肿瘤特异性锚定型α-gal模拟表位真核表达质粒的构建及功能研究

Construction and function research of eukaryotic expression plasmid encoding tumor-specific anchored α-gal mimic epitope

目的构建Survivin启动子调控的、α-gal模拟表位序列和GPI锚定序列融合基因的真核表达质粒,研究该质粒在肺癌A549细胞表面锚定表达α-gal模拟表位及其抗肺癌细胞效应。方法 PCR法扩增gal-GPI融合基因,以pSGFP质粒为载体,构建Survivin启动子调控的pSGFP-gal-GPI真核表达质粒。将其用脂质体瞬时转染A549细胞后,用荧光显微镜观察GPI锚定表达功能,分别用免疫组化法和Western-blot法对锚定表达的α-gal模拟表位进行定位和免疫反应性分析,并用新鲜人血清对表达α-gal模拟表位的A549细胞进行补体介导的溶细胞效应研究。结果 pSGFP-gal-GPI质粒转染细胞的绿色荧光不均匀分布在细胞膜周围,免疫细胞化学分析表明重组融合蛋白表达在细胞膜上,Western-blot结果表明重组融合蛋白与人IgG有良好的免疫反应性;人新鲜血清对表达该表位的A549细胞具有明显的溶细胞效应。结论成功构建了能在A549细胞膜锚定表达α-gal模拟表位的pSGFP-gal-GPI真核表达质粒,为肺癌等靶向基因治疗奠定了基础。

This paper is aimed to construct the eukaryotic expression plasmid of the fusion gene of α-gal mimic epitope and GPI anchored sequence,and to study the expression of tumor-specific anchored α-gal mimic epitope as well as its cytolysis effect in A549 cells. The gal-GPI fusion gene was amplified by PCR,then the pSGFP-gal-GPI eukaryotic expression plasmid was constructed,which was modulated by tumor-specific survivin promoter. Then the recombinant plasmid was transfected into A549 cells by Lipofec-tamine 2000. Fluorescence microscopy showed that the fluorescence of transfected A549 distributed around the cell membrane unevenly; immunocytochemistry indicated the recombinant protein was around the A549 cell membrane; while Western blotting confirmed that the protein was successfully expressed in A549 cells,which can react with human IgG in serum. Furthermore,the transfected A549 cells could react with normal human serum （anti-gal） and cause the cytolysis effect induced by complement. In this study,the pSGFP-gal-GPI eukaryotic expression plasmid which can express anchored α-gal mimic epitope on A549 cell was constructed successfully,which provide a basis for further study of targeting gene therapy of tumor.