单链抗体2F5和4E10的原核表达、纯化和鉴定  

The Prokaryotic Expression,Purification and Identification of Single Chain Antibodies 2F5 and 4E10

作　　者：刘秀侠[1] 杨雄[1] 陈红英[1] 

机构地区：[1]西北农林科技大学生命科学学院,杨陵712100

出　　处：《生物技术通报》2013年第10期177-183,共7页Biotechnology Bulletin

基　　金：教育部新世纪优秀人才计划(NCET-09-0625)

摘　　要：为深入研究2F5和4E10抗体,构建了特异性识别HIV-1病毒MPER区域的2F5和4E10单链抗体的重组质粒,进行了融合蛋白的表达、纯化及鉴定。利用重叠PCR扩增及基因重组技术,获得2F5-scFv和4E10-scFv基因,将其插入原核表达载体pET-28a(+)上,获得表达质粒。转入大肠杆菌BL21(DE3)及Rosetta-gami2(DE3)pLysS中,IPTG诱导表达,SDS-PAGE检测发现,2F5-scFv和4E10-scFv在两种表达菌中均以包涵体形式存在,重组蛋白分子量分别约为27 kD、29 kD。变性条件下,利用镍离子螯合层析方法对包涵体蛋白进行纯化,经复性后获得可溶性单链抗体。ELISA检测表明,制备的单链抗体与原核和真核表达的MPER抗原都有特异性结合活性。For further study of 2F5 and 4E10 antibody, 2F5-scFv and 4E10-scFv were expressed and purified from E.coli cells. The 2F5scFv and 4E10-scFv genes were amplified by overlapping PCR. Recombinant vectors pET28a/2F5-scFv and pET28a/4E1-scFv were constructed and transformed into E. coli BL21（DE3）and Rosetta-gami2（DE3）pLysS, and protein expression was induced with IPTG. 2F5-scFv and 4E10-scFv were expressed, with the molecular weight of 27 kD and 29 kD, respectively. The formed inclusion bodies in E. coli. Denatured single chain antibody proteins, which were purified by nickel chelating chromatography and refolded by dialysis, showed specific reactivates to both MPER antigens prepared from prokaryotic and eukaryotic expression systems.

关 键 词：HIV-1 GP41 MPER 2F5 4E10 单链抗体 

分 类 号：R392[医药卫生—免疫学]