细胞周期蛋白D1重组质粒的构建及其在鼻咽癌细胞中的表达

目的克隆人细胞周期蛋白D1基因,构建真核表达载体,诱导并鉴定其表达。方法利用RT-PCR法,以低分化鼻咽癌CNE-2Z细胞mRNA为模板,扩增周期蛋白D1基因,构建重组表达载体pIRES2-EGFP-D1和pEGFP-C2-D1,用脂质体LipofectamineTM 2000将重组质粒转染到低分化鼻咽癌CNE-2Z细胞,诱导其表达,免疫荧光、Western blotting等鉴定其表达。结果电泳获得918bp预期片段,重组质粒经酶切、PCR和测序分析证实含有周期蛋白D1完整读码框,将重组质粒导入CNE-2Z细胞并成功表达,目的蛋白经免疫荧光和Western blotting鉴定具有生物学活性。结论成功克隆并构建含周期蛋白D1基因的真核表达GFP质粒,为进一步研究周期蛋白D1在鼻咽癌CNE-2Z细胞中的作用奠定基础。

Construction of cyclin D1 recombinant plasmids and its expression in human nasopharyngeal carcinoma cells

Objective To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells(CNE-2Z cells).Methods The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR.The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion,PCR and sequencing.The recombinant vectors were transfected into CNE-2Z cells via LipofectamineTM 2000,and the expression of cyclin D1 in the ce...