论文部分内容阅读
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。
Objective To construct an eukaryotic expression plasmid containing Survivin promoter-regulated α-gal mimotope sequence and GPI anchored sequence fusion gene and study the expression of α-gal mimotope on lung A549 cells and its anti-lung cancer cells effect. Methods The gal-GPI fusion gene was amplified by PCR. The pSGFP plasmid was used as the vector to construct the eukaryotic expression plasmid pSGFP-gal-GPI regulated by Survivin promoter. After transient transfection of A549 cells with liposome, the GPI anchored expression was observed by fluorescence microscopy. The anchored α-gal mimotopes were located and immunoreactive with immunohistochemistry and Western-blot respectively Sex analysis and complement-mediated cytolysis of A549 cells expressing a-gal mimotopes with fresh human serum were studied. Results The green fluorescence of pSGFP-gal-GPI transfected cells was unevenly distributed around the cell membrane. Immunocytochemical analysis showed that the recombinant fusion protein was expressed on the cell membrane. Western-blot results showed that the recombinant fusion protein had good immunoreactivity with human IgG Human fresh serum had obvious cytolytic effect on A549 cells expressing this epitope. Conclusion The eukaryotic expression plasmid pSGFP-gal-GPI that can express α-gal mimic epitope in A549 cell membrane has been successfully constructed, which lays a foundation for targeted gene therapy such as lung cancer.