Mapping Of IgG-Binding Epitopes On Buffalo β-lactoglobulin

Milk allergy is one of the most common food allergies. This allergy has been estimated to affect 1-2% of the adult population and up to 8% of children below the age of three.β- lactoglobulin(β-lg)is well known as one of the major milk allergens to which 82% of milk allergy people are sensitive. A cross-reactivity exists between buffalo and bovineβ-lg. because of high homologous amino acid sequences. Therefore, it is necessary to investigate buffaloβ-lg and better understand the mechanism of allergy caused by buffaloβ- lactoglobulin.The present research was composed of four parts including the purification of buffaloβ-lg, antigenicity evaluation of buffaloβ-lg, mapping of linear and conformational epitopes on buffaloβ-lg. The main methods, results and conclusions are as follows.1. After buffalo whey was fractionated by column chromatography, the target product were collected and identified by SDS-PAGE,IEF-PAGE and ESI-Q-TOF-MS. The results showed that buffaloβ-lg could be separated and purified by the combination of DEAE-Sepharose Fast Flow ion- exchange chromatography and SephadexG-75 gel- chromatography. The antigenicity of buffaloβ-lg preserved well and the purity was above 90%.2. Specific rabbit antibody was raised against buffaloβ-lg /bovineβ-lg from Japanses rabbits immunized with a routine method. The antigenicity of buffaloβ-lg was evaluated by indirect ELISA, competitive inhibition ELISA and western blotting with anti-buffalo and anti-bovineβ-lg rabbit serum. The results showed that the buffaloβ-lg isolated was highly antigenic, and there was a cross-reactivity between buffalo and bovineβ-lg with 69.27% cross reactivity ratio.3. In order to collect specific rabbit antibody with high purity, buffaloβ-lg was coupled to CNBr-activated Sepharose 4B, which eluated with 3 mol/L MgCl₂.The results showed highly purified specific rabbit antibody was identified by SDS-PAGE, and the protein content was 5.8mg/mL and the titre was 1:10⁹ tested by ELISA. The method can be applied to purifying the specific IgG from rabbit serum.4 .To predict the linear epitopes on buffaloβ-lg, bioinformatics research was cconducted using LaserGene software and web service with Kyte-Doolittle to mapping the regions of hydrophilicity of amino acids, Jameson-wolf to predicting antigen index, Emini to describing Surface Probability and Chou-Fasman and Garnier-Robson to identifying its second structure, such as flexibility, turn ,coil. Based on these analysis, five possible regions on buffaloβ- lactoglobulin, AA30-45, AA67-78, AA97-115, AA124-141 and AA150-156 were predicted as epitopes.5. Ninety-nine positive clones were identified among 300 radom clones derived from the panning of Heptapeptide phage display peptide library with purified anti-buffaloβ-lg rabbit sera. Through amino acid sequence analysis using the web tool of MIMOX , six mimic epitope regions on buffaloβ-lg, AA14-20, AA26-32, AA36-42, AA70-80, AA82-86 and AA149-156 were mapped. Among them, AA70-80 and AA149-156 were major epitope regions.6. The immunodominant rabbit IgG-binding epitopes on buffaloβ-lg were mapped using array of overlapping peptides synthesized on activated cellulose membranes. Based on the prediction and panning results, senven linear rabbit IgG-binding epitopes on buffaloβ-lg were mapped, these being AA21-30(SLAMAASD IS ),AA25-34 (AASDIS LLDA) , AA29-38 (ISLLDAQSAP) , AA77-86 (KIPAVFKIDA) ,AA87-96 (LN ENKVLVLD) ,AA134-143 (EKFDKALKAL) and AA150-159 (AFNPTQLEGQ) .7. Compared with the results of prediction and panning, 30% of predicted epitope regions accorded with panned epitope regions, however, 40% of panned epitopes were not predicted by bioinformatics analysis. Additionally, two rabbit IgG-binding epitopes on buffaloβ-lg were found not to be reported, which are AA134-143 (EKFDKALKAL) and AA150-159 (AFNPTQLEGQ) .8. Two conformational IgG binding epitopes on buffaloβ-lg,Ser-Pro-Leu-Thr-Glu -Asn-Lys and Pro-Leu-Asn-Glu-Asn-Lys-Asp, were mapped in the analysis of the conformational mimic peptide using the web tool of MIMOX.9. The method of mapping conformation epitopes was established by Disulfide Constrained Phage Display Peptide Library combined with the web tool of MIMOX, which can be applied to mapping the conformational epitopes on food allergens.