乙肝病毒大包膜蛋白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 of HBV

构建包含编码乙肝病毒大包膜蛋白(L蛋白)基因的植物表达载体,并转化根癌农杆菌,为进一步制备转基因植物口服乙肝疫苗奠定基础。通过聚合酶链反应(PCR),以质粒pVAX1-L为模板扩增出L基因片段,插入到含有玉米特异性启动子的pCR2.1载体上;再用PCR技术扩增globulin-1-L融合片段,定向克隆到经过加工修饰的含有抗除草剂基因bar的双元表达载体pCAMBIA1300上,将重组质粒转化至农杆菌LBA4404中。HindⅢ和XbaⅠ双酶切证实,globulin-1-L融合片段已经插入到pCAMBIA1300上,测序结果说明克隆的靶基因序列与GenBank上公布的序列完全吻合。

To construct plant expression plasmid containing gene encoding large envelope protein of HBV, and to transform the recombinant plasimid into Agrobacterium turnefaciens LBA4404, we used pVAX1-L as template to amplify L gene by polymerase chain reaction （PCR） and inserted it into vector pCRG. The fusion fragment of promoter globulin-1 and the target gene L which get using pCRGL as template by PCR was subcloned into pCAMBIA1300 , transformed recombinant plasimid pCAMG-L into LBA4404 . Restriction enzyme digestion demonstrated that the inserted gene fragments were confirmed, DNA sequence analysis revealed that the target gene sequences were totally consistent with the GeneBank reported. In conclusion, we have successfully constructed expression plasmid containing genes encoding L protein of HBV and transformed into LBA4404,and lays a foundation for a new effective edible vaccine against HBV.