Hadi Nahal, Nazarian Shahram, Rouhi Saber, Hosseini Seyed Edris, Fathi Javad
Department of Bacteriology and Virology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran.
Heliyon. 2024 Feb 16;10(4):e26361. doi: 10.1016/j.heliyon.2024.e26361. eCollection 2024 Feb 29.
is a major problem in developing countries. Immunoglobulin Y (IgY) can be used for prophylaxis and neutralize bacteria. The aim of this study was to produce IgY against the chimeric protein containing IpaD, StxB, and TolC antigens from , investigate its prophylactic and neutralizing effects against Stx and . The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in BL21 (DE3). Protein expression was confirmed by SDS-PAGE and the recombinant protein was purified by Ni-NTA affinity chromatography. The 150 μg of chimeric protein was mixed with Freund's adjutant and injected into laying hens (Leghorn). IgY was purified using PEG6000 precipitation. Antibody titer in the serum and egg yolk was evaluated by ELISA. IgY challenge against 1,10 and 50 LD of Stx and was investigated. A 60.6 kDa recombinant protein was confirmed by SDS-PAGE. ELISA showed that the antibody titer was significantly increased. MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] showed that at 16 μmol/L, IgY protected HeLa cells against Stx. Treatment of mice with 1000 and 1500 μg IgY leads to complete survival of the mice against 1LD toxin and 4000 μg of IgY led to complete survival against 1LD, also 70% and 30% survival against 10 and 50 LD. This study showed that IgY produced against Stx and virulence factors could cause high protective effects against bacteria and toxins.
在发展中国家是一个主要问题。免疫球蛋白Y（IgY）可用于预防和中和细菌。本研究的目的是制备针对含有来自[具体来源未提及]的IpaD、StxB和TolC抗原的嵌合蛋白的IgY，研究其对志贺毒素（Stx）和[另一物质未明确写出]的预防和中和作用。将与嵌合蛋白对应的核苷酸序列克隆到pET28a质粒中并在BL21（DE3）中表达。通过SDS-PAGE确认蛋白表达，并通过镍-亚氨基二乙酸（Ni-NTA）亲和层析纯化重组蛋白。将150μg嵌合蛋白与弗氏佐剂混合后注射到产蛋母鸡（来航鸡）体内。使用聚乙二醇6000（PEG6000）沉淀法纯化IgY。通过酶联免疫吸附测定（ELISA）评估血清和蛋黄中的抗体效价。研究了IgY对1、10和50个致死剂量（LD）的Stx和[另一物质未明确写出]的攻击作用。通过SDS-PAGE确认了一种60.6 kDa的重组蛋白。ELISA显示抗体效价显著升高。噻唑蓝（MTT）法[3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐]显示，在16μmol/L时，IgY可保护HeLa细胞免受Stx侵害。用1000和1500μg IgY处理小鼠可使小鼠在面对1个致死剂量的毒素时完全存活，4000μg IgY可使小鼠在面对1个致死剂量时完全存活，在面对10和50个致死剂量时分别有70%和30%的存活率。本研究表明，针对Stx和[另一物质未明确写出]毒力因子产生的IgY可对细菌和毒素产生高度保护作用。
