| Hepatitis C virus (HCV) is the main cause of acute and chronic liver diseases, including chronic active hepatitis, as well as liver cirrhosis and hepatocellular carcinoma in our country. HCV nonstructural proteins3 (NS3) contains 631 amino acids with molecular weight of 70 Kda and is an important cause of chronic liver disease leading to cirrhosis and hepatocellular carcinoma.Part I: To investigate the biological function of hepatitis C virus NS3 protein (HCV NS3), we used yeast-two hybrid to screen proteins in leukocytes which can interact with HCV NS3. The HCV NS3 gene was amplified by polymerase chain reaction (PCR) and HCV NS3 bait plasmid was constructed by using yeast-two hybrid system 3, then the vector was transformed into yeast AH109. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2 X YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu~His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-a-gal for selecting two times and screening.After extracting and sequencing of plasmid from blue colonies, we underwent analysis by bioinformatics. The results showed that among eighteen sequenced colonies, four colonies were eukaryotic translation elongation factor 2 (EEF2), two colonies were immunoglobulin A. light chain, one colony was B-actin (ACTB), one colony was ferritin, light polypeptide (FTL), one colony was mitogen-activated protein kinase kinase 3 (MAPKK3), two colonies were myosin IF (MY01F), one colony was interleukin 2 receptor, IL2RB), one colony was splicing factor, arginine/serine-rich 6 (SFRS6), one colony was cathepsin S (CTSS), one colony was 2'-5'-oligoadenylate synthetase-like (OASL), one colony was DAZ (deleted in azoospermia) associated protein 2 (DAZAP2), one colony was calponin 2 (CNN2), and one colony was a new gene. From this part of experiment, we successfully cloned the genes of HCV NS3 interacting proteins in leukocytes and the present results brought some new clues for studying the biological functions of HCVNS3 and associated proteins.Part II: In order to explore the new target genes transactivated by HCV NS3, we conducted microarray assay on the hepatoblastoma HepG2 and HepG2 transfected by NS3 expressive vector. The results will pave the way for elucidating the pathogenesis of HCV infection. Sequence -specific primers were designed and synthesized according to the HCV-H strain of virus sequence. PCR was performed to amplify the NS3 coding gene for the construction of expression vector pcDNA3. 1-NS3. Hepatoblastoma cell line HepG2 was transfected with plasmid DNA of pcDNA3. 1-NS3, andtotal RNA was extracted from it. Reverse transcribed cDNA were subjected for microarray assay. The coding gene trasactivated by HCV NS3 was cloned by bioinformations methods. The results showed that the expression vector has been constructed and approved correct. The RNA has been purified from HepG2 and HepG2 cells transfected with pcDNA3-NS3, respectively. The cDNA derived has been subjected for microarray assay. New gene named NS3TP6 has been cloned in combination of molecular biological and bioinformatics methods. Our results indicated that HCV NS3 is a potential transact ivator and microarray is an efficient and convenient method for analysis of differentially expressed genes. A new gene has been recognized as the new target transactivated by HCV NS3 protein. These results will help to study on the transactivation of HCV NS3 protein.To investigate the biological function of NS3TP6, a gene transactivated by non-structural protein 3 (NS3) of hepatitis C virus (HCV), we performed yeast-two hybrid to screen proteins in leukocytes interacting with HCV NS3TP6. The HCV NS3TP6 gene was amplified by PCR and HCV NS3TP6 bait plasmid was constructed by using yeast-two hybrid system 3, then the constructed vector was transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing leukocytes cDNA library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic drop...
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