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
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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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