pEGFP-C2/LAT-ORF3真核表达载体在Vero细胞中的表达特性及其对细胞活性的影响  

Expression Characteristics of Eukaryotic Expression Plasmid pEGFP-C2/LAT-ORF3 in Vero Cells and Its Effect on Cells Activity

作　　者：薛礼长[1,2] 杨慧兰[1] 张发洲[1,2] 龙朝钦[1] 

机构地区：[1]广州军区广州总医院皮肤科,广东广州510010 [2]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代生物医学进展》2010年第22期4245-4248,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(30972666);全军医学科学技术研究"十一五"计划资助项目(06J008)

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3(ORF3)的表达特点及其对细胞活性的影响。方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF3真核表达载体pEGFP-C2/LAT-ORF3的可用性,并将其转染入vero细胞,通过荧光和RT-PCR检验其在细胞中的表达,用MTT法进行细胞活性分析。结果:融合蛋白在细胞核中大量集中,且影响了绿色荧光蛋白在细胞中的分布;重组质粒对Vero细胞没有损伤作用。结论:HSV-2 LAT ORF3可抵消空质粒对细胞的损伤作用;其作用靶点可能主要存在于细胞核中,为阐明HSV-2 LAT ORF3在潜伏复发中的功能提供了实验基础。Objective：To investigate the expression characteristics of cells activity of HSV-2 LAT ORF3 and the effect on Vero cells in vitro.Methods：The availability of recombination eukaryotic vector pEGFP-C2/LAT-ORF3 was identified by restriction endonu-clease digestion and DNA sequencing.Then the recombinant plasmid was transferred into Vero cells mediated by Xfect.The expression of the recombination plasmid in the cells was detected by fluorescence microscope 48 hours later,and the expression of ORF3 was de-tected by RT-PCR.The location of fluorescence was observed by fluorescence microscopy in Vero cells and cell activity were analyzed by MTT.Results：There was a lot of fusion protein of GFP and HSV-2 LAT ORF3 in nucleus,which affect the distribution of green fluo-rescent protein in cells.The recombinant plasmid had no harmful to the Vero cells.Conclusions：HSV-2 LAT ORF3 eliminate the dam-age of empty plasmid and the target may be mainly in the nucleus.The role of ORF3 in Vero cells may be the basis for further research on the mechanism of HSV-2 LAT latency and reactivation.

关 键 词：单纯疱疹病毒Ⅱ型 LATORF3 绿色荧光蛋白 VERO细胞 

分 类 号：Q75[生物学—分子生物学] Q78