Cloning Of E2 Gene Of Bovine Viral Diarrhea Virus Strain Of China And Study On Antigenic Specificity Of Recombinant E2 Protein

The E2 gene of two Chinese bovine viral diarrhea virus (BVDV) isolates, cc!84 and ZM-95, was amplified by RT-PCR and nest-PCR. The PCR products were then cloned into pGEM-T-Vector followed by sequencing. The results showed the fragment of E2 gene of two isolates is 1122bp for cc!84 and 1125bp for ZM-95. These two strains are classified into pestivirus type I as identified by homological analysis of the genes using the DNASIS software. Strain cc!84 and strain Osloss, between which share 91.6% identity, are divided into to subtype Ib. Strain ZM-95, Strain NADL and CV24, among which share 69.9% identity, are divided into subtype la. The deduced amino acid sequence showed that glycoprotein E2 of of strain ZM-95 has an insertion of histidine at 55th amino acid residue site and has three lysine residues at this region caused by an A and AG insertions at its 167th-176th nt of the nucleotide acid sequence while other strain of BVDV not.Two prokaryotic expression plasmids, pET28a-BE2 and pBV220-BE2, were constructed by sub-cloning the E2 gene of strain Cc 184 into pET28a(+) and pBV220, respectively. The E2 was expressed inductively in pET28a-BE2-transformed BL21(DE3)pLysS and pBV220-BE2-transformed DH5a resulting in the yield of 6.25%and 3.28% of the total bacterial protein, respectively. However, when the transmembrane sequence of E2 gene was deleted from the two plasmids the expression yield of the E2 was increased to 35.7% and 8.96% of the total bacterial protein, respectively.Rabbits were immunized with the purified recombinant E2 protein and hyperimmuno-serum was prepared from the immunized rabbits. There is no46cross-reactivity between the recombinant CSFV E2 protein and the hyperimmuno-serum against the recombinant BVDV E2 and as well as between the recombinant DVBV E2 protein and the hyperimmuno-serum against the recombinant CSFV E2 as demonstrated by indirect ELISA. The results indicated there is the possibility that indirect ELISA with those two recombinant protein as antigen can differentiate the antibody of pigs infected with either CSFV or BVDV.