鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰  

Modification of the Full-length cDNA Clone of Newcastle Disease Virus of Goose Origin

作　　者：刘玉良[1] 胡顺林[1] 张艳梅[1] 吴艳涛[1] 刘秀梵[1] Rmer-Oberdrfer Angela Weits Jutta Lange Martina 黄勇[1] 龙进学[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,扬州225009 [2]德国Friedrich-Lffler研究所国家动物病毒病研究中心分子生物学实验室

出　　处：《微生物学通报》2005年第6期1-6,共6页Microbiology China

基　　金：国家自然科学基金项目(No.39893290);国家"973"项目(No.G19990199)

摘　　要：将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6.5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT-PCR技术从尿囊液中扩增NDV基因组F/HN基因区域部分片段,利用限制性内切酶BsmB I将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基插入突变成功,F/HN基因区域碱基突变均得以纠正。以上cDNA克隆的修饰与替换为该毒株的反向遗传研究打下了基础。The 6.5kb specific fragment containing the T7 promoter and the transcription vector was cut down from the full-length cDNA clone of Newcastle disease virus strain ZJI of goose origin, and thereafter it was self-ligated to form the high guality plasmid for mutagenesis. Site-directed mutagenesis technique was used for inserting three additional G nucleotides （nts） into the region between the T7 promter and the leader sequence of the NDV. The RT-PCR was employed to amplify the F/HN genes fragements, and then they were ligated by the shairing restriction enzyme BsmBI and finally the corresponding fragment in the mutatant full-length cDNA was substituted by the new one. The sequencing results showed that the three additional G nts were successfully inserted and the mutant nts in the full-length cDNA were corrected and all these studies lay a foundation for the research on the reverse genetics of NDV strain ZJI.

关 键 词：鹅源新城疫病毒 基因定点突变 基因组cDNA克隆 改造 

分 类 号：S852.65[农业科学—基础兽医学]