玉米特异启动子驱动下结核Hsp65与Esat-6融合基因表达载体的构建及鉴定  

Construction of Vector for Expression of Hsp65 and Esat-6 Genes Driven by a Maize Endosperm-specific Promoter

作　　者：李君武[1] 王珊珊[1] 宋东[1] 刘艳[1] 黄清华[1] 

机构地区：[1]暨南大学医学院,微生物与免疫学教研室,广东广州510632

出　　处：《华北农学报》2009年第3期28-31,共4页Acta Agriculturae Boreali-Sinica

基　　金：广东省科技计划重大专项(2006A20101006);广东省科技计划重点项目(2004B31201019)

摘　　要：利用转基因玉米研制新型结核口服疫苗。构建植物双元表达质粒pCAMBIA1300GHLE,并转化农杆菌LBA4404。以本实验室构建的pEGHLE为模板经聚合酶链反应(PCR)扩增出Hsp65-Esat6基因,连接到含有玉米特异性启动子globulin-1的pCR2.1载体上;将globulin1-HLE联合片断切下连到含有抗除草剂基因bar的pCAMBIA1300载体中;电击法将重组质粒转化到农杆菌LBA4404中。成功构建了pC1300GHLE质粒,酶切鉴定得到3.3 kb和8.6 kb 2条带,测序分析表明克隆的Hsp65和Esat6序列与NCBI上公布序列一致;成功转化到农杆菌中,酶切从农杆菌中所提的质粒,条带大小与预期结果相符合。成功构建和转化了pC1300GHLE表达载体,为成功研制利用转基因植物生产抗结核口服疫苗奠定了基础。To construct the plant expression plasmid containing Mycobacterium tuberculosis Hsp65 and Esat-6 genes, and transform the recombined vector into Agrobacterium tumerfaciens LBA4404. The fusion DNA fragment of Hsp65 and Esat-6 were amplied from pEGHLE by polymerase chain reaction（PCR）were cloned into the vector pCRG.The combine fragment of promoter globulin-1 and the target gene HLE which get from doubled enzymes digestion of the recombined plasmid pCRGHLE was inserted into the plant expression vector pCAMBIA1300 which contain the gene bar for herbicide resistance. The recombinant plasmid was analyzed by restriction enzyme digestion and the inserted target genes in the pC1300GHLE were verified by nucleotide sequencing. Then transformed pC1300GHLE vector into Agrobacterium tumerfaciens LBA4404 by electroporation. The binary expression plasmid, which could express HSP65 and ESAT-6 ,was correctly constructed. The recombinant vector containing Mycobacterium tuberculosis Hsp65 and Esat-6 genes is constructed successfully and transformed into LBA4404, and lays a foundation for further study on its immunity effectiveness against MTB.

关 键 词：结核分支杆菌 热休克蛋白65 ESAT-6 globulin-1 

分 类 号：Q786[生物学—分子生物学]