Screening Of Classical Swine Fever Virus NS2 Interaetive Proteins In Swine Umbilical Vein Endothelial Cell Line By Yeast Two-hybrid

Classical swine fever (CSF)which caused by classical swine fever virus(CSFV), was classified as a notifiable (previously list A) disease by the OIE. Because CSFV is difficult to cultivate in vitro and does not cause cytopathogenic effect, most researchers focus on the development of detection methods and novel vaccines at home and abroad,only few pay attention to the molecular pathology of CSFV. NS2 protein is one of non-structural protein of CSFV. Available information indicates that, NS2 protein play certain regulatory role in the replication of CSFV by the way to interact with the host cell's proteins.NS2 also has protease activity, which can enzymolysis NS2-3 to relaes NS3 monomer and induces accumulation of NS3 monomer in host cell and thus casues cytopathogenic effect. But it is still not very clear to that how NS2 interact with the cell and regulate the replication of viral RNA.To elucidate the interaction between CSFV NS2 and host cell's, yeast two-hybrid technology was adopted to screening interactive proteins of swine umbilical vein endothelial cell(SUVEC). From the experiments, the following results were obtained:(1)The carboxyl terminal 360 bp fragment of NS2 was amplified by PCR using plasmid pEGFP-NS2 as a template to construct the pGBKT7-NS2-C360 bait vector which has non self-activation and toxicity to yeast cell .(2)Isolated and purified double strand cDNA successfully from swine umbilical vein endothelial cell to construct the prey library.(3)Co-transformed yeast competent cells with pGBKT7-NS2-C360 bait vector, double-strand cDNA and prey vector pGADT7-Rec, we obtained 16 positive clones after screening.Throught PCR ,sequencing and alignment analysis,8 known protein were obtained, namely: cytosol nonspecific dipeptidase (CNDP2), heat shock protein 40 (Hsp40), CFTR associated ligand (CAL), E3 ubiquitin-protein ligase, ATP synthase 6 (ATPase6), NRAS-related protein, modulator of apoptosis 1(MOAP1)and ribonucleoprotein H1.Among screened eight known proteins, Hsp40 protein,which was recorded that involvs in regulation of viral RNA replication,has high reproducibility relatively. Therefore, the Hsp40 protein will be emphasised in further study.