Seneca recombinant virus of recombinant A-type foot-and-mouth disease virus VP1 gene, recombinant vaccine strain and preparation method and application of recombinant vaccine strain

A foot-and-mouth disease virus and recombinant vaccine technology, applied in the field of Seneca recombinant virus, can solve problems such as indistinguishability, and achieve the effects of reducing pathogenicity, improving biological safety, and ensuring pertinence

Active Publication Date: 2020-11-27
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Seneca recombinant virus of recombinant A-type foot-and-mouth disease virus VP1 gene, recombinant vaccine strain and preparation method and application of recombinant vaccine strain
  • Seneca recombinant virus of recombinant A-type foot-and-mouth disease virus VP1 gene, recombinant vaccine strain and preparation method and application of recombinant vaccine strain
  • Seneca recombinant virus of recombinant A-type foot-and-mouth disease virus VP1 gene, recombinant vaccine strain and preparation method and application of recombinant vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Acquisition of VP1 Gene of Type A Foot-and-Mouth Disease Virus

[0093] The A / GDMM / 2013 strain is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Veterinary Bureau of the Ministry of Agriculture, and the public can obtain it through the letter of entrustment approved by the Veterinary Bureau of the Ministry of Agriculture. According to the gene sequence of A / GDMM / 2013 strain (Genebank: KF450794), the primers for synthesizing and amplifying VP1 were designed:

[0094] AVP1-F0:

[0095] 5'-gatgcaatcaggcgacgtcgagaccaaccctggccctatgaccaccgccaccggggaa-3' (SEQ ID NO. 3);

[0096] AVP1-F:

[0097] 5'-ccca gcat gct tccctttcgcagctacaagcagaagatgctgatgcaatcaggcgacgtc-3' (SEQ ID NO. 4);

[0098] AS-R: 5'-caggatcgggttgtcagaagcgggtccagggttggactcaac-3' (SEQ ID NO. 5).

[0099] In the above specific primers, the upstream primers AVP1-F and AVP1-F0 used to amplify the AVP1 fragments introduce the SphI restriction site and the SVA gene sequenc...

Embodiment 2

[0102] Construction of Seneca Virus Infectious Clones of Recombinant Foot-and-Mouth Disease Virus Type A VP1 Gene

[0103] 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), design synthetic amplification primers:

[0104] SVA-1F0:

[0105] 5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3' (SEQ ID NO. 6);

[0106] SVA-1F: 5'-ataggt ttaattaa tgttaagcgtctgatgagtccgtgaggacgaaactatagga-3'

[0107] (SEQ ID NO. 7);

[0108] SVA-1R: 5'-gggaa gcatgc tggggcaccaggcac-3' (SEQ ID NO. 8);

[0109] SVA-2F1: 5'-gttgagtccaaccctggacccgcttctgacaacccgatcctg-3' (SEQ ID NO. 9);

[0110] SVA-2R: 5'-ttttctaga gcggccgc t 38 -3' ...

Embodiment 3

[0120] Rescue of Seneca virus with recombinant foot-and-mouth disease virus type A VP1 gene and culture characteristics of different cells

[0121] 3.1 Rescue of recombinant Seneca virus

[0122] use Plasmid Plus Maxi Kit (QIAGEN company) prepares the recombinant plasmid prSVV / FJ-M-AVP1 that obtains by embodiment 2, is used for transfection when BHK-21 cell grows to 80%, in liposome Lipofectamine TM Under the mediation of 2000 (Invitrogen), 4 μg of recombinant plasmids were transfected into BHK-21 cells, and liposome controls and normal cell controls were set up at the same time, and placed in 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, and harvest the virus when about 90% of the cells are pathological, freeze and thaw repeatedly 3 times and then again Inoculate BHK-21 cells until the virus can stably produce cytopathic changes, and the cells be...

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Abstract

The invention provides a Seneca recombinant virus of a recombinant A-type foot-and-mouth disease virus VP1 gene, a recombinant vaccine strain and a preparation method and application of the recombinant vaccine strain, and relates to the technical field of gene engineering. The invention provides a Seneca recombinant nucleic acid, the Seneca recombinant virus containing the recombinant nucleic acid, the Seneca recombinant vaccine strain containing the Seneca recombinant virus, and a preparation method and application of the vaccine strain. An SVV/FJ/001 strain is subjected to gene deletion mutation transformation, the VP1 gene of the A-type FMDV is fused into cDNA of the SVV/FJ/001 strain to obtain the Seneca recombinant virus, the recombinant virus can express the fused gene, and an expression product has good reactogenicity; and the obtained vaccine strain is high in antigen productivity, the pathogenicity is remarkably reduced, even no pathogenicity is caused to pigs, the immune response of SVA can be stimulated after animals are immunized by the inactivated vaccine, the immunocompetence aiming at fusion genes can be generated, and the vaccine strain can be used for preventing and controlling Seneca virus and one or more non-Seneca virus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Seneca recombinant virus recombined with the VP1 gene of type A foot-and-mouth disease virus, 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 t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N15/42C12N7/01C12N15/85A61K39/125A61K39/135A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/85C12N2770/32021C12N2770/32034C12N2770/32052C12N2770/32122C12N2770/32134C12N2800/107
Inventor 郑海学杨帆曹伟军蒋保余朱紫祥魏婷郑敏田宏张克山张伟刘永杰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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