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.
出处
《基础医学与临床》
CSCD
北大核心
2004年第3期331-334,共4页
Basic and Clinical Medicine