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目的 制备 6A8α 甘露糖苷酶羧基端单抗并进行鉴定 ,以确定 6A8α 甘露糖苷酶在细胞的表达部位。方法 用基因重组技术构建 6A8cDNA 3′端DNA片段的重组表达载体 ,在原核细胞表达其编码蛋白 ,杂交瘤技术制备单抗 ,以 3D5细胞为靶细胞 ,间接免疫荧光染色后 ,用激光共聚焦显微镜及流式细胞仪鉴定单抗。结果 6A8cDNA 3′端DNA在原核细胞获得表达 ,制备了抗 6A8α 甘露糖苷酶羧基端的单抗 ,观察到 6A8α 甘露糖苷酶不仅表达于 3D5细胞胞浆 ,也表达于胞膜。结论 成功地制备了抗 6A8α 甘露糖苷酶羧基端单抗 ,6A8α 甘露糖苷酶不仅表达于 3D5细胞胞浆中 ,也表达于胞膜
Objective To prepare and identify the carboxy-terminal McAb of 6A8α-mannosidase to determine the expression site of 6A8α-mannosidase in cells. Methods Recombinant expression vector of DNA fragment of 6A8 cDNA was constructed by gene recombination technique. The expressed protein was expressed in prokaryotic cells. The monoclonal antibody was prepared by hybridoma technique. 3D5 cells were used as target cells. After indirect immunofluorescence staining, And flow cytometry identification of monoclonal antibodies. Results The 3’-end DNA of 6A8 cDNA was expressed in prokaryotic cells. The monoclonal antibody against 6A8α-mannosidase was prepared. The 6A8α-mannosidase was not only expressed in the cytoplasm of 3D5 cells but also expressed in the cell membrane. Conclusion The McAb against 6A8α-mannosidase was successfully prepared. 6A8α-mannosidase was expressed not only in the cytoplasm of 3D5 cells but also in the membrane