Screening proteins in hepatocytes interacting with the middle surface protein of hepatitis B virus using the yeast-two hybrid technique / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVES</b>To screen proteins in hepatocytes interacting with hepatitis B virus surface antigen middle protein (MHBs) with yeast-two hybrid technique for studying the biological functions of MHBs.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used to amplify the gene of MHBs from the plasmid A7 containing the whole fragment of adr subtype of HBV and the PCR product was cloned into pGEM-T vector and then evaluated by sequencing. The gene of MHBs was cut by EcoRI and BamH I from pGEM-T vector and then cloned into the yeast expression plasmid pGBKT7. MHBs bait plasmid was constructed by ligating MHBs gene with yeast expression vector pGBKT7 with yeast-two hybrid system 3 and then was transformed into yeast AH109 (a type). The transformed yeast cells were mated with yeast Y187 (alpha type) containing liver cDNA library plasmid 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 and screening. After extracting and sequencing the plasmid from true positive blue colonies, the results were analyzed by bioinformatics.</p><p><b>RESULTS</b>A pGBKT7- MHBs yeast expressed vector was successfully constructed. Two colonies were sequenced. One colony was Homo sapiens aldolase B fructose-bisphosphate, the other was a new gene with unknown function, which was named MHBs-binding protein 1.</p><p><b>CONCLUSION</b>MHBs gene was successfully cloned. Two genes of MHBs interacting proteins in hepatocytes were obtained by yeast-two hybrid system 3. Our results brought some new clues for studying the biological functions of MHBs and the mechanisms of HBV carcinogenesis.</p>