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Recombinant porcine reproductive and respiratory syndrome virus as well as preparation method and application thereof

A respiratory syndrome and recombinant virus technology, applied in biochemical equipment and methods, antiviral agents, virus antigen components, etc., can solve the problems of strong virulence, general effect, and insufficient protection of highly pathogenic virus strains. Achieve high safety and good preventive effect

Active Publication Date: 2013-05-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the attenuated vaccine prepared by the classic strain is not comprehensive enough to protect the highly pathogenic strain due to the sequence difference between the classic strain and the highly pathogenic strain; The effect makes the effect of the humoral immune response mediocre; the attenuated vaccine prepared from the highly pathogenic virus strain through continuous subculture has the risk of returning to strong virulence, and cannot be well distinguished from the wild-type strain, making it impossible to determine whether the virus is infected after the outbreak. Is it caused by the wild-type strain or the virulence of the attenuated vaccine strain?

Method used

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  • Recombinant porcine reproductive and respiratory syndrome virus as well as preparation method and application thereof
  • Recombinant porcine reproductive and respiratory syndrome virus as well as preparation method and application thereof
  • Recombinant porcine reproductive and respiratory syndrome virus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1, Isolation and Identification of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus HV Strains

[0045] 1. Isolation of highly pathogenic porcine reproductive and respiratory syndrome virus HV strain

[0046] In June 2007, the viscera of sick pigs were retrieved from the pig farm where highly pathogenic "PRRS" broke out in Beijing, my country, as disease materials, ground into powder with liquid nitrogen, resuspended by adding appropriate amount of RPMI1640 culture medium, and then frozen and thawed three times , centrifuge to get the supernatant, and then filter with a 0.22 μm filter to obtain the supernatant of the disease material. Inoculate the supernatant into porcine alveolar macrophages (PAMs), culture at 37°C, collect the virus when 60%-80% of the cells show lesions, and continue to passage to obtain the virus supernatant.

[0047] 2. Identification of HV strains of highly pathogenic porcine reproductive and respiratory syndrome viru...

Embodiment 2

[0056] Embodiment 2, the preparation of recombinant porcine reproductive and respiratory syndrome virus

[0057] 1. Construction of recombinant plasmid carrying HV strain genome

[0058] The full-length genome sequence (sequence 1) of the HV strain measured in Example 1 was analyzed, and 6 restriction sites for splicing the full-length genome sequence were selected, namely SmiI, XhoI, AflII, and ClaI , EcoRV and NotI. The full-length genome sequence of the HV strain (sequence 1) contains restriction sites XhoI (3400th and 6901st of sequence 1), AflII (6465th of sequence 1), ClaI (8798th of sequence 1 position) and the recognition sequences of EcoRV (12071st position of sequence 1), but not the recognition sequences of restriction sites SmiI and NotI. The full length of the genome is divided into 5 fragments through the four restriction sites on the genome, and the restriction sites SmiI and NotI are added to the two ends of the viral genome respectively, and then the 5 fragm...

experiment example 3

[0094] Experimental example 3, the comparison of three kinds of virus in vitro traits

[0095] The wild-type virus HV (WT), the virus rHV (CK) rescued from the recombinant plasmid pcDNA3.1M-HV, and the attenuated vaccine rHV-GFP rescued from the recombinant plasmid pcDNA3.1M-△HV-GFP (experimental group) Inoculate alveolar macrophages (porcine alveolar macrophages, PAMs) with the same toxic dose (MOI=0.1). Among them, the wild-type virus HV (WT) adopts the fifth generation (P5), the virus rHV (CK) adopts the fifth generation (P5), and the attenuated vaccine rHV-GFP (experimental group) adopts the fifth generation (P5). After that, the following two aspects of detection are carried out:

[0096] 1. Determination of virus titer

[0097] Cell supernatants were collected 12 hours, 24 hours, 36 hours and 48 hours after virus inoculation. The cell supernatant was serially diluted 10 times and inoculated into alveolar macrophages cultured in a 96-well plate to detect the virus tite...

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Abstract

The invention discloses a recombinant porcine reproductive and respiratory syndrome virus as well as a preparation method and an application thereof. The recombinant porcine reproductive and respiratory syndrome virus provided by the invention is the recombinant virus which is obtained by carrying out substitution or deletion on a wild recombinant porcine reproductive and respiratory syndrome viral genome RNA; the substitution means carrying out substitution on all or a part of six structural proteins including a structural protein ORF2a, a structural protein ORF2b, a structural protein ORF3, a structural protein ORF4, a structural protein ORF5a and a structural protein ORF5 in the wild recombinant porcine reproductive and respiratory syndrome viral genome RNA as coding RNAs of labelled proteins; and the deletion means carrying out deletion on all or a part of coding RNAs of the six structural proteins in the wild recombinant porcine reproductive and respiratory syndrome viral genome RNA. The experiments prove that the recombinant virus has a good prevention effect on a high pathogenicity porcine reproductive and respiratory syndrome and has great importance to a porcine reproductive and respiratory syndrome viral vaccine.

Description

technical field [0001] The invention relates to a recombinant porcine reproductive and respiratory syndrome virus and its preparation method and application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is an acute viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. The disease can infect pigs of any age and is clinically characterized by reproductive impairment, respiratory disease, high morbidity and mortality of suckling piglets, persistent infection and immunosuppression, and has caused great harm to the world's pig industry . A 2005 report indicated that the disease costs the U.S. swine industry $560 million a year, and the global swine industry will be even more lost. At present, PRRSV has become a global pandemic and has become one of the main disease factors affecting the pig industry, causing huge economic losses to the pig industry worldwide. [0003] PRRSV is a single-st...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/10C12N15/63C12N1/15C12N1/19C12N1/21A61K39/12A61P31/14C12R1/93
Inventor 封文海王良海侯隽高丽郭雪坤余志彬朱耀华刘翼浩唐军
Owner CHINA AGRI UNIV
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