CP4-EPSPS转基因蛋白的适体筛选与亲和性分析

Screening and Affinity Analysis of the Aptamers against the Protein of CP4-EPSPS Gene

目的:通过筛选获得CP4-EPSPS转基因蛋白的最优适体,为应用于检测作准备。方法:体外合成长度为78个碱基的随机ssDNA文库,应用指数富集配基的系统进化(SELEX)技术,以CP4-EPSPS转基因蛋白为靶目标进行15轮筛选,将筛选到的适体群进行克隆、测序,用DNAMAN软件对每个适体的保守序列和二级结构进行分析并应用生物素-抗地高辛碱性磷酸酶显色系统进行亲和性分析。结果:成功获得靶目标的最优适体,即D04号适体,结合率最高(2.2745)为最低的5.9倍。结论:筛选流程合理、可行,实验结果较理想。

Objective:Aptamers against protein of CP4-EPSPS gene were screened.Method:An in vitro synthesized 78 mer random DNA library was subjected to 15 rounds of selection using SELEX method against CP4-EPSPS transgenic protein and the screened aptamers were cloned and sequenced.The conserved sequences and secondary structures of every aptamer were analysed.Using the DNAMAN package and the affinity was visualized by biotin-streptavidin-anti-digoxigenin-ap system.Result:The result shower that aptamers which binding to the target were obtained successfully and their first and secondary structures closely related with affinity.Conclusion:The selection was effective and feasible.