抗A型肉毒毒素人源单链抗体的表达、纯化及结合活性分析

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pET22b表达载体构建重组表达质粒，在大肠杆菌BL21(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：pET22b可稳定表达人源单链抗体基因，表达产物占全茵蛋白的25％，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。

Expression,purification and binding activity analysis of human ScFv specific against botulinum neurotoxin type A

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.