抗产气荚膜梭菌ε毒素Phage-ScFv库的构建、筛选与鉴定

Isolation and identification of Phage-ScFv library against Clostridium perfringens ε toxin

利用本实验室已诱导并纯化后的重组毒素蛋白制成类毒素免疫BALB/c小鼠,提取小鼠脾脏mRNA。通过RT-PCR和SOE-PCR获得由抗体重链可变区(VH)-Linker-轻链可变区(VL)构成的750bp左右单链抗体(ScFv)基因片段。随后利用噬菌体展示技术成功构建了抗产气荚膜梭菌ε毒素噬菌体单链抗体(Phage-ScFv)库。并通过"吸附-洗脱-扩增",5轮高效的淘洗筛选系统,最终筛选出特异性强的噬菌体抗体。最后,对强阳性的单链抗体侵染E.coli HB2151进行表达,SDS-PAGE结果显示其相对分子质量为30 000左右,与预期结果一致,并且能以分泌形式表达。本试验为抗产气荚膜梭菌ε毒素中毒治疗及其分子致病机制的研究奠定了基础。

ε-toxin,produced by Clostridium perfringens type D is responsible for diverse pathologies in humans and animals,resulting in food poisoning and enterotoxemia,especially in cattle and sheep,posing agreat risk to the development of animal husbandry and human health.However,using traditional methods to make monoclonal antibodies needs a long time and high cost.In addition,the conditions of cell culturing are harsh and human always reject these heterologous antibodies.In this study,we used the protein which has been induced and purified immunized BALB/c mice.Extract the mRNAs of spleens from mice.Single-chain antibody(ScFv)gene of about750 bp which constituted by heavy chain variable region(VH)-Linker-light chain variable region(VL)was obtained by RT-PCR and SOE-PCR.Then using phage display technology successfully constructed anti-Clostridium perfringensεtoxin phage antibody(Phage-ScFv)libraries.And through the 'adsorption-elution-amplification',five efficient screening system selected specificity phage antibody.The strongly positive single chain antibody was expressed by infected of E.coli HB2151 and consistent with the expected results.The research lays the foundation for the cure ofε-toxin and the study on its molecular pathogenic mechanism.