含幽门螺杆菌CagA、UreB蛋白与霍乱肠毒素B亚单位（CTB）融合基因的植物表达载体的构建及遗传转化

幽门螺旋杆菌(Helicobacter pylori,Hp)感染是慢性胃炎和消化性溃疡的主要病因,与胃癌和胃粘膜相关淋巴组织(MALT)淋巴瘤的发生也密切相关。目前所用的疫苗制造成本高、运输费用贵,然而利用转基因植物生产的植物疫苗成本低廉、服用方便是现有疫苗的良好替代品。将Hp相关蛋白与免疫佐剂CTB的融合基因(ctb-linker-cagA和ctb-linker-ureB)利用PCR、酶切、连接等一系列方法从载体p1300-WxCLCN和p1300-WxCLUN中重组到载体pCAMBIA2301中(含35S启动子),重组载体分别命名为p2301-35SCLCN和p2301-35SCLUN。通过冻融法将载体导入农杆菌EHA105菌株中,以农杆菌介导的方法,将重组载体p2301-35SCLCN和p2301-35SCLUN转化烟草黄苗榆和心叶烟,获得了具有卡那霉素抗性再生植株,经过酶切、PCR、GUS染色和PCR-Southern鉴定结果表明,目的基因分别正确插入载体中并稳定整合到植株中,为利用植物反应器生产幽门螺杆菌疫苗奠定了坚实的基础。

Construction of plant expression vectors with fusion gene of Helicobacter pylori cagA,ureB and ctb and its genetic transformation in tobacco

Helicobacter pylori (Hp) is the principal cause of most chronic active gastric and peptic ulcer disease, and also is closely related with gastric cancer and MALT lymphoma. Current vaccines are expensive to produce and deliver, however, transgenic plants expressing recombinant vaccine immunogens offer an attractive and potential inexpensive alternative to vaccination and injection. In this study, plant expression vectors which harbor Hp related proteins CagA and UreB were constructed. Fusion gene ctb-linker-cagA and ctb-linker-ureB were cut from vectors p1300-WxCLCN and p1300-WxCLUN, and then constructed into vector pCAMBIA2301 which was under the control of the CaMV 35S promoter by series molecular methods. Those reconstructed vectors were named p2301-35SCLCN and p2301-35SCLUN and were introduced into Agrobacterium tumefaciens strain EHA105.Tobacco was transformed by co-cultivating leaf discs with Agrobacterium strains harboring fusion genes. The regenerated Kanamycin-resistant transforms were selected, elongated, rooted and transferred for flowering in greenhouse. Recombinant plant expression vectors were confirmed by digestion and PCR and transgenic plants were analyzed by PCR, GUS histochemical assays, PCR-Southern blot. The results show that more than 80% transgenic plants are confirmed to be positive ones and these results also indicate that ctb-linker-cagA and ctb-linker-ureB are integrated into the genomic DNA of the tobacco which laid a solid foundation for the research of establishing transgenic plants as bioreactors to carry microbe antigen and Hp transgenic plant vaccines.