含GFP报告基因的伪狂犬病病毒上海株gE-gI基因部分缺失株的构建  

Construction of Transfer Plasmid Vector of gE-gI Gene Partial Deletion Mutant of Pseudorabies Virus SH Strain Including GFP Report Gene

作　　者：姜焱[1] 侯玉峰[2] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点实验室,南京210095 [2]南京出入境检验检疫局,南京210001

出　　处：《农业生物技术学报》2002年第4期363-366,共4页Journal of Agricultural Biotechnology

基　　金：上海市农委重点攻关课题

摘　　要：在伪狂犬病病毒(pseudorabies virus,PRV)上海株gI基因和gE基因克隆鉴定的基础上,进一步构建了转移载体质粒,为PRV-SH的gE-gI基因部分缺失毒株的构建作准备。将gI和gE基因克隆入pUC18中构建pgEI载体,利用gE基因中的酶切位点缺失掉gE基因5’端363 bp,并把绿色荧光蛋白(GFP)基因表达盒插入到缺失部分。通过酶切鉴定,表明GFP基因已插入到pgEI中,构建了含GFP的缺失转移载体pgEI-GFP。用DOTAP试剂将pgEI-GFP转染感染了PRV-SH株的兔肾(RK)细胞,待出现80％以上的细胞病变时收获病毒,并以GFP为标志,通过蚀斑法得到纯化重组病毒,命名为PFDE/DI。On the basis of cloning and identification of gE-glgene of pseudorabies virus SH strain, transferring plasmid vector was constructed in order to get the partial gE-gI deletion mutant. gE and gI genes were cloned into pUC18 respectively and the corresponding recombinant pgEI vector was obtained. Then, 363 bp fragment of 5'terminal sequence of gE gene was deleted by using restrict endonuclease and the green fluorescence protein (GFP) expressing cassette was inserted into the deleting site. The recombinant plasmid pgEI including GFP report gene of deleted part of gE-gI gene was constructed. Rabbit kidney (RK) cell which was infected with PRV-SH for 1-2 h was transfected with the complex of pgEI-GFP and DOTAP.The virus was harvested when 80% cell pathogenic effect (CPE) appeared. A deletion mutant was selected and purified 3-4 times in RK cell through GFP and named PFDE/DI.

关 键 词：GFP报告基因 伪狂犬病病毒上海株 gE-gI基因 部分缺失株 构建 GE基因 转移载体质粒 绿色荧光蛋白 家畜 

分 类 号：S852.659.1[农业科学—基础兽医学]