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

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.