Establishment of a Stable Cell Line Coexpressing Dengue Virus-2 and Green Fluorescent Protein for Screening of Antiviral Compounds
Establishment of a Stable Cell Line Coexpressing Dengue Virus-2 and Green Fluorescent Protein for Screening of Antiviral Compounds
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DOI：
10.1177/1087057111426903
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发表时间：
2012-03-01
影响因子：
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通讯作者：
Sirigulpanit, W.
中科院分区：
文献类型：
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作者：
Leardkamolkarn, V.;Sirigulpanit, W.
本研究旨在生成一种稳定的细胞系，该细胞系含有亚基因组登革热病毒复制子和绿色荧光基因（DENV/GFP），用于基于细胞的模型筛选抗DENV化合物。DENV-2的基因编码包膜蛋白被删除，然后被GFP基因片段和口蹄疫病毒2a衍生的切割位点所取代。在5′端和3′端分别添加人巨细胞病毒即时早期和反义丁型肝炎病毒核酶序列。内核糖体进入位点和新霉素耐药基因被放置在NS1基因的上游和旁边。重组质粒在哺乳动物细胞系中繁殖。从6个克隆中筛选出绿色荧光蛋白最亮、病毒蛋白和RNA表达最高的稳定细胞系。然后使用两种已知的抗病毒药物作为对照，用从当地植物中分离的化合物检测该克隆在抗病毒药物筛选试验中的有效性。两种新的黄酮类化合物PMF和TMF被发现具有抑制denv的特性。这些数据通过传统的斑块滴定试验得到验证。结果表明，该新建立的细胞系可作为抗denv化合物初步筛选的细胞模型。
This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.