Construction method of porcine delta coronavirus infectious clone plasmid

A technology of infectious cloning and coronavirus, which is applied in the prevention and control of animal infectious diseases and the field of biology, and can solve problems such as long cycles

Active Publication Date: 2021-05-28
HUAZHONG AGRI UNIV
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Problems solved by technology

In addition, most of the existing reverse genetic operating systems based on the BAC system use multi-step homologous recombination as construction strategies, and the cycle is relatively long

Method used

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  • Construction method of porcine delta coronavirus infectious clone plasmid
  • Construction method of porcine delta coronavirus infectious clone plasmid
  • Construction method of porcine delta coronavirus infectious clone plasmid

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Embodiment 1

[0061] 1. Formulas of main test materials, vectors, plasmids, reagents and related media or solutions:

[0062] PDCoV CHN-HN-2014 strain: isolated, identified and preserved by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Dong N, Fang L, Yang H, Liu H, Du T, Fang P, Wang D, Chen H, XiaoS .Isolation, genomic characterization, and pathogenicity of a Chinese porcinedeltacoronavirus strain CHN-HN-2014.Vet Microbiol.2016,196:98-106). The whole genome sequence is shown in GenBank accession number: KT336560.

[0063] pBAC-AJ1102 is an infectious clone containing the full-length cDNA of PEDV AJ1102 strain: it was constructed and preserved for the Virus Division of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0064] LLC-PK1 cells: purchased from American Type Culture Collection (ATCC).

[0065] Chloramphenicol (product number: BS049B): purchased from Biosharp Company.

[0066] Various restrictio...

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Abstract

The invention discloses a construction method of porcine delta coronavirus infectious clone plasmids, and belongs to the technical field of animal infectious disease prevention and treatment and biology. According to the invention, a reverse genetic system of a recombinant virus of a PDCoV CHN-HN-2014 strain is rescued based on a BAC system and a homologous recombination one-step cloning method, wherein five gene segments of the strain are cloned to an intermediate vector pBAC-M-PDCoV in one step, and a recombinant plasmid pBAC-CHN-HN-2014 containing full-length cDNA of the PDCoV CHN-HN-2014 strain is obtained, and the recombinant virus rCHN-HN-2014 is rescued after cells are transfected; therefore, a porcine delta coronavirus reverse genetic manipulation platform is established, and a foundation is laid for in-depth study on infection and pathogenic mechanisms of PDCoV. Meanwhile, aiming at the practical problem that no available PDCoV vaccine exists in China at present, the PDCoV infectious cloning platform disclosed by the invention can also be used for developing a safer and more efficient novel PDCoV genetic engineering vaccine. The platform can also be used as a live virus vector and is used for developing a multi-combined vaccine, so that the PDCoV infectious cloning platform disclosed by the invention has a great value for the research related to the virus.

Description

technical field [0001] The invention belongs to the prevention and treatment of animal infectious diseases and the fields of biology. In particular, it relates to a method for constructing an infectious cloning plasmid of porcine delta coronavirus. Background technique [0002] Porcine deltacoronavirus (porcine deltacoronavirus, PDCoV) belongs to the Coronaviridae family, a member of the genus Deltacoronavirus, has an envelope and a genome of single-stranded positive-sense RNA, and is currently the only deltacoronavirus that has been successfully isolated. Woo et al first discovered porcine deltacoronavirus (Woo P.C.Y, Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the genesource of gammacoronavirus and deltacoronavirus. J. Virol. 2012, 86: 3995-4008). In early 2014, PDCoV infection first broke out in the United States, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/50C12N7/00C12R1/93
CPCC12N15/85C07K14/005C12N7/00C12N2800/107C12N2800/204C12N2830/50C12N2770/20022C12N2770/20052Y02A50/30
Inventor 方六荣方谱县肖少波夏思进任杰张健淞张慧畅
Owner HUAZHONG AGRI UNIV
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