Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.
Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.
Mol Immunol. 2024 Sep;173:53-60. doi: 10.1016/j.molimm.2024.07.002. Epub 2024 Jul 24.
Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella.
The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10 mg/kg) under various time conditions.
The recombinant protein, weighing 82 kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5 mg/ml of egg yolk and the purity of the extracted IgY was over 90 %. The results of the ELISA showed that at least 19 ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10 mg/kg IgY leads to 80 % survival of the mice against 10 LD50 S. dysenteriae.
Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.
志贺氏菌病是一种全球性的胃肠道疾病，发病率和死亡率都很高，但目前尚无抗志贺氏菌疫苗。在志贺氏菌病的治疗中使用抗生素会加剧抗生素耐药性。抗体，特别是卵黄抗体（IgY），为解决这一挑战提供了一种有前途的方法。本研究旨在研究针对包含志贺氏菌免疫原 IpaD、IpaB、StxB 和 VirG 的重组嵌合蛋白产生的 IgY 的预防效果。
嵌合蛋白由 IpaD、IpaB、StxB 和 VirG 组成，在 E. coli BL21 中表达，并使用 Ni-NTA 柱进行纯化。用 PEG-6000 法从鸡卵黄中提取 IgY，并用 SDS-PAGE 和 ELISA 技术进行分析。随后，通过用 10LD50 的 S. dysenteriae 挑战小鼠，并在不同的时间条件下给予不同浓度的 IgY（1.25、2.5、5 和 10mg/kg）来评估 IgY 的预防功效。
纯化的重组蛋白分子量为 82kDa，并用 Western blot 进行了验证。IgY 的浓度确定为 9.5mg/ml 的卵黄，提取的 IgY 的纯度超过 90%。ELISA 的结果表明，至少 19ng 的纯抗体可识别重组蛋白并与之反应。用 IgY 和志贺氏菌进行的挑战试验表明，存活率与抗体浓度之间存在直接相关性，随着浓度的增加，死亡率降低。用 10mg/kg 的 IgY 治疗小鼠可使 80%的小鼠对 10LD50 的 S. dysenteriae 存活。
我们的研究结果表明，IgY 可能具有治疗志贺氏菌感染和对抗抗生素耐药性的潜力。
