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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 full-length C1INH gene of human complement was obtained by PCR. The fragment of antigenic epitope was amplified by PCR and cloned into pET32a (+) vector. The positive clones were screened and transformed into Escherichia coli. IPTG induced Expression, the recombinant strain BL21 culture was lysed by ultrasonic wave, and then the New Zealand rabbit was immunized to prepare the antiserum. SDS-PAGE analysis showed that the pET32a-C1 fusion protein was highly expressed in E. coli BL21 (DE3) as a inclusion body. The titer of the obtained antisera was 1: 400 by indirect ELISA. Western- The antiserum specifically binds to the C1INH protein expressed in prokaryotic cells, which lays the foundation for the clinical detection of C1INH in order to further detect the eukaryotic expression protein and study the functional activity of rhC1INH.