口蹄疫病毒O/China/99株多基因植物组成型表达载体的构建及序列分析

目的构建包含FMDV结构蛋白P1、非结构蛋白2A、3C以及部分2B的基因片段P12X3C的植物组成型表达载体。为FMDV可饲疫苗的研制奠定基础。方法通过PCR扩增法，从pGEM／P12A质粒和pGEM／3C质粒中扩增P12A及部分2B基因（P12X）、3C基因，将获得的P12X与3C片段经Bam HI／SpeⅠ和SpeⅠ／SalⅠ双酶切后，与经Bam HI／SalⅠ双酶切后的pUC18质粒大片段连接，得到重组质粒pUC18／P12X3C，pUC18／P12X3C质粒经Bam HI／SalⅠ双酶切后的大片段与经同样双酶切的植物表达载体pBin438连接。转化子经酶切、PCR及测序鉴定后。获得包含FMDVO／China／99株P12X3C片段的植物组成型表达载体pBin438／P12x3C，最后通过三亲交配法，将表达载体pBin438／P12x3C导入农杆菌。结论成功构建FMDV多基因的植物组成型表达载体。

Construction and sequence analysis of the plant constitutive expression vector with multi-genes of foot and mouth disease virus(FMDV) O/China/99 strain

In order to construct the plant constitutive expression vector with multi-genes of foot and mouth disease4 virus(FMDV) including the gene fragments coding the structural protein P1,non-structural protein 2A,3C and part of 2B of FMDV O/China/99 strain,the P12A,part of 2B gene (P12X) and 3C gene were amplified from the recombinant plasmid pGEM/P12A and pGEM/3C by PCR.After being digested by restriction enzymes BamHI/Spe I and Spe I/Sal I respectively,the gene P12X and 3C were cloned into the pUC18 vector that was digested by BamHI/Sal I.and then after being digested by restriction enzyme Bam HI/Sal I for recombinant plasmid pUC18/P12x3C,the P12x3C gene was obtained and then cloned into the expression vector pBin438 that was digested by the same enzyme.Recombinant plasmid was checked by restriction enzyme analysis,PCR and nucleic acid sequencing. The recombinant vector pBin438/P12x3C was then transferred to Agrobacterium tumefaciens by tri-parental mating.Experimental result showed thatthe plant constitutive expression vector pBin438/P12x3C for FMDV multi-genes including the full length of P1,2A,3C and part of 2B was successfully constructed.These results could offer an experimental basis for the preparation of FMD vaccine.