噬菌体展示技术筛选人肠道病毒71型3A 蛋白相互作用蛋白

摘要

目的：利用 T7噬菌体展示技术筛选与人肠道病毒71型3A 蛋白相互作用的蛋白。方法构建3A 蛋白的原核表达载体，表达并纯化3A 蛋白，以3A 蛋白为靶蛋白，应用 T7噬菌体展示技术对人肝细胞 cDNA 文库进行筛选，对筛选到产物进行DNA 序列分析及同源性研究。结果表达并纯化了3A 蛋白，经过4轮筛选后，选择37个阳性克隆，采用 T7特异性引物扩增插入片段，将 PCR 产物送公司进行测序。经过同源性分析，确定了2个与人肠道病毒3A 蛋白相互作用蛋白。结论通过 T7噬菌体展示技术可以得到与3A 蛋白相互作用蛋白，通过研究此蛋白的功能就可以初步推测出3A 蛋白可能的功能，为进一步研究人肠道病毒的致病机制鉴定了基础。%Objective To screen and identify protein interacted with enterovirus 71 (EV71)3A protein by means of T7-phage display system.Methods The prokaryotic expression vector of 3A protein was constructed for expressing and purifying 3A pro-tein.With 3A protein as the target protein,the T7-phage display system was adopted to screen the human hepatocyte cDNA library. The screened products were performed the DNA sequence analysis and the homologous research.Results 3A protein was expressed and purified,after 4 rounds of biopanning,37 positive clones were selected.The inserted fragments were amplified by PCR using specific T7 primer.The products were sequenced,2 proteins were identified to interact with 3A protein by homologous analysis. Conclusion Trough the T7 phage display system,two proteins interacted with 3A are obtained.The possible function of 3A protein can be speculated by researching the function of these two proteins,which lays the foundation for the further studying the pathogen-ic mechanism of EV71.