反转录病毒载体RCASBP介导的EGFP基因在DF-1细胞中的表达  

Expression of Enhanced Green Fluorescent Protein(EGFP)Gene Carried by a Lentiviral Vector RCASBP in DF-1 Cell

作　　者：秦建如 黎娜铭 许长剑 廖明[1] 曹伟胜[1] 

机构地区：[1]华南农业大学兽医学院农业部兽用疫苗创制重点实验室,广州510642

出　　处：《中国畜牧兽医》2015年第10期2538-2543,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家肉鸡产业技术体系(CARS-42-G09);农业部公益性行业科研专项(201203055);广东省科技计划项目(2012A020100001)

摘　　要：为建立反转录病毒载体RCASBP介导的增强型绿色荧光蛋白(EGFP)基因在DF-1细胞中的表达体系,本研究将PCR获得的EGFP基因插入反转录病毒载体RCASBP,构建重组反转录病毒载体RCASBP-EGFP,之后将重组载体转染DF-1细胞;用基于禽白血病病毒(ALV)p27抗原的ELISA检测盲传至第4代的细胞上清,ELISA阳性结果说明重组病毒拯救成功;荧光显微镜观察发现80%以上DF-1细胞都有明显的绿色荧光信号,证明DF-1细胞表达绿色荧光蛋白;对DF-1细胞基因组进行特异性PCR检测,扩增出特异性条带说明重组反转录病毒载体RCASBP-EGFP携带的EGFP基因整合到DF-1细胞的基因组中。本研究建立的RCASBP介导反转录病毒表达体系为研究ALV基因的结构和功能奠定了基础。To construct a lentiviral vector RCASBP carrying the enhanced green fluorescent protein（EGFP）gene which could be expressed stably in DF-1cell,EGFP gene was amplified by PCR and then inserted into the lentiviral vector RCASBP after digested with the restriction endonuclease ClaⅠto construct recombinant lentiviral vector RCASBP-EGFP.The recombinant vector was transfected into DF-1cells by LipofectamineTM2000.Avian leukosis virus（ALV）p27antigen ELISA test was performed after four passages of the transfected cells and the positive results of ELISA suggested the success rescue of the virus.The expression of EGFP was observed in more than 80 percentages of DF-1cells under fluorescence microscope.The proviral genome PCR showed EGFP gene carried by the recombinant lentiviral vector RCASBP-EGFP had been integrated into the genome of DF-1cells.The RCASBP lentiviral-mediated expression system provided a basis for study of the structure and function of ALV genes.

关 键 词：禽白血病病毒 反转录病毒载体RCASBP 增强型绿色荧光蛋白基因 DF-1细胞 

分 类 号：Q785[生物学—分子生物学]