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A kind of Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus vp1 gene, recombinant vaccine strain and its preparation method and application

A foot-and-mouth disease virus and recombinant virus technology, applied in the field of genetic engineering, can solve problems such as indistinguishability

Active Publication Date: 2021-09-10
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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  • A kind of Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus vp1 gene, recombinant vaccine strain and its preparation method and application
  • A kind of Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus vp1 gene, recombinant vaccine strain and its preparation method and application
  • A kind of Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus vp1 gene, recombinant vaccine strain and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Acquisition of VP1 Gene of Type O Foot-and-Mouth Disease Virus

[0090] The O / BY / 2010 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 a letter of entrustment approved by the Veterinary Bureau of the Ministry of Agriculture. According to the gene sequence of O / BY / 2010 strain (Genebank: JN998085), the primers for synthesizing and amplifying VP1 were designed:

[0091] OVP1-F0: 5'-gatgcaatcaggcgacgtcgagaccaaccctggccctatgaccacttcgacgggcgag-3' (SEQ ID NO. 3);

[0092] OVP1-F: 5'-ccca gcatgc ttccctttcgcagctacaagcagaagatgctgatgcaatcaggcgacgtc-3' (SEQ ID NO. 4);

[0093] OS-R: 5'-caggatcgggttgtcagaagcgggcccagggttggactccac-3' (SEQ ID NO. 5).

[0094] Among the above specific primers, the upstream primers OVP1-F and OVP1-F0 used to amplify the OVP1 fragment were introduced into the SphI restriction site and the SVA gene sequence, and the dow...

Embodiment 2

[0097] Construction of Seneca Virus Infectious Clones of Recombinant Type O Foot-and-Mouth Disease Virus VP1 Gene

[0098] 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:

[0099] SVA-1F0: 5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3' (SEQ ID NO. 6);

[0100] SVA-1F: 5'-ataggt ttaattaa tgttaagcgtctgatgagtccgtgaggacgaaactatagga-3' (SEQ ID NO. 7);

[0101] SVA-1R: 5'-gggaa gcatg ctggggcaccaggcac-3' (SEQ ID NO. 8);

[0102] SVA-2F0: 5'-gtggagtccaaccctgggcccgcttctgacaacccgatcctg-3' (SEQ ID NO. 9);

[0103] SVA-2R: 5'-ttttctaga gcggcc gct 38 -3' (SEQ ID NO. 10);

...

Embodiment 3

[0112] Rescue of Seneca Recombinant Virus with Recombinant Type O Foot-and-Mouth Disease Virus VP1 Gene and Culture Characteristics of Different Cells

[0113] 3.1 Rescue of recombinant Seneca virus

[0114] use Plasmid Plus Maxi Kit (QIAGEN company) prepares the recombinant plasmid prSVV / FJ-M-OVP1 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, 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 ...

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Abstract

The present invention provides a restructuring of the Seneca reorganization of virus, reorganization vaccine, and its preparation methods and applications of the A -type mouthpiece vaccination virus VP1 gene, which involves the field of genetic engineering technology.The present invention conducts genetic lack of mutation of SVV / FJ / 001 plants, and at the same time integrates the VP1 gene of the O -type hoof virus in SVA CDNA to obtain the Seneca reorganization virus.Gene, and the expression of the product has a good original response; the vaccine plant has high antigen production capacity, the pathogenicity is significantly reduced, or even no pathogenicity to the pig.The immune activity of the fusion gene is generated, the vaccine plant significantly improves biological safety, and can be used for the prevention and control of Symba virus and one or more non -Senda 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 O-type foot-and-mouth disease virus VP1 gene, a recombinant vaccine strain and a preparation method and application thereof. 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, n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/41C12N15/42C12N7/01C12N15/85A61K39/295A61K39/125A61K39/135A61P31/14
CPCA61K39/12A61K2039/552A61K2039/70A61P31/14C07K14/005C12N7/00C12N15/85C12N2770/32021C12N2770/32022C12N2770/32034C12N2770/32122C12N2770/32134
Inventor 郑海学杨帆朱紫祥魏婷曹伟军刘华南郑敏张伟田宏张克山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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