Mre11多克隆抗体的制备及初步鉴定

Prokaryotic expression of human mre11 and preparation of its polyclonal antibody

目的原核表达人Mre11蛋白,并制备出Mre11蛋白的多克隆抗体,为研究Mre11蛋白功能奠定基础。方法以人cDNA文库为模板,构建出原核表达质粒PET41a-Mre11,然后转化受体菌E.coli BL21,再经IPTG诱导表达,通过采用GST亲和纯化获得GST-融合蛋白,将该融合蛋白免疫兔以制备出Mre11多抗血清,最后采用western-blot及免疫荧光等方法检测其在细胞中的特异性。结果在受体菌E.coli BL21中成功诱导表达出了GST-Mre11融合蛋白,免疫新西兰大白兔后获得了高效价的Mre11蛋白抗血清,IP、western-blot及免疫荧光等多种方法检测证实该抗血清能够特异性识别293T和U2OS细胞株中Mre11蛋白,并能够正确清楚检测293T和U2OS细胞株中的Mre11表达情况,具有良好的特异性和高效性。结论成功制备出了兔抗人Mre11蛋白的多克隆抗体,为深入研究Mre11蛋白在DNA损伤中的功能奠定了基础。

Objective To express and purify the protein of Mrell in prokaryocytes and to prepare rabbit anti- Mrell antibody. Methods Amplified Mrell from human cDNA library, and then constructed recombinant expression vector PET41a- Mrell. GST-Mrell fusion protein was expressed in E. coli BL21 after being induced with isopropylthiosoprgalactoside（IPTG） and then used as the immunogen. Mrell expression in the liver cell lines were analyzed with Western blot and Immunofluorescence. Results The GST- Mrell fusion protein was expressed in E. coli BL21 and specific polyclonal antibody was obtained after the immunization. Mrell protein can be recognized in 293T cell lines by Western blot and in U2OS cell lines by Immunofluorescence. Conclusion The polyclonal antibody to Mrell was successfully prepared, which will provide a basis for further exploring of the role of Mrell in the DNA damage response.