重组骆驼源单域抗体的原核表达及纯化  

Prokaryotic Expression and Purification of Recombinant Camelid Single Domain Antibody

作　　者：刘超[1] 彭媛[1] 宁保安[1] 高志贤[1] 

机构地区：[1]军事医学科学院卫生学环境医学研究所天津市环境与食品安全风险监控技术重点实验室,天津300050

出　　处：《生物技术通讯》2016年第3期354-357,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472985;21207161);国家科技支撑计划(2012BAK08B06);国家重大科学仪器设备开发专项(2013YQ14037106)

摘　　要：目的:原核表达重组骆驼源单域抗体并纯化。方法:将雌二醇特异性的单域抗体基因片段克隆到pET32a载体中,经IPTG诱导表达后对形成的包涵体进行纯化,利用间接ELISA法检测获得的单域抗体的活性,并以孕酮、雌酮和雌三醇为对照,鉴定雌二醇单域抗体的特异性。结果:重组雌二醇单域抗体的相对分子质量约为34×103,通过大肠杆菌BL21(DE3)-p ET32a构建的原核表达体系实现了雌二醇单域抗体的高水平表达,并通过包涵体纯化获得了可溶性的高纯度单域抗体抗体,经间接ELISA检测,该雌二醇特异性抗体的50%抑制浓度(IC50)为20.03 ng/mL,对孕酮和雌酮2种化合物的交叉反应率分别为29.78%和1.63%,特异性较好。结论:通过构建原核表达体系,可以获得功能性的骆驼源单域抗体。Objective：To establish the prokaryotic expression and purification system for camelid single domain antibody.Methods：The gene segment of anti-E2 VHH antibody was cloned into pET32a plasmid,IPTG was used to induce the expression.Anti-E2 VHH antibody was purified from the inclusion body and the activity was determined by indirect ELISA.Results：Molecular weight of anti-E2 VHH was 34 kD.High level expression of interest protein was achieved through established prokaryotic expression.By indirect ELISA,the 50％ inhibiting concentration of anti-E2 VHH was 20.03 ng/mL.The cross-reactivity values of progesterone and estrone were 29.78％and 1.63％ respectively.Conclusion：By establishing the prokaryotic system,the functional camelid single domain antibody was successful acquired.

关 键 词：骆驼源单域抗体 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]