Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene

A nucleocapsid protein, porcine circovirus technology, applied in the direction of antiviral agents, genetic engineering, virus antigen components, etc., can solve the problems of difficult to cure PMWS, unclear mechanism, and no clinical symptoms of the virus, and achieve replication. The effect of high efficiency and large clone space

Inactive Publication Date: 2008-10-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
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Problems solved by technology

PMWS caused by PCV2 mixed infection with PPV and PRRSV is difficult to cure. Foreign studies have shown that mixed infection of PCV2 and these two viruses can reproduce typical PMWS under experimental conditions, but infection with these three viruses alone does not show clinical symptoms, so Some people think that PCV2 may have a synergistic effect with other pathogens, but the mechanism is still unclear

Method used

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  • Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene
  • Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene
  • Recombination virus particles for expressing 2-typed porcine circovirus nucleocapsid protein Cap gene

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Embodiment Construction

[0044] 1. Construction and application of PPV VP2 virus-like particles expressing foreign genes

[0045] 1) Obtain the ΔVP2 target gene and clone it into pMD19-T vector

[0046] Using the NADL-2 gene sequence in GenBank with accession number NC: 001718 as a template, primers were designed with the software Primer 5.0: KpnI and NotI restriction sites were added, respectively. Attenuated PPV strain (NADL-2) was purchased from China Veterinary Drug Control Institute.

[0047] P1 5′-gaggtaccgggggggttggtgtgt-3′

[0048] P2 5′-cgggcggccgcctagtataattttcttgg-3′

[0049] With P1, P2 primer amplifies the fragment containing VP2 (1740bp) gene C-terminal 1640bp ( figure 1 ). After the amplified PCR product was electrophoresed on 1% agarose gel, it was recovered and connected with pMD19-T Vector to transform E.coli DH 5 α, Plasmid was extracted by alkaline lysis method, identified by KpnI and Not I double enzyme digestion, and a positive plasmid (pTΔVP2) was obtained.

[0050] 2) Con...

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Abstract

The invention relates to construction and application of recombinant nucleocapsids of Cap genes of porcine circovirus expression type 2 nucleocapsid protein, belonging to the genetic engineering bacterin field. C-terminal gene fragments of porcine parvovirus (PPV) VP2 genes are cloned into a type 5 adenovirus shuttle vector of the human beings, and recombinant adenovirus rAd-deltaVP2 is obtained; deltaVP2 proteins are expressed successfully and highly efficiently and can be self-assembled into the nucleocapsids [PPV:VLPs]; the PPV VP2 nucleocapsids are used as antigen transport vectors and 165 to 200 sites of amino acid (deltaCap) genes of the porcine circovirus type 2 (PCV2) nucleocapsid proteins (Cap) are embedded into an N-terminal (deltaVP2) of the PPV VP2, and then recombinant adenovirus rAd-deltaCap-deltaVP2 is obtained; embedded VP2 (deltaCap-deltaVP2) proteins are expressed successfully and highly efficiently and can be self-assembled into nucleocapsids [PPV:VLP(PCV2)]. The invention also relates to application of the recombinant virus and recombinant PPV VP2 nucleocapsids of the expression Cap genes of the recombinant virus in the aspects of bacterin immunity and so on.

Description

1. Technical field [0001] The invention relates to the construction and application of recombinant virus-like particles expressing type 2 porcine circovirus nucleocapsid protein Cap gene in an adenovirus expression system and belongs to the field of genetic engineering vaccines. 2. Background technology [0002] PPV (Porcine Parvovirus) is not only one of the main pathogens causing reproductive disorders in sows, but also can cause a variety of pig diseases. VP2 is the main component of virions, accounting for about 80%. The coat protein of parvoviruses can be assembled autonomously in the natural state. VP2 has good immunogenicity, and many scientists have used the good immune characteristics of VP2 to construct recombinant vaccines to prevent this disease, and achieved good results. At the same time, it is also a substance with hemagglutination activity. Martinez et al. cloned the PPV VP2 gene into a baculovirus expression system and successfully expressed it in insect ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/34C12N15/861A61K39/12A61P31/12
Inventor 何孔旺潘群兴温立斌张雪寒倪艳秀俞正玉郭容利吕立新茅爱华肖琦黄克和
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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