重组马立克病毒通用转移载体的构建及初步应用

Construction and Primary Application of the Universal Transferring Vector of Marek’S Disease Virus Expressing GFP Gene

本实验以独立启动子控制的增强型绿色荧光基因(GFP)作为报告基因,同时将CMV启动子及其多克隆位点与之连接,构成外源基因表达盒,插入到马立克病毒(MDV)复制非必需区基因(短独特区US2等)构成的同源臂中,构建成重组马立克病毒的通用载体。鉴定正确后,将转移载体与提取的MDV基因组共转染鸡胚成纤维细胞(CEF),同源重组获得具有感染性的重组病毒,待病毒蚀斑出现后,荧光显微镜下观察,可见到明显的绿色荧光病毒蚀斑,经三次筛选,初步分离到重组病毒。结果表明,转移载体与MDV基因组共转染可获得感染性病毒,US2基因可作为重组病毒构建中的外源基因插入位点,证实通用转移载体的构建是可行的,为重组马立克病毒新型疫苗的研究奠定物质基础。

The expressing cassette containing the gene of GFP as the reporter gene, the CMV promoter and an enhancer,nulitiple cloning sites（MCS） and SV40 poly（A） signal was cloned into recombinant plasmid（M2） to yield the universal transferring vector （M2-CMV-GFP）. The 2.6kb homologous flank（ORF3 US2 and US3） was amplified from Marek＇s disease virus（MDV） genome DNA and subcloned to construct the plasmid M2. The recombinant virus, designated rMDV, was generated by co-transfection of CEF with the transferring vector and the MDV （MDV1 vaccine CVI988/Rispens virus） genome DNA. The green fluorescence could be seen in the rMDV plaque, then the recombinant virus was selected and purified by the green fluorescence. The results showed that GFP gene could be expressed correctly in rMDV and the US2 gene of MDV could be an insertion site for the construction of rMDV. The data showed that the recombinan MDV transferring vector was successfully constructed, providing the basis for further studying of recombinant MDV vaccine expressing foreign gene vaccine.