The Polyclonal Antibody Preparation Of Classical Swine Fever Virus C And NS3 Protein & Preliminary Researching On The Interaction Between CSFV And IRG6 Of Porcine

Classical swine fever virus (CSFV) is the pathogen of classical swine fever, a highly contagious and lethal disease of swine. CSFV infection can lead to severe leukopenia, immune suppression, extensive thrombosis and endothelial injury in swine, thus bringing a great threat and economic losses to the swine industry. CSFV is a small-envelope and single stranded RNA virus, belonging to Pestivirus, Flaviviridae, with the genome about 12.5kb. It only has a open reading frame (ORF), and all the structural and non-structural proteins are encoded by the ORF. The encoded polyprotein has about 4000 amino acids, with the molecular weight of about 438 kDa, and can form 12 kinds of mature viral proteins under the processing and modification with cellular and viral proteases.Previous studies of our reseach group showed that the mRNA expression level of Inflammatory response gene 6 protein (IRG6) was in a very high level during the whole infection process of CSFV. Through the NCBI BLAST system, IRG6 was found to have a very high homology to 83.7% with viperin in other species in genetic structure, and the homology of the main functional area reached up to 90%. The structure and function of protein are interconnected and IRG6's high degree of homology with viperin in structure suggests IRG6's high degree of similarity with viperin in function. In the process of IRG6 infection, the high mRNA expression level of IRG6 also shows its close relationship with the CSFV.In this study, using the method of subcloning, the pET-C and pET-NS3 prokaryotic expression plasmids were constructed on the basis of the pET-32a(+) prokaryotic expression vector. pET-C and pET-NS3 were then transformed into e. coli Rosetta (DE3) for induced expression and C and NS3 recombinant proteins were successfully obtained. With the Ni2+ affinity chromatography, these C and NS3 recombinant proteins were purified and a soluble antigen was prepared with the renaturation of the recombinant protein NS3 expressing in the form of inclusion body. Protein polyclonal antibodies against C protein and NS3 protein were prepared with the mice immunized by C and NS3 recombinant proteins. The Western blot method was used to detect the protein polyclonal antibodies against C protein and NS3 protein, both of which can respectively have a good reaction with the recombinant proteinsC and NS3. The specificity and reactivity of polyclonal antibodies were further detected. And the protein polyclonal antibodies against C protein and NS3 protein could combine with the C protein and NS3 protein of the naturally constructed CSFV, with the optimal antibody concentration of 1:2000 and 1:2000 respectively. It was proved that both of the two prepared polyclonal antibodies have a good reactivity and specificity.The detection on PK-15 cells, the PK-15 cell line stably transformed with the peGFP eukaryotic expression vector, and the PK-15 cell line stably transformed with the pIRG6-eGFP eukaryotic expression vector, with the real-time fluorescence quantitative PCR, Western blot and IFA, showed that IRG6 gene decreased the levels of RNA, protein and titer of CSFV, possibly suggesting a kind of inhibition effect of IRG6 on the biological activity of virus replication.