Cloning Of The Gene Of Protein Interacting With HCV Polyprotein And Functional Study Of Hcbp6

Cloning of the gene of protein interacting with HCV polyprotein and functional study of Hcbp6LiKe PhD CandidateTutor: Zhang Ling-xia Cheng JunHepatitis C virus (HCV) is a small enveloped RNA virus which causes acute and chronic liver disease, including chronic active hepatitis, as well as liver cirrhosis and hepatocellular carcinoma. HCV infects an estimated 170 million persons worldwide and thus represents a viral pandemic. Ten polypeptids produced by cleavage of the HCV H strain polyprotein, core, El, E2, P7, NS2, NS3, NS4a, NS4b, NS5a, NS5b, play important role in HCV replication and propagation. The pathogenesis of HCV is not clear. The polypeptides interacting with proteins of hepatocytes may be contribution to the diseases caused by HCV. To investigate the effects of HCV polypeptides on hepatocytes, we used yeast two hybrid system developed by Gietz in 1992 to clone the genes of proteins of hepatocytes that interact with peptides of HCV expressed by the plasmid of liver cDNA library.Plasmid pBRTM/HCV-1 containing full-length HCV cDNA(9401nt) was used to design polymerase chain reaction (PCR) primers for NS2(2769-3419nt), NS3(3420-5312nt) and core(342-914nt ) of HCV. The products of PCR were cloned into pGEM-T. After being verifyed it accurate, the sequences of the genesof HCV peptides was ligated into "bait" plasmid - pGBKT? and transformed into yeast strain AH 109. Then, using Western blotting to prove the expression of HCV polypeptides, we performed yeast two hybrid by mating AH 109 containing HCV genes with Y187 containing plasmids of human liver cDNA library. The diploidy cells was selected with quadro-dropout and triple-dropout media containing x- a -gal. After 2-3 weeks, the blue colonies were considered harboring the protein interacting with HCV peptides. The cDNA library plasmids were isolated and sequenced. The gene whose function has not been known was deposited in GenBank. In vitro transcription/translation and in vitro bingding assays and second yeast two hybrid assay was performed by using the interesting genes and the genes of HCV peptides to confirm the true interactions.The results showed that the proteins of Hcbpl (AY032593, HcbpG (AY032594), Hcbpl2 (AF395068) and translin protein could interact with HCV core protein. Translin protein plays an important role in certain lymphoma. The prevalence of lymphoma in patients infected with HCV was obviously higher than that in people without HCV infection, our report indicated a molecular mechanism that the interaction between HCV core protein and translin protein may trigger the B-cell progressing into lymphoma in patients infected with HCV. How the interaction between the HCV core protein and translin protein causes chromosomal translocation or rather, causes lymphoma, more experiments are necessary toelucidate it.In order to investigate the function of Hu Hcbp6, we firstly monitor the gene expression and protein localization in vivo and in situ by expression of the protein fused with green fluorescent protein in HepG2, COS-7, NIH 3T3 cells. We also found that this protein could stimulate the growth of HepG2, COS-7, NIH 3T3cells. Whether the protein is related to the tumorigenesis, more experiments are needed. The function of Hu Hcbpl2 is not clear.The successful cloning of these genes may be provide some clues for the pathogenesis of HCV, especially, Hu Hcbp6 that could facilitate the growth of cells, perhaps have relation with the tumorigenesis caused by HCV infection. The interaction between HCV core protein and translin protein could partly interpret the molecular mechanism of hepatocellular carcinoma and lymphoma.