Screening and cloning of hepatitis C virus non-structural protein 4B interacting protein gene in hepatocytes

Abstract

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. 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. 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). 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.