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目的筛选并克隆人肝细胞中与乙型肝炎病毒e抗原(HBeAg)相互作用的蛋白质基因。方法将重组诱饵质粒pGBKT7eAg转化酵母细胞AH109后与预转了人肝cDNA文库质粒的酵母细胞Y187进行配合,在营养缺陷型培养基上进行双重筛选阳性菌落,提取质粒后转化大肠埃希菌并进行序列测定,进行生物信息学分析。结果配合后筛选出既能在4缺(SD/TrpLeuAdeHis)培养基又能在铺有Xα半乳糖(Xαgal)的4缺培养基上生长并变成蓝色的真阳性菌落245株。完成了101株克隆的测定,经过序列分析排除了重复同源序列后,最终确定其中有41株不同的基因,其中人类同源基因35条,其余6株为未知基因。结论成功克隆出肝细胞中的HBeAg结合蛋白基因,为进一步研究HBeAg的功能提供了新线索。
Objective To screen and clone the gene of protein interacting with hepatitis B virus e antigen (HBeAg) in human hepatocytes. Methods Recombinant bait plasmid pGBKT7eAg was transformed into yeast AH109 and then mixed with yeast cell Y187 pre-transferred into human liver cDNA library. The positive colonies were screened on auxotrophic medium. The plasmids were extracted and transformed into Escherichia coli Sequence determination, bioinformatics analysis. As a result, 245 true-positive colonies that grew on 4-deficient (SD / TrpLeuAdeHis) medium and 4-medium-deficient Xαgal-plated medium and became blue were screened. 101 clones were sequenced, and 41 homologous genes were identified after eliminating the repeated homologous sequences. Among them, 35 were human homologous genes and the remaining 6 were unknown genes. Conclusion The successful cloning of HBeAg-binding protein gene in hepatocytes provides new clues for the further study on the function of HBeAg.