LT-B转基因烟草植株的建立

The establishment of LT-B transgenic tobacco plants

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 plant tr an sformation vector containing Escherichia coli heat-labile enterotoxin B sub unit (LT-B) gene and establish LT-B transgenic plants producing large amount o f LT-B proteins. Methods The LT-B coding sequence was amplified by PCR with specific primers and plasmid pMMB68 used as a templete, subcloned into middle v ector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB. On the T-DNA regions of the pBI-LTB binary vector contained constitutive Caul iflower mosaic virus (CaMV) 35S promoter, nopaline synthase terminator, and neom ycin phosphotransferase npt II gene, which allowed the selection of transfor med plants against kanamycin. The tobacco (Nicotiana tobacum L.Cuttivar Xanthi ) plants were transformed by co-cultivating leaf discs method via Agrobacte rium tumefaciens LBA4404 harboring the plant expression vector. The regenerate d transgenic tobacco plants were selected with kanamycin, and confirmed by PCR, Southern blot, SDS-PAGE Western blot and ELISA techniques. Results PCR and Southern blot analyses confirmed stable int egration of the LT-B gene into the tobacco genome in 12 stains of the transform ed plants. SDS-PAGE analysis showed that the expressed protein was about 45 ku in MW, consistent with the pentamer of LT-B in 9 strains of transfor med tobacco plants. Western blot verified the protein of interest being reactive with the antibody against LT-B, implying that the given protein was LT-B. The levels of LT-B expression varied from 3.36～10.56μg·g -1 total soluble tobacco leaf protein by ELI SA assay. Conclusion Transgenic LT-B tobacco plants are generated, a nd LT-B protein is expressed effectively in transgenic tobacco plants.