反转录病毒载体RCASBP介导的EGFP基因在DF-1细胞中的表达

doi:10.16431/j.cnki.1671-7236.2015.10.004

›› 2015, Vol. 42 ›› Issue (10): 2538-2543.doi: 10.16431/j.cnki.1671-7236.2015.10.004

反转录病毒载体RCASBP介导的EGFP基因在DF-1细胞中的表达

秦建如, 黎娜铭, 许长剑, 廖明, 曹伟胜

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

收稿日期:2015-04-01 出版日期:2015-10-20 发布日期:2015-10-23
通讯作者: 曹伟胜 E-mail:caoweish@scau.edu.cn
作者简介:秦建如(1989-),女,河南浚县人,硕士生,研究方向:动物传染病,E-mail:jianru890526@126.com
基金资助:
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)

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

QIN Jian-ru, LI Na-ming, XU Chang-jian, LIAO Ming, CAO Wei-sheng

Key Laboratory of Veterinary Vaccine Development, Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China

Received:2015-04-01 Online:2015-10-20 Published:2015-10-23

摘要/Abstract

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。

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

Abstract: To construct a lentiviral vector RCASBP carrying the enhanced green fluorescent protein (EGFP) gene which could be expressed stably in DF-1 cell,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-1 cells by Lipofectamine^TM 2000.Avian leukosis virus (ALV) p27 antigen 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-1 cells 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-1 cells.The RCASBP lentiviral-mediated expression system provided a basis for study of the structure and function of ALV genes.

Key words: avian leukosis virus; lentiviral vector RCASBP; enhanced green fluorescent protein (EGFP) gene; DF-1 cell

中图分类号:

Q785

秦建如, 黎娜铭, 许长剑, 廖明, 曹伟胜. 反转录病毒载体RCASBP介导的EGFP基因在DF-1细胞中的表达[J]. , 2015, 42(10): 2538-2543.

QIN Jian-ru, LI Na-ming, XU Chang-jian, LIAO Ming, CAO Wei-sheng. Expression of Enhanced Green Fluorescent Protein (EGFP) Gene Carried by a Lentiviral Vector RCASBP in DF-1 Cell[J]. , 2015, 42(10): 2538-2543.

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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

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