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机构地区:[1]军事医学科学院微生物流行病研究所,北京100071
出 处:《军事医学科学院院刊》2004年第5期414-416,共3页Bulletin of the Academy of Military Medical Sciences
基 金:国家重点基础研究项目" 973"课题资助(2 0 0 2CB5 13 2 0 5 )
摘 要:目的 :实现特异性抗A型肉毒毒素人源单链抗体 (ScFv)的表达与纯化 ,并进行结合活性分析。方法 :克隆单链抗体基因ScFv(VH Linker Vk) ,利用pET2 2b表达载体构建重组表达质粒 ,在大肠杆菌BL2 1(DE3)中进行IPTG化学诱导 ,固相亲和层析纯化蛋白 ,ELISA测定其特异结合活性。结果 :pET2 2b可稳定表达人源单链抗体基因 ,表达产物占全菌蛋白的 2 5 % ,表达蛋白全部以包涵体形式存在于胞内。经IMAC纯化 ,蛋白纯度大于 95 %。竞争性ELISA测定结果表明 ,重组抗毒素在体外具有良好的活性 ,可竞争肉毒马血清与肉毒毒素结合。结论 :采用原核表达系统可实现抗A型肉毒毒素人源单链抗体的高效表达 ,重组人源单链抗体具有抗原特异结合活性。Objective: To express and purify human ScF v specific against botulinum neurotoxin type A(BoNT/A), and analyze its binding a c tivity. Methods: ScFv gene was cloned into prokaryotic exp ression vector pET22b to construct a recombinant expression plasmid, and ScFv w a s expressed in E.coli BL21(DE3) by IPTG induction. Recombinant ScFv was puri fied by IMAC and identified by ELISA. Results: Human ScFv was expressed stably as inclusion body by vector pET22b in E.coli and its ex pression level was beyond 25% of total bacteria protein. Purity of recombinant S cFv (rScFv)protein was above 95% after a simple IMAC. rScFv Showed good bindin g activity to BoNT/A to compete with antitoxin serum in ELISA. Conclu sion: Recombinant human ScFv specific against BoNT/A could be overexp ressed in E.coli, and showed good binding activity to BoNT/A.
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