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目的构建表达绿色荧光蛋白的巨细胞病毒Towne株,检测其活性,研究其在抗巨细胞病毒药物筛选中的应用价值。方法将PHM673质粒扩增纯化,采用脂质体转染法转入原代培养的人胚肺成纤维细胞,感染人巨细胞病毒,获得重组病毒GFP-HCMV,采用空斑法纯化重组病毒,采用PCR法鉴定,获得携带绿色荧光蛋白的重组巨细胞病毒。用抗病毒药物更昔洛韦进行干预,检测重组病毒荧光表达强度与药物浓度的关系。结果成功构建携带绿色荧光蛋白的重组巨细胞病毒,荧光病毒的活性与原始病毒的活性差异无统计学意义(P>0.05),生长繁殖并没有受到影响。重组病毒的荧光表达强度与抗巨细胞病毒药物更昔洛韦抑制病毒的效价有关。结论该荧光病毒可以作为研究病毒致病机制和抗病毒药物筛选的新型工具。
Objective To construct the cytomegalovirus Towne strain expressing green fluorescent protein (GFP) and test its activity in the screening of anti-cytomegalovirus drugs. Methods The PHM673 plasmid was amplified and purified. The recombinant plasmid was transformed into primary cultured human embryo lung fibroblasts by lipofection. The recombinant plasmid was infected with human cytomegalovirus (GFP-HCMV) and purified by plaque purification. PCR method to identify, obtain green fluorescent protein-carrying recombinant cytomegalovirus. The antiviral drug ganciclovir was used to intervene to detect the relationship between the intensity of recombinant virus fluorescence and drug concentration. Results Recombinant cytomegalovirus carrying green fluorescent protein was successfully constructed. There was no significant difference between the activity of the fluorescent virus and the original virus (P> 0.05). The growth and reproduction were not affected. The fluorescence intensity of the recombinant virus is related to the anti-cytomegalovirus ganciclovir inhibiting the titer of the virus. Conclusion The fluorescent virus can be used as a new tool to study the pathogenesis of virus and the screening of antiviral drugs.