Screening Of Proteins Binding Gene2 Tranactivated By Hepatitis C Virus NS3 Protein From Liver CDNA Library By Yeast Two-hybrid Technique

Background and objective:Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma. Up to date, HCV infects an estimated 170 million persons worldwide,but there is no effective way to cure these diseases,only interferon and ribavirin are used on to these related diseases and the curative effect is limited.The research on the mechanism of HCV is improtent to virus hepatitis treatment.Its RNA genome codes for a polyprotein,which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products,including Core,E1,E2,P7,NS2,NS3,NS4A,NS4B,NS5A,NS5B. The nonstructural protein 3 codes for 631 amino acids and is about 70 KD,It has many functions act as the serine proteinase,RNA helicase and NTPase,so it is essential for the reproduction of the virus and the artifaction of the polypeptide which coded by the virus.Dang xiaoyan and other scolars had screened and cloned the new target genes transactivated by hepatitis C virus (HCV) nonstructural protein 3(NS3) by suppression subtractive hybridization (SSH) technique,one of these the sequence was a new gene with unkown functions, e reverse transcription PCR(RT-PCR)was used to amplify the new gene, named as NS3TP2, from the mRNA of HepG2 cells ,and has been deposited into Genebank. The accession of NS3TP2 is AY116969.In order to study the pathogenesis of HCV and the biological functions of a novel HCV Non-structural protein 3 transregulated protein (NS3TP2), we vestigate the gene expression of HCV NS3TP2 in yeast and Screen the genes of proteins binding with NS3TP2 by yeast two- hybrid technique..Methods:NS3TP2 bait plasmid was constructed by ligating the NS3TP2 gene with yeast expression plasmid pGBKT7, then transformed into yeast cells AH109(a type). Theyeast protein was isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting.Thereafter, the transformed yeast cells were ampli- fied and mated with yeast cells Y187(atype) containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and containing x-α-gal for selecting two times. Plasmids of true positive blue colonies were extracted. Then we continue select the results above through the crossback experiment and make the extrem results analysed by DNA sequencing and blast in genbank.Results:HCV NS3TP2 protein was successfully expressed in yeast expression system,and its molecular is about 70KD.And through the yeast two-hybrid technique we obtain 6 pieces of gene, including Homo sapiens vacuolar protein sorting 13 homolog A, Homo sapiens acyl-CoA synthetase long-chain family member 5 (ACSL5), acyl-Coenzyme A binding protein , Homo sapiens ring finger protein 130, Homo sapiens mitochondrial DNA , Homo sapiens eukaryotic translation elongation factor 1 alpha 1.Conclusions :1. The bacterial-yeast shuttle plasmid pGBKT7-NS3TP2 was constructed successfully so that the bait protein of the two hybrid system was obtained.2. HCV NS3TP2 protein was successfully expressed in yeast expression system for the first time.3. Several positive clones were obtained through the two-hybrid system successfully.Amony the proteins which react with the NS3TP2 protein ,one type of them are some ones related with protein metabolism,the others are some ones related with fat metabolism.4. NS3TP2 transactivated by HCV NS3 may play a role in fat metabolism and protein metabolism, especially in the fat metabolism. it is presumed that it take a part in the change of fat metabolism which may induced by the HCV NS3 protein and the happeness of fatty liver disease. All these brought some new clues for studying the biological functions of the gene NS3TP2 and the pathogenesis of HCV.