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.
- Author:
Guo-feng CHEN
1
;
Lin WANG
;
Jun CHENG
;
Ling-xia ZHANG
;
Li LI
;
Jian ZHANG
;
Qing SHAO
;
Dong JI
Author Information
- Publication Type:Journal Article
- MeSH: Cloning, Molecular; DNA-Directed DNA Polymerase; chemistry; genetics; metabolism; Gene Library; Hepatitis B virus; enzymology; genetics; Humans; Liver; metabolism; Plasmids; genetics; Protein Binding; Receptors, Virus; genetics; isolation & purification; metabolism; Transformation, Genetic; Two-Hybrid System Techniques; Viral Proteins; chemistry; genetics; metabolism
- From: Chinese Journal of Experimental and Clinical Virology 2005;19(1):84-86
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase.
METHODSTP 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.
RESULTSForty-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.
CONCLUSIONGenes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.
