Loc Nguyen Hoang, Bach Nguyen Hoang, Kim Tae-Geum, Yang Moon-Sik
Institute of Resources, Environment and Biotechnology, Hue University, Hue, Viet Nam.
Protein Expr Purif. 2010 Jul;72(1):82-6. doi: 10.1016/j.pep.2010.02.010. Epub 2010 Feb 20.
The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a non-toxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. We synthesized a gene encoding the LTB adapted to the optimized coding sequences in plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. The synthetic LTB gene was located into a plant expression vector under the control of CaMV 35S promoter and was introduced into Peperomia pellucida by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification method. The assembly of plant-produced LTB was detected by western blot analysis. The amount of LTB protein produced in transgenic P. pellucida leaves was approximately 0.75% of the total soluble plant protein. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers.
大肠杆菌不耐热肠毒素（LTB）的B亚基是一种无毒但具有强大生物学特性的分子，是一种强大的黏膜和非肠道佐剂，可诱导针对共同给药或偶联抗原的强烈免疫反应。我们合成了一个编码适应植物优化编码序列的LTB的基因，并与内质网保留信号SEKDEL融合，以提高其在植物中的表达水平和蛋白质组装。合成的LTB基因位于CaMV 35S启动子控制下的植物表达载体中，并通过基因枪转化法导入透明椒草。通过基因组DNA PCR扩增法证实了合成的LTB基因整合到转基因植物的基因组DNA中。通过蛋白质印迹分析检测植物产生的LTB的组装。转基因透明椒草叶片中产生的LTB蛋白量约占植物总可溶性蛋白的0.75%。酶联免疫吸附测定表明，植物合成的LTB蛋白与GM1神经节苷脂特异性结合，GM1神经节苷脂是细胞表面LTB的受体，这表明LTB亚基形成了生物活性五聚体。
