去除Etag标记的抗CD3/抗Pgp微型双功能抗体原核表达载体的构建及表达  

Construction and expression of diabody [CD3×Pgp] without Etag

作　　者：刘娟妮[1] 高瀛岱[1] 王金宏[1] 邵晓枫[1] 范冬梅[1] 纪庆[1] 熊冬生[1] 杨纯正[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所血液病医院实验血液学国家重点实验室,天津300020

出　　处：《细胞与分子免疫学杂志》2007年第10期946-949,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30400558);天津市科技发展计划项目(05YFGPGX06900)

摘　　要：目的:构建和表达不带Etag标记的抗CD3/抗Pgp微型双功能抗体(Diabody[CD3×Pgp]),并测定其生物学活性。方法:利用PCR方法构建不带Etag的Diabody[CD3×Pgp]原核表达载体,进行原核表达,制备抗抗CD3scFv亲和层析柱进行纯化,SDS-PAGE鉴定。通过间接免疫荧光法和竞争性免疫荧光结合流式细胞术(FCM)检测生物学活性。结果:无Etag的Diabody[CD3×Pgp]表达载体经测序证实其序列正确。SDS-PAGE电泳显示2条带,相对分子质量(Mr)分别为25000和26000,与预期Mr相符。去除Etag的Dia-body[CD3×Pgp]与K562/A02和Jurkat细胞结合阳性率分别为89.87%和83.95%。竞争结合实验中,与K562/A02和Jurkat细胞竞争后结合率分别为50.25%和43.78%。结论:成功构建了不带Etag的Diabody[CD3×Pgp]表达载体、进行原核表达及纯化。不带Etag的Diabody[CD3×Pgp]能特异性结合CD3和Pgp靶抗原,Etag标记去除后其生物学活性没有降低。AIM： To construct and express a diabody [ CD3×Pgp] without Etag and analyse its biological activity. METHODS： In this study, the diabody [ CD3×Pgp] was obtained by PCR and restriction cleavage, and expressed in E. coli16C9. The product was punied by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry （FCM） was used to analyse the bingding properties and competitive bingding capacity. RESULTS： The sequence of diabody [ CD3×Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight（25 kD and 26 kD） in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively. CONCLUSION： The diabody [ CD3 ×Pgp ] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesnt decrease.

关 键 词：PGP CD3 DIABODY 基因工程抗体 

分 类 号：Q786[生物学—分子生物学]