摘要
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 。
Objective To produce monoclonal antibody to the C terminal part of 6A8 α mannosidase and to determine the sites on 3D5 cell where 6A8 α mannosidase exists using the McAb. Methods Gene recombinant technique was used to construct an expression vector of the 3′ fragment of 6A8 cDNA. Monoclonal antibody to 6A8 protein was produced by immunization of mouse with the expressed protein. Immunofluorescent staining was used for screening of McAbs in a laser scan confocal microscopy and a flowcytometer. Results A 67 kDa GST 6A8 fusion protein was expressed in protokaryotic cells. A monoclonal antibody (3E12) to the C terminal part of 6A8 α mannosidase was prepared. With this antibody as a probe we found that 6A8 α mannosidase expressed not only in cell plasma but also on cell membrane. Conclusion 6A8 α mannosidase expressed both in the cytoplasm and on the cell membrane of 3D5 cell.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第4期413-416,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 (396 30 30 0 )
