Cloning And Expressing In Insect Cells Of Classical Swine Fever Virus E2 Gene

The paper compared the differation of E2 gene nucleotide of four reference strains and 9 prevalent strains classical swine fever virus (CSFV) ; the other is E2 gene of CSFV was expressed in insect cell．(1) The E2 gene of nine prevalent virulent strains of CSFV were amplified by reverse transcription polymerase chain reaction (RT-PCR) and the nested PCR(nPCR),Then them were cloned and sequenced, comparered with the published sequences of CSFV C-strain, Brescia,Alfort and Shime strain. The phylogenetic tree was constructed based on sequence comparion and analysis. The result indecated that there were at least two subgroups, GXNN, GXGL, LNAS , SDJN and C-strain, Brescia, Alfort, Shime strain all blonged to one subgrop. The nucleotide homology among them were 94.0% to 99.4%; and that of amino acid were 93.7% to 99.5%. HNLS, SXZZ, HNCA, HNXY and HNDX belonged to another subgroup. The nucleotide homology among them were 81.9% to 99.7％;and that of amino acid were 88.2% to 99.1%. The nucleotide homology of all the 13 strains were 81.9% to 99.7% also; and that of amino acid were 88.2% to 99.5%. It was showed epitopes of all strains were not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains．(2) Based on E2 gene sequence date, a fragment 1033bp in E2 gene (Guangxi Yuling Strain) cloned in plasmid pMD18-T was amplified by polymerase chain reaction(PCR).The E2 fragment was subcloned into baculovirus transfer vector and recombinant baculovirus vector pFBHT-E2 was extracted.Then it was transferred into E.coli DH10Bac and recombinant bacmid was constructed. The recombinant baculovirus was obtained after the tranferation of the recombinant bacmid into sf9 cell,Thetechniques of SDS-PAGE, Western-blotting and ELISA were used to detect and identify the products expressed in sf9 cell by recombinant baculovirus. The results indicated the E2 gene was properly expressed in sf9 cell, and the expressed protein could be recognized by the postive serum of CSFV.