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

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.