RESUMO
OBJECTIVE: To investigate biological functions of non-structural protein 4B (NS4B) of hepatitis C virus (HCV), yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4B. METHODS: HCV NS4B bait plasmid was constructed by ligating the NS4B gene with carrier plasmid pGBKT7 and transformed into yeast cells AH109 (type alpha). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selecting two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent Escherichia coli and analysed by DNA sequencing and bioinformatics. RESULTS: Five genes in eight positive colonies were obtained. There were one NADH dehydrogenase subunit 3, one cytochrome c oxidase subunit III, one retinol binding protein 4, one reticulon 3-A (RTN3) and one fibrinogen gamma polypeptide (FGG). CONCLUSION: Genes of HCV NS4B interacting proteins in hepatocytes were successfully cloned and the results paved the way for studying the biological functions of NS4B and associated proteins.
Assuntos
Hepatócitos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismo , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Biblioteca Gênica , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
OBJECTIVES: To identify and clone human genes transactivated by HCV F protein by constructing a cDNA subtractive library using the suppression subtractive hybridization technique. METHODS: Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV F protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1 (-)-F or with pcDNA3.1(-) empty vector as a control, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 or adaptor 2. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 alpha. The cDNA was sequenced and analyzed in GenBank with blast search after PCR. RESULTS: The subtractive library of genes transactivated by HCV F protein was constructed successfully. The amplified library contains 71 positive clones. Colony PCR shows that 56 clones contain 200-1000 bp inserts. Sequence analysis was performed on 28 clones randomly, and the full length sequences were obtained with using the bioinformatics method. Altogether 19 coding sequences were obtained, consisting of 17 known and 2 unknown. CONCLUSIONS: The obtained sequences may be target genes transactivated by HCV F protein, and some gene coding proteins are those involved in cell cycle regulation, metabolism, and cell apoptosis.