Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo

Nahal Hadi; Shahram Nazarian; Saber Rouhi; Seyed Edris Hosseini; Javad Fathi

doi:10.1016/j.heliyon.2024.e26361

Production of egg yolk antibody (IgY) against a chimeric protein containing IpaD, StxB, and TolC antigens from Shigella: An investigation of its prophylactic effects against Shiga toxin (Stx) and Shigella dysenteriae in vitro and in vivo

Heliyon. 2024 Feb 16;10(4):e26361. doi: 10.1016/j.heliyon.2024.e26361. eCollection 2024 Feb 29.

Authors

Nahal Hadi¹, Shahram Nazarian², Saber Rouhi³, Seyed Edris Hosseini³, Javad Fathi¹

Affiliations

¹ Department of Bacteriology and Virology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
² Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran.
³ Resident of Large Animal Internal Medicine, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Iran.

Abstract

Shigella 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 Shigella, investigate its prophylactic and neutralizing effects against Stx and Shigella dysenteriae. The nucleotide sequence corresponding to the chimeric protein was cloned into pET28a plasmid and expressed in E. coli 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 LD₅₀ of Stx and S. dysenteriae 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 1LD₅₀ toxin and 4000 μg of IgY led to complete survival against 1LD₅₀, also 70% and 30% survival against 10 and 50 LD₅₀S. dysenteriae. This study showed that IgY produced against Stx and Shigella virulence factors could cause high protective effects against bacteria and toxins.

Keywords: IgY; IpaD; Prophylaxis; Shigella; StxB; TolC.