细胞内转录拯救Asia1型口蹄疫病毒.pdfVIP

中国农业科学 2009,42(2):688-693 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2009.02.038 细胞内转录拯救Asia1型口蹄疫病毒 李平花，白兴文，卢曾军，孙 普，郭建宏，李 冬，曹伟军，陈应理，刘湘涛，殷 宏，刘在新 （中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/农业部畜禽病毒学重点实验室/ 国家口蹄疫参考实验室，兰州 730046 ） 摘要： 【目的】建立口蹄疫病毒（FMDV）感染性分子克隆的体内拯救技术平台，为深入研究该病毒基因组的 结构与功能奠定基础。【方法】将置于 T7 启动子下的 FMDV Asia1/JS/China/2005 株全长 cDNA 的真核重组质粒 pFMDV-A 经 Not I 线化后和表达 T7 RNA 聚合酶的真核质粒 pcDNAT7P 共转染 BHK-21 细胞，进行病毒体内拯救的 研究。【结果】转染细胞盲传第3代48 h 后出现致细胞病变（CPE），第4代10 h出现典型的CPE。RT-PCR、间 接免疫荧光、电镜等均检测到了拯救的FMDV，而且存在分子标签，表明构建的FMDV全长cDNA分子克隆在BHK-21 细胞内成功包装出FMDV。拯救毒与亲本毒对乳鼠的致病力（LD50）差异不显著，具有相似的生物学特征。共转染 试验重复多次，均拯救到FMDV病毒。【结论】成功建立了基于质粒为基础的FMDV的体内拯救方法。 关键词：口蹄疫病毒；感染性分子克隆；病毒拯救；T7 RNA聚合酶 Rescue of Infectious Foot-and-Mouth Disease Virus in vivo from Full-Length cDNA Clone of Asia1 Type LI Ping-hua, BAI Xing-wen, LU Zeng-jun, SUN Pu, GUO Jian-hong, LI Dong, CAO Wei-jun, CHEN Ying-li, LIU Xiang-tao, YIN Hong, LIU Zai-xin (State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Virology of Ministry of Agriculture/National Foot-and-Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Lanzhou 730046) Abstract: 【Objective 】The aim of the study was to develop a reverse genetics platform of infectious foot-and-mouth disease (FMDV) in vivo, which is a base for the construction and function researches of viruses. 【Method 】 The full-length genome of FMDV Asia1/JS/China/2005 strain was assembled into a mammalian expression vector downstream of a T7 promoter. The recombinant plasmids, pFMDV-A were linearized with NotI enzyme and cotransfected into BHK-21 cells with pcDNAT7P plasmids that co