摘要
目的筛选干扰素α(IFN α)启动子DNA结合蛋白,探讨IFN α基因表达调节的分子生物学机制。方法应用噬菌体展示技术,以IFN α启动子的聚合酶链反应产物作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行5轮“吸附-洗脱-扩增”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,随机挑选40个克隆,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IFN α启动子结合的肝细胞蛋白,筛选到17种与IFN α启动子具有结合作用的蛋白。结论多种具有不同生理、生化功能的蛋白与IFN α启动子具有结合作用。
Objective To investigate the interferon alpha regulation mechanisms by screening binding proteins of interferon alpha promoter by phage display. Methods PCR product of interferon-alpha promoter was incubated with a phage display cDNA library that expressed a library of human liver proteins on the surface of bacteriophage T7. Unbound phages were washed off and the phages bound to the interferon alpha promoter were amplified. Positive plaques were amplified by PCR and cloned into a pGEM-Teasy vector in order to perform DNA sequence analysis and subsequent computer blasting analysis. Results Positive phage-displayed proteins binding with interferon alpha promoter were enriched after five rounds of biopanning. We found that the following proteins were relevant to interferon alpha: mitochondrial ribosomal protein, chromosome clone, fibrinogen A alpha polypeptide, enoyl coenzyme A hydratase short chain, eukaryotic translation elongation factor 1 alpha, PI-3-kinase-related kinase SMG-1-like, xeroderma pigmentosum C group, and Homo sapien activity-dependent neuroprotector (ADNP). Conclusions Many proteins with different functions could bind with interferon alpha promoter.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第7期520-523,共4页
Chinese Journal of Hepatology
基金
第35批中国博士后科学基金(2004035283)
