携带GFP绿色荧光标记的重组BAC-HSV-1 HF株的构建及其子代病毒的特性研究  

Construction of a Recombinant BAC-HSV-1 Strain HF with a GFP Reporter Gene and Characterization of Its Infectious Progeny Virus

作　　者：刘新静[1] 宋波[1] 卢甲盟[1] 王青志[1] 韩志强[1] 许予明[1] 

机构地区：[1]郑州大学第一附属医院神经内三科,郑州450052

出　　处：《病毒学报》2011年第3期238-243,共6页Chinese Journal of Virology

基　　金：河南省卫生厅医学科技攻关项目(200801003);河南省卫生厅医学科技攻关项目(200702005);河南省教育厅河南省基础与前沿技术研究计划项目(082300450270);河南省教育厅自然科学研究计划项目(2009A320045)

摘　　要：本研究旨在构建由细菌人工染色体(Bacteria artificial chromosome,BAC)携带的单纯疱疹I型病毒质粒及携带绿色荧光蛋白(Green fluorescent protein,GFP)的重组型BAC-HSV-1感染性子代病毒。构建了携带HSV-1同源臂的质粒C223-UL43左臂-UL47右臂。将该质粒线性化后与HSV-1基因组共转染至Vero细胞,通过真核细胞内同源重组产生了含有GFP报告基因的BAC-HSV-1重组病毒,噬斑纯化筛选出阳性重组病毒,并再次感染Vero细胞,Hirt法提取BAC-HSV-1环形基因组并将其电穿孔入DH10B感受态细胞,由PCR和酶切法鉴定BAC-HSV-1质粒。为研究BAC-HSV-1子代病毒的生物学特性,将实验组和对照组细胞分别给予BAC-HSV-1质粒和HSV-1基因组DNA,收取病变细胞的上清液,以MOI=0.1再次感染Vero细胞,半数组织培养感染剂量(50% tissue culture infective dose,TCID50)法测定两组的病毒滴度。PCR和酶切法分别鉴定BAC-HSV-1,结果示BAC-HSV-1构建成功。TCID50法测定实验组和对照组病毒滴度,经统计学分析两组病毒滴度间差异无统计学意义(P>0.05)。本研究成功地构建了真核细胞和原核细胞间穿梭的HSV-1-BAC重组病毒/质粒。To construct the plasmid of BAC-HSV-1 with GFP reporter gene and research the biological property of its infectious progeny virus.We constructed the plasmid C223-UL43-left-arms-UL47-right-arms which carried the homologous sequences of HSV-1.Liposome embedding method was used to transfect HSV-1 genome and the plasmid C223-UL43-left-arms-UL47-right-arms linearized by Mlu I digestion into Vero cells.After the successful homologous recombination in the eukaryotic cells,the recombinant BAC-HSV-1 with GFP reporter gene was generated.Then,the positive CPE were taken by plaque purification and by hirt extraction during the moment of the circularization of HSV-1 DNA,and the plasmid of BAC-HSV-1 was acquired.Electroporation was used to transfect the BAC-HSV-1 into DH10B,and then the single colonies of interest were confirmed both by MluI digestion and PCR.Experimental group and the control group cells were given BAC-HSV-1 plasmid and HSV-1 genomic DNA respectively to produce the BAC-HSV-1 and HSV-1 progeny virions.Vero cells were inoculated with the progeny virions at MOI = 0.1 and then a TCID50 assay was performed to determine the titers of virons in the two groups at 48 hours post inoculation.The plasmid BAC-HSV-1 was successfully constructed by the restriction enzyme analysis and the PCR.The titers of progeny virions were calculated by the TCID50 assay.No significant difference in the titers of virions between two groups was observed（P0.05）.The infectious BAC-HSV-1 shuttle virus/plasmid between eukaryotic and prokaryotic cells was successfully constructed.

关 键 词：细菌人工染色体 单纯疱疹病毒I型 同源重组 

分 类 号：R373.9[医药卫生—病原生物学]