PubMed · 12472956
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S K Sarin, S K Satapathy, R Chauhan. 2002. Hepatitis B e-antigen negative chronic hepatitis B.. https://doi.org/10.1046/j.1440-1746.17.s3.20.x
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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. CONCLUSION: 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.
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