Analysising Complete Nucleotide Sequence Of Swine Vesicular Disease And Expression Of VP2 Gene In E.Coli

We can study deeply virus by researching and analyzing preliminary structure ofvirus. Protein expressed by technique of gene engineering is basic of diagnosisingSVDV. This study includes two sides of contents: 1.Analyzing complete sequence of swine vesicular disease virus(SVDV):Acquiring six overlapping DNA fragments from SVDV RNA by RT-PCR。Thesefragments were sequenced。The length of complete nucleotide sequence of SVDVHK'70 is 7 401nt,the length of ORF is 6 555nt,encoding polyprotein that is2185aa.Lengthes of 5'-NCR and 3'-NCR are 743nt and 103nt respectively,Lengthesof 1A,1B,1C and 1D are 207nt ,783nt ,714nt and 849nt respectively；Lengthes of2A,2B and 2C are 450nt,297nt,987nt respectively；Lengthes of 3A,3B,3C and3D are 267nt,66nt,549nt,1386nt respectively. Comparing and analysising ofhomologies of nucleotide and animo acid sequence by computer software.The DNAsequence homologies were 98.4% with SVDV J1'73,98.2% with SVDV H/3'76,98.2% with SVDV(Seechurn.P, et al),91.0% with SVDV NET/1/92,76.0%～80.4%withCVB1,CVB2,CVB3,CVB4,CVB5,CVB6;The amino acid sequence homoiogieswere 98.9% with SVDV J1'73,98.9% with SVDV H/3'76,99.0% with SVDV( Seechurn.P,et al ) 96.9% with SVDV NET/1/92,76.0% ～ 80.4% withCVB1,CVB2,CVB3,CVB4,CVB5,CVB6. 2.Expressing VP2 gene of SVDV in E. coli: The VP2 gene of Swine vesiculardisease was amplified by RT-PCR and nPCR. The amplified fragments were clonedinto the expression vector pGEX 4T-1. The insert position ,the size and the readingframe were right by PCR,restriction digestion and the sequence analysis of therecombinant plasmids. SDS-PAGE indicated that the recombinant plasmids couldexpress the VP2 gene of Swine vesicular disease. Western blot indicated that thepositive serum of SVDV could acted expressed protein.