GFAP特异性启动IκBα突变型基因真核表达载体的构建和鉴定

Construction and Identification of Eukaryotic Expression Vector that Express Mutated IκBα under the Control of GFAP Promoter

目的构建GFAP特异性启动的、能稳定表达IκBα突变型基因的真核表达载体。方法用基因工程的方法将潮霉素抗性基因和GFAP特异性启动的IκBα突变型基因定向克隆入载体pBluescript-SK中,酶切反应鉴定重组载体SK-hyg和SK-hyg-GFAP/IκBαM。用脂质体法分别将载体SK-hyg和SK-hyg-GFAP/IκBαM转入永生化星形胶质细胞株和PC12细胞株中,经潮霉素(150μg/mL)筛选,挑选阳性克隆并扩大培养,Westernblot检测阳性细胞株中IκBα的表达,以NF-κB特异性启动表达的荧光素酶报告基因检测细胞中NF-κB的活性。结果限制性酶切分析证实重组载体成功,稳定转染SK-hyg和SK-hyg-GFAP/IκBαM的PC12细胞内IκBα蛋白质表达及细胞中NF-κB活性无差异,稳定转染SK-hyg-GFAP/IκBαM的永生化星形胶质细胞内有突变型IκBα的蛋白质表达,而转染SK-hyg的永生化星形胶质细胞内则没有,且前者细胞内NF-κB活性下降。结论GFAP特异性启动的、能稳定表达IκBα突变型基因的真核表达载体构建成功。

Objective To establish eukaryotic expression vector that can stably express mutated IkBα under the control of glial fibrillary acidic protein（GFAP） promoter. Methods The bygromycin resistance gene and the mutated IkBα under the control of GFAP promoter were cloned properly into pBluescript-SK by genetic engineering. The recombinant plasmids SK hyg and SK-byg-GFAP/IkBαM were identified by restriction enzyme digestion. The immortalized astrocyte and PC12 cell strain was transfected with plasmids SK-byg and SK-byg- GFAP/IkBαM. Ceils containing stable transformants were selected and isolated by the ability of resistance to hygromycin （150 μg/mL）. Western blot was used to detect the expression of IkBα protein. Luciferase assay system was used to detect the activity of NF-kB in transfected cells. Results It was established by restriction enzyme digestion that the hygromycin resistance gene and the mutated IkBα under the control of GFAP promoter were recombinated to the vector successfully. There was no difference in the expression of IkBα protein and the NF-kB activity between the SK-hyg transfected and SK-hyg-GFAP/IkBαM transfected PC12 cell. Mutated IkBα protein was expressed in SK-hyg-GFAP/IkBαM transfected immortalized astrocyte and the NF-kB activity was also down-regulated in the cell. But mutated IkBα protein was not espressed in SK-hyg transfected immortalized astrocyte. Conclusions Eukaryotic expression vector that can stably express mutated IkBα under the control of GFAP promoter has been successfully established.