Prokaryotic Expression Of ISG15 And Polyclonal Antibody Preparation, And A Preliminary Study Of The Interaction Between ISG15 And CSFV

Classical swine fever virus (CSFV) is a pathogen for highly infectious diseases as swine fever swine, belonging to Flaviviridae pestivirus genus.It is a positive and single strand of RNA virus and can cause persistent infection in pigs. ISG15 is a kind of antiviral protein induced by type I interferon. Some studies shown that ISG15 possessed antiviral activity to some viruses, such as HIV-1, B-type influenza virus. In the previous study of our laboratory, gene chip was used to detect the gene expression profile changes in peripheral blood lymphocytes of classical swine fever virus infection pigs. The results showed that ISG15 mRNA were 2.63,4.51,4.43,2.18 folds change in peripheral blood lymphocytes of pigs post infection CSFV in 1,3,6,9 days. In this study,we further to verify if the expression of ISG15 protein is increased in CSFV infected PK-15 cells, and to explore the interaction between the ISG15 and CSFV in the host cell, laying the foundation for further discover the molecular mechanism of persistent infection of CSFV.Recombinant human interferon IFN a-2b induced PK-15 cell to transcript mRNA of ISG15 gene, then the ISG15 gene was amplified by RT-PCR, and cloned into pMD18-T vector. The ISG15 gene was subcloned into the prokaryotic expression vector pGEX-4T-1 and eukaryotic expression vector pCDNA 3.0. Prokaryotic expression vector pGEX-ISG15 was transformed into BL21 strain, expression of ISG15 recombinant protein under IPTG induced and purified by Ni-NTA resin. The purified recombinant protein ISG15 was emulsified with Freund's adjuvant and was immunized to mice for preparing anti-ISG15 polyclonal antibody. Eukaryotic expression vector pCDNA-ISG15 was used to transfect into PK-15 cells mediated liposome. to obtain The PK-15 cell line which the ISG15 protein is stably expressed was obtained with the antibiotic neomycin G418 selection.The ISG15 protein expression in PK-15 cells was dectected with Western-blot and indirect immunofluorescence analysis post CSFV infected 0,3, 6,12,24,48 and 60h. ISG15 protein increased expression in the CSFV infected PK-15 cells. Presumably ISG15 protein mediated of viral protein ubiquitination hydrolysis pathway is blocked by classical swine fever virus.