Patent ID: 11883482
Assignee: LANZHOU VETERINARY RESEARCH INSTITUTE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES
Field: Pharmaceuticals (Chemistry)
Classification: CPC A  C | IPC A  C

Claim 4:
5. A method of constructing the recombinant Seneca valley virus according to claim 3, comprising:
(1) using a cDNA of SW/FJ/001 strain as template, a S1 fragment and a S2 fragment of SW/FJ/001 strain are generated using specific primer pairs respectively; the specific primer pair for amplifying the S1 fragment comprise two forward primers and a reverse primer SVA-1R, wherein the forward primers comprises SVA-1F0 and SVA-1F; the nucleotide sequence of the forward primer SVA-1F0 is set forth in SEQ ID NO:2, the nucleotide sequence of the forward primer SVA-1F is set forth in SEQ ID NO:3, and the nucleotide sequence of the reverse primer SVA-1R is set forth in SEQ ID NO:4;
a specific primer pair for amplifying the S2 fragment comprises a forward primer SVA-2F and a reverse primer SVA-2R, wherein the nucleotide sequence of the forward primer SVA-2F is set forth in SEQ ID NO:5, and the nucleotide sequence of the reverse primer SVA-2R is set forth in SEQ ID NO:6;
(2) ligating the S1 fragment and S2 fragment with pMD20 T vector respectively to obtain subclone plasmids PMD-S1 and PMD-S2;
(3) using the subclone plasmid PMD-S1 as a template, amplifying the PMD-S1 with mutation primers SVA-m5UTRF and SVA-m5UTRR to obtain subclone plasmid PMD-mS1, wherein the nucleotide sequence of SVA-m5UTRF is set forth in SEQ ID NO:7, and the nucleotide sequence of SVA-m5UTRR is set forth in SEQ ID NO:8;
(4) after digesting the plasmid PMD-mS1 with PacI and SphI, and digesting the plasmid PMD-S2 with SphI and NotI, collecting the gene fragments and inserting into eukaryotic transcription plasmid prO/CHA/99 digested with PacI and NotI, thus obtaining the recombinant plasmid prSVV/FJ-M; and
(5) transfecting the Seneca valley virus sensitive cells with the obtained eukaryotic transcription plasmid prSVV/FJ-M to obtain the recombinant Seneca valley virus.