表达GFP和gpt基因的重组CVI988病毒的构建  

Construction of Recombinant CVI988 Virus Expressing GFP and Escherichia coli gpt Genes

作　　者：邱亚峰[1] 葛菲菲[2] 曹瑞兵[3] 周斌[3] 马志永[1] 陈溥言[3] 

机构地区：[1]中国农业科学院上海兽医研究所兽医公共卫生研究室,上海200232 [2]上海市动物疫病预防控制中心,上海201103 [3]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京210095

出　　处：《农业生物技术学报》2009年第2期183-188,共6页Journal of Agricultural Biotechnology

基　　金：2007年人事部高层次留学人才回国资助项目;国家自然科学基金(No.30700593)资助

摘　　要：以马立克氏病病毒(MDV)CVI988株基因组为模板,利用PCR技术扩增出约2.7和3.0kb的基因片段,将上述片段同时插入pUC19中,获得约5.5kb MDV同源重组臂;以该基因片段的US2区的BglⅡ为插入位点,分别插入基因表达盒CMV-gpt-ployA和CMV-gfp-polyA,构建转移载体pUS-gpt-GFP,将该载体瞬时转染CHO细胞,在荧光显微镜下,可以观察到绿色荧光蛋白的表达;将该转移载体转染已用MDV CVI998株感染的次代CEF细胞,利用MX-HAT培养基筛选重组病毒,并用荧光显微镜挑选表达绿色荧光蛋白的蚀斑,结果获得重组病毒rMDV gptGFP。通过PCR检测和病毒生长测定,证明重组病毒获得纯化。Two fragments in the non-essential region of Marek＇s disease virus （MDV） were amplified by PCR from the genome of MDV CVI988 strain, and cloned into pUC 19 for generating the 5.5 kb homologous recombinant arms. A transfer vector pUS-gpt-GFP was constructed by inserting the expressing cassettes of the CMV-gpt-ployA or CMV-GFP-polyA, respectively, into the unique Bgl Ⅱ site of US2 region in the recombinant arms. The transfer vector was transiently transfected into CHO cells and the expression of the green fluorescence protein was observed under the fluorescence microscope. This transfer vector was then transfocted into CEF infected With MDV CVI988 strain. The recombinant CVI988 viruses expressing foreign genes, named rMDVgptGFP, were cloned by purifying the plagues expressing GFP in the MX-HAT selection medium. The purified recombinant viruses were further confirmed by PCR detection and growth of recombinant virus in selective and nonselective media.

关 键 词：马立克氏病病毒(MDV) 转移载体 绿色荧光蛋白 转染 重组病毒 

分 类 号：S188[农业科学—农业基础科学]