携带乙肝大包膜蛋白L基因与结核Esat6融合基因植物表达载体的构建及鉴定  

Construction and Identification of the Expression Plasmid Containing Genes Encoding the Large Envelope Protein L of HBV and Esat-6 Gene of Mycobacterium tuberculosis

作　　者：李君武[1] 宫君原[1] 刘鑫[1] 叶秋萍[1] 黄清华[1] 

机构地区：[1]暨南大学医学院,微生物与免疫教研室,广东广州510632

出　　处：《华北农学报》2011年第2期1-6,共6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引导项目(2004B31201019);国家重大科技专项(2008ZX1002-009)

摘　　要：构建包含编码结核杆菌Esat6基因和乙肝病毒大包膜蛋白(L蛋白)基因的植物双元表达载体,并转化根癌农杆菌LBA4404。分别以质粒pPIC9K-L和结核杆菌基因组为模板进行PCR扩增,获得L和Esat6基因,然后运用部分重叠聚合酶链式反应扩增出L-Esat6融合基因片段,连接到有玉米特异性启动子globulin-1的pEGG载体上,将G-L-Esat6融合基因片段酶切下,连接到含有抗除草剂基因bar的双元表达载体pCAMBIA1300上,电击法将重组质粒转化到农杆菌LBA4404中。构建了真核表达重组质粒pCAMG-L-Esat6,测序分析表明,克隆的L和Esat6序列与NCB I上公布序列一致。成功构建与转化了包含编码乙肝病毒大包膜蛋白L基因和结核杆菌Esat6基因的植物表达载体,并将其转化入根癌农杆菌LBA4404中,为成功研制利用转基因植物生产抗乙肝和结核联合口服疫苗奠定了基础。To construction and identification of the expression plasmid containing genes encoding East6 gene of Mycobacterium tuberculosis and the large envelope protein L of HBV,and transform the recombinant vector into Agrobacterium tumefaciens LBA4404.The L and East-6 gene were amplied from pPIC9K-L and Mycobacterium tuberculosis genome by PCR,then the fusion DNA fragment of L and East6 were amplied by Splicing by Overlap Extension were cloned into the vector pEGG.The combine fragment of promoter globulin-1 and the target gene L-East6 which get from doubled enzymes digestion of the recombined plasmid pEG-G-L-Esat6 was inserted into the plant expression vector pCAMBIA1300 which contain the gene bar for herbicide resistance.Then transformed recombinant plasimid pCAM-G-L-Esat6 into Agrobacterium tumerfaciens LBA4404.We have successfully constructed eukaryotic expression recombinantplasmid pCAMG-L-Esat6 and it is showed that the cloned sequence of L and Esat6 is correct by sequencing.We have successfully constructed expression plasmid containing genes encoding L protein of HBV and Esat6 gene and transformed into LBA4404,and lays a foundation for construction combined gene vaccine against HBV and MTB.

关 键 词：结核分枝杆菌 乙肝病毒 大包膜蛋白L基因 Esat6基因 globulin-l 

分 类 号：Q786[生物学—分子生物学]