【摘要】 为了建立新城疫病毒(NDV)Mukteswar毒株的反向遗传操作系统,根据已测定的NDV Muk-teswar毒株的全基因组序列设计了5对引物,扩增出包含全基因组cDNA的5条片段,并按一定顺序依次克隆入pSL1180载体中,然后在cDNA 5′末端插入T7启动子序列,3′末端导入具有自我剪切功能的丁肝病毒核酶序列和T7转录终止信号,构建了能转录具有精确5′和3′末端全基因组cDNA的转录载体pNDVT7。将表达核蛋白、磷酸蛋白及转录大蛋白的辅助表达载体pCIneoNP、pCIneoP和pCIneoL与pNDVT7按一定比例混合共转染BRS-T7细胞,4 d后将细胞悬液接种9日龄SPF鸡胚,结果获得了高效价的重组病毒。表明本研究建立的反向遗传系统能高效、快速拯救重组新城疫病毒毒株。更多还原

【Abstract】 A reverse genetic system was developed to rescue Newcastle disease virus(NDV) from cloned cDNA.A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned in a transcription plasmid which was driven by T7 RNA polymerase.The full-length viral cDNA was flanked by T7 promoter and hepatitis delta virus ribozyme(HdvRz) sequences,resulted in the synthesis of antigenomic RNA with exact terminus.With the supply of T7 RNA polymerase,infectious NDV could be generated efficiently in BRS-T7 cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP,P and L proteins from cotransfected helper plasmids.Four days later,the supernatant was injected into the allantoic cavities of 9-day-old embryonated eggs to amplify the viruses.The results demonstrated that the new system could rescue recombinant NDV quickly and efficiently.更多还原