Clin Exp Vaccine Res.  2015 Jul;4(2):177-183. 10.7774/cevr.2015.4.2.177.

Production of specific IgY Helicobacter pylori recombinant OipA protein and assessment of its inhibitory effects towards attachment of H. pylori to AGS cell line

• Borhani K
• Mobarez AM
• Khabiri AR
• Behmanesh
• Khoramabadi N

• KMID: 1965402
• DOI: http://doi.org/10.7774/cevr.2015.4.2.177

Abstract

PURPOSE
The common triple therapy for Helicobacter pylori is challenged by the increasing cases of antibiotic resistant infections, raising the need to explore alternative therapies. Oral administration of egg yolk immunoglobulin Y (IgY) has been previously reported as a means of passive immunization therapy for H. pylori infections. In this work, we investigated the inhibitory effect of IgY on the attachment of H. pylori to AGS cell line.
MATERIALS AND METHODS
Recombinant OipA was prepared. Hens were immunized with recombinant protein three times. IgY was purified from egg yolks of immunized hens using polyethylene glycol precipitation method. The inhibitory effect of the specific immunoglobulin was evaluated in AGS cell line infected with H. pylori.
RESULTS
The presence of recombinant OipA (30 kD) was confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunization of hens was confirmed using enzyme-linked immunosorbent assay. The purified IgY from egg yolks were assessed using SDS-PAGE and confirmed by western blot.
CONCLUSION
The results showed that IgY-OipA had inhibitory effect on attachment of H. pylori to AGS cell line and may be utilized as a therapeutic or prophylaxis material.

Keyword

Helicobacter pylori; IgY; OipA; AGS cell line; Co-culture techniques

MeSH Terms

Administration, Oral
Blotting, Western
Cell Line*
Complementary Therapies
Egg Yolk
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Helicobacter pylori*
Helicobacter*
Immunization
Immunization, Passive
Immunoglobulins
Polyethylene Glycols
Sodium Dodecyl Sulfate
Immunoglobulins
Polyethylene Glycols
Sodium Dodecyl Sulfate

Figure

• 

  Fig. 1 Detection and purification of expressed OipA. (A) OipA was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Lane M: low molecular weight standard protein size marker (kDa); lane 1: non-induced OipA; lane 2, 3, 4, and 5: induced OipA with 1 mmol/L IPTG at t = 0, 1, 2, and 3 hr, respectively. 30-kD band were detected in induced lanes. T = 3 hr was the best time for induction. (B) Purified OipA was detected using SDS-PAGE. Lane M: low molecular weight standard protein size marker (kDa); lane 1: 30-kD band was detected as purified OipA.

• 

  Fig. 2 The reaction between IgY-OipA produced in serum and recombinant OipA. The titration of serum in the first hen having the highest affinity toward recombinant protein was shown (No. 1). Serum of non-immunized hens (phosphate buffered saline injected) were used as negative control.

• 

  Fig. 3 Purified IgY detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After purification by polyethylene glycol precipitation method, IgY was detected by SDS-PAGE. Lane IgY: purified IgY; light chain (25 kDa) and heavy chain (60 kDa); M: low molecular weight standard protein size marker (kDa).

• 

  Fig. 4 Specific IgY against rOipA detection by western blot analysis. Lane IgY-OipA: rOipA band (30 kDa) was detected; lane M: low molecular weight standard protein size marker (kDa). Western blot analysis using IgY against OipA showed a single 30-kDa protein on nitrocellulose paper. Purified IgY was used as first layer, and rabbit-anti-IgY conjugated with horseradish peroxide enzyme was used as second-layer antibody.

• 

  Fig. 5 Inhibition of Helicobacter pylori attachment to AGS cell line by IgY-OipA. Attahment of H. pylori SS1 to AGS cells were assessed by co-culture and enzyme-linked immunosorbent assay. Treatments of bacteria with ≥ 0.5 mg/mL concentration of IgY-OipA inhibited attachment of bacteria to the AGS cell line.

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