Construction Of A Recombinant CSFV Expressing BVDV E2 Gene And Cloning, Expression Of Porcine IRF3/7

Classical swine fever (CSF), which causes by classical swine fever virus (CSFV), is a contagious and fatal viral disease of pigs. It is one of the major viral diseases which have serious effect on the pig industry in China. Bovine Viral Diarrhea Virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae and is closely related to the classical swine fever virus. BVDV can naturally infect pigs and causes the same clinical symptom with CSFV, and there is high rate of BVDV antigen positive of swine in China. It brings new problem for the control and eradication of CSF. In this study, we aim to generate a recombinant CSFV which expressed BVDV E2 gene by reverse genetics technique and study the molecular mechanism of inhibition of type I interferon by CSFV.First, different fragments of genome of CSFV were amplified by RT-PCR and PCR, and then cloned into the pMD18T vector. A full-genome cDNA of CSFV was generated by ligation these fragments together. Marker gene EGFP was inserted into the Npro gene of the full genome cDNA by fusion with Npro protein. Based on the full-length cDNA/EGFP, EGFP gene was replaced with BVDV E2 gene in order to construct a recombinant CSFV expressed the BVDV E2 protein. All the full-length cDNA constructs were flanked with T7 promoter and HDV/T7 terminor. The full-length cDNAs were transfected into PK-15 cells pre-infected with MVA-T7 which can express T7 RNA polymerase. A recombinant CSFV expressed EGFP was obtained but unstable.In order to study the molecular mechanism of inhibition of type I IFN by classical swine fever virus, some genes maybe related to it was cloned. Swine interferon regulatory factor 3 and 7 (IRF3/7) of those genes were expressed in E.coli, and rabbit against IRF3/7 polyclonal antibodies were obtained by immunization with purified IRF3/7 proteins. Reactionogenicity of IRF3 and IRF7 proteins was confirmed by western blotting. Indirect immunefluorescence and western blot demonstrated that expression of IRF3 protein in PK-15 cell was down-regulated by CSFV, but no effect on IRF7. It is very important to futher study of mechanisms of classical swine fever virus inhibit IFN synthesis.