Expression Of CSFV E2 Gene In PK-15 Cells And Autologous Animal Immunity Test

Classical swine fever (CSF) is a kind of acute and contagious swine disease, which does damage to swine industry. Classical swine fever virus (CSFV) can cause degeneration and necrosis in small vascular and capillary endothelial cells of swine, appearing disseminated hemorrhage in systemic organs and tissues of swine. CSFV belongs to genera Pestivirus of Flaviviridae family; its genome total length is 12.3kb, including a big ORF and encoding 11 structural and nonstructural proteins.The gp55 protein, encoded by CSFV E2 gene, is a major protective antigen of CSFV, and the best source of genes used for engineering vaccine at present. Based in affinities of genetic codes in gene expression, the E2 gene is expressed in PK-15 cells line to increase productions of recombinant proteins expressed on one hand, and the post-processing in PK-15 cells from swine has obvious priority contrast to other host cells on the other. Thus, the immunogenic and immunity protective capacity of the productions would be increased, which offers basis for putting CSFV gene engineering vaccine into actual application.This study consists of three parts. The first, E2 gene of CSFV shimen strain is cloned by reverse transcript PCR (RT-PCR) and nest PCR (nPCR), comparatively homology of nucleotide and amino acid sequence is analyzed, and the phylogenetic tree is rendered so as to acquire hereditary variation of CSFV shimen strain E2 gene during multiple passages, which offers molecular biology basis for CSF gene engineering vaccine. The second, the E2 gene encoding envelope protein into genome of PK-15 cells by retrovirus vehicle is integrated, and the E2 envelope protein on cells membrane of PK-15 cells is expressed. The third, Blab/c rats and pigs are immunized by PK-15 cells expressing E2 envelope protein, and pigs for virus-challenge test are vaccinated, thus establishing a basis for application of CSF gene to engineer vaccine.1. The E2 gene of CSFV shimen strain was cloned and amplificated by RT-PCR and nPCR, then cloned into pGEM-T Easy vehicle, and its nucleotide sequence was determinated. Homology of nucleotide and amino acid sequence was compared, and the phylogenetic tree was rendered. The results indicate that the length of CSFV shimen strain of the E2 gene tested was 1 170bp, encoding amino acid sequences, which all included a complete signal peptide sequence and a partial translation membrane sequence which was made up by 384 amino...