重组马立克病病毒CVI988/Rispens的构建

根据Ⅰ型马立克病病毒（MDV）强毒GA株基因序列,设计两对引物,用PCR方法扩增出CV1988／Rispens株的US10及其侧翼序列,分别克隆入pUC18载体中。经测序检测正确后,进一步插入含CMV启动子的绿色荧光蛋白（EGFP）基因表达盒,获得了含EGFP报告基因的转移载体质粒pPUC18-US10-EGFP。通过同源重组,成功地筛选出表达EGFP的重组病毒rCV1988-EGFP,经传代证明重组rCV1988-EGFP在感染的CEF细胞中能稳定表达EGFP。结果表明：构建的重组转移载体质粒正确,US10是MDV复制非必需片段,为进一步利用US10区构建重组MDV多价基因工程疫苗奠定了基础。

Construction of recombinant Marek's disease virus vaccine strain CVI988/Rispens

The US10 and its flanks of CVI988/Rispens strain were amplified by PCR with primers designated according to the sequence of "virulent" GA strain of Marek's disease virus (MDV) serotype I and subcloned into pUC18 plasmid, named as pUC-US10. The expression cassette including EGFP gene controlled by CMV promoter and the enhancer was then cloned into pUC-US10 vector as a marker of the transfer vector pUC18-US10-EGFP. A stable recombinant Marek's disease virus (rMDV) expressing EGFP was obtained by homologous recombination with virus of CVI988/Rispens strain in CEF. These results proved that the US10 of MDV is one of non-essential sites in the viral genome for viral growth in vitro. It facilities the construction of recombinant MDV expressing foreign genes in future.