单链二硫键稳定抗体靶向超抗原SEA(D227A)分子的表达、纯化及鉴定  

Expression, purification, and characterization of single-chain disulfide-bond Fv(ScdsFv) antibody fused with targeted superantigen SEA(D227A)

作　　者：郝淮杰[1] 郑玉玲[1] 余多慰[1] 马茹[2] 姜永强[2] 

机构地区：[1]南京师范大学生命科学学院分子生物学实验室,江苏南京210097 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《细胞与分子免疫学杂志》2005年第3期269-272,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(No.30171091;30271478)

摘　　要：目的:提高抗肝癌靶向超抗原SEA(D227A)的产量和稳定性,构建单链二硫键稳定抗体靶向超抗原分子scdsFv-SEA(D227A)。方法:构建scdsFv SEA(D227A)表达质粒,用IPTG诱导其在大肠杆菌BL21plusS表达。包涵体经洗涤和复性后,用QSepharoseHP和Hiprep26/60SephacrylS-200HR纯化。纯化蛋白经AMS烷化处理并结合PAGE电泳来检测二硫键形成情况,并通过ELISA和MTS分别检测重组蛋白与肝癌细胞的结合活性、稳定性和细胞杀伤活性。结果:重组蛋白以包涵体的形式表达,表达量占菌体总蛋白的30%以上。通过复性及QSepharoseHP离子交换柱和Hiprep26/60SephacrylS-200H凝胶过滤两步纯化后,获得目的蛋白,每升诱导菌液的产量高达60mg。活性测定结果表明,相比与单链抗体靶向超抗原和二硫键抗体靶向超抗原SEA,二硫键抗体靶向超抗原在不影响结合活性和杀伤效果的情况下,稳定性有了较大提高。结论:建立了一种新的稳定的免疫毒素和提高其产量的方法,为hscFv25抗体靶向超抗原的临床应用奠定了基础,同时为其他低稳定性或低产量抗体的改造提供了借鉴的方法。AIM: To express, purify, and characterize scdsFv antibody fused with superantigen SEA(D227A). METHODS: The expression plasmid of scdsFv-SEA(D227A) was constructed by standard molecular cloning procedures. The recombinant protein was induced to express in E.coli BL21plusS by IPTG and purified by Q Sepharose HP column and Hiprep 26/60 Sephacryl S-200 HR column. Formation of the intramolecular disulfide bond of the purified protein was analysed by AMS alkylation and PAGE electrophoresis. The binding activity, stability and killing activity of the purified protein were assayed by ELISA and MTS, respectively. RESULTS: The recombinant protein was expressed as inclusion body, accounting for more than 30% of total bacterial protein. After purification by Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR, the yield of the purified protein was 60 mg per liter of induced culture. AMS alkylation and PAGE electrophoresis analysis showed that intramolecular disulfide bond formed correctly in the recombinant protein. The purified protein had similar binding affinity as dsFv fused SEA and scFv fused SEA have and similar killing activity as native SEA has to human hepatoma cell line, but more stable, in vitro, as compared with dsFv fused SEA and scFv fused with SEA. CONCLUSION: The scdsFv fused with SEA, as a novel form of immunotoxin, might be used in cancer treatment.

关 键 词：单链二硫键稳定抗体 产量 超抗原 SEA(D227A) 稳定性 

分 类 号：Q78[生物学—分子生物学]