Expression of cholera toxin B subunit oligomers in transgenic potato plants
Expression of cholera toxin B subunit oligomers in transgenic potato plants
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DOI：
10.1023/a:1018487401810
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发表时间：
1997-11-01
影响因子：
3
通讯作者：
Langridge, WHR
中科院分区：
文献类型：
--
作者：
Arakawa, T;Chong, DKX;Langridge, WHR
将编码霍乱毒素 B 亚基蛋白 (CTB) 的基因与内质网 (ER) 保留信号 (SEKDEL) 融合，插入植物表达载体中双向甘露碱合酶 P2 启动子附近，该载体含有与 P1 启动子连接的细菌荧光素酶 AB 融合基因 (luxF)。通过携带该载体的根癌农杆菌转化马铃薯叶外植体，并再生出卡那霉素抗性植物。通过聚合酶链式反应扩增，在生物发光植物的基因组DNA中鉴定出CTB-SEKDEL融合基因。免疫印迹分析表明，植物来源的CTB 蛋白与细菌CTB 蛋白在抗原上无法区分，并且寡聚CTB 分子（M-r 类似于50 kDa）是从转基因马铃薯叶和块茎组织中分离出的主要分子种类。与细菌 CTB 类似，植物合成的 CTB 在热或酸处理过程中解离成单体（M-r 类似于 15 kDa）。在生长素诱导的转基因马铃薯叶和块茎组织中检测到的CTB蛋白的最大量约为总可溶性植物蛋白的0.3%。酶联免疫吸附测定方法表明，植物合成的 CTB 蛋白与霍乱毒素的天然膜受体 G(M1)-神经节苷脂特异性结合。在 SEKDEL 信号存在的情况下，CTB 蛋白在马铃薯组织中积累，并组装成保留天然生化和免疫学特性的寡聚体形式。寡聚CTB蛋白的表达与可食用植物中的天然CTB蛋白具有相同的免疫学和生化特性，这为制备廉价的食用植物口服疫苗开辟了道路，以预防世界各地流行地区的霍乱和其他病原体。
A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M-r similar to 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M-r similar to 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to G(M1)-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.