二型谷氨酸羧肽酶稳定表达细胞株的建立  

Estahlishment of a cell strain stably expressing glutamate carboxypeptidase Ⅱ

作　　者：田士强[1] 王任直[1] 李桂林[1] 王欣[2] 张波[1] 姚勇[1] 窦万臣[1] 孔燕国[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院神经外科,北京100730 [2]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出　　处：《基础医学与临床》2004年第3期331-334,共4页Basic and Clinical Medicine

摘　　要：为了深入研究二型谷氨酸羧肽酶 (glutamatecarboxypeptidaseⅡ ,GCPⅡ )功能和了解其在兴奋性氨基酸代谢过程中作用 ,拟构建GCPⅡ的表达载体 ,建立稳定表达细胞株 ;采用聚合酶链反应 ,酶切连接 ,和蓝白筛选的方法 ,构建表达载体 ;磷酸钙共沉淀的方法转染HEK2 93细胞 ,G4 18筛选阳性细胞克隆 ;逆转录聚合酶链反应和免疫荧光细胞染色的方法鉴定阳性细胞株。采用PCR方法扩增出GCPⅡ ;构建含有GCPⅡ基因的表达载体———pcDNA3 1 NA ;建立了细胞株HEK 2 93 NA。成功构建了GCPⅡ的表达载体 ,建立了稳定表达细胞株 ,为深入研究该酶的功能和兴奋性氨基酸毒性作用奠定了基础。To construct a glutamate carboxypeptidase Ⅱ(GCP Ⅱ)expressing vector and to establish a stable expression cell line to facilitate recognition of the function of GCP Ⅱ and the role on excitatory amino acid metabolism. Polymerase chain reaction,digestion with endonucelease and ligation ,blue-white selection were carried to construct the expression vector; Ca 3(PO 4) 2 method was used to transfect HEK293 cell,G418 to select positive cell clone; reverse-transcription PCR and immunofluresene cell staining were used to identify positive cell line. GCP Ⅱwas amplified by PCR method;the expression vector containing GCP Ⅱ——pcDNA3.1-NA was constructed;the cell strain-HEK 293-NA was established.The expressive vector for GCPⅡ was constrcucted and the stable expression cell strain was successfully established to support further researches on GCPⅡand the role on excitotoxicity.

关 键 词：二型谷氨酸羧肽酶 稳定表达细胞株 表达载体 聚合酶链反应 

分 类 号：R32924[医药卫生—人体解剖和组织胚胎学]