葡萄糖调节蛋白78真核表达载体及稳定转染人视网膜色素上皮细胞株的构建

背景：葡萄糖调节蛋白78是存在于内质网上最多的分子伴侣蛋白，具有保护细胞对抗细胞调亡的作用。目的：构建携带并正确表达外源性人葡萄糖调节蛋白78基因的重组真核表达载体，建立葡萄糖调节蛋白78基因稳定转染的人视网膜色素上皮细胞系。方法：扩增人葡萄糖调节蛋白78全长编码序列，将该目的基因与真核表达载体PEGFP-N1进行定向连接，其产物转化细菌感受态细胞，以PCR方法鉴定阳性克隆，并进行测序和分析比对，获得融合蛋白表达质粒载体pEGFP-grp78。通过阳离子脂质体介导将重组质粒pEGFP-grp78转染入体外培养的人视网膜色素上皮细胞，经G418筛选，建立稳定表达细胞系。结果与结论：体外培养的人视网膜色素上皮细胞株作为真核细胞表达系统，经荧光显微镜和RT-PCR方法检测，G418筛选的稳定转染细胞系中GFP大量表达，葡萄糖调节蛋白78 mRNA的表达量是正常视网膜色素上皮细胞的4倍左右，表示脂质体介导的pEGFP-grp78质粒成功转染视网膜色素上皮细胞，并高效稳定表达葡萄糖调节蛋白78。

Construction of human glucose regulated protein 78 eukaryotic expression vector and expression in retinal pigment epithelium cells

BACKGROUND:Glucose regulated protein 78 (grp78) is a molecular chaperone protein, which is mostly located on the endoplasmic reticulum, and protects cells from cell apoptosis.OBJECTIVE:To construct the pEGFP-grp78 fusion protein eukaryotic expression vector and to detect their expression in human retinal pigment epithelium (RPE) cell lines.METHODS:The encoding fragment of human grp78 gene was obtained from a recombinant plasmid POTB7-grp78 using PCR, and it was digested by restriction enzymes Hind Ⅲ and KpnⅠ. Then the purified grp78 gene fragment was inserted into the GFP expression vector PEGFP-N1, and the recombinant plasmid PEGFP-grp78 was identified by PCR analysis and DNA sequencing. The recombinant plasmids were transfected into RPE cells by lipofectamin. RPE cell lines with grp78 stable expression were established by G418 selection. The expression of grp78 gene was detected by RT-PCR. The expression of fusion proteins was assayed by fluorescence microscopy.RESULTS AND CONCLUSION:We successfully constructed the expression vector of pEGFP-grp78 fusion protein and the sequence results were matched to the cDNA of human grp78. Stably transfected RPE cell line was established and grp78 was expressed successfully.