Immunological comparison of native and recombinant egg allergen, ovalbumin, expressed in Escherichia coli.
Chicken ovalbumin is one of the major egg white allergens which causes IgE-mediated food hypersensitivity. A gene encoding for chicken ovalbumin (Gad dI) was isolated from chicken oviduct by PCR amplification and was cloned under the control of T5 promoter fused with a six-histidine tag at the N-terminal end. Escherichia coli harbouring this construct expressed high quantities of the recombinant protein in the form of soluble fraction. The protein was purified using affinity chromatography on a Ni(2+)-nitrilotriacetic acid agarose column and was further purified to homogeneity by ion exchange chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot and secondary conformation analysis. The reactivity of the recombinant and native protein was tested against six egg allergic human patient's sera and the IgE and IgG binding activity was tested using both Western blot and ELISA. When compared to native ovalbumin, the recombinant protein had similar binding activity in immunoblotting, but slightly increased activity by ELISA. Circular dichroism revealed that the recombinant protein had a slightly less compact structure than the native form. Both antigens exhibited a similar immunogenicity in mice.
Rupa
Prithy
P
Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Mine
Yoshinori
Y
D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D004527:Egg Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010047:Ovalbumin / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D000485:Allergens; D004527:Egg Proteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D010047:Ovalbumin