HSV-2潜伏相关转录体开发读码框2载体的构建及其在SH-SY5Y细胞中的表达  

Construction of HSV - 2 LAT ORF2 eukaryotic expression vector and its expression in SH- SY5Y cells

作　　者：杨慧兰[1] 白利利[1,2] 樊建勇[1] 

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

出　　处：《中国麻风皮肤病杂志》2010年第4期233-235,共3页China Journal of Leprosy and Skin Diseases

基　　金：国家自然科学基金项目;编号:30972666;全军医学科学技术研究"十一五"计划课题项目;编号:06J008

摘　　要：目的:构建单纯疱疹病毒2型(HSV-2)潜伏相关转录体(LAT)的开放读码框2(ORF2)基因的真核表达载体pEGFP-ORF2,并检测其在SH-SY5Y细胞中的表达。方法:采用亚克隆技术构建HSV-2 LAT ORF2与EGFP基因融合的真核细胞表达载体pEGFP-ORF2,双酶切及测序鉴定;脂质体法转染试剂将重组载体转染人神经母细胞瘤细胞SH-SY5Y,G418筛选20天挑取表达pEGFP-ORF2的抗性克隆扩大培养,传代。荧光显微镜直接观察重组体在细胞中的表达。结果:重组质粒pEGFP-ORF2序列和方向正确;EGFP-ORF2融合基因在SH-SY5Y细胞中获得表达。结论:成功建立稳定表达pEGFP-ORF2的SH-SY5Y细胞株,为进一步研究HSV-2 LAT ORF2基因的功能奠定了实验基础。Objective： To construct pEGFP- ORF2 eukaryo/ic expression vector anti to establish stable transfected SH - SY5Y cell line expressing pEGFP - ORF2 tusion protein related to HSV - 2, and to detect it＇ s expression in SH - SY5Y cells. Methods： The eukaryotic HSV - 2 LAT ORb2 and EGFP fusion gene expression vector pEGFP- ORb2 was constructed by gene sub- clone ,nethod. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing. The recombination vector was transfected into human neuronal cell line SH - SY5Y by liposome transfection reagent. Anti - clones stably expressing HSV - 2 LAT ORF2 were observed by fluo- rescence microscope.-Results： The sequenee and direction of reeombinant plasmid pEGFP- ORF2 was correct anti the expression of EGFP - ORF2 was observed with green fluorescent protein. Conclusion： Eukaryotic expression vector pEGFP - ORBS2 has been constructed suecesst＇ully, which forms a basis fi）r further study of the function of HSV - 2 LAT ORF2 gene.

关 键 词：HSV-2 LAT ORF2 绿色荧光蛋白 SH-SY5Y 

分 类 号：R752.11[医药卫生—皮肤病学与性病学]