-02
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PRRS inactivated vaccine has the advantages of safety and easy storage, but its disadvantages are weakened antigenicity, low immune efficiency, large dosage, etc., and the immune effect on heterologous strains is not ideal; attenuated vaccine has good immune effect and long duration of immunity, but Its safety is still worrying, which brings great difficulties to the prevention and control of porcine reproductive and respiratory syndrome in my country

Method used

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  • CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof
  • CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof
  • CDNA (Complementary Deoxyribonucleic Acid) infectious clone of porcine reproductive and respiratory syndrome virus vaccine strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of full-length cDNA sequence clone of porcine reproductive and respiratory syndrome virus vaccine strain CH-1R genome

[0024] 1. Materials and methods

[0025] 1.1 Viruses, vectors and main reagents

[0026] PRRSV attenuated vaccine strain CH1R, its microorganism preservation number: CGMCC NO.1883; the cloning vector pBeloBAC11 is preserved by our institute; virus RNA extraction kit, DNA gel recovery kit, plasmid small extraction kit and large extraction kit are purchased from QIAGEN Company; reverse transcription kit SuperScriptIII First-Strand Synthesis System was purchased from Invitrogen Company; high-fidelity DNA polymerase Phusion High-fidelity DNA Polymerase was purchased from NEB Company; pZeroBack vector kit was purchased from Tiangen Company; all restrictions Both endonuclease and T4 DNA ligase were purchased from NEB Company.

[0027] 1.2 Primers

[0028] According to the whole genome sequence of PRRSV CH1R strain (GenBank accessio...

Embodiment 2

[0050] Example 2: Rescue and Identification of Porcine Reproductive and Respiratory Syndrome Virus

[0051] 1. Materials and methods

[0052] 1.1 Viruses, cells and main reagents

[0053] PRRSV attenuated vaccine strain CH1R was attenuated and preserved by our laboratory, its microbial preservation number: CGMCC NO.1883; Marc-145 cells were preserved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; DNA transfection reagent X-tremeGENE HP was purchased from Roche company; DMEM medium and fetal bovine serum (FBS) were purchased from GIBICO Company; anti-PRRSVN protein monoclonal antibody was provided by Associate Researcher Li Xi, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; FITC-labeled goat anti-mouse fluorescent secondary antibody was purchased from Sigma company.

[0054] 1.2 Transfection of infectious clone plasmids

[0055] Culture a monolayer of Marc-145 cells in a 6-well plate, and transfec...

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Abstract

The invention provides a cDNA (complementary Deoxyribonucleic Acid) infectious clone of a porcine reproductive and respiratory syndrome virus vaccine strain and application thereof, and discloses a recombinant vector, which is an infectious clone of a porcine reproductive and respiratory syndrome virus vaccine strain CH1R constructed by adopting a reverse genetics approach. The infectious clone comprises a full-length gene cDNA sequence of the porcine reproductive and respiratory syndrome virus vaccine strain CH-1R. The invention further discloses an artificially cloned attenuated vaccine strain, which is a cloned strain of the porcine reproductive and respiratory syndrome virus vaccine strain CH-1R, wherein a marked restriction enzyme locus is introduced in a structural protein gene sequence. Through performing genetic engineering reconstruction of the infectious clone, a better vaccine can be researched, and the prevention and control of the porcine reproductive and respiratory syndrome are facilitated.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a porcine reproductive and respiratory syndrome virus vaccine strain cDNA infectious clone and application thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Reproductive disorders such as mummified fetuses, respiratory symptoms and high mortality in pigs of all ages are the main features. PRRS was first discovered in the United States in 1987, and the pathogen was first isolated and identified in the Netherlands in 1991, and named Lelystad virus (LV). In 1992, the PRRS pathogen was also isolated in the United States, named VR-2332. Guo Baoqing and others in my country isolated PRRSV from 4 aborted fetuses in 1996. Since then, the virus ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/01A61K39/12A61P31/14C12R1/93
Inventor 翁长江蔡雪辉李江南王承宝刘永刚黄丽石文达
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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