一种鉴定MNSs红细胞血型单克隆抗体类型的方法的建立  

Establishment of a Method Used in Identification of the MNSs Blood Group Monoclonal Antibody

作　　者：陈琦[1] 曹慧[1] 张海燕[1] 高晓飞[1] 李厚达[1] 

机构地区：[1]扬州大学医学院生理学教研室,扬州225001

出　　处：《中国生物化学与分子生物学报》2007年第5期419-422,共4页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：为鉴定MNSs血型单克隆细胞株6D7C9分泌的抗体类型,通过克隆、亚克隆、细胞转染等分子生物学技术建立了血型糖蛋白GPA、GPB的异源表达系统,并将其作为抗原,通过ELISA、Western印迹法确定6D7C9分泌的McAb.结果显示,RT-PCR技术成功克隆获得了GPA、GPB血型糖蛋白编码基因,通过分别构建其重组逆转录病毒表达载体pEGZ/GPA及pEGZ/GPB,转染包装细胞293T,再感染L929细胞,经zeocin筛选2周后,RT-PCR及流式细胞仪分析证实,L929/GPA和L929/GPB转基因细胞中分别有GPA、GPB目的基因的转录和蛋白表达.用稳定高表达GPA、GPB的转基因细胞通过ELISA和Western印迹法证实单克隆细胞株6D7C9分泌的是抗GPA/GPBMcAb.本研究成功地建立了血型糖蛋白GPA、GPB的异源表达系统,为MNSs血型McAb的检测及GPA、GPB蛋白的功能学研究奠定了基础.In order to determine the type of the McAb which is a kind of MNSs blood group secreted by 6D7C9 cells line, the heterologous systems expressing GPA and GPB blood group antigens were constructed by molecular biological technology such as cloning, subcloning and cell transfection. These transgenic cells were used as antigens to determine the McAb secreted by 6D7C9 through ELISA and Western blot analysis. The results showed that the GPA and GPB genes could be amplified by RT-PCR successfully. By inserting amplified fragments into retrovirus vector pEGZ, the constructed recombinant vectors were transfected into the package cell 293T and then infected L929 cells. The L929 cell clones stably expressing the GPA and GPB respectively were obtained in the presence of zeocin （500 μg/ml）. The transcripts and proteins of GPA and GPB in L929/GPA and L929/GPB transgenic cell lines were further confirmed by RT-PCR and flow cytometry. ELISA and Western blot analysis showed that the 6D7C9 cells line was capable to secrete the McAb against GPA/GPB, The results indicated that a heterologous system stably expressing GPA and GPB at a high level was successfully constructed. The method might be used for the detection of MNSs blood group McAb and also for the study of the function of GPA and GPB proteins.

关 键 词：血型糖蛋白A 血型糖蛋白B 单克隆抗体 基因表达 

分 类 号：Q987.2[生物学—遗传学]