摘要
从肝星状细胞(HSC-T6)中获得瞬时受体电位通道7(TRPM7)蛋白的cDNA,采用EcoR I和BamH I双酶切,用T4连接酶将该cDNA连接pEGFP-C2质粒将构建成功的pEGFP-C2-TRPM7质粒经PCR、限制性内切酶酶切和测序鉴定后转染非洲绿猴肾成纤维细胞(COS-7),荧光显微镜下观察绿色荧光蛋白表达,PCR鉴定基因表达。结果显示进行双酶切鉴定该质粒可见TRPM7片段,转染重组质粒后可观察到绿色荧光蛋白的表达,PCR结果也可检测到TRPM7基因表达。表明成功构建重组pEGFP-C2-TRPM7表达裁体,TR-PM7蛋白可与绿色荧光蛋白在COS-7细胞中融合表达。
Get the cDNA of transient receptor potential melastatin 7（TRPM 7） from HSC-T6.Then TRPM7 cDNA was amplified by PCR and cut with the double enzyme EcoR I and BamH I,then insert into the eukaryotic expression vector pEGFP-C2 with T4 enzyme.The recombinant vector was verified by PCR,restriction enzymes cut and sequencing identified.Then transfected into renal fibroblasts COS-7 cells and the expression of pEGFP-C2-TRPM7 was monitored by fluorescence and confocal microscope and PCR.Deal with recombinant pEGFPC2-TRPM7 with double digestion,then can see fragments of TRPM7,also GFP can be detected in the transfected renal fibroblasts COS-7 cells.TRPM7 gene expression can detected by PCR.Successfully constructed eukaryotic expression vector of TRPM7,the fusion expression of TRPM7 protein and GFP can be detected in COS-7.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第1期89-93,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81072686)
