单纯疱疹病毒Ⅱ型潜伏相关转录体真核表达载体的构建及表达  

Construction and expression of a herpes simplex virus type II latency-associated transcript eukaryotic expression vector

作　　者：吕芳彪[1] 杨慧兰[2] 

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

出　　处：《中国组织工程研究》2012年第42期7866-7870,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30972666)~~

摘　　要：背景:单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1真核表达载体的构建可以为研究单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1在病毒潜伏复发中的功能奠定基础。目的:构建含存单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1的真核表达载体,并通过转染非洲绿猴肾细胞(Vero),验证载体的体外表达情况。方法:根据单纯疱疹病毒Ⅱ型潜伏相关转录体开放读码框1基因序列设计一对引物,以单纯疱疹病毒Ⅱ型333标准株基因组为模板PCR法扩增出开放读码框1基因,克隆至真核表达载体上,并进行酶切鉴定以及测序;利用X-fect转染试剂盒将重组质粒pEGFP-C2/潜伏相关转录体开放读码框1转染vero细胞,RT-PCR检测其mRNA水平的表达,并用荧光倒置显微镜观察融合蛋白的表达。结果与结论:开放读码框1基因的体外扩增目的片段为741bp,所构建的真核表达载体pEGFP-C2/潜伏相关转录体开放读码框1经双酶切鉴定,与预期大小一致,测序结果与NCBI收录的开放读码框1基因序列一致;重组质粒pEGFP-C2/潜伏相关转录体开放读码框1转染vero细胞后,RT-PCR验证有目的基因的转录,荧光显微镜观察到融合蛋白在转染的Vero细胞中表达。表明成功构建pEGFP-C2/潜伏相关转录体开放读码框1真核表达载体,并且实现其在非洲绿猴肾细胞(Vero)中的表达。BACKGROUND：The construction of herpes simplex virus（HSV-II） latency-associated transcript（LAT） open reading frame 1（ORF1）（pEGFP-C2/LAT ORF1） eukaryotic expression vector can lay a foundation for the study of the function of HSV-2 LAT ORF1 in the virus latency and recurrence.OBJECTIVE：To construct pEGFP-C2/LAT ORF1 eukaryotic expression vector and to verify its expression in vitro through the transfection of African green monkey kidney cells.METHODS：A PCR primer was designed according to DNA sequencing of pEGFP-C2/LAT ORF1.HSV-Ⅱ333 standard plant genome was used as the template and ORF1 gene was amplified by PCR method and subcloned into the pEGFP-C2 eucaryotic experssion vector.And then enzyme digestion and DNA sequencing were used for identifying the recombinant plasmid pEGFP-ORF1.Finally,the recombinant plasmid pEGFP-C2/LAT ORF1 was transfected into Vero cells in vitro by X-fect transfection kits.Fusion green fluorescent protein expression was observed by inverted fluorescence microscope and its mRNA expression levels were detected by reverse transcription PCR.RESULTS AND CONCLUSION：In vitro amplified targeted DNA fragment was 741 bp in length and the size of pEGFP-C2/LAT ORF1 eucaryotic experssion vector constructed was conformed to the expectation,moreover,its sequencing results were conformed to those of ORF1 gene sequencing included by NCBI.Recombinant plasmid pEGFP-C2/LAT-ORF1 was verified by reverse transcription PCR after transfected into Vero cells,and green fluorescent protein expression in the transfected Vero cells was determined by fluorescence microscope.These results suggest that the pEGFP-C2/LAT ORF1 eucaryotic experssion Vector have successfully constructed and realized its expression in African green monkey kidney cells（Vero cells）.

关 键 词：单纯疱疹病毒Ⅱ型 潜伏相关转录体 开放读码框1 真核表达载体 转染 体外表达 

分 类 号：R318[医药卫生—生物医学工程]