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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 screen interferon α (IFN α) promoter DNA binding protein and investigate the molecular biological mechanism of IFN α gene expression. Methods Phage display technology was used to carry out 5 rounds of “adsorption-elution-amplification” process of the phage human hepatocyte cDNA library by using polymerase chain reaction product of IFN α promoter as solid screening molecule. After plaque PCR amplification Afterwards, the cloning vector was constructed. Finally, DNA sequencing and homology search of the selected clones were carried out. Results After phage enrichment, 40 clones were randomly selected and cloned into vector. After sequencing, homologous search was performed to identify the hepatocyte proteins that bind to IFNα promoter. Seventeen IFNγ promoter Protein with binding. Conclusion A variety of proteins with different physiological and biochemical functions have a binding function with IFNα promoter.