Production of Chicken Egg Yolk Immuno-globulin (IgY) against Recombinant TolC Pro-tein From Shigella flexneri Bacteria

Abstract

Background: Growing antibiotic resistance is a global concern for the treatment of shigellosis. The development of efflux pumps such as the AcrAB-TolC pump has increased antibiotic resistance in Shigella flexneri strains. The aim of this study was to produce IgY against the recombinant immunogen TolC and evaluate the binding of the antibody to the recombinant immunogen Materials and Methods: A construct containing immunogen regions of TolC protein was designed and synthesized; then, the recombinant plasmid pET28a-tolC was transferred to E. coli BL21 (DE3) competent cells. Expression of the recombinant protein in E. coli was induced with 1 mM IPTG. The recombinant protein was purified using affinity chromatography by Nickel-Nitrilotriacetic acid (Ni-NTA) resin and evaluated by western blotting. Leghorn chickens were immunized with 200 micrograms of the recombinant protein. IgY was purified using PEG precipitation, and the antibody titer was determined in immunized chicken’s serum and egg yolk by ELISA method compared to the control group. Results: Recombinant protein TolC was expressed with a molecular weight of 40 kDa and an expression yield of 10 mg/l of the culture media. The IgY concentration was determined to be 9 mg/ml of egg yolk. The antibody titer in serum against the recombinant protein after the third immunization was 1: 6400 (P<0.05). In the ELISA method, at least seven nanograms of antibody reacted with the recombinant protein. Conclusion: Antibodies against TolC recombinant immunogen can be investigated as a source of passive immunity in the disease caused by Shigella flexneri.

Keywords

• shigella flexneri
• efflux pump
• tolc
• recombinant protein
• igy