Screening And Identification Of Interaction Cellular Proteins With Classical Swine Fever NS3Protein

Classical swine fever virus (CSFV) is the causative agent of Classical swine fever, a devastating disease of pigs worldwide which is classified as a notifiable disease by the OIE. There is a low titre in CSFV infected cells cultured in vitro but it has previously been recognized that the NS3protein has important role in the (cytopathic effect) CPE induced by the CSFV and there is a relationship between the expression of the NS3and the CPE occured in the porcine cell line. Meanwhile NS3protein of CSFV, Shimen isolate, has capacity of higher level of NS3expression quantity is necessary for its CPE. NS3of CSFV is a sign of pathopoiesis and is related to the pathogenicity of PK-15.To elucidate the interaction between CSFV NS3and host cell protein, yeast two-hybrid system was applyed to screening interactive proteins of swine umbilical vein endothelial cell. According to the experiments, the following results were obtained:The NS3gene fragment of classical swine fever virus was amplified by RT-PCR and confirmed by sequencing and restriction digestion. The NS3gene was then subcloned into pGBKT7to construct the bait vector according to the reading frame.The reconstructed bait vector pGBKT7-NS3was transformed into Y2H Gold yeast strain, its self-activation and toxic effect was tested by the phenorype assay. The expression of pGBKT7-NS3was analysed by western blot. As a result, the successfully reconstructed bait vector pGBKT7-NS3would not self-activication the reporter gene and no toxic effect to the yeast.which layed the foundation of using yeast two-hybrid screen protein interaction.It was to establish the yeast two hybrid swine vascular endothelial cells cDNA library, and then to a provide good experimental system for the research of swine fever virus gene function and pathogenesis provide. In vitro cultivation of swine vascular endothelial cells, total RNA were extracted from swine umbilical vein endothelial cells (SUVEC) by trizol reagent, mRNA was purified by Oligotex mRNA Mini Kit, and double-stranded cDNA(ds cDNA) was synthesized using SMART technique. The ds cDNA purified by CHROMA SPINTM TE-400Column and the linearized pGADT7-Rec vector were co-transformed into competent cells of yeast strain Y187. A SUVEC yeast two-hybrid cDNA library was constructed in yeast cells by homologous recombination. The library capacity was1.2×107cfu/mL with insert size ranging from400bp to2000bp, and the average length of inserts was approximately1000bp with the recombination rate of100%.These results showed that a high-quality cDNA library of SUVEC could be constructed, which could be used for screening host cell protein interaction with the protein of classical swine fever virus.Among screened eight known proteins. Among screened we obtained six proteins, including35SLC35B1, ATF4, ATP5A1, GMPR, EF2, and Parp8. With co-immunoprecipitation assays it is verified that NS3protein interact with the screening ATF4protein.