抗克伦特罗单链抗体可溶性表达及特性鉴定  

Soluble Expression and Characterization of Single Chain Fv Against Clenbuterol

作　　者：王弘[1] 潘科[1] 杨金易[1] 张宏斌[2] 王捷[2] 武婕[2] 雷红涛[1] 孙远明[1] 

机构地区：[1]广东省教育厅食品质量安全重点实验室华南农业大学食品质量与安全研究所,广东广州510640 [2]广州军区广州总医院医学试验科,广东广州510010

出　　处：《食品科学》2007年第12期332-335,共4页Food Science

基　　金：国家自然科学基金项目(30400354;20477012);教育部高等学校博士点基金项目(20050564011);广东省自然科学基金项目(04300502)

摘　　要：目的:表达抗克伦特罗(CBL)可溶性单链抗体(scFv)并进行抗原结合活性鉴定。方法:利用呈现scFv的重组噬菌体感染大肠杆菌HB2151,IPTG诱导可溶性scFv表达。渗透休克法提取菌体细胞周质腔中scFv。经SDS-PAGE、Western-Blotting以及间接ELISA法分析检测可溶性scFv的表达,并采用间接竞争ELISA法鉴定scFv的抗原结合活性。结果:细菌培养上清及周质腔中均有抗CBL可溶性scFv表达,其分子量约为29kD;上清液及周质腔中scFv效价分别为1:80,1:1600,能够被CBL竞争性抑制,IC50分别为0.78、0.95ng/ml。结论:可溶性抗CBLscFv在大肠杆菌HB2151中获得成功表达,表达的scFv与CBL具有很好的结合活性,可用于进一步建立CBL相应的免疫学检测方法。Objective： To express and characterize the soluble single chain Fv （scFv） against clenbuterol （CBL）. Methods： The soluble anti-CBL scFv was expressed by E.coli. HB2151 infected by recombinant phages after being induced by IPTG and extracted from the periplasmic through osmotic shock. The characterization and antigen-binding activity of the scFv were identified by SDS-PAGE, Western-Blotting, and indirected by ELISA and competitive ELISA. Results： The scFv with MW 29kD is expressed both in supernatant and periplasmic. The titer of scFv in supematant is 1：160 and that in periplasmic extracts 1：3200. The indirect competitive ELISA showed that the scFv in both supernatant and periplasmic can competitively combine with CBL, whereas IC50 is 0.78 ng/ml and 0.95 ng/ml respectively. Conclusion： The soluble scFv directed against CBL is expressed successfully and shows the ideal binding activity to CBL. The results showed that the corresponding immnunoassay methods can be set up by this scFv.

关 键 词：克伦特罗 单链抗体 可溶性表达 

分 类 号：Q78[生物学—分子生物学]