双峰驼IgA基因的原核表达及抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Bactrian Camel IgA Gene

为制备抗双峰驼IgA的抗体,将双峰驼IgA重链恒定区基因克隆至pET-28a(+)上,构建重组蛋白质粒pET-28a-IgA-H,再进行IPTG诱导表达、分析和纯化,以及SDS-PAGE和Western blot验证。用以获得的目的蛋白制备兔抗双峰驼IgA多克隆抗体,并通过ELISA和Western blot检测效价及其特异性。结果表明,获得的重组目的蛋白大小约为39 ku,最佳诱导表达条件为IPTG 23.83μg/mL、诱导时间6 h,且重组蛋白主要以包涵体的形式表达。ELISA检测显示抗体效价达1∶32000,Western blot鉴定抗体能特异性识别原核表达的pET-28a-IgA-H蛋白,并能有效地将双峰驼IgA与IgG区别开来,表明成功获得了双峰驼IgA重链恒定区的重组蛋白及特异性良好的多克隆抗体。

In order to prepare anti-Bactrian camel IgA antibody,the recombinant protein plasmid of pET-28a-IgA-H was built by cloning the heavy chain constant region gene of Bactrian camel IgA to pET-28a(+),and then expressed by IPTG induction.Meanwhile,this prepared pET-28a-IgA-H was analyzed and identified by the SDS-PAGE and Western blot.The rabbit anti-Bactrian camel IgA polyclonal antibody was prepared with purified protein,then the antibody titer and specificity were detected by ELISA and Western blot.The results showed that the molecular weight of recombinant protein was 39 ku,the optimal induced condition was IPTG 23.83μg/mL,the induction time was 6 h,and the expression formation of this recombinant protein was inclusion body.In addition,the antibody titer was 1∶32000 tested by ELISA,and the specificity of prokaryotic expression antibody from the prepared pET-28a-IgA-H protein was identified by Western blot.The results demonstrated that the Bactrian camel IgA heavy chain constant recombinant protein and good specificity polyclonal antibody were successfully obtained in this study.