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.
出处
《华北农学报》
CSCD
北大核心
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)
关键词
口蹄疫病毒
疫苗株
感染性cDNA克隆
病毒拯救
Foot-and-mouth disease virus（FMDV）
Vaccine strain
Infectious cDNA clones
Virus rescue
作者简介
曹伟军（1983-），男，甘肃天水人，硕士，主要从事口蹄疫反向遗传学研究。
通讯作者：刘在新（1964-），男，甘肃景泰人，研究员，主要从事动物病毒免疫学和分子生物学研究。
