人补体C1INH基因的原核表达及其抗血清的制备

Prokaryotic Expression of Human C1 Inhibitor and Its Antiserum Preparation

采用PCR技术获得人补体C1INH基因全长,以其为模板扩增出抗原表位基因片段命名为C1,然后将C1片段重组到pET32a(+)载体中,筛选阳性克隆,转化大肠杆菌;IPTG诱导表达,用超声波裂解重组菌BL21培养物,纯化蛋白后免疫新西兰兔制备抗血清。SDS-PAGE分析表明,pET32a-C1融合蛋白在大肠杆菌BL21(DE3)菌株中以包涵体形式高效表达;获得的抗血清经间接ELISA法检测效价为1∶6 400;Western-blot检测显示,抗血清可与原核表达的C1INH蛋白进行特异性结合,为进一步检测真核表达蛋白及研究rhC1INH的功能活性,并为C1INH的临床检测奠定基础。

The complete gene of human C1INH by gene cloning techniques was obtained and C1INH was chosen as templates to obtain the fragment of hC1INH which was named as C1,then cloning the fragment C1 into pET32a（＋） vector,and screening positive cloning.Transform the recombinant plasmid into E.coli BL21（DE3） and induce it with IPTG for expressing;split the recombinant strain culture solution and purify the fusion protein and immunize the New Zealand rabbits so as to prepare the antisera.SDS-PAGE analysis showed that pET32a-C1 was expressed in the form of inclusion bodies in E.coli BL21（DE3）;The titer of the rabbit antibody which prepared by pET32a-C1 was 1：6400;Western-blot analysis showed that the antisera could bind to C1INH protein of prokaryotic expression specifically,which laid a foundation for further study on the detection of C1INH expression in the eukaryotic syetem and its function,as well as for the clinical research of human complement C1INH.