H3N2亚型猪流感病毒反向遗传操作技术平台的建立  

Establishment of reverse genetics of H3N2 subtype swine influenza virus

作　　者：宫晓倩[1] 汪秀会 阮宝阳 刘晓敏[1] 周艳君[1] 郑浩[1] 童武[1] 李泽君[1] 童光志[1] 于海[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国兽医科学》2015年第8期781-786,共6页Chinese Veterinary Science

基　　金：国家自然科学基金青年科学基金项目(31201916);上海市自然科学基金青年项目(12ZR1453500);中央级公益性科研院所基本科研业务费项目(2015JB07);中国农业科学院创新工程科研团队"动物流感病毒病原生态学"项目

摘　　要：为了构建猪流感病毒A/swine/Heilongjiang/1/2005(H3N2)的反向遗传操作技术平台,利用RT-PCR扩增了H3N2亚型猪流感病毒的8个基因片段,分别连接到pBD双向转录表达载体上。将8个重组质粒纯化后,共转染293T细胞,48h后收集上清,接种MDCK细胞。当MDCK细胞呈现明显病变时,收集上清,做血凝试验,检测出有血凝现象。通过MDCK细胞病变情况、生长曲线、空斑形态等验证拯救病毒的可靠性,结果发现:野生毒株和拯救毒株在MDCK细胞上引起的病变情况基本一致,生长曲线的测定结果没有明显差异,空斑形态也极其相似。H3N2亚型猪流感病毒的成功拯救为猪流感病毒的致病机理、传播机制、基因功能研究以及疫苗研发奠定了基础。To rescue A/swine/Heilongjiang/1/2005 （H3N2） of H3N2 subtype swine influenza virus, the eight gene segments of H3N2 were amplified by RT-PCR and separately cloned into the vector of pBD. And the eight plasmids were purified and co-transfected into 293T cells. The supernatant of 293T cells was collected after 48 hours and inoculated into the Madin-Darby canine kidney（MDCK） cells. When MDCK cells appeared cytopathic effect（CPE）,virus titer was detected by hemagglutination test and the result was positive. The reliability of the rescued virus was identified by comparing the rescued virus＇s and the wild type virus＇s CPE,growth curves and plaque morphology on MDCK. Their CPE on MDCK cells was almost the same,there was no obvious difference between the growth curve of the wild type virus and rescued virus. Besides that,their plaque morphologies were also very alike. The establishment of rescued H3N2 provided the foundation for further studies on pathogenesis,mechanism of transmission, genetic function and new influenza vaccine.

关 键 词：猪流感 H3N2亚型 反向遗传操作技术 生长曲线 空斑形态 

分 类 号：S852.659.5[农业科学—基础兽医学]