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Recombinant O-type foot-and-mouth disease virus epitope gene Seneca recombinant virus, recombinant vaccine and its preparation method and application

A technology of foot-and-mouth disease virus and recombinant virus, applied in the field of genetic engineering, to achieve good reactogenicity, high virus titer, and reduced pathogenicity

Active Publication Date: 2021-07-13
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

However, there is currently no research on the application of FMDV-related genes to the preparation of Seneca recombinant virus or recombinant vaccines

Method used

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  • Recombinant O-type foot-and-mouth disease virus epitope gene Seneca recombinant virus, recombinant vaccine and its preparation method and application
  • Recombinant O-type foot-and-mouth disease virus epitope gene Seneca recombinant virus, recombinant vaccine and its preparation method and application
  • Recombinant O-type foot-and-mouth disease virus epitope gene Seneca recombinant virus, recombinant vaccine and its preparation method and application

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preparation example Construction

[0082] The present invention also provides a preparation method of the Seneca recombinant vaccine of the recombinant O-type foot-and-mouth disease virus epitope gene, comprising the following steps:

[0083] 1) Inoculating the Seneca recombinant virus into susceptible cells for proliferation and culture to obtain the Seneca recombinant virus liquid;

[0084] 2) Inactivating and emulsifying the Seneca recombinant virus in the Seneca recombinant virus liquid to obtain the Seneca recombinant vaccine.

[0085] 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.

[0086] 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...

Embodiment 1

[0095] Acquisition of B-cell Epitope and T-cell Epitope Genes of Type O Foot-and-Mouth Disease Virus

[0096] According to the gene sequence of the O / BY / 2010 strain (Genebank: JN998085), a flexible Linker (Gly-Gly-Gly- Gly-Ser) in series for three times, and at the 3' end, link the 21-35 amino acid coding gene of FMDV 3A in series, and the 2A gene in series at the same time, to obtain the series O-type FMDV B cell epitope, 3A T cell epitope and 2A Gene, the recombinant epitope gene OB-3A. At the same time, according to the sequence of the SVV / FJ / 001 strain, the gene sequence for synthesizing SVA on the flanks includes the 5' end SphI to the 3' end NheI (NheI is an enzyme cleavage site introduced after undergoing amino acid synonymous mutation according to the sequence of SVA), synthesized The gene containing recombinant O-type foot-and-mouth disease virus epitope gene OB-3A is the nucleotide sequence shown in SEQ ID NO.1.

Embodiment 2

[0098] Construction of Seneca Virus Infectious Clones of Recombinant Type O Foot-and-Mouth Disease Virus Epitope Gene

[0099] 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:

[0100] SVA-1F0:

[0101] 5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3' (SEQ ID NO. 3);

[0102] SVA-1F: 5'-ataggtttaattaatgttaagcgtctgatgagtccgtgaggacgaaactatagga-3' (SEQ ID NO. 4)

[0103] SVA-1R: 5'-gggaagcatgctggggcaccaggcac-3' (SEQ ID NO.5)

[0104] SVA-2F1: 5'-gccctgctagcgacaacccgatcctg-3' (SEQ ID NO. 6)

[0105]SVA-2F: 5'-ccccagcatgcttccctttcgcagc-3' (SEQ ID NO.10)

[0106] SVA-2R: 5'-t...

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Abstract

The invention provides a Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus epitope gene, a recombinant vaccine, a preparation method and application thereof, and relates to the technical field of genetic engineering. The present invention obtains the full-length cDNA of SVV / FJ / 001 strain, and performs deletion and mutation transformation on its 5'UTR, and at the same time fuses the recombinant epitope gene of O-type FMDV into the SVA cDNA to construct the recombinant foot-and-mouth disease antigen epitope The Seneca recombinant virus, which can express the fused foot-and-mouth disease B-cell epitope and T-cell epitope, and the expression product has good reactogenicity, and the pathogenicity of the recombinant virus is significantly reduced or even non-pathogenic to pigs It can significantly improve the biological safety of the strain; the prepared inactivated vaccine has good immunogenicity, and can effectively stimulate SVA neutralizing antibodies while also producing specific antibodies against FMDV, which can be used for Seneca virus and Control of one or more non-Seneca viruses.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Seneca recombinant virus, a recombinant vaccine, and a preparation method and application of the recombinant O-type foot-and-mouth disease virus epitope gene. 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 sympt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/41C12N15/42C12N7/01C12N15/85A61K39/125A61K39/135A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70A61P31/14C07K14/005C12N7/00C12N15/85C12N2770/32022C12N2770/32034C12N2770/32121C12N2770/32122C12N2770/32134
Inventor 郑海学杨帆曹伟军朱紫祥宁建刚马坤郝荣增张伟郑敏魏婷蒋保余田宏张克山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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