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目的 筛选与乙型肝炎病毒 (HBV)DNA聚合酶-N末端蛋白 (TP)相互结合的肝细胞蛋白 ,进一步探讨TP的生物学功能。方法 用聚合酶链反应 (PCR)技术扩增TP片段 ,连接入酵母表达载体pGBKT7中构建诱饵质粒 ,转化酵母细胞AH 10 9并在其内表达 ,然后与转化了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合 ,在营养缺陷型培养基和X-α-半乳糖苷酶 (X-α-gel)上进行双重筛选阳性菌落 ,DNA序列测定并进行生物信息学分析。结果 成功获得了 4 7个与TP特异性结合的阳性克隆 ,包括人类固醇调节元件结合蛋白 1(SREBP1)、RNA聚合酶Ⅱ亚单位 (hsRPB7)、血浆铜蓝蛋白(CP)等 2 1种已知功能蛋白质基因和 19个假设蛋白基因。结论 HBVDNA聚合酶-TP可以与某些已知和未知功能蛋白相互结合 ,提示其具有较广泛的生物学功能
Objective To screen the hepatocyte proteins that bind to hepatitis B virus (HBV) DNA polymerase-N-terminal protein (TP) and further explore the biological function of TP. Methods The TP fragment was amplified by polymerase chain reaction (PCR) and inserted into the yeast expression vector pGBKT7 to construct the bait plasmid. The recombinant plasmid was transformed into yeast AH 10 9 and expressed in E. coli. Cells Y187 were mated to double screen positive colonies on auxotrophic medium and X-alpha-gel for DNA sequencing and bioinformatic analysis. RESULTS: Forty-seven positive clones that specifically bound to TP were successfully obtained, including 21 steroid regulatory element binding protein 1 (SREBP1), RNA polymerase subunit 2 (hsRPB7) and ceruloplasmin Known functional protein genes and 19 hypothetical protein genes. Conclusion HBVDNA polymerase-TP can interact with some known and unknown functional proteins, suggesting that it has a wider range of biological functions