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目的构建携带有绿色荧光蛋白(GFP)基因的逆转录病毒载体,观察该报告基因在动物体内外表达情况以及对靶细胞生物特性的影响。方法以质粒pEGFP-C1为模板进行PCR反应,获得增强型绿色荧光蛋白(EGFP)基因片段,定向插入逆转录病毒载体pLXSN获得重组逆转录病毒载体pLXSN-EGFP,转染PA317包装细胞后收集具有感染能力的病毒颗粒,转染人多型性脑胶质瘤细胞SW038-C2,通过荧光显微镜和流式细胞仪观察EGFP作为报告基因在体内外的表达情况以及对靶细胞生物特性的影响。结果成功克隆携带有EGFP基因的逆转录病毒载体,该载体经包装细胞包装后可有效转染人多型性脑胶质瘤细胞SW038-C2并在体内外稳定表达,对靶细胞及其所成肿瘤的生长无抑制。高表达GFP的靶细胞形态变得细长,FACS检测约有98%的细胞表达GFP,体外培养6个月荧光强度没有明显的衰减,而低表达GFP的靶细胞形态无变化,FACS检测有30.7%细胞表达GFP。结论该载体可在体内外成功表达,对靶细胞的生长无明显抑制,高表达GFP的靶细胞有一定形态上的变化;使用GFP作为报告基因,检测到的病毒载体转染率比实际偏低。
Objective To construct retroviral vector carrying green fluorescent protein (GFP) gene and observe its expression in vivo and in vitro as well as its effect on the biological characteristics of target cells. Methods The plasmid pEGFP-C1 was used as a template to obtain enhanced green fluorescent protein (EGFP) gene fragment. The recombinant plasmid pLXSN-EGFP was inserted into the retroviral vector pLXSN and transfected into PA317 cells. Ability of the virus particles transfected human multi-type glioma cells SW038-C2, observed by fluorescence microscopy and flow cytometry EGFP as a reporter gene in vitro and in vivo expression and biological characteristics of target cells. Results The retroviral vector carrying EGFP gene was cloned successfully. The vector was transfected into human multi-type glioma cells SW038-C2 by packaging cells and stably expressed in vitro and in vivo. No inhibition of tumor growth. The morphology of target cells with high expression of GFP became slender. About 98% of the cells expressed GFP by FACS. There was no obvious attenuation of the fluorescence intensity in vitro after 6 months of culture, while the morphology of target cells with low expression of GFP did not change. The FACS assay showed 30.7 % Cells express GFP. Conclusion The vector can be successfully expressed both in vitro and in vivo without any significant inhibition on the growth of target cells. The target cells with high expression of GFP have some morphological changes. Using GFP as the reporter gene, the transfection rate of virus vector was lower than the actual one .