Sedlin蛋白稳定表达细胞株的建立

Establishment of a stable cell strain expressing Sedlin protein

目的建立稳定表达Sedlin蛋白的细胞株,研究Sed-lin蛋白在细胞内的分布。方法扩增SEDL基因全长编码序列,经双酶切后克隆至真核表达载体pCDGFP中,将重组表达质粒转染至HEK293细胞,用G418筛选阳性克隆,荧光显微镜和Western blot检测Sedlin蛋白的表达,并观察Sedlin蛋白在HEK293细胞中的分布。结果经测序鉴定,成功构建了SEDL基因的真核表达载体,转染HEK293细胞后,Western blot检测,证明SEDL基因能够稳定有效的表达,荧光观察显示,Sedlin蛋白在细胞核和细胞质中都有表达。结论成功建立了稳定表达Sedlin蛋白的细胞株,Sedlin蛋白广泛分布于整个细胞中。

Objective To establish a stable cell strain expressing protein Sedlin continuously and to study the distribution of Sedlin in HEK 293 cells. Methods PCR methods were used to amplify the open reading frame of SEDL from pAS-SEDL. Then the fragment was inserted into pCDGFP vector after digested by BamH Ⅰ and EcoR Ⅰ to construct expression vector pCDGFP-SEDL. HEK 293 ceils were transfected with plasmid pCDGFP-SEDL, then the cells were selected under the pressure of G418, fluorescent microscopy and Western blot were performed to detect the expression of Sedlin protein. Results An eukaryotic expression vector encoding GPF-Sedlin fusion protein was constructed. The results of western blot demonstrated that GFP-Sedlin fusion protein could express stably. Immunofluorescence showed Sedlin localized both in nucleus and cytoplasm. Conclusion A stable cell strain expressing Sedlin protein has been established. Sedlin is found to be widely distributed in the whole cell.