The Study On Genetic Variation And In Vitro Expression Of Gene E2 Of Classical Swine Fever Virus

The E2 gene of CSFV C strain derived from rabbit spleen tissue was amplified by reverse transcription (RT) and nested Polymerase Chain Reaction (nPCR), then was cloned and sequenced. Compared with the published sequences of CSFV other virulent strains, the nucleotide homology of C strain from rabbit spleen tissue with strain C adapted to SK6 cell, Shimen, Brescia, Alfort strain were 98.87%, 94.58%, 91.00% and 80.78% respectively, and the amino acid homology were 98.95%, 94.22%. 91.60% and 89.23% respectively. There were little difference in the amino acid composition of antigen domain of A, B and C between C strain from rabbit spleen tissue and virulent strains above. This indicated that the prevalence of CSF and immunal failure may be not caused by the antigen variation of C stain vaccine virus.The E2 genes of 51 CSFV prevalent virulent strains isolated from 12 provinces in China were amplified and sequencd. The phylogenetic tree was constructed based on sequence comparison and analysis. The result showed that C strain derived from rabbit spleen tissue, C strain adapted to SK6 cell, Shimen virulent strain all belonged to group 1.1, and the prevalent virulent strains belonged to subgroup 2.1 or subgroup 2.2. This indicated there were at least two subgroups, which were prevailing in China, and the prevalent virulent strains were diverse far from C. strain vaccine virus.The E2 genes above of the prevalent Strain (Guangxi Yulin Strain) were cloned respectively into secreted expression vector pPIC9K of eukaryotic expression system P.Pastoris and transformed into P.Pastoris by electroporation after linearization, 25 high-copied transformants were obtained by G418 screening. It was proved that the E2 genes were integrated stably into chromosome of P.Pastoris by Dot blot and DNA sequencing. The results of SDS-PAGE and Western blot showed that the culture supernatant of transforments contained the correct glycosylated E2 protein and the recombinants could still secretedly express the specific proteins after culturing for 8generations. Studies on immune activity showed that the E2 protein expressed in P.Pastoris could induce animals to produce CSFV specific antibodies, the titre amounts to 1:128-1:256. This suggests proved that the E2 protein could protect animals against virulent virus of CSFV. Its the first time that the CSFV E2 protein was expressed successfully in eukaryotic expression system P.Pastoris.In order to improve the expression level of E2 gene in P.Pastoris, 24 low -usage codons for P.Pastoris were substitued by directed mutagnesis based on PCR. The optimized E2 gene was cloned into P.Pastoris expression vector pPIC9K and transformed into P.Pastoris SMD1168 host cell. The transformants were induced with methanol to express the interested gene .The results of SDS -PAGE and Western blot showed that E2 protein was expressed at high level, up to 1.08g/L.The expression conditions of E2 gene in P.Pastoris were optimized, the results indicated that the peak obtained after 72 hours;pattern of inhibition/ induction could improve expression level; The best pH value were between 7.5 and 8.0 and the optimized methanol-induced concentration was 2%-3%The E2 genes of the prevalent Strain (Guangxi Yulin Strain) and C strain derived from rabbit spleen tissue were amplified and cloned into E.coli the expression vector pPROEX-HTb respectively, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained and then were transformed into the DH5a E.coli competent bacteria respectively, the recombinant bacteria could express the major antigen region of E2 gene, the expression yields amount to 35% and 38% repectively.The signal peptide sequence which was used for secretedly expressing divergent gene in mammary gland cells was ligated to the E2 gene, the E2 gene with signal sequence was obtained by PCR .The gene resisting kanamycin was cut down from pGFP-Cl vector and inserted into P22 vector, a P22 vector with gene resisting kanamycin was constructed, it was tried to construct an expression vector for trans...