[Screening of HBeAgTP interacting proteins in hepatocytes with yeast-two hybrid technique].
OBJECTIVE
To screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) with yeast-two hybrid technique for investigating the biological functions of HBeAgTP.


METHODS
Suppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HBeAg. The HBeAgTP gene was amplified by polymerase chain reaction (PCR) and HBeAgTP bait plasmid was constructed with yeast-two hybrid system 3, and then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver 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-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics.


RESULTS
HBeAgTP gene was successfully cloned and expressed in yeast cells. Fifteen genes in twenty-four positive colonies were obtained using yeast-two hybrid technique.


CONCLUSIONS
HBeAgTP conjugated protein genes were successfully cloned, along with the genes involved in transcription and translation of proteins, immunoloregulation, materials and energy metabolism in vivo.
Wang
Jian-Jun
JJ
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, Beijing 100039, China. awjia@126.com
Cheng
Jun
J
Liu
Min
M
Yang
Qian
Q
Lin
Shu-Mei
SM
Liu
Yan
Y
D006513:Hepatitis B e Antigens / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000276:immunology; D022781:Hepatocytes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D006513:Hepatitis B e Antigens; D011498:Protein Precursors