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Vaccine strains of infectious clones of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof

A technology for respiratory syndrome and pig breeding, applied in the field of bioengineering, can solve problems such as large immunization dose of inactivated vaccines, unsatisfactory immunization effect of heterologous strains, safety concerns, etc., and achieve an effect that is conducive to prevention and control

Active Publication Date: 2011-03-09
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

[0004] Although all major pig-raising countries in the world have paid enough attention to PRRS, since the discovery of PRRSV in 1987, people have made unremitting explorations around the biological characteristics, immunological characteristics, pathogenic mechanisms and vaccines of PRRSV pathogens. Some good progress has also been made in the research of genome structure, virus-encoded protein characteristics, host cell proteins interacting with virus, and anti-viral infection immunity. However, because PRRSV is prone to mutation, can cause persistent infection and immunosuppression, Moreover, the characteristics of antibody-dependent enhancement (ADE) and so on have become the main obstacles to the control of PRRS.
In addition, the PRRSV strains currently existing in my country present a situation of diversification and coexistence, that is, there are not only classic strains without genome deletions, but also strains with nucleotide insertions in the coding region of the NSP2 gene, and strains with nucleotides in the coding region of the NSP2 gene. Mutant strains with different numbers of missing acids, etc., complicate the epidemic situation caused by PRRSV in my country, which not only increases the difficulty of preventing and controlling PRRS, but also brings new challenges to the research of PRRSV
[0005] At present, the means of controlling the disease mainly rely on vaccine immunization, which can effectively reduce the spread of the disease. Among them, the inactivated PRRS vaccine has the advantages of safety, easy storage and transportation, etc., and its disadvantage is that it is easy to prepare the inactivated vaccine. The antigenicity of pathogens is weakened, which reduces the immune efficiency of vaccines, and the immune dose of inactivated vaccines is large, the cost is high, and the immune effect on heterologous strains is not ideal
The weak live vaccine has good immune effect and long duration of immune response, but its safety is still worrying, especially for the highly pathogenic porcine reproductive and respiratory syndrome in my country. Prevention and Control of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome in China Brings Great Difficulties

Method used

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  • Vaccine strains of infectious clones of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof
  • Vaccine strains of infectious clones of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof
  • Vaccine strains of infectious clones of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of full-length cDNA clone of porcine reproductive and respiratory syndrome virus vaccine strain HuN4-F112 genome

[0048] 1. Materials and methods

[0049] 1.1 Viruses, vectors and reagents

[0050] The PRRSV vaccine strain F112 was attenuated and preserved by the laboratory (Tong Guangzhi et al. 2007; Tong GZ et al., 2007; Zhou YJ et al., 2008; Tian ZJ et al., 2009), cloning vector pBulescriptII SK(+) Was purchased from Fermentas; 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; DL-15000, DL-2000 DNA Marker were purchased from TakaRa; other chemical reagents were imported or domestically pure.

[0051] 1.2 Primer

[0052] According to the whole genome sequence of PRRSV HuN4 strain (GenBank accession number: EF6350...

Embodiment 2

[0070] Example 2 Rescue and identification of porcine reproductive and respiratory syndrome virus

[0071] 1. Method

[0072] 1.1 RNA synthesis and transfection in vitro

[0073] Linearize the plasmid pSK-HuN4-Vac containing the full-length cDNA using the SwaI restriction site introduced by the 3'end Poly(A) tail, and synthesize viral RNA according to the instructions of the mMessage High Yield Capped RNA Transcription kit in vitro transcription kit. Inoculate BHK21 cells and perform transfection according to the instructions of DMRI-C transfection reagent when the cell density is 70% to 90%.

[0074] 1.2 Identification of rescue virus

[0075] 1.2.1 RT-PCR detection of genomic markers (genomic marker)

[0076] The RNA of the clonal virus cHuN4-Vac and the parental virus HuN4-F112 infected cells was extracted, reverse transcribed, and the fragments containing MIulI were amplified by primers JD1 and JD2, and the MIulI was used for restriction digestion identification, and the pMD18-T vec...

Embodiment 3

[0086] Example 3 Animal Test

[0087] 1. Animal test methods:

[0088] Fifteen 40-day-old healthy piglets negative for PRRS virus and antibodies, and negative for PRV, PCV2 and CSFV, were randomly divided into 3 groups, namely the HuN4-F112 vaccine control group, the artificial clonal virus cHuN4-Vac vaccine group and the blank control group. There are 5 pigs in each group, and each pig in the artificial clonal virus vaccine group has a dose of 10 5.0 TCID 50 In the HuN4-F112 vaccine control group, each pig was vaccinated according to the vaccine instruction manual, and the blank control group was vaccinated with DMEM. 28 days after inoculation, all test pigs attacked HuN4-F5 virulent 3×10 each 4.0 TCID 50 Observe the clinical manifestations and temperature every day after the challenge. Blood samples are taken on days 0, 1, 3, 7, 14, 21, and 28 to detect antibody levels, isolate the virus and perform differential diagnosis tests on it, and perform pathological tissues on the test ...

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Abstract

The invention discloses an artificially cloned attenuated vaccine strains. The vaccine strains are strains cloned from attenuated vaccine strains HuN4-F112 of highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), and marked restriction enzyme sites are introduced in the structural protein gene sequences of the strains. The invention also discloses a recombinant vector which comprises a full-length gene cDNA sequence of the attenuated vaccine strains HuN4-F112 of the highly pathogenic PRRSV. The 5'-end of the full-length gene cDNA sequence is additionally provided witha transcription promoter and the full-length gene cDNA sequence is internally introduced with the marked restriction enzyme sites. The artificially cloned attenuated vaccine strains of the invention can not only provide completely safe immune protection for resistance of an immune pig to the highly pathogenic PRRSV, but also effectively distinguish an immune pig of the PRRSV from a naturally infectious pig of the PRRSV, thus being beneficial to preventing and controlling the highly pathogenic PRRSV.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to an artificially cloned attenuated vaccine strain with infectious and highly pathogenic porcine reproductive and respiratory syndrome virus and its application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV) due to pregnancy sows abortion, stillbirth, weak fetus, mummified fetus and other reproductive disorders, and respiratory symptoms and symptoms of pigs at various ages. An infectious disease characterized by high mortality, commonly known as "Porcine Reproductive and Respiratory Syndrome", is one of the seven major swine diseases legally reported by OIE. Since it was first discovered in 1996, it has been popular in my country, causing huge economic losses to the pig industry in my country. In 2006, a "swine hyperthermia syndrome" spread rapidly in our pig herd, with an incidence rate of 50-100% and a mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/40C12N7/04C12Q1/70C12Q1/68A61K39/12A61P31/14
Inventor 童光志周艳君张善瑞姜一峰李国新于海
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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