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Preparation method and application of full-length infectious clone of porcine SVV (Seneca Valley Virus)

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: 2019-05-28
YANGZHOU UNIV
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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 full-length infectious clone of porcine SVV (Seneca Valley Virus)
  • Preparation method and application of full-length infectious clone of porcine SVV (Seneca Valley Virus)
  • Preparation method and application of full-length infectious clone of porcine SVV (Seneca Valley Virus)

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Embodiment Construction

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

[0028] 1 Materials 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 (porcine testis cells), pEGFP-C3 vector, Escherichia coli TOP10 competent cells are preserved by our laboratory.

[0031] 1.2 Main reagents and instruments

[0032] Restriction enzymes, Prime START DNA Polymerase, Taq TM 、TaKaRa MiniBEST ViralRNA / DNA Extraction Kit、PrimeScript TM 1st Strand cDNA Synthesis Kit and DL5000 DNAmarker were purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.; Agarose Gel DNA Purification Kit was purchased from Axygen; Gibson Assembly Kit was purch...

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Abstract

The invention relates to a preparation method and an application of a full-length infectious clone of a porcine SVV (Seneca Valley Virus), comprising the following steps: (1) amplification of a full sequence of an SVV / GD05 strain genome; (2) construction of infectious clone plasmid of a pSVV-GD05 virus; (3) construction of pSVV-GD05-iLOV recombinant plasmid; and (4) rescue of a parental virus (SVV-GD05) and a recombinant virus (SVV-GD05-iLOV). The method provided by the invention utilizes a strain of the SVV isolated from a Chinese pig herd to construct a viral infectious clone pSVV-GD05 withbacterial plasmid as a skeleton, and can successfully rescue the virus. At the same time, a reporter gene iLOV is inserted into an SVV infectious clone virus genome, and the recombinant SVV virus capable of expressing the reporter gene is successfully rescued. The preparation method and application of the invention provide an effective platform for deep and basic research and application of SVV, and have important scientific application value.

Description

technical field [0001] The invention relates to a preparation method and application of a full-length infectious clone of porcine seneca virus. Background technique [0002] Seneca virus (Seneca Valley Virus, SVV) is the only member of the genus Senecavirus in the Picorna viridae family. Its genome is a single-stranded positive-sense 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 jointly encode a long polyprotein. This polymeric protein follows the L-4-3-4 pattern and can be processed into 12 structural and non-structural proteins after proteolytic cleavage, namely L, 1A(VP4), 1B(VP2), 1C(VP3), 1D (VP1), 2A, 2B, 2C, 3A, 3B, 3C, 3D. [0003] SVV-VP1 gene sequence analysis shows that the existing SVV strains can be divided into...

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Application Information

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Patent Type & Authority Applications(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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