摘要
目的 :通过筛选乙型肝炎病毒 (HBV)核心启动子 (corepromoter ,CP)结合蛋白 ,为HBV复制机制和调控机制的研究探索新的途径。方法 :应用噬菌体展示技术 ,以HBV的核心启动子的聚合酶链反应 (PCR)产物作为固相筛选分子 ,对噬菌体人肝细胞cDNA文库进行 4轮“吸附 -洗脱 -扩增”筛选过程 ,经噬斑的PCR扩增后 ,构建克隆载体 ,最后对所筛选克隆进行DNA序列分析和同源性搜索。结果 :噬菌体经富集后 ,随机筛选得到 7个阳性克隆 ,成功构建了克隆载体。序列测定后经过同源性搜索 ,确定了和HBV启动子结合的肝细胞蛋白基因序列 ,分别编码 β2微球蛋白和 3种未知功能蛋白。 结论 :用噬菌体人肝cDNA文库筛选得到了与HBV核心启动子结合的蛋白 ,分析了该蛋白的编码基因 。
Objective:The function of core promoter(CP) of hepatitis B virus (HBV) is complicated. To screen the HBV CP DNA-binding protein, and to investigate their potential role in the regulation of replication and expression of HBV DNA. Methods: By using HBV biotinylated CP promoter DNA as the selective molecule, the T7 select phage human liver cDNA library was biopanned and positive clones were selected. After screening, positive plaqueswere performed to amplify for inserted DNA fragment and cloned into pGEM-Teasy vector.Results:Positive plaques were obtained for DNA sequencing. They encode 4 proteins respectively. They are β 2 microglobulin and three kinds of homo sapiens hypothetical protein. The binding protein of HBV CP DNA wasidentified by phage display screening. Conclusion: These results suggested that these proteins could bind to CP DNA. These proteins need to befurther studied and this approach development a new way to the study of the molecular biology mechanism between HBV and hepatocyte.
出处
《中西医结合肝病杂志》
CAS
2004年第4期209-211,215,共4页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金资助项目No:C03011402
C30070689
军队回国留学人员启动基金项目No:98H038
军队"十五"科技攻关青年基金项目No:01Q138
军队"十五"科技攻关面上项目No:01MB135
