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目的 构建大肠杆菌热不稳定肠毒素B亚单位 (LT B)基因的植物表达载体 ,并建立LT B转基因烟草植株。方法 用PCR从pMMB6 8扩增LT B编码基因 ,将其克隆于 pUCmT和 pBI12 1载体 ,进而构建植物表达双元载体pBI LTB ,LT B基因由CaMV 35S启动子控制表达 ;将pBI LTB用电穿孔法导入根瘤农杆菌LBA4 4 0 4 ;采用叶盘共培育法经根瘤农杆菌介导转化烟草 ,获得转基因烟草植株 ;用PCR、Southern、Westernblot和ELISA检测转基因植株。结果 经PCR及Southern杂交分析 ,共发现 12株烟草的基因组中有LT B基因整合 ;Westernblot和ELISA分析表明有 9株转基因烟草能有效表达LT B蛋白 ,其表达量为烟草叶片总可溶蛋白的 3.36～ 10 .5 6 μg·g-1TSP。结论 建立的LT B转基因烟草植株可成功表达LT B ,并具有五聚体结构 ,为进一步生产预防婴幼儿大肠杆菌腹泻可食用疫苗及黏膜免疫佐剂奠定了基础。
Objective To construct a plant expression vector for the heat-labile enterotoxin B subunit (LT B) gene of E. coli and establish LT B transgenic tobacco plants. Methods LTB encoding gene was amplified from pMMB6 8 by PCR and cloned into pUCmT and pBI12 1 vector to construct plant expression binary vector pBI LTB. LTB gene was controlled by CaMV 35S promoter. The pBI LTB was electroporated Method was introduced into Agrobacterium tumefaciens LBA4404. Transgenic tobacco plants were transformed by Agrobacterium tumefaciens mediated by leaf disk co-culture method. Transgenic plants were detected by PCR, Southern blot and ELISA. Results LTB gene integration was found in the genomes of 12 tobacco plants by PCR and Southern blot analysis. Western blot and ELISA analysis showed that 9 transgenic tobacco plants could express LTB protein effectively, the expression level of which was LTB 3.36 ~ 10 .5 6 μg · g-1TSP. Conclusion The LTB transgenic tobacco plants can successfully express LTB and have pentameric structure, which lays the foundation for further production of edible vaccine and mucosal immune adjuvant for preventing E. coli diarrhea in infants and young children.