Recombinant nucleic acid of seneca virus, recombinant vaccine strain as well as preparation method and application thereof

A recombinant nucleic acid, recombinant vaccine technology, applied in the field of genetic engineering, can solve problems such as indistinguishability, achieve high titer, achieve innovation, and achieve the effect of great application value

Active 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

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

Method used

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  • Recombinant nucleic acid of seneca virus, recombinant vaccine strain as well as preparation method and application thereof
  • Recombinant nucleic acid of seneca virus, recombinant vaccine strain as well as preparation method and application thereof
  • Recombinant nucleic acid of seneca virus, recombinant vaccine strain as well as preparation method and application thereof

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preparation example Construction

[0081] The present invention also provides a preparation method of the Seneca recombinant vaccine, comprising the following steps:

[0082] 1) Inoculating the Seneca recombinant virus into susceptible cells for proliferation and culture to obtain the Seneca recombinant virus liquid;

[0083] 2) Inactivating and emulsifying the Seneca recombinant virus in the Seneca recombinant virus liquid to obtain the Seneca recombinant vaccine.

[0084] The susceptible cells described in step 1) of the present invention preferably include BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells, more preferably BHK-21 suspension cells or ST suspension cells.

[0085] In the present invention, when carrying out the proliferation culture described in step 1), the virus titer of the Seneca recombinant virus is preferably not less than 10 6.5 TCID 50 / mL, more preferably 10 6.5 TCID 50 / mL~10 10.0 TCID 50 / mL. The Seneca recombinant virus strain of the ...

Embodiment 1

[0095] Construction of infectious clones of Seneca recombinant virus:

[0096] The SVV / FJ / 001 strain is preserved in the China Center for Type Culture Collection (microorganism collection number: CCTCCNO.V201802), (disclosed in the authorized patent "Seneca Valley virus vaccine and its preparation method and application" ZL201810003888.2, which The full text is incorporated in this application by reference), according to the SVA genome sequence (Genebank: KY747510), design synthetic amplification primers:

[0097] SVA-1F0: 5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3' (SEQ ID NO. 2);

[0098] SVA-1F: 5'-ataggt ttaattaa tgttaagcgtctgatgagtccgtgaggacgaaactatagga-3' (SEQ ID NO. 3);

[0099] SVA-1R: 5'-gggaa gcatgc tggggcaccaggcac-3' (SEQ ID NO. 4);

[0100] SVA-2F: 5'-cccca gcatgc ttccctttcgcagc-3' (SEQ ID NO. 5);

[0101] SVA-2R: 5'-ttttctaga gcggccgc t 38 -3' (SEQ ID NO. 6);

[0102] SVA-m5UTRF: 5'-gttctagcctactcgttttttcccctactcactcattcgtgttgtaact...

Embodiment 2

[0109] Rescue of Seneca Recombinant Virus and Culture Properties of Different Cells

[0110] 2.1 Rescue of Seneca recombinant virus

[0111] use Plasmid Plus Maxi Kit (QIAGEN company) prepares the recombinant plasmid prSVV / FJ-M that obtains by embodiment 1, is used for transfection when BHK-21 cell grows to 80%, under the mediation of liposome LipofectamineTM2000 (Invitrogen) Transfect 4 μg of the recombinant plasmid into BHK-21 cells, and set up liposome control and normal cell control at the same time, and place them in a place containing 5% CO 2 In a 37°C incubator, discard the supernatant after 6 hours of transfection, add MEM medium, continue to culture, observe the cell state and cell pathological changes, harvest the virus when about 90% of the cells are pathological, and freeze and thaw three times. BHK-21 cells were inoculated again until the virus could stably produce cytopathic changes, and the cells became round and shed, gradually forming plaques and disintegra...

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Abstract

The invention provides a recombinant nucleic acid of a seneca virus, a recombinant vaccine strain as well as a preparation method and application thereof and relates to the technical field of gene engineering. The invention provides a recombinant nucleic acid of a seneca virus, a seneca recombinant virus containing the recombinant nucleic acid, a seneca recombinant virus coded by the recombinant nucleic acid, a seneca recombinant vaccine strain containing the seneca recombinant virus as well as a preparation method and application of the seneca recombinant vaccine strain. According to the invention, the vaccine strain having the characteristics of high antigen productivity, remarkably reduced pathogenicity and even no pathogenicity for pigs, strong antibody response, high immune protectionrate and the like is prepared, and the vaccine strain remarkably improves the biosafety and may be used for preventing and controlling the seneca virus in China and surrounding countries.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Seneca virus recombinant nucleic acid, a recombinant vaccine strain and a preparation method and application thereof. Background technique [0002] Senecavirus A (Senecavirus A, SVA), also known as Senecavirus (Senecavirus), Seneca valley virus (Seneca valley virus, SVV), belongs to the small RNA virus family (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 persis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N7/01C12N15/85A61K39/125A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2770/32022C12N2770/32034A61K2039/552A61K2039/5254C12N15/66C12N7/00C12N2770/32021C12N2770/32051C12N2770/32063A61K39/125A61K2039/5252A61K2039/53C12N15/102
Inventor 郑海学杨帆朱紫祥曹伟军田宏张克山魏婷郑敏张伟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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