人视网膜母细胞瘤蛋白真核表达载体的构建及其在前列腺癌细胞中的表达与定位

Construction, expression and location of eukaryotic expression vector CMV-FLAG-RB in human prostate cancer PC-3 cells

目的构建人视网膜母细胞瘤(retinoblastoma 1,RB-1)基因真核表达载体,并在前列腺癌细胞株PC-3中表达,为进一步研究其在前列腺癌发病中的作用和机制打下基础。方法采用PCR方法扩增RB-1基因编码区的全长序列,并加上FLAG标签,利用分子克隆技术将其重组于CMV载体中。利用酶切、序列分析鉴定克隆正确性。转染PC-3细胞,用Western Blot检测其表达,用免疫荧光检测其在细胞中的定位。结果克隆的RB-1真核表达载体完全正确,Western Blot检测到RB-1有表达,免疫荧光检测其主要定位于细胞核内。结论成功地构建了RB-1真核表达载体,其能够在PC-3中高效表达,并主要定位于细胞核内。

Objective To construct an eukaryotic recombinant expression vector for retinoblastoma 1 gene （RB-1） and investigate the role of RB-1 in prostate cancer. Methods The coding sequence of RB-1 gene tagged with FLAG was amplified from the plasmid CMV-RB by PCR method. The fragment was cloned into CMV expression vector and identified by restriction enzyme digestion and sequence analysis. Western Blotting was used to detect RB-1 expression and immunofluorescence was used to observe RB-1 distribution in PC-3 cells transfected with the recombinant. Results The expression vector CMV-FLAG-RB was successfully constructed as confirmed by PCR, endonuclease digestion and DNA sequence analysis. RB-1 protein was highly expressed and showed a nuclear distribution in PC-3 cells transfected with the recombinant. Conclutions The eukaryotic expression vector for RB-1 has been successfully constructed and can be efficiently expressed in PC-3 cells. The expression of RB-1 is located in the cell nuclei.