噬菌体展示技术筛选DNA错配修复蛋白MutS高亲和性抗体  

High affinity antibody on DNA mismatch repair protein MutS through method of phage display

作　　者：刘俊[1] 全智勇[1] 刘建华[1] 

机构地区：[1]上海交通大学生命科学与技术学院,上海200240

出　　处：《复旦学报（医学版）》2006年第4期544-546,共3页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(30170211)项目资助

摘　　要：目的制备MutS的高亲和抗体,以有利于MutS的检测及应用。方法利用IPTG的诱导作用,在E.coli中诱导表达目标蛋白MutS。利用亲和层析方法获得目标蛋白,并利用凝胶阻滞实验分析其活性。将MutS作为抗原,在Tomlinson抗体库中利用噬菌体展示技术来筛选MutS的高亲和力噬菌粒。将其转化到大肠杆菌后,诱导表达并通过亲和层析的方法来获得可溶性的抗体片段,并检测其亲和力。结果纯化后的MutS具有识别、结合错配DNA双链的活性。ELISA分析表明,利用噬菌体展示技术可获得MutS高亲和力的噬菌粒。将其转化到大肠杆菌HB2151后,诱导表达并纯化到抗体片段,其亲和力为0.9×108L/mol。结论利用噬菌体展示技术筛选到MutS高亲和力的抗体。Purpose To prepare high affinity antibody on DNA mismatch repair protein MutS, so that to benefit its detection and application. Methods Protein MutS was expressed in E. coli with induction of IPTG and purified through method of affinity chromatography. Activity of purified MutS for recognizing and binding mismatch duplex DNA was detected by gel retardation assay. Through method of phage display with MutS as antigen, we selected high affinity phagmid against MutS in Tomlinson library. After phagemids transformation into E. coli, soluble antibody fragment was induced, expressed and purified, whose affinity was detected through ELISA. Rmults Enzyme assay showed that purified MutS had the activity for recognizing and binding mismatch duplex DNA. High affinity phagemid after selection was checked through ELISA. After aim phagmid was transformed into E. coli strain HB2151, soluble antibody fragment were induced, expressed and purified that had high affinity of 0.9 × 10^8 L/mol. Conclusions Results showed that high affinity antibody against MutS was obtained through method of phage display.

关 键 词：噬菌体展示 DNA错配修复蛋白MutS 可溶性抗体片段 

分 类 号：Q81[生物学—生物工程]