表达绿色荧光蛋白的重组CVI988病毒的构建及特性

提取马立克氏病毒Ⅰ型疫苗毒株CVI988的总DNA为模板，利用PCR技术扩增出病毒生长非必需的US2基因并克隆入T—easy载体。将CMV启动子和增强子控制的含GFP基因表达盒克隆入US2基因中，成功构建了含GFP基因的转移质粒载体pGUS2GFP。用脂质体将其与CVI988株共转染CEF细胞，用96孔板稀释法得到纯化的表达绿色荧光蛋白的重组CVI988病毒株rCVIGFP，并分别测定其在体内和体外的生长情况。表达EGFP基因的重组病毒在细胞上生长曲线与亲本毒CVI988类似，体外实验表明，1日龄腹腔接种该重组毒后，可以从鸡体内分离到表达绿色荧光的病毒。

Construction and Characterization of Recombinant CVI988 Expressing the Green Fluorescent Protein

The US2 gene nonessential for viral replication was amplified by polymerase chain reaction(PCR) with DNA from CEF cells infected by MDV vaccine strain CVI988 as template, and was cloned into T-easy vector; The expression cassette including GFP gene controlled by CMV promoter and enhancer was cloned into US2 gene to give rise to a transfer vector pGUS2GFP, then the complex of pGUS2GFP and DOTAP was transfected into CEF cells infected CVI988 strain virus. Recombinant rCVIGFP expressing the green fluorescence protein was selected and purified. The characterization of recombinant virus was evaluated in vivo and in vitro. The growth curve of recombinant virus having EGFP gene are similar to that of the parent virus in CEF cells. The recombinat virus expressing GFP could infect chicken and be reisolated from the chickens following intra-abdominal inoculation of rCVIGFP.