口蹄疫病毒O/China/99株VP1基因植物种子特异性表达载体的构建及农杆菌的导入  

Construction of plant seed-specific expression vector with VP1 of FMDV O/China/99 and transfer to Agrobacterium tumefaciens

作　　者：潘丽[1] 张永光[1] 王永录[1] 王宝琴[1] 王文秀[1] 方玉珍[1] 蒋守田[1] 王炜[1] 孙元[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《中国兽医科技》2005年第6期413-417,共5页Chinese Journal of Veterinary Science and Technology

基　　金：国家高技术研究发展计划(863)项目(2001AA213071)

摘　　要：根据植物组成型表达质粒pBin438的限制性酶切位点及FMDVVP1的核苷酸序列设计并合成引物,从种子特异性启动子7S质粒pU82质粒中扩增7S启动子,经HindⅢ和BamHⅠ酶切,与经同样双酶切的植物组成型表达载体pBin438大片段连接,经酶切、PCR鉴定及序列分析,种子特异性表达载体p7SBin438构建完成。从FMDVVP1基因的pGEMVP1质粒中扩增VP1基因,经BamHⅠ和SalⅠ双酶切后,与p7SBin438质粒酶切后的大片段连接,经酶切、PCR及测序鉴定,FMDVVP1基因已置于种子特异性启动子7S下游,成功构建了FMDVVP1基因的种子特异性表达载体p7SBin438VP1。通过三亲交配法,将表达载体p7SBin438VP1导入根癌农杆菌,为研究FMD可饲疫苗奠定了基础。A pair of primers containing HindⅢ and BamHⅠrestriction site was designed to amplify the 7S promoter from plasmid pUC18 containing seed-specific promoter. After digestion by restriction ~enzyme HindⅢ and BamHⅠ respectively, the gene 7S was cloned into the plant constitutive-expression vector pBin438 digested by the same enzymes. The recombinant plasmid was checked by restriction enzyme analysis ,PCR and nucleic acid sequencing. The identification results showed that the seed-specific expression vector p7SBin438 was constructed . The VP1 gene ligated with restriction enzyme site was isolated from the recombinant plasmid pGEM-VP1 by PCR. After the recombinant plasmid pGEM-VP1 was digested by restriction enzyme BamHⅠ and SalⅠ respectively, the VP1 gene was cloned into the p7SBin438 vector digested by the same enzyme. The recombinant plasmid was checked by restriction enzyme analysis,PCR and nucleic acid sequencing.The vector p7SBin438-VP1 was transferred to Agrobacterium tumefaciens by triparental mating.It was concluded from the results that the plant seed-specific expression vector p7SBin438-VP1 had been constructed, which will provide an important experimental base for the development of transgenic plant vaccine of FMDV.

关 键 词：口蹄疫病毒 植物组成型表达载体 种子特异性表达载体 根癌农杆菌 VP1基因 疫苗 

分 类 号：S852.659.6[农业科学—基础兽医学]