Jani D, Singh N K, Bhattacharya S, Meena L S, Singh Y, Upadhyay S N, Sharma A K, Tyagi A K
Department of Plant Molecular Biology, University of Delhi South Campus, 110021 New Delhi, India.
Plant Cell Rep. 2004 Feb;22(7):471-7. doi: 10.1007/s00299-003-0711-9. Epub 2003 Sep 27.
Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 microg recombinant CTB was injected intradermally into Balb/c (H2K(d)) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G(M1) ganglioside.
将编码霍乱弧菌霍乱毒素B亚基（CTB）的基因通过农杆菌介导的转化方法导入烟草品种Samsun中，该基因经修饰后含有编码内质网滞留信号（SEKDEL）的序列，其受花椰菜花叶病毒35S启动子的控制。从转基因叶片组织中分离出总蛋白，并将含有5微克重组CTB的等分试样皮内注射到Balb/c（H2K(d)）小鼠体内。在给予植物表达的或天然纯化的CTB的动物中检测到CTB特异性血清IgG。使用脾细胞进行了T细胞增殖研究，并对从免疫致敏动物中分离出的巨噬细胞进行体外刺激后产生的上清液中的细胞因子进行了测定。在从注射天然或植物表达的CTB的动物中分离出的脾T淋巴细胞中观察到T淋巴细胞增殖受到抑制。从用天然或植物表达的CTB免疫的小鼠中分离出的巨噬细胞显示白细胞介素-10的分泌增强，但脂多糖诱导的白细胞介素-12和肿瘤坏死因子α的分泌受到抑制。这些研究表明，就对T细胞增殖和细胞因子水平的影响而言，植物表达的蛋白表现得与天然CTB相似，这表明植物表达系统适合用于生产可作为食用疫苗的细菌抗原。发现转基因在后代中得以遗传，并表达产生CTB的五聚体形式，这可通过其与G(M1)神经节苷脂的相互作用得以证明。
