【摘要】 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。更多还原

【Abstract】 Objective To construct fluorescent protein-expressing human cytomegalovirus(HCMV-Towne-GFP)and to study its application in screening antiviral agents.Methods The plasmid PHM673 was amplified and purified,and then was transfected into primary culture HELF via LipofectamineTM 2000.Then GFP-HCMV was purified by plaque assays and the presence of the recombinant virus was confirmed by PCR. The antiviral agent ganciclovir was screened using the GFP-based antiviral assay.Results GFP-HCMV was successfully constructed and there was no significant difference in activity between the recombinant virus and original virus.The growth and reproduction of recombinant virus were not affected.Fluorescence expression of the experimental group was related to the virus-inhibitory effects of the specific antiviral agent ganciclovir.Conclusion GFP-HCMV may be a new tool to research the pathogenic mechanism and screen antiviral agents.更多还原