Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Mol Immunol. 2022 Jul;147:81-89. doi: 10.1016/j.molimm.2022.02.010. Epub 2022 Apr 30.
Enterotoxigenic Escherichia coli is the most important bacterial agent causing traveller's diarrhea in developing countries. Enterotoxins (LT & ST) and colonization factors (CFs) are two important factors in the ETEC pathogenesis. In the present study, a recombinant four-part fusion protein containing CFAB*ST, CFAE, and LTB (CCL) was expressed in hairy roots of Nicotiana tabacum. The synthetic gene sequence and gene order were designed based on bioinformatics analysis that predicted the best arrangement for antigenicity and stimulation of the immune response. Codon usage was optimized for expression in tobacco plant, under the control of promoter CaMV35S in plasmid pBI121. CCL was efficiently expressed in tobacco hairy roots to yield 1.11% soluble protein as determined by quantitative ELISA and Western blot. In this study, mice were immunized with purified CCL via the oral and subcutaneous route. Humoral immunity especially mucosal immunity with antigen specific IgG and IgA detected in serum and feces. The ability of CCL to elicit neutralizing antibodies was evaluated in the rabbit ileal loop model, using anti-CCL antibodies derived from immunized mice, and co-incubated with ETEC strains. A decrease in fluid accumulation in the intestinal lumen of rabbit ileal loops challenged with ETEC LT and ST positive strains, correlated with the presence of anti-CCL antibodies capable of toxin neutralization. The ability of these antibodies to neutralize toxin confirmed the recognition of epitopes, either linear or conformational, displayed by the recombinant chimeric protein expressed in transgenic tobacco hairy roots. Transgenic plants containing multivalent immunogenic vaccine candidates have the potential to be used for immunization with protection against gastrointestinal pathogens like ETEC.
肠产毒性大肠杆菌是发展中国家引起旅行者腹泻的最重要的细菌病原体。肠毒素（LT 和 ST）和定植因子（CFs）是 ETEC 发病机制中的两个重要因素。在本研究中，一种含有 CFAB*ST、CFAE 和 LTB（CCL）的重组四部分融合蛋白在烟草毛状根中表达。基于生物信息学分析设计了合成基因序列和基因顺序，预测了抗原性和免疫反应刺激的最佳排列。根据烟草植物表达的密码子使用情况进行了优化，在质粒 pBI121 中受 CaMV35S 启动子的控制。通过定量 ELISA 和 Western blot 测定，CCL 在烟草毛状根中高效表达，可产生 1.11%的可溶性蛋白。在这项研究中，通过口服和皮下途径用纯化的 CCL 免疫小鼠。在血清和粪便中检测到针对抗原的体液免疫特别是黏膜免疫，具有特异性 IgG 和 IgA。用免疫小鼠产生的抗 CCL 抗体在兔回肠环模型中评估 CCL 引发中和抗体的能力，并用 ETEC 菌株共同孵育。用 ETEC LT 和 ST 阳性菌株挑战时，兔回肠环肠腔中液体积累的减少与能够中和毒素的抗 CCL 抗体的存在相关。这些抗体中和毒素的能力证实了表达在转基因烟草毛状根中的重组嵌合蛋白展示的表位（线性或构象）的识别。含有多价免疫原性疫苗候选物的转基因植物具有用于免疫接种的潜力，以预防 ETEC 等胃肠道病原体。
