Seneca virus infectious clone based on single plasmid rescue system, construction method and application

A rescue system and simple substance technology, applied in the field of genetic engineering, can solve the problems of increased transmission speed and pathogenicity, complex strains, etc., and achieve the effect of simple operation, important application value, efficient and stable rescue efficiency

Pending Publication Date: 2020-07-10
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Seneca virus has successively caused epidemics in the United States, Canada, Brazil, Colombia, China, Thailand and other countries. It was introduced into my country in 2015. It first appeared in Guangdong and spread rapidly. In Fujian, Guangxi, Hubei, Henan, Hebei Provinces such as Shandong, Liaoning and other provinces have caused a large number of pig farms to become sick, and the transmission speed and pathogenicity have increased compared with previous reports. Studies have shown that the current epidemic strains of SVA in my country are complex, and the prevention and control situation is still grim.

Method used

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  • Seneca virus infectious clone based on single plasmid rescue system, construction method and application
  • Seneca virus infectious clone based on single plasmid rescue system, construction method and application
  • Seneca virus infectious clone based on single plasmid rescue system, construction method and application

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preparation example Construction

[0044] The present invention uses the cDNA of the SVV / FJ / 001 strain as a template to amplify the A gene of the SVV / FJ / 001 strain with a pair of specific primers; the pair of specific primers includes an upstream primer and a downstream primer SVA-AR; the upstream Primers include SVA-1F0 and SVA-1F, the nucleotide sequence of the upstream primer SVA-1F0 is shown in SEQ ID NO.1, the nucleotide sequence of the upstream primer SVA-1F is shown in SEQ ID NO.2 The nucleotide sequence of the downstream primer SVA-AR is shown in SEQ ID NO.3. The Seneca virus strain SVV / FJ / 001 strain described in the present invention is preferably preserved in the China Type Culture Collection Center, the preservation number is SVV / FJ / 001 of CCTCC NO.V201802, and the patent "Seneca virus" has been authorized in China. Valley virus vaccine and its preparation method and application” have been disclosed in ZL201810003888.2. When amplifying the A gene, the present invention preferably includes: using the...

Embodiment 1

[0064] Construction of Infectious Clones of Seneca Recombinant Virus

[0065] The SVV / FJ / 001 strain used is preserved in the China Center for Type Culture Collection (microorganism preservation number: CCTCC NO: V201802), (disclosed in the authorized patent "Seneca Valley virus vaccine and its preparation method and application" ZL201810003888.2 , which is incorporated in this application by reference in its entirety), according to the SVA genome sequence (Genebank: KY747510), through comparative analysis, design the amplification primers that introduce the rescue element and cover the whole genome of SVA:

[0066] SVA-1F0: 5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3' (SEQ ID NO. 1);

[0067] SVA-1F: 5'-ataggt ttaattaa tgttaagcgtctgatgagtccgtgaggacgaaactatagga-3' (SEQ ID NO. 2);

[0068] SVA-AR: 5'-gttgtc gctagc agggccagggttggtctc-3' (SEQ ID NO. 3);

[0069] SVA-BF: 5'-cccca gctagc gacaacccgatcctg-3' (SEQ ID NO. 4);

[0070] SVA-2R: 5'-ttttctaga g...

Embodiment 2

[0075] Construction of full-length cDNA infectious clone of Seneca virus

[0076] The schematic diagram of the construction method is as follows figure 2 Shown: Plasmid PMD-A was double-digested with PacI and NheI, and plasmid PMD-B was double-digested with NheI and NotI, and the target fragments were respectively recovered, and then the polymerase promoter gene, polymerase terminator, ribozyme The sequenced eukaryotic plasmid was double-digested with PacI and NotI, the vector fragment was purified and recovered, ligated by T4 ligase, transformed into JM109 competent cells, positive clones were identified by enzyme digestion and sequencing, and the modified SVV / FJ / 001 was obtained The eukaryotic transcription plasmid prSVV / FJ of the full-length gene.

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Abstract

The invention provides a single plasmid rescue system aiming at a positive strand RNA virus, a Seneca virus infectious clone based on the single plasmid rescue system, a construction method and an application, and relates to the technical field of gene engineering. The invention provides the single plasmid rescue system aiming at the positive strand RNA virus and an application of the single plasmid rescue system in directional design of an SVA reverse recombinant vaccine and construction of an SVA directional mutation strain in researches of virus gene functions, virus variation, pathogenesisand the like. According to the invention, a single plasmid Seneca virus rescue system is constructed by using elements like promoters, terminators and ribozyme of polymerase I and polymerase II, so editing of a virus genome on a plasmid level is realized, and a technical platform is provided for directional design, construction and rescue of viruses.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Seneca virus infectious cloning and construction method and application based on a single plasmid rescue system. Background technique [0002] Seneca virus A (SenecavirusA, SVA), also known as Senecavirus (Senecavirus), Seneca valley virus (Seneca valley virus, SVV), belongs to Picornaviridae (Picornaviridae) Senecavirus genus (Senecavirus) , the only member of the genus. The virus infects pigs and can cause primary vesicular disease in pigs, which is indistinguishable from clinical symptoms caused by foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. SVA infection can cause vesicular lesions in weaned piglets, nursery, fattening and breeding pigs of all ages, accompanied by clinical symptoms such as lameness, fever, anorexia, lethargy, etc. In addition to the above symptoms, newborn piglets may also have persistent diarrhea , d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/66C12N7/01A61K39/125A61P31/14
CPCA61K39/12A61K2039/53A61P31/14C12N15/66C12N15/86C12N2770/32034C12N2770/32043
Inventor 郑海学杨帆朱紫祥曹伟军田宏张克山魏婷张伟郑敏党文马旭升李丹茹毅何继军郭建宏刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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