INHA基因的克隆表达以及多克隆抗体的制备

Expression of INHA in E. coli and Preparation of Polyclonal Antibody to INHA

目的INHA的表达、纯化,以及多克隆抗体的制备。方法利用基因重组技术获得INHA基因以及去信号肽的INHA基因,将其都分别构建到原核表达系统并分离纯化得到相对分子质量为58000的带有组氨酸标签的重组INHA融合蛋白。用分离纯化后的INHA融合蛋白作为抗原免疫,获得抗INHA的多克隆抗体。结果成功地获得INHA基因,并且以包涵体的形式表达于大肠杆菌中,用间接酶联免疫吸附(ELISA)方法,Westernblot检测结果显示,该抗体能特异性地与INHA的抗原以及胎盘组织产生明显免疫亲和反应。结论建立原核高效稳定INHA表达系统,获得具有生物学活性的蛋白,为进一步获得高特异性的抗体奠定基础。

Objective To express and purify the recombinant protein from the constructed expression plasmids pET32a-INHA. Methods The coding sequences of INHA with or without the signal peptide were obtained by PCR. The expression plasmids were then constructed by inserting the coding sequences into pET32a. Plasmids were then induced by IPTG. The expressed proteins were allowed to re-fold by using dialysis method. Target protein was purified using Ni＋ -NTA agarose and molecular sieve. The purified INHA was used to immunize rats, and the polyclonal antibody against INHA was obtained. Results INHA was cloned and expressed in E. coli successfully. INHA was found in the form of insoluble inclusion body, and the amount of the expressed protein was about 80% of total protein, ELISA and Western blot analysis showed that the rat immune serum was specific for INHA. Conclusion pET32a-INHA expression plasmid was constructed. The expressed INHA1 is bioaetive and the induced antiserum is specific for INHA1. The recombinant protein can be used in the preparation of highly specific antibody.