鸽源新城疫病毒JS/07/04/Pi株感染性分子克隆的构建及病毒拯救  

Construction and Rescue of Infectious cDNA Clone of Pigeon-Origin Newcastle Disease Virus Strain JS/07/04/Pi

作　　者：朱艳梅[1] 胡增垒[1] 宋庆庆[1] 段志强[1] 顾敏[1] 胡顺林[1] 王晓泉[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院农业部畜禽传染病学重点开放实验室,扬州225009

出　　处：《病毒学报》2012年第1期67-72,共6页Chinese Journal of Virology

基　　金：国家自然科学基金项目(30630048);国家蛋鸡产业体系项目(CARS-41-K07)

摘　　要：参照GenBank上公布的鸽源Ⅵb亚型新城疫病毒JS/07/04/Pi株基因组全序列设计9对引物,RT-PCR扩增目的片段后,依次亚克隆至TVT7/R转录载体,成功构建出含JS/07/04/Pi株基因组全长cDNA的转录载体TVT/071204。然后将其与三个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,60h后将转染细胞及其上清接种鸡胚,收集死亡鸡胚尿囊液进行HA与HI试验。结果显示死亡鸡胚尿囊液HA呈阳性,并能被ND阳性血清所抑制,表明该病毒已成功拯救,且拯救病毒rNDV/071204在细胞上的生长增殖能力同母本病毒相似。该病毒的成功拯救为鸽源Ⅵb亚型NDV感染的宿主特异性及鸽用ND新型疫苗的研究提供了理想的生物材料。Based on the complete genome sequence of pigeon-origin Newcastle disease virus strain JS/07/04/Pi（genotype VIb）,nine overlapped fragments covering its full-length genome were amplified by RT-PCR.The fragments were connected sequentially and then inserted into the transcription vector TVT7/R resulting in the TVT/071204 which contained the full genome of strain JS/07/04/Pi.The TVT/071204 was cotransfected with three helper plasmids pCI-NP,pCI-P and pCI-L into the BSR cells,and the transfected cells and culture supernatant were inoculated into 9-day-old SPF embryonated eggs 60h post-transfection.The HA and HI tests were conducted following the death of embryonated eggs.The results showed that the allantoic fluids obtained were HA positive and the HA could be inhibited by anti-NDV serum which indicated that the strain JS/07/04/Pi was rescued successfully.The rescued virus rNDV/071204 showed similar growth kinetics to its parental virus in CEF.The successful recovery of this strain would contribute to the understanding of the host-specificity of pigeon-origin NDV and to the development of the novel vaccines against the NDV infection in pigeons.

关 键 词：鸽 新城疫病毒 共转染 拯救 

分 类 号：S852.65[农业科学—基础兽医学]