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

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

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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Getting of the A-type mouth-of-mouth disease virus VP1 gene

[0093] The A / GDMM / 2013 poison is designated by the Agricultural Veterinary Bureau to designate the national foot-and-mouth disease reference laboratory, and the public can be obtained through the entrustment letter instructions from the Veterinary Bureau of the Ministry of Agriculture. The primer of the A / GDMM / 2013 strain (GeneBank: KF450794) is designed to synthesize an amplified VP1:

[0094] AVP1-F0:

[0095] 5'-gatgcaatcaggcgcgtcgagaccaaccctggcccttructgaccccccccgggguaa-3 '(seqid No.3);

[0096] AVP1-F:

[0097] 5'-CCCA gcatgct TccctTTCGCAGCTACAAGCAGAGATGCTGATGCAATCAGGCGACGTC-3 '(SEQ ID No.4);

[0098] AS-R: 5'-CaggigggttgtcagaagcgggtccagggtTggACTCAAC-3 '(SEQ ID No.5).

[0099] In the above specific primers, the upstream primers AVP1-F used in the AVP1 fragment, and the SPHI enzyme digestion and SVA gene sequence were introduced, and the downstream primers were AS-R for amplification and SVA-S20. Fusio...

Embodiment 2

[0102] Construction of Seneca virus infective cloning of recombinant Type Apertureh disease virus VP1 gene

[0103] The SVV / FJ / 001 strain used is deposited in China Typical Culture Warehouse (Microorganism Number: CCTCC NO: V201802), (disclosed in the authorized patent "Seneka Vaccine and its preparation method and application" ZL201810003888.2 The full text is incorporated herein by reference in its entirety in this application, design synthetic amplification primers according to SVA genomic sequences (GeneBank: KY747510):

[0104] SVA-1F0:

[0105] 5'-gtgaggacgaaActataggaaaggaattcctatagtcttgaaaggggggctggccc-3 '(seqid No.6);

[0106] SVA-1F: 5'-Ataggt TTAATTAA TGTTAAGCGTGATGATGAGTCCGTGAGGACGAAACTATAGGA-3 '

[0107] (SEQ ID NO.7);

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

[0109] SVA-2F1: 5'-gttgagtccaaccctGacccgcttctgacaacccgatcctg-3 '(SEQ IDNO.9);

[0110] SVA-2R: 5'-TTTTTCTAGA GCGCCGC t 38 -3 '(SEQ ID NO.10);

[0111] SVA-M5UTRF: 5'-gttcctagecctcgtt...

Embodiment 3

[0120] Rescue of Seneca viruses in recombinant A-type mouthpiece disease vp1 gene and cultivation of different cells

[0121] 3.1 Rescue of recombinant Seneca virus

[0122] use Plasmid Plus Maxi Kit (QIAGEN) was prepared from the recombinant plasmid PRSVV / FJ-M-AVP1 obtained in Example 2, which is used for transfection when BHK-21 cells grow to 80%, in liposome LIPOFECTAMINE TM The BHK-21 cells were transfected with 4 μg of recombinant plasmids under the mediated mediated mediation, and a liposome control and normal cell control were placed in 5% CO. 2 In the 37 ° C incubator, the supernatant was discarded after 6 hours, and the MEM medium was added, and the cell state and cytopathic changes were continued. When the cells occurred in the cells, the virus was harvested, repeatedly frozen 3 times BHK-21 cells are inoculated until the virus can stably produce cytometry, and there is a cytometry, fall off, gradually forming air spots, and disintegrates the lesion. The resulting rec...

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Abstract

The invention provides a Seneca recombinant virus of recombinant type A foot-and-mouth disease virus VP1 gene, a recombinant vaccine strain, a preparation method and application thereof, and relates to the technical field of genetic engineering. The invention provides Seneca virus recombinant nucleic acid, Seneca recombinant virus containing said recombinant nucleic acid, Seneca recombinant vaccine strain containing said Seneca recombinant virus, and preparation method and application thereof. The present invention transforms SVV / FJ / 001 strain gene deletion mutation, and fuses the VP1 gene of type A FMDV into its cDNA to obtain Seneca recombinant virus, which can express the fused gene, and the expression product has good Reactogenicity: The obtained vaccine strain has high antigen production capacity, significantly reduced pathogenicity and even no pathogenicity to pigs. After immunizing animals with inactivated vaccines, it can not only stimulate the immune response of SVA, but also produce immune activity against the fusion gene, which can be used For the control of Seneca virus and one or more non-Seneca viruses.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and more particularly to a recombinant VP1 gene of recombinant A-type open-and-mouth disease virus VP1 gene, a recombinant vaccine, and a preparation method and application thereof. Background technique [0002] Seneca virus A (Senecavirus A, SVA), also known as Senecavirus, Seneca Valley Virus, SVV, belonging to the small RNAviridae Seneca virus (Senecavirus) ), The only member of the belonging. The viral infection pigs can cause primary blisters of pigs, and it is difficult to distinguish between clinical symptoms caused by foot-and-mouth disease, pigmentation and blistersqueral inflammation. After SVA infection, the pigs can cause blisters in all ages of weaning pigs, conservation, fattening and reproduction. At the same time, with clinical symptoms such as lame, fever, anorexia, lethargy, and newborn pigs may have sustained diarrhea in addition to the above symptoms. , Dehydration and ot...

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
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/85C12N2770/32021C12N2770/32034C12N2770/32052C12N2770/32122C12N2770/32134C12N2800/107
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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