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Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof

A technology for respiratory syndrome and attenuated vaccines, applied in the field of highly pathogenic porcine reproductive and respiratory syndrome virus genetically engineered attenuated vaccine strains, can solve the problem of inability to effectively distinguish between natural infection and vaccine-immunized animals, control and purify highly pathogenic Problems such as PRRSV obstruction, to achieve the effect that is conducive to prevention and control

Active Publication Date: 2013-02-27
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the existing attenuated vaccine still has the important problem of not being able to effectively distinguish between natural infection and vaccine-immunized animals during use, which will bring serious obstacles to more effective control and purification of highly pathogenic PRRSV in the future. Therefore, it is necessary to develop a vaccine that can achieve Novel Genetically Engineered Marked Attenuated Vaccine for Differential Diagnosis Purpose

Method used

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  • Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof
  • Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof
  • Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus Gene Deletion Strain

[0047] 1. Materials and methods

[0048] 1.1 Viruses, vectors and reagents

[0049] The PRRSV vaccine strain HuN4-F112 was attenuated and preserved by our laboratory, and the infectious cloning vector pSK-Hun4-F112 was constructed and preserved by our laboratory (Tong Guangzhi et al. 2007; Tong GZ et al., 2007; Zhou YJ et al., 2008; TianZJ et al., 2009), RNeasy Plus Mini Kit was purchased from QIAGEN; gel recovery kit was purchased from Shanghai Huashun Biotechnology Co., Ltd.; SupersciptIII reverse transcriptase and pfx DNA polymerase was purchased from Invitrogen; RNase H, T4 DNA ligase and restriction endonuclease were purchased from NEB Company; DL-15000 and DL-2000 DNA Marker were purchased from TakaRa; other chemical reagents were imported or domestically produced analytically pure.

[0050] 1.2 Primer design

[0051] According to the Nsp2 ge...

Embodiment 2

[0080] Example 2 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus Genetic Engineering Marked Attenuated Vaccine Strain

[0081] 1. Materials and methods

[0082] 1.1 Viruses, vectors and reagents

[0083] Newcastle disease virus LaSota strain was provided by the Avian Infectious Disease Laboratory of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, PRRSV vaccine strain and its infectious cloning vector pSK-Hun4-F112 were constructed and preserved by our laboratory (Tong GZ et al., 2007; ZhouYJ et al., 2008), the Newcastle disease virus NP protein monoclonal antibody was provided by the Avian Paramyxovirus Research Group of the Poultry Ward, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the RNeasy Plus Mini Kit was purchased from QIAGEN; the gel recovery kit was purchased from Shanghai Hua Shun Biotechnology Co., Ltd.; SupersciptIII reverse transcriptase and pfx DNA polym...

Embodiment 3

[0114] Example 3 Porcine Reproductive and Respiratory Syndrome Virus Genetic Engineering Marked Attenuated Vaccine Strain Immune Protection Test

[0115] 1. Materials and methods

[0116] 1.1 Selection and grouping of test pigs

[0117] Select 30 35-day-old PRRSV, CSFV, PCV2 and other pathogenic and antibody-negative healthy piglets, and randomly divide them into 4 groups. Two cloned viruses inserted into NP49 obtained in Example 2 are used as experimental genetic engineering marker vaccine viruses. The experimental animals were divided into: rHN4-Δ52+NP49 (group A) test group, rHN4-Δ25+NP49 (group B) test group, HuN4-F112 (group F) vaccine control group, DMEM (group K) control group. Five pigs in each group were kept in isolation.

[0118] 1.2 Immunization of Genetic Engineering Marked Attenuated Vaccine Strains

[0119] Adjust the virus titer of genetic engineering marked attenuated vaccine strain and HuN4-F112 vaccine control group to TCID 50 10 5 / ml, were inoculated ...

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Abstract

The invention discloses a genetic engineering marked attenuated vaccine strain of a porcine reproductive and respiratory syndrome virus (PRRSV). The attenuated vaccine strain comprises a genomic nucleic acid of a porcine reproductive and respiratory syndrome virus attenuated vaccine strain HuN4-F112; the HuN4-F112 genome includes a mutation in a genetic region for coding an Nsp2 protein, and the mutation is as follows: a nucleotide sequence for coding a Newcastle disease virus NP protein is inserted to a lacking region of a nucleotide sequence for coding 480-532-site amino acid of the Nsp2 protein; or the nucleotide sequence for coding the Newcastle disease virus NP protein is inserted to the lacking region of a nucleotide sequence for coding 508-532-site amino acid of the Nsp2 protein. The invention also discloses an application of the genetic engineering marked attenuated vaccine strain. The genetic engineering marked attenuated vaccine strain of the porcine reproductive and respiratory syndrome virus provided by the invention not only can provide completely safe immune protection to resist high-pathogenicity PRRSV after the porcine is immunized, but also can effectively distinguish the immunized porcine of the porcine reproductive and respiratory syndrome vaccine with the naturally infected porcine of the field virus.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a highly pathogenic porcine reproductive and respiratory syndrome virus genetic engineering marked attenuated vaccine strain and its application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the infectious diseases that seriously endanger the pig industry at present. The disease is infected by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Its main clinical features are serious reproductive disorders such as abortion, premature birth and stillbirth in pregnant sows, and obvious respiratory disorders in piglets and finishing pigs. So far, PRRS is still prevalent in most parts of the world and is one of the main infectious diseases of pigs. [0003] Since May 2006, the outbreak of highly pathogenic porcine reproductive and respiratory syndrome caused by the Ame...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/40C12N15/63G01N33/569A61K39/12A61P31/14
Inventor 童光志周艳君徐彦召张善瑞
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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