Expression Of Envelope Protein Gene E0 Of Classical Swine Fever Virus And Prepartion Of The Polyclonal Antibody Against E0

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), which show high fever and extensive hemorrhagic lesions of tissues and organs in clinical.According to the published sequence of Classical swine fever virus (CSFV) HCLV strain E0 in Genbank, a pair of specific primers was designed. Envelope gene of CSFV HCLV strain were amplified by Reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGEM-T-easy vector. Sequence analysis results indicated that the homology between these two sequences was 99.9%.The fragment of E0 was respectively subcloned into the prokaryotic expression vector pET32a(+), and transformed into the competent cells of BL21 (DE3) E. coli. After 4h incubation, expression of the E0 protein was induced by 1.0mmoL IPTG. at 37℃for 5 hours. After brief sonication and centrifuge, the supernatant of the cell lysate was collected for SDS PAGE analysis. Results showed E0 gene was high-level expression and existed in inclusion. The expression product was soluble by lowering temperature to 25℃. E0 expression product of CSFV could react with CSFV antiserum from swine in Western blot analysis.The fragment of E0 was subcloned into baclovirus tranfer vector pFastBac1 to produce pFastBac1-E0. Then the pFastBac1-E0 was transformed into DH10Bac E.coli cells by site-specific transposition. E0 gene was integrated into Bacmid and produce recombinant shuttle vector,which was named Bacmid-E0. The recombinant baculovirus, designated as reBac-E0, was obtained from Sf9 cells transfected with the shuttle vector Bacmid-E0 mediated by lipofectin. rBac-E0 was expressed in the Sf9 cells at 96h post-infection with reBac-E0, which was identified by IFA and Western blot with the sera from the mice immunized with E0 ptotein from the minced gel of SDS-PAGE. These results suggested that the E0 expressed protein could be used to develop monoclonal antibodies and investigate the characterization of CSFV.