GFP-SUMO-3融合蛋白的重组及其在LNCaP细胞中的表达和定位

Recombination of GFP-SUMO-3 fusion protein and its expression and localization in LNCaP cells

目的:构建以绿色荧光蛋白(GFP)为报告基因的重组真核表达载体pEGFP-N1-SUMO-3,观察GFP-SUMO-3融合蛋白在人前列腺癌细胞LNCaP中的表达与定位。方法:采用PCR扩增SUMO-3基因全长cDNA编码区序列,克隆入pEGFP-N1真核表达载体。重组质粒pEGFP-N1-SUMO-3通过酶切和测序鉴定正确后,脂质体法将重组质粒转染至LNCaP中,利用GFP抗体通过Western blotting检测GFP-SUMO-3融合蛋白的表达,通过荧光显微镜观察其在细胞中的表达及分布。结果:重组质粒pEGFP-N1-SUMO-3经BamHⅠ和XhoⅠ双酶切鉴定,得到大小分别为4 700和312bp的条带,与预期结果一致。测序结果显示,GFP在N端,是阅读框正确的SUMO-3的基因序列。在LNCaP细胞中过表达GFP-SUMO-3融合蛋白,Western blotting得到相对分子质量为39 000的蛋白表达条带,与GFP-SUMO-3大小相符。荧光显微镜观察,转染重组质粒pEGFP-N1-SUMO-3的细胞中呈绿色荧光,主要在细胞核内表达,与DAPI重合。结论:成功构建重组质粒pEGFP-N1-SUMO-3,并表达代绿色荧光蛋白的SUMO-3融合蛋白,鉴定出SUMO-3在LNCaP细胞中的核定位性。

Objective To construct the fusion gene eukaryotic expression vector pEGFP-N1-SUMO-3,and to study the expression and localization of GFP-SUMO-3 fusion protein in human prostate cancer cell line LNCaP.Methods The encoding region of SUMO-3 was obtained by PCR,and cloned into eukaryotic expression vector pEGFP-N1.Then the pEGFP-N1-SUMO-3 plasmid was transfected into LNCaP cells by liposome.The expression of the fusion protein was detected by Western blotting,and the subcellular localization was observed by fluorescence microscope.Results The recombinant plasmid pEGFP-N1-SUMO-3 was identified by BamHⅠ and XhoⅠ double digestion.As expected,it showed two bands of 4 700 and 312 bp.The recombinant plasmid also showed right sequence by the full length sequencing.The pEGFP-N1-SUMO-3 plasmid was transfected into prostate cancer LNCaP cells.A specific protein expression band at molecular weight of 39 000 was detected with using GFP-antibody by Western blotting method.When observed by fluorescence microscope,the GFP-SUMO-3 fusion protein was mainly located in the nucleus of LNCaP cells.Conclusion The recombinant plasmid pEGFP-N1-SUMO-3 is successfully constructed,and GFP-SUMO-3 fusion protein is successfully expressed in mammalian cells and mainly located in the nucleus.