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目的:通过筛选HBsAg基因启动子Ⅰ(SP Ⅰ,surface promoter Ⅰ)结合蛋白,为HBV复制机制的研究探索新的途径. 方法:应用噬菌体展示技术,以HBsAg基因启动子Ⅰ的聚合酶链反应(PCR)产物DNA作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行4轮“黏附-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,从随机筛选的14个克隆中得到8 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表面抗原基因启动子Ⅰ特异结合的肝细胞蛋白,共编码7种蛋白.其中3个克隆编码未知功能蛋白;其余的克隆分别编码4-氨基丁酸氨基转移酶、触珠蛋白、复制蛋白等蛋白. 结论:用噬菌体人肝cDNA文库筛选得到HBsAg基因启动子Ⅰ的结合蛋白,分析了该蛋白的编码基因.
OBJECTIVE: To explore a new approach for the study of HBV replication mechanism by screening HBsAg gene promoter Ⅰ (SP Ⅰ, surface promoter Ⅰ) binding protein.Methods: Using phage display technology, the polymerase chain reaction of HBsAg gene promoter Ⅰ PCR) product DNA as a solid-phase screening molecule, the phage human hepatocyte cDNA library was subjected to 4 rounds of “adhesion-elution-amplification” screening process. After plaque PCR amplification, a cloning vector was constructed. Finally, DNA sequence analysis and homology search were performed.Results: After phage enrichment, 8 positive clones were obtained from 14 randomly selected clones, and the cloning vector was successfully constructed.After homology search, HBV surface antigen gene promoter Ⅰ specific binding of hepatocyte proteins, encoding a total of seven kinds of proteins.One of the three clones encoding unknown protein function; the rest of the clones encoding 4-aminobutyric acid aminotransferase, haptoglobin, replication protein Protein.Conclusion: The binding protein of HBsAg promoter Ⅰ was screened by phage human liver cDNA library, and the gene encoding the protein was analyzed.