口蹄疫病毒O/HN/93疫苗株的拯救及病毒活性鉴定  

Rescue and Identification of Virus Activity of Foot-and-Mouth Disease Virus Strain O/HN/93 from Full-length cDNA Clone

作　　者：曹伟军[1,2] 李平花[1] 白兴文[1] 卢曾军[1] 孙普[1] 刘在新[1] 

机构地区：[1]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开放实验室,国家口蹄疫参考实验室,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《华北农学报》2010年第3期32-37,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家支撑计划(2006BAD06A12);国家“973”项目(2005CB523201)

摘　　要：利用定点突变方法,构建含有预期突变的口蹄疫病毒O/HN/93株全长cDNA克隆,将全长cDNA的重组质粒线化后,与表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,转染细胞盲传至第3代50 h后出现典型致细胞病变效应(CPE),第4代10 h出现典型的CPE。对收获的病毒分别用RT-PCR、分子标签测序、间接免疫荧光和电镜观察等进行鉴定,均证实成功拯救到了O/HN/93株FMDV。成功获得O/HN/93株的感染性分子克隆,为进一步研究抗原谱广、免疫原性好的候选疫苗株奠定了骨架基础。We construct containing the expected mutations in foot-and-mouth disease virus（FMDV） vaccine strain O/HN/93 full-length cDNA clone by using site-directed mutagenesis.The recombinant plasmids containing O/HN/93 full-length genome were linearized with Not I enzyme and cotransfected into BHK-21 cells with pcDNAT7P plasmids that could express T7 RNA polymerase to rescue virus.The transfected cells were serily passaged,and the third pas-sage showed apparent cytopathogenicity effect（CPE） within 50 h,the CPE were observed after 10 h in the fourth pas-sage.The results of the RT-PCR,indirect immunofluorescence,electron microscope confirmed the success of the rescue out of FMDV O/HN/93 strain.The recovered virus contained genetic tags.Successfully obtained infectious full-length cDNA clone of O/HN/93 strain,It lay a foundation for further study of the candidate vaccine strain which covers a wi-der spectrum of antigens over pandemic strain.

关 键 词：口蹄疫病毒 疫苗株 感染性cDNA克隆 病毒拯救 

分 类 号：Q78[生物学—分子生物学]