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Preparation method and application of porcine seneca virus full-length infectious clone

An infectious cloning and virus technology, which is applied in the field of preparation and application of full-length infectious clones of swine Seneca virus, can solve problems such as lack of prevention and control measures, and achieve the effect of important scientific application value

Active Publication Date: 2019-11-05
YANGZHOU UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, there is still a lack of effective prevention and control measures

Method used

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  • Preparation method and application of porcine seneca virus full-length infectious clone
  • Preparation method and application of porcine seneca virus full-length infectious clone
  • Preparation method and application of porcine seneca virus full-length infectious clone

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Embodiment Construction

[0027] The present invention will be further described below in conjunction with the drawings and specific embodiments of the specification, but the embodiments do not limit the present invention in any form.

[0028] 1Materials and methods

[0029] 1.1 Viruses, cells, strains and reference sequences

[0030] The SVV / GD05 strain (GenBankaccession numbers: MH316116) was isolated from a pig farm in Guangdong at the end of 2017 and was isolated and preserved by our laboratory. BHK-21 cells, ST-R cells (pig testicular cells), pEGFP-C3 vector, and E. coli TOP10 competent cells are kept in our laboratory.

[0031] 1.2 Main reagents and instruments

[0032] Restriction enzymes, Prime STARTM DNA Polymerase, Taq TM , TaKaRa MiniBEST ViralRNA / DNA Extraction Kit, PrimeScript TM 1st Strand cDNA Synthesis Kit and DL5000DNAmarker were purchased from Baori Biotech (Beijing) Co., Ltd.; Agarose Gel DNA Purification Kit was purchased from Axygen; Gibson Assembly Kit was purchased from NEB; VP1 monoc...

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Abstract

The invention relates to a preparation method and application of a full-length infectious clone of porcine seneca virus, comprising the following steps: (1) amplification of the full genome sequence of SVV / GD05 strain; (2) construction of pSVV‑GD05 viral infectious clone plasmid ; (3) Construction of pSVV‑GD05‑iLOV recombinant plasmid; (4) Rescue of parental virus (SVV‑GD05) and recombinant virus (SVV‑GD05‑iLOV). The present invention utilizes an SVV strain isolated from a Chinese pig herd to construct a viral infectious clone pSVV‑GD05 with a bacterial plasmid as a backbone, and can successfully rescue the virus. At the same time, the reporter gene iLOV was inserted into the genome of the SVV infectious clone virus, and the recombinant SVV virus capable of expressing the reporter gene was successfully rescued. The invention provides an effective platform for in-depth basic and applied research on SVV, and has important scientific application value.

Description

Technical field [0001] The invention relates to a preparation method and application of a full-length infectious clone of swine Seneca virus. Background technique [0002] Seneca Valley Virus (SVV) belongs to the only member of the Senecavirus genus of Picorna viridae (Picorna viridae). Its genome is a single-stranded positive-stranded RNA with a length of about 7.28kb. The viral genome consists of a 5'non-coding region (0.66Kb), an open reading frame (ORF, 6.54Kb), a 3'non-coding region (0.07Kb) and polyadenylic acid (polyA). ORF is composed of L gene, P1 structural protein gene, P2 and P3 non-structural protein genes, which together encode a long polyprotein. This polymerized protein follows the L-4-3-4 model and can be processed into 12 structural and non-structural proteins after being cleaved by protease, namely L, 1A (VP4), 1B (VP2), 1C (VP3), 1D (VP1), 2A, 2B, 2C, 3A, 3B, 3C, 3D. [0003] SVV-VP1 gene sequence analysis showed that the existing SVV strains can be divided i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/41C12N15/86C12Q1/70C12N7/01
Inventor 陈振海王敏敏孙怀昌张鑫宇周晓慧牟春晓
Owner YANGZHOU UNIV
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