Mer免疫球蛋白样区原核蛋白表达和单克隆抗体制备  

Expression of Mer IgG-like Domain and Its Monoclonal Antibody

作　　者：范磊[1] 沈飞[1] 谢丽倩[1] 周梦云[1] 阮长耿(指导)[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,江苏苏州215006

出　　处：《苏州大学学报（医学版）》2007年第1期4-7,共4页Suzhou University Journal of Medical Science

基　　金：江苏省卫生厅135重点学科开放课题(135XY0603);苏州大学欧莱雅医学发展基金资助项目(EE122522);苏州大学优秀博士论文资助项目(23320624)

摘　　要：目的利用原核蛋白表达载体,体外表达Mer的IgG样区,并利用此重组蛋白制备抗Mer的单克隆抗体。方法从K562细胞中抽提总RNA,利用RT-PCR扩增Mer的IgG样区片断。经过测序将所得片断与PQE30原核表达载体连接并转化到大肠杆菌M15中,挑选阳性克隆,经诱导和纯化后获得原核蛋白。利用所得的重组蛋白免疫Balb/c小鼠,取其脾脏和小鼠杂交瘤细胞融合制备针对Mer的IgG样区的特异性抗体。利用ELISA方法筛选阳性克隆,Westernblot方法进行验证。结果体外成功表达MerIgG样区的原核蛋白,片断大小经修饰后约为26kd,表达量占菌体总蛋白的15%,经Ni-NTAagrose柱纯化后得到纯化蛋白并免疫Balb/c小鼠。将小鼠脾脏细胞和杂交瘤细胞融合后得到1株特异性抗MerIgG样区的单克隆抗体——SZ-128,Westernblot显示此单抗可以和表达的重组蛋白和Mer胞外区真核蛋白反应。结论成功制备1株抗MerIgG样区的特异性单克隆抗体,为研究Mer的功能提供了有力的工具。Objective To express Mer IgG-like domain protein in E. coli and prepare its monoclonal antibody（McAb）. Methods K562 total RNA was extracted by Trizol and Mer IgG-like fragment was amplified by RT-PCR. After sequence analysis, the interested fragment was inserted into expression vector-PQE30, the recombinant vector was transfected into E. coli M15 and induced by IPTG. Purified recombinant Mer IgG-like proterin was used to immunize Balb/c mice. After fusion of immunized mouse spleen cells and hybridoma SP2/0, ELISA and westernblot were used to select the positive clone and identify the antibody. Results The recombinant Mer IgG-like domain was about 26 kd, it nearly accounted for 15% of the total bacteria protein. Purified recombinant protein was obtained by chromatographyon Ni-NTA agrose, and after fusion of immunized mouse spleen cells and hybridoma SP2/0, a McAb against Mer, named SZ-128 was developed, which can bind both prokaryotic and eukaryotic fragment of Mer by westemblot. Conclusion A McAb against Mer IgG-like domain is successfully developed, which will be useful on its fuction research.

关 键 词：MER IgG样区 原核表达 单克隆抗体 

分 类 号：R392.2[医药卫生—免疫学]