稳定表达重组小鼠FAPα蛋白细胞株的构建  

Establishment of a Cell Line Stably Expressing Recombinant Mouse FAPα

作　　者：张欢[1] 方瑞[2] 黄思超[3] 杨倩之[1] 杜军[2] 蔡绍晖[1] 

机构地区：[1]暨南大学药学院,广州510632 [2]中山大学药学院,广州510006 [3]珠海市人民医院药剂科,珠海519000

出　　处：《生物技术通报》2012年第5期93-98,共6页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(30973565)

摘　　要：采用RT-PCR方法从BALB/c胚鼠成纤维细胞克隆FAPα基因,将其连接至表达载体pTd-FL-N,转化Stbl3感受态。筛选重组质粒,经酶切、PCR检测及测序鉴定,证实表达质粒构建正确。将表达载体转染HEK293细胞,经G418筛选单克隆阳性细胞,采用Western blot技术证实稳定表达FAPα细胞株构建成功。通过流式细胞术确定FAPα蛋白可定位表达于细胞膜上。The fibroblast activation protein a （ FAPa ）gene was ampfified from BALB/c mouse embryo fibroblasts （ EF ） by RT-PCR and cloned into expression plasmid pTd-FL-N. Subsequently, the recombinant plasmid pTd-FL-N/FAPa was transformed in E.coli Stbl3. The recombinant colonies were screened and identified by restriction enzyme digestion, PCR detection and sequencing. These results showed that the expression plasmid was correctly constructed. The human embryonic kidney cells （ HEK293 ） were transfected with the expression plasmid. G418 screening and Western blot displayed that the stable cell line which could highly express the FAPa protein was established successfully. Meantime, the expression of FAPa on the cell membrane was confirmed by flow cytometry.

关 键 词：FAPα pTd-FL-N表达载体 G418筛选 稳定细胞株 

分 类 号：R341[医药卫生—基础医学]