Kang Tae-Jin, Loc Nguyen-Hoang, Jang Mi-Ok, Jang Yong-Suk, Kim Young-Sook, Seo Jo-Eun, Yang Moon-Sik
Basic Sciences Research Institute, Chonbuk National University, Jeonju 561-756, South Korea.
Transgenic Res. 2003 Dec;12(6):683-91. doi: 10.1023/b:trag.0000005114.23991.bc.
Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.
由于具有独特优势，转基因叶绿体已成为用于异源基因表达的有吸引力的系统。在此，我们报告了一项通过烟草叶绿体转化生产大肠杆菌不耐热肠毒素（LTB）无毒B亚基的可行性研究。通过PCR和基因组Southern印迹分析在转化植物中证实了LTB基因稳定位点特异性整合到叶绿体基因组中。免疫印迹分析表明，植物来源的LTB蛋白是寡聚体，煮沸后解离。五聚体LTB分子是从转基因烟草叶组织中分离出的LTB中的主要分子形式。在转质体烟草叶中检测到的LTB蛋白量约占植物总可溶性蛋白的2.5%，比通过核转化产生的植物中高约250倍。GM1-ELISA结合试验表明，叶绿体合成的LTB蛋白与GM1-神经节苷脂受体结合。在可食用植物叶绿体中具有与天然LTB蛋白相同生化特性的LTB蛋白为生产用于人类和动物的廉价、安全且有效的植物源可食用疫苗开辟了道路。
