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目的:脂质体介导重组起搏基因pIRES2-EGFP-HCN2转染HEK293细胞,观察转染效率,探讨其构建生物起搏器的可行性。方法:对含hHCN2cDNA的PTR载体进行转化和扩增,将所得hHCN2基因定向克隆到真核表达载体pIRES2-EGFP中,双酶切来鉴定克隆的正确性。将重组质粒用脂质体转染HEK293,荧光显微镜观察EGFP的表达并计算转染效率。结果:构建了重组质粒pIRES2-EGFP-HCN2。荧光显微镜下可见转染后的HEK293呈绿色荧光,其转染效率可达90%以上。结论:成功构建了重组质粒pIRES2-EGFP-HCN2,并用脂质体转染的方法导入HEK293细胞,为生物起搏的研究奠定实验基础。
OBJECTIVE: Liposome-mediated recombination pacing gene pIRES2-EGFP-HCN2 was transfected into HEK293 cells to observe the transfection efficiency and to explore its feasibility of constructing biological pacemaker. Methods: The hHCN2 cDNA containing PTR vector was transformed and amplified. The resulting hHCN2 gene was cloned into the eukaryotic expression vector pIRES2-EGFP, and double-digested to identify the correctness of the cloning. The recombinant plasmids were transfected into HEK293 by lipofectamine. The expression of EGFP was observed by fluorescence microscopy and the transfection efficiency was calculated. Results: The recombinant plasmid pIRES2-EGFP-HCN2 was constructed. Under the fluorescence microscope, HEK293 transfection showed green fluorescence, the transfection efficiency of up to 90%. CONCLUSION: The recombinant plasmid pIRES2-EGFP-HCN2 was successfully constructed and transfected into HEK293 cells by lipofectamine transfection, which laid the experimental foundation for the study of biological pacing.