[Screening and cloning of the genes of protein interacting with the N-terminal protein of hepatitis B virus DNA polymerase by yeast-two hybrid technique].

Abstract:

OBJECTIVE: To screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase. METHODS: TP was amplified by polymerase chain reaction (PCR) and TP bait plasmid was constructed by using yeast two-hybrid system 3, then transformed into yeast AH 109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) and that containing X-alpha-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics. RESULTS: Forty-seven clones were obtained, these clones included human P36956 sterol regulatory element binding protein-1, RNA polymerase II subunit hsRPB7 mRNA, asialoglycoprotein receptor 2, transcript variant 3, ceruloplasmin (ferroxidase), transmembrane 4 superfamily member 2 and 19 of the hypothetical proteins and so on. CONCLUSION: Genes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.

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