新城疫病毒基因Ⅲ型强毒JS/7/05/Ch的拯救  

Rescue of Genotype Ⅲ Velogenic Newcastle Disease Virus Strain JS/7/05/Ch

作　　者：宋庆庆[1] 朱杰[1] 丁平云[1] 段志强[1] 屠颉 刘晓文[1] 刘秀梵[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,江苏扬州225009

出　　处：《中国畜牧兽医》2013年第10期37-41,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31172338);现代农业产业技术体系建设专项资金(nycytx-41-G07);公益性行业(农业)专项经费(201003012)

摘　　要：新城疫病毒Ⅰ系苗Mukteswar株为中等毒力,野外分离株JS/7/05/Ch的基因组核苷酸与其具有高于99%的相似性,但其毒力却明显强于前者。为了给基因Ⅲ型NDV的遗传进化机制研究提供基础,本研究成功构建了JS/7/05/Ch株的反向遗传平台。首先根据GenBank上公布的基因Ⅲ型新城疫病毒JS/7/05/Ch株基因组全序列设计并合成8对引物,RT-PCR扩增目的片段后,分别依次亚克隆至pCR2.1载体中构建含有JS/7/05/Ch全长基因组cDNA的克隆质粒pJS/7/05/Ch,然后再通过特异性酶切位点将JS/7/05株全长基因组cDNA转移到TVT7R(0.0)转录载体中,成功构建出含JS/7/05/Ch株基因组全长cDNA的转录质粒pTVT/JS705。然后将该质粒与3个辅助质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,转染60h后将该细胞及其上清接种鸡胚;血凝(HA)试验和RT-PCR结果表明JS/7/05/Ch株新城疫病毒被成功拯救。The genome of JS/7/05/Ch isolate shared more than 99% nucleotide identity with that of Mukteswar strain.However,the pathogenicity of JS/7/05/Ch was much stronger than that of Mukteswar strain.In order to provide a good foundation for the further related research,we built the rescue system of JS/7/05/Ch in this study.Based on the genomic sequence of JS/7/05/Ch strain,eight pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pCR2.1vector,to construct the plasmid pJS/7/05/Ch which contained the full-length cDNA of NDV.Then the full-length cDNA was transferred to TVT7R（0.0）vector to construct full-length NDV infectious clone,pTVT/JS705,using specific enzymes.The infectious clone together with three helper plasmids（pCI-NP,pCI-P and pCI-L）were cotransfected into BSR-T7/ 5cell, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus.The results of HA and RT-PCR indicated that JS/7/05/Ch strain of NDV was successfully rescued.

关 键 词：新城疫病毒 基因Ⅲ型 反向遗传技术 拯救 

分 类 号：S852.65[农业科学—基础兽医学]