小鼠钙网蛋白的原核表达和多克隆抗体的制备

Expression of mouse calreticuin protein and preparation of its polyclonal antibodies

钙网蛋白(calreticulin,CRT)是存在于哺乳动物细胞具有高度保守性和多种生物学活性的蛋白,为了更好的研究其生物学活性,本课题组通过PCR方法扩增CRT截短基因,将其克隆到原核表达载体PET-28a(+)中,经大肠杆菌表达并纯化CRT蛋白。以纯化的CRT蛋白抗原免疫BALB/c小鼠,制备多克隆的CRT血清。进一步采用Western blot、ELISA、流式细胞术等技术对制备的抗体进行初步鉴定。结果显示:原核表达重组质粒在大肠杆菌中能高效表达CRT蛋白;获得多抗血清应用Western blot鉴定几种细胞株中CRT,流式细胞术检测CRT的外翻现象。因此我们得到结论——成功制备了CRT蛋白及其抗体,并有很好的特异性,能识别人鼠两种源性的CRT。

In order to investigate the biological properties of calreticulin（CRT）,a protein present in the mammalian cells with highly conservative and multiple biological properties,a short fragment of mouse CRT was amplified through RT-PCR method and then was cloned into prokaryotic expression vector pDET28a（＋）.The recombinant protein CRT was induced by IPTG in E.coli BL21（DE3） and the expressed protein was detected by Western blotting.The purified protein was used to immune mouse to prepare polyclonal antibody and the specificity and titer of the antibody were detected by ELISA,Western blotting and FCM.It was demonstrated that the recombinant prokaryotic expression plasmid pET28a（＋） /CRT was successfully constructed and the CRT protein was expressed in E.coli BL21（DE3） with high efficiency.Western blot assay showed that this recombinant protein was characterized with its antibody.Mouse immunized with the purified protein produced high titer of antibody.Western blot assay displayed that the CRT protein was highly expressed in some of eukaryotic cells and could specifically combine with the antibody.FCM assay displayed that the antibody could also specifically combine with the membrane extracellular region of CRT.It is evident that the preparation of recombinant CRT and its polyclonal antibodies have a strong specificity to match with the CRT protein from mouse and human.