Construction Of Baculovirus Transfer Vector Containing E2 Gene Of CSFV And Researches About Apoptosis Of Porcine Kidney Cells Induced By CSFV

Classical swine fever virus (CSFV) is a member of the genus Pestivirus and belongs to the family Flaviviridae. The CSFV genome has a single positive strand of nonpolyadenylated RNA, approximately 13 kilobases in length, and includes a single large open reading frame. CSFV causes an economically devastating disease in swine and many countries employ surveillance and/or eradication programs to limit infection. The disease is characterized by a peracute, acute, subacute, chronic, atypical, or unapparent course. So, a novel, effective gentic engineering vaccine against CSF and elucidating the pathogenesis of CSF will be beneficial for control and eradication program of CSF.E2 glycoprotein of CSFV,which is the protecting antigen and induces the neutralizing antibody during the infection of CSFV,is 1119 base pairs in length and encodes 373 amino acid residues. In this study, two baculovirustransfer vectors pACWZE2 and pACSME2 were constructed. The completed E2 gene of Guangxi Wuzhou (GXWZ), SHIMEN (SM) strain of CSFV were amplified by Reverse transcription-polymerase chain reaction (RT-PCR) and then cloned into the pMD18-T plasmid. The generated recombinant plasmid designated as pMDSME2 and pMDWZE2 were identified harboring E2 completed gene by PCR , restriction endonuclease digestion and sequencing. The nucleotide sequences of these two fragments were sequenced and the amino acid sequences were deduced. Compared with the corresponding region of standard SM and GXWZ strain of CSFV, the nucleotide sequence homology is 99.1% and 99.5 % respectively, and the amino acid sequence homology is 99.2% and 99.5% respectively. The complete coding region of E2 gene of SM and GXWZ was then subcloned into baculovirus transfer vector pACSG2. The recombinant baculovirus transfer vector containing E2 gene in correct orientation were designated as pACSME2, pACWZE2 and identified by restriction endonuclease digestion.Furthermore,apoptosis of the porcine kidney cell line PK-15 % PK-IBRS2 cells infected with hog cholera lapinized virus (HCLV), GXWZ, SM strain of CSFV in vitro was studied in this paper. The cells were collected at different time post-infection and analyzed by DNA agarose gel electrophoresis. No typical DNA ladders were observed and there were no significant difference between the negative control and cells experimentally infected with CSFV. This suggests that GXWZ, SM, HCLV strain of CSFVinduces no typical apoptosis in the host cells of PK-15N PK-IBRS2 in vitro. These results above will lay the foundation for expressing E2 gene in the baculovirus expression vector system, studying the function of E2 gene and researching CSFV recombinant subunit vaccine and provide the theratical knowledge of the cytopathgencity effect and pathogenesis of CSFV.