Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America.
PLoS One. 2013;8(1):e54708. doi: 10.1371/journal.pone.0054708. Epub 2013 Jan 23.
Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.
结核分枝杆菌引起的结核病是导致死亡的主要传染病之一。世界卫生组织已将结核病疫苗的开发视为一项重大公共卫生优先事项。在这项研究中，三种候选抗原，即 ESAT-6（6 kDa 早期分泌抗原靶标）和 Mtb72F（来自两个结核抗原 Mtb32 和 Mtb39 的融合多蛋白）与霍乱毒素 B 亚单位（CTB）和 LipY（细胞壁蛋白）融合，在烟草和/或生菜叶绿体中表达，以促进生物包封/口服传递。通过 Southern 印迹分析证实了转基因在叶绿体基因组中的定点整合。在叶绿体转化体叶片中，CTB 融合蛋白以预期大小的可溶性单体或多聚体形式存在，其表达水平随叶片发育阶段和收获时间而变化，在下午 6 点收获的成熟叶片中积累水平最高。CTB-ESAT6 和 CTB-Mtb72F 的表达水平分别达到成熟烟草叶片总可溶性蛋白的 7.5%和 1.2%。叶绿体转化体 CTB-ESAT6 生菜植物中积累的总叶蛋白最高可达 0.75%。在室温下储存长达六个月的冻干生菜叶片的 Western blot 分析表明，CTB-ESAT6 融合蛋白稳定，可长时间保持正确的折叠、二硫键和五聚体组装。此外，冻干后每克叶组织中的抗原浓度增加了 22 倍。用纯化的 CTB-ESAT6 蛋白进行的溶血试验表明红细胞发生部分溶血，并证实了 ESAT-6 抗原的功能。GM1 结合试验表明，CTB-ESAT6 融合蛋白形成五聚体与 GM1-神经节苷脂受体结合。在叶绿体转化体中表达功能性结核分枝杆菌抗原，应有助于开发具有成本效益和可口服的结核病加强疫苗，并具有在室温下长期储存的潜力。据我们所知，这是在叶绿体中表达结核病疫苗抗原的首次报道。
