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目的:构建巨噬细胞特异性启动子调控的靶向巨噬细胞的真核表达载体。方法:PCR方法合成巨噬细胞特异性启动子,并将其插入带有绿色荧光蛋白(GFP)基因的真核表达载体pEGFP-N1中,替代CMV启动子。利用脂质体转染法,将其与红色荧光蛋白真核表达载体(pERFP-N1)共转染巨噬细胞和非巨噬细胞,通过荧光显微镜观察GFP和RFP在不同细胞中的表达水平来鉴定启动子的靶向性。结果:成功构建了巨噬细胞特异性启动子调控的真核表达载体,并且能在巨噬细胞中高效特异性地表达报告基因。结论:构建的靶向巨噬细胞的真核表达载体能提高目的基因的表达特异性,为提高基因治疗胞内菌感染的靶向性及降低毒副作用奠定了实验基础。
OBJECTIVE: To construct eukaryotic expression vector targeting macrophages regulated by macrophage-specific promoter. Methods: Macrophage specific promoter was synthesized by PCR and inserted into eukaryotic expression vector pEGFP-N1 with green fluorescent protein (GFP) gene instead of CMV promoter. The liposome transfection method was used to co-transfect red fluorescent protein eukaryotic expression vector (pERFP-N1) into macrophages and non-macrophages. The expression levels of GFP and RFP in different cells were observed by fluorescence microscopy Identify promoter targeting. Results: The eukaryotic expression vector regulated by macrophage-specific promoter was successfully constructed and the reporter gene was efficiently and specifically expressed in macrophages. CONCLUSION: The constructed eukaryotic expression vector targeting macrophages can improve the specificity of the target gene expression, and lay a foundation for improving the targeting of gene therapy for intracellular bacteria infection and reducing the side effects.