咪唑啉受体抗血清选择性蛋白的表达、纯化及单克隆抗体制备

Expression,purification of IRAS protein and preparation of its monoclonal antibody

目的:表达和纯化咪唑啉受体抗血清选择性蛋白(imidazoline receptor antiserum-selected protein,IRAS pro-tein),制备IRAS蛋白的单克隆抗体。方法:采用基因重组技术在大肠杆菌表达IRAS蛋白;金属镍螯合的Ni-NTA亲和层析柱进行蛋白纯化;杂交瘤技术建立分泌IRAS单克隆抗体的杂交瘤细胞株;以间接ELISA方法筛选分泌特异性IRAS单克隆抗体的杂交瘤细胞;采用蛋白免疫印迹、间接免疫荧光方法鉴定单克隆抗体的特异性。结果:成功表达并纯化了IRAS重组蛋白,纯度达到95%。共筛选出5株分泌IRAS单克隆抗体的杂交瘤细胞株,制备腹水并纯化了IRAS的单克隆抗体,腹水中单克隆抗体效价分别为1∶8×106,1∶2×106和1∶5×106,属于IgG1亚型。该抗体能与原核及真核系统表达的IRAS重组蛋白发生特异性反应,间接免疫荧光显示IRAS蛋白主要定位于细胞质中。结论:建立了稳定分泌IRAS单克隆抗体的杂交瘤细胞株,成功制备了特异性好的IRAS单克隆抗体,为研究IRAS的功能提供了有力的研究工具。

Objective：To express and purify the imidazoline receptor antiserum-selected protein （IRAS） and prepare its monoclonal antibody （mAb）. Method ：The IRAS protein was expressed with gene recombinant technique in E. coli and purified with nickel chelate-nitrilotriacetic-acid （Ni-NTA） affinity chromatograph column. Hybridoma cell lines that secreted anti-IRAS mAb were established by cell fusion and screened by indirect enzyme linked immunosorbent assay （ELISA）. The specificity of anti-IRAS mAb was indentified by Western blot, and indirect immunofluorescent staining. Results： Recombinant plasmid pET-43, la-IRAS was expressed in E. coli BL21. Five hybridoma cells were selected and anti-IRAS mAb was purified from mouse ascites. The titers of three anti-IRAS mAb in ascites were 1：8 × 10^6, 1：2 × 10^6and 1：5 × 10^6 ,respectively. The isotypes of the mAb were IgG1. Anti-IRAS mAb specifically reacted with recombinant IRAS protein in prokaryotic and eukaryotic systems. The efficient expression of IRAS fusion protein was observed and the fluorescence was mainly located in the cytoplasm. Conclusion：Stable hybridoma cell lines that secreted anti-IRAS mAb were established and anti-IRAS mAb was successfully prepared with high specificity, which provides a powerful reagent for the function study of IRAS.