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。 In order to study the function of glutamate carboxypeptidaseⅡ (GCPⅡ) and its role in the metabolism of excitatory amino acids, GCPⅡexpression vector was constructed and stable cell lines were constructed. The expression of GCPⅡwas determined by polymerase chain reaction Positive cells were screened by G418, and positive cells were identified by reverse transcription-polymerase chain reaction and immunofluorescent staining. GCPⅡ was amplified by PCR method. The expression vector containing GCPⅡ gene was constructed, pcDNA3 1 NA. The cell line HEK 2 93 NA was established. The GCPⅡexpression vector was successfully constructed and a stable expression cell line was established, which lays a foundation for further study on the function of this enzyme and the toxic effect of excitatory amino acids.