稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体GluA1亚基单克隆细胞株的构建

Establishment of monoclonal cell strain stably expressingα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluA1

目的构建稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体GluA1亚基的细胞株。方法PCR扩增GluA1目的基因,并将其装载至慢病毒表达载体PLV. 0-绿色荧光蛋白(GFP),采用4质粒系统转染HEK293T细胞包装慢病毒。嘌呤霉素抗性筛选细胞,挑选单克隆细胞株用Real-time PCR、Western blotting、免疫荧光和全细胞膜片钳鉴定GluA1的表达与功能。结果菌落PCR鉴定扩增产物与GluA1分子量(2724 bp)一致,经测序插入慢病毒载体序列与GluA1序列一致;慢病毒感染并抗性筛选的HEK293T细胞经Real-time PCR、Western blotting和免疫荧光实验证明,GluA1在过表达GluA1组的HEK293T细胞正确表达,空载体组不表达。单克隆挑选的稳定细胞株免疫荧光染色显示,各细胞膜表面GluA1蛋白表达相对一致。单克隆细胞在电压钳模式下,细胞外液加10 mmol/L谷氨酸诱导,空载体组检测不到电信号,而过表达GluA1组可记录5~40 p A电信号。结论成功构建了稳定表达AMPA受体GluA1亚基的HEK293T细胞株。

Objective To build a cell line stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid（ AMPA） receptor GluA1 subunit. Methods The GluA1 gene was amplified by PCR and loaded into the lentiviral expression vector PLV. 0-green fluorescent protein（ GFP）. HEK293 T cells were transfected by using the four plasmid system. Puromycin was used for screening stable cell lines. The monoclonal cells were identified by Real-time PCR,Western blotting,immunofluorescence and whole cell patch clamp. Results The amplified product identified by colony PCR was consistent with the molecular weight of GluA1（ 2724 bp）. Further sequencing result showed that the sequence inserted into the lentiviral vector was consistent with the GluA1 sequence; HEK293 T stable cell lines screened by puromycin selection were confirmed by Real-time PCR,Western blotting and immunofluorescence. GluA1 expression was correct in the experimental group,but not expressed in the control group. Immunofluorescent staining showed that the expression of GluA1 protein was relatively uniform on each cell surface. Stable cell lines were detected in voltage clamp mode with 10 mmol/L glutamate in extracellular fluid,and no electrical signal was detected in the empty vector group,whereas 5-40 p A electrical signal was recorded in the GluA1 overexpressing group. Conclusion We successfully established a HEK293 T monoclonal stable cell strain expressing GluA1 subunit.