Ⅰ类新城疫病毒NDV08-004株微型基因组的构建及其功能鉴定  

Construction and Identification of Mini-genome of Class Ⅰ Newcastle Disease Virus NDV08-004 Strain

作　　者：陈云霞[1,2] 管峰[1] 赵云玲[1] 郑东霞[1] 张维[1] 刘华雷[1] 王志亮[1] 

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]广西大学动物科学学院,广西南宁530005

出　　处：《中国动物检疫》2011年第9期27-31,共5页China Animal Health Inspection

基　　金：国家自然科学基金(30901079)

摘　　要：目的本研究旨在构建Ⅰ类新城疫病毒NDV08-004的微型基因组和辅助质粒,为构建病毒拯救体系奠定基础。方法根据Ⅰ类新城疫病毒NDV08-004的全基因组序列,采用SOE方法将增强绿色荧光蛋白基因(eGFP)插入NDV08-004的3’-Leader和5’-Trailer区域之间后反向插入转录载体pOLTV5中,构建了NDV08-004的微型基因组pLGT-03。将pLGT-03转染辅助病毒NDV08-004感染的表达T7RNA聚合酶BSRT7/5细胞进行功能鉴定。采用RT-PCR将NDV08-004的NP、P和L蛋白基因亚克隆入表达载体pCI-neo中,分别构建了三个辅助质粒pCI-NP、pCI-P、pCI-L。通过构建的微型基因组pLGT-03和三个辅助质粒按照一定的比例共转染BSRT7/5细胞来验证辅助质粒的功能。结果微型基因组pLGT-03转染辅助病毒感染的BSRT7/5细胞后可见明显荧光,表明微型基因组构建成功。微型基因组和辅助质粒共转染可见荧光表达,表明三个辅助质粒均具有相应的功能。结论表明微型基因组和辅助质粒均具有相应的功能,为建立NDV08-004的反向遗传操作平台奠定了基础。Objective To construct the mini-genome system of Class I Newcastle disease virus NDV08-004 strain, providing a foundation of reverse rescue system of the virus. M^aod The mini-genome plasmid, named as pLGR-03, containing the 3＇ and 5＇ terminal regions of class I Newcastle disease virus NDV08-004 isolate linking eGFP as the reported gene in the order of 3＇ Leader-eGFP-5＇ Traile were constructed by over-lap extension （SOE）, and then sub-cloned into transcription vector pOLTV5. The pLGR-03 was transfected into BSR T7/5 cells, infected with NDV08-004 as the helper virus, to identify the function of the mini-genome. Three helper plasmids, pCI-NP, pCI-P and pCI-L,were constructed by sub-cloning the complete ORFs of N-P,P,and L gene into pCI-neo vector respectively. Co-transfections of three helper plasmids together with pLGR-03 were conducted to evaluate the fimction of the helper plasmids. The reporter gene could be expressed after it was tmnsfected into the BSR T7/5 cells infected with helper virus NDV08-004. The BSR T7/5 cell transfected by pLGR-03 and three helper plasmids indicated the eGFP expressed as expected. Concision The work mentioned above will accelerate greatly the rescue of class I NDV and other relative research.

关 键 词：Ⅰ类新城疫病毒 辅助性质粒 微型基因组 病毒拯救 

分 类 号：S852.659.5[农业科学—基础兽医学]