玉米特异表达启动子驱动下结核esat-6抗原基因植物表达载体的构建及鉴定

以本实验室构建的pEGHLE为模板经聚合酶链反应(PCR)扩增出Esat-6基因,连接到含有玉米特异性启动子globulin-1的pCR2.1载体上,将globulin-1-Esat6联合片断切下连到含有抗除草剂基因bar的pCAMBIA1300载体中,构建植物双元表达质粒pCAMBIA1300GEsat-6,电击法将重组质粒转化到农杆菌LBA4404中.通过对pC1300GEsat-6质粒酶切鉴定和测序分析表明克隆的Esat-6与预期相符,酶切从农杆菌中所提的质粒,条带大小与预期结果相符合.

Construction of Vector for Expression of Esat-6 Gene Driven by a Maize Endosperm-specific Promoter

To construct the plant expression plasmid containing Mycobacterium tuberculosis Esat-6 genes,and transform the recombined vector into Agrobacterium tumerfaciercs LBA4404.The DNA of Esat-6 amplied from pEGHLE by polymerase chain reaction(PCR) were cloned into the vector pCRG2.1,The combine fragment of promoter globulin and the target gene Esat-6 which get from doubled enzymes digestion of the recombined plasmid pCRGEsat-6 was inserted into the plant express vector pCAMBIA1300 which contain the bar gene for herbicide resistance.The recombinant plasmid was analyzed by restriction enzyme digestion and the inserted target genes in the pC1300GEsat-6 were verified by nucleotide sequencing.Then transformedpC1300GEsat-6 into Agrobacterium tumerfaciercs LBA4404 by electroporation.The binary expression plasmid,which could express ESAT-6,was correctly constructed by digestion and sequencing.Digestedplasmid which was extracted from Agrobacterium tumerf aciercs,fragments were also correct.The recombinant vector containing Mycobacterium tuberculosis Esat-6 is constructed successfully and transformed into LBA4404,and lays a foundation for further study on its immunity effectiveness against MTB.