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HSV-2LATORF1在Vero细胞中的表达特性及其对细胞活性的影响

Expression and Characteristics of HSV-2 LAT ORF1 in Vero Cells and Its Effects on Cells Activities
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摘要 目的:研究单纯疱疹病毒Ⅱ型(HSV-2)潜伏相关转录体(LAT)开放读码框1(ORF1)的表达特点及其对Vero细胞活性的影响。方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF1真核表达载体pEGFP-ORF1,并以转染试剂盒Xfect介导其转染至Vero细胞,通过RT-PCR和绿色荧光蛋白检验其在细胞中的表达,用MTT法进行细胞活性分析。结果:重组质粒表达的融合蛋白主要集中细胞核,而空质粒表达的绿色荧光蛋白在细胞核和细胞质中分布均匀;重组质粒对Vero细胞没有损伤作用。结论:HSV-2 LAT ORF1影响了绿色荧光蛋白的分布,可降低空质粒对细胞的损伤作用;其作用位点可能主要定位在细胞核中,为阐明HSV-2 LAT ORF1在潜伏复发中的功能奠定了实验基础。 Objective： To study the expression and characteristics of HSV-2 LAT ORF1 and its effects on Vero cells activities in vitro.Methods： The laboratory constructed HSV-2 LAT ORF1 eukaryotic expression vector pEGFP-ORF1 was verified by double en-zymes digestion and sequencing.Then the recombinant plasmid was transfected into Vero cells mediated by the kit Xfect.The expression of the recombination plasmid in the cells was observed by fluorescence microscopy and the expression of ORF1 was verified by RT-PCR.The cells activities were analyzed by MTT.Results： Fusion protein expressed by the recombinant plasmid is mainly exists in the nucleus,while the empty plasmid expressing green fluorescent protein distributed evenly in the nucleus and cytoplasm.The recombinant plasmid had no harmful to the Vero cells.Conclusion： HSV-2 LAT ORF1 can reduce the damage caused by empty plasmid and the target may be mainly exists in the nucleus.The function of ORF1 in Vero cells may has lay the basis for further research on the mechanism of HSV-2 LAT latency and reactivation.
作者 吕芳彪 杨慧兰 樊建勇 刘艳华 张丽 王颖
机构地区 广州军区广州总医院皮肤科 华南理工大学生物科学与工程学院
出处 《现代生物医学进展》 CAS 2013年第4期607-610,共4页 Progress in Modern Biomedicine
基金 国家自然科学基金项目(30972666) 国家自然科学基金项目(81071401) 广东省科技计划项目(2009B080701091)
关键词 单纯疱疹病毒Ⅱ型 LAT ORF1 转染 绿色荧光蛋白 HSV-2 LAT ORF1 Transfection GFP
分类号 Q78 [生物学—分子生物学] R373 [医药卫生—病原生物学]
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参考文献20

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二级参考文献25

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现代生物医学进展
2013年 第4期

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