Cloning And Procaryote Expression E2 Gene Of Classical Swine Fever Virus

The paper has two parts, one is nucleotide different in the main antigen domain E2 gene of the C-strain and the Guangxi prevalent strains classical swine fever virus (CSFV); the other is expression of the major antigen region of E2 gene of CSFV in E.coli, the fused E2 protein was highly expressed in E.coli prokaryotic expression system in the experiment -s and provided the basic materials for the CSF diagnose.(1) The E2 gene of three prevalent virulent strains of CSFV from Guangxi China were amplified by reverse transcription(RT) and the nested polymerase chain reaction (nPCR). The amplifed E2 fragments of three CSFV strains were all 1170bp in length by agarose gel electrophoresis.Three E2 fragments were cloned respectively into PMD-18T vector. 1170bp cDNA fragment of three prevalent virulent E2 gene were sequence and based on the amino acid sequences of Brescia and Alfort strain 384 residues amino acid sequences of E2 were deduced. The nucleotide homology among three prevalents virulents were 90.10% to 98.54 %;and that of amino acid were 89.59% to 97.92%. The nucleotide identity of C- strain from spleen tissue of rabbit with that of three Chinese prevalent virulent strains were 82.87 %to83.99%,and that of amino acid were 86.98% to 90.10%. There revealed some differences in gp55 of C-strain and the 3 prevalent virulent strains.(2)The major antigen region E2 gene of CSFV (Guangxi Yuling Strain) and Chinese C-strain virus derived from hog and rabbit spleen tissue, was amplified by RT-PCR and nPCR.After the amplified fragments were cloned into the expression vector pPROEX-HTb, The recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained. The insert position , the size and the reading frame were right by PCR,restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the reciepient germs transducted and induced by the recombinant plasmids pPROEX-GXYL and pPROEX-C could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of CSFV. The content of the expressed protein in the induced bacteria protein was 35 % and 38 % respectively.