Construction and Characterization of Recombinant HSV-1 Expressing Early Growth Response-1.
Construction and Characterization of Recombinant HSV-1 Expressing Early Growth Response-1.
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表达早期生长反应 1 的重组 HSV-1 的构建和表征。
DOI：
10.1155/2014/629641
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Hsia,Victor
中科院分区：
文献类型：
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作者：
Bedadala,Gautam;Chen,Feng;Figliozzi,Robert;Balish,Matthew;Hsia,Victor
早期生长反应-1(Egr-1)是一种具有多种生物学功能的转录因子。已被证明通过招募不同的辅因子复合体来调节HSV-1基因在不同细胞环境中的表达和复制。以往的研究表明，HSV-1感染可以诱导角膜细胞产生Egr-1，但与其他类型的细胞相比，Egr-1的水平较低。本研究的主要目的是构建重组HSV-1Egr-1，并研究病毒在不同细胞类型或在Egr-1过表达动物体内的复制调控。采用的方法是将Egr-1导入含有HSV-1(F)全基因组的BAC系统。为了辅助Egr-1的插入，构建了一个基因盒，该基因盒包含Egr-1基因，两侧有loxP位点。在该克隆中，Egr-1在CMV即刻早期启动子控制下表达，另一个基因盒在延伸因子1α(EF-1α)启动子控制下表达增强型绿色荧光蛋白。构建的重组病毒将Egr-1基因整合到病毒基因组中，并通过qRT-PCR和Western印迹分析对重组病毒的表达进行了鉴定。我们的结果表明，重组病毒感染Vero和兔角膜细胞SIRC后，可以产生和积累Egr-1的转录本和蛋白。因此，这种独特的病毒对于研究Egr-1在HSV-1复制过程中的作用以及对上皮细胞和神经元的基因调控是有用的。
Early Growth response‐1 (Egr‐1) is a transcription factor that possesses a variety of biological functions. It has been shown to regulate HSV‐1 gene expression and replication in different cellular environments through the recruitment of distinct cofactor complexes. Previous studies demonstrated that Egr‐1 can be induced by HSV‐1 infection in corneal cells but the level was lower compared to other cell types. The primary goal of this report is to generate a recombinant HSV‐1 constitutively expressing Egr‐1 and to investigate the regulation of viral replication in different cell types or in animals with Egr‐1 overexpression. The approach utilized was to introduce Egr‐1 into the BAC system containing complete HSV‐1 (F) genome. To assist in the insertion of Egr‐1, a gene cassette was constructed that contains the Egr‐1 gene flanked by loxP sites. In this clone Egr‐1 is expressed under control of CMV immediate‐early promoter followed by another gene cassette expressing the enhanced green fluorescent protein (EGFP) under the control of the elongation factor 1α(EF‐1α) promoter. The constructed recombinant viruses were completed containing the Egr‐1 gene within the viral genome and the expression was characterized by qRT‐PCR and Western blot analyses. Our results showed that Egr‐1 transcript and protein can be generated and accumulated upon infection of recombinant virus in Vero and rabbit corneal cells SIRC. This unique virus therefore is useful for studying the effects of Egr‐1 during HSV‐1 replication and gene regulation in epithelial cells and neurons.