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目的构建血管内皮细胞生长因子(VEGF)基因和铜绿假单胞菌外毒素衍生物(PE38)基因的真核融合表达载体,并研究其表达产物对U251细胞的影响。方法通过PCR获得本实验需要的VEGF165基因片段,通过酶切pRB391质粒获得铜绿假单胞菌外毒素衍生物PE38基因,再通过适当的酶切及连接反应,构建VEGF165与PE38融合基因的真核表达载体pIRES2-VEGF165-PE38-EGFP,经酶切及DNA测序证实构建成功后,用Lipofectamine2000脂质体转染293细胞,然后用RT-PCR及Elisa法检测VEGF165-PE38融合蛋白在293细胞的表达,并利用转染细胞后的培养上清体外测定VEGF165-PE38融合蛋白对U251的选择性细胞毒作用。结果酶切分析及DNA测序证实,VEGF165-PE38融合基因被成功克隆入真核表达质粒载体pIRES2-EGFP,RT-PCR及Elisa法检测证实重组基因可在293细胞表达。细胞转染上清对U251具有较强的细胞抑制效应。结论VEGF165-PE38融合基因构建,表达及其功能的初步研究,为进一步研究其对胶质瘤的靶向毒性及临床应用奠定基础。
Objective To construct a eukaryotic fusion expression vector of vascular endothelial growth factor (VEGF) gene and Pseudomonas aeruginosa exotoxin derivative (PE38) gene and study its effect on U251 cells. Methods The VEGF165 gene fragment obtained by this experiment was obtained by PCR. The PE38 gene of Pseudomonas aeruginosa exotoxin derivative was obtained by digesting the plasmid pRB391. The eukaryotic expression of fusion gene VEGF165 and PE38 was constructed by appropriate restriction enzyme digestion and ligation The recombinant plasmid pIRES2-VEGF165-PE38-EGFP was successfully constructed and transfected into 293 cells by Lipofectamine 2000. The expression of VEGF165-PE38 fusion protein in 293 cells was detected by RT-PCR and Elisa. The selective cytotoxic effect of VEGF165-PE38 fusion protein on U251 was determined by using the culture supernatant after transfected cells. Results Enzyme digestion analysis and DNA sequencing confirmed that VEGF165-PE38 fusion gene was successfully cloned into eukaryotic expression plasmid vector pIRES2-EGFP. The recombinant plasmid was confirmed by RT-PCR and Elisa assay in 293 cells. The cell transfection supernatant has a strong cytostatic effect on U251. Conclusion The preliminary study on the construction, expression and function of VEGF165-PE38 fusion gene lays the foundation for further study of its targeted toxicity and clinical application in gliomas.