基于纳米抗体CDR3区抗原表位展示制备生存素抗体及其鉴定

Preparation and Identification of Anti-Survivin Antibody Based on Display of Antigenic Epitopes in CDR3 Region of Nanobody

目的:通过纳米抗体CDR3区展示生存素N端表位的方式,探索纳米抗体在抗原表位展示中的作用。方法:通过基因合成方法将生存素N端起始表位(氨基酸序列1~15)插入纳米抗体CDR3区,再构建到原核表达载体pET24a中,IPTG诱导表达,用带His标签的填料纯化,获得高纯度的目的蛋白,免疫雌性BALB/c小鼠,间接ELISA检测5次免疫后的效价,用抗原偶联纯化介质纯化免疫多抗,Western印迹检测多抗特异性。结果:IPTG诱导后,目的蛋白主要以包涵体形式存在,亲和层析获得纯度大于96%的目的蛋白,包涵体经复性后免疫小鼠,效价可达1∶512000,West?ern印迹特异性检测显示免疫多抗能够特异性结合生存素。结论:纳米抗体CDR3区生存素抗原N端表位展示的方法可用于抗生存素抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。

Objective: To verify the role of nanobodies in epitope display by displaying the N-terminal epitope of survivin in the CDR3 region of nanobodies. Methods: The N-terminal epitope of survivin(amino sequence 1~ 15) was inserted into the CDR3 region of the nanobody by gene synthesis method. The newly synthesized gene was constructed into the prokaryotic expression vector pET24a. The expression was induced by IPTG and purified by His tag filler. The purified target protein was immunized to female BALB/c mice. After five immunizations, indi? rect ELISA titer was detected and the polyclonal antibody was purified by antigen-coupled purification medium. Western blotting was used to detect the specificity of multi-antibodies, clinical serum samples were collected, and indirect ELISA was used to further detect the effect of multi-antibodies. Results: The target protein existed main? ly in the form of inclusion bodies by induction of IPTG, and its purity reached over 96% via affinity chromatography. After refolding, the inclusion bodies were immunized with a titer of 1∶512 000. Western blotting showed that the immune polyclonal antibody could specifically bind to survivin. Conclusion: The method of displaying the N- terminal epitope of survivin antigen in CDR3 region of nanobody can be used for the preparation of survivin anti? body, laying a foundation for the future research on displaying the epitope of nanobody.