Aptamers Targeting, Enzymolysis And Desensitization Research On Allergenic Epitopes Of Beta-lactoglobulin

Milk allergy is a common food allergy, bovine beta-lactoglobulin is considered one of the most important allergen protein. In this paper, using β-Lg targeted binding aptamers,the p-Lg allergen epitopes cover processing, in order to reducing the β-Lg sensitization effect. Conclusions of the study are as follows:1 The computer-assisted aptamer design and screen of β-lactoglobulin allergen epitopeIn this research, β-lactoglobulin was chosen as the template, the targeting aptamers based on the sense and antisense strand complementary principle from different allergen epitopes can be designed. The targeting aptamers which can bind P-lactoglobulin specifically were screened through the computer simulation software of docking. According to the minimum energy principle are selected from the QWVW, RRSL, GWGLP, RDGY and WVDL of the five groups of aptamers.2 Optimization of two protease hydrolyzing β-lactoglobulin conditionsNeutral protease and trypsin selected to hydrolyze β-lactoglobulin. Through the single factor and orthogonal experiment, Amino acid-N content was determined by formaldehyde titration after protein hydrolyzed to filter out the optimum conditions of the hydrolysis of two kinds of enzymes of P-lactoglobulin. The experimental results show that, neutral protease hydrolysis optimum substrate concentration was at 1mg/mL, the hydrolysis temperature was at 50℃, pH was at 7.2, enzyme and substrate mass ratio was at 3%, the hydrolysis time in 6h. Trypsin Hydrolysis optimum substrate concentration was at lmg/mL, the hydrolysis temperature was at 45 ℃, pH was at 8.0, enzyme and substrate mass ratio was at 2%, the hydrolysis time in 4h.3 Purification and sensitization sites hydrolysis separation β-lactoglobulin hydrolysateTest using Sephadex G-15 as a separation medium, for the separation of beta lactoglobulin, neutral protease hydrolysis of beta-lactoglobulin, Trypsin Hydrolysis of beta lactoglobulin product. The experimental results show that, the optimal separation conditions for phosphate buffer ionic strength 0.03mol/L and pH8.0, constant flow rate was 0.7ml/min. Through the determination of free amino acid found, aptamer QWVW docking beta-lactoglobulin by protease hydrolysis, decreased the content of free amino acid in different degree, the beta-lactoglobulin hydrolysis degree decreased.4 Influence of hydrolysis product of β-lactoglobulinsensitization in miceThrough the mice ear immediate active skin allergy test and oral sensitized mice intestinal permeability test showed that, after the beta lactoglobulin sensitized mice, compared with the blank group, Evans blue extravasation rate and intestinal index increased significantly. Aptamer QWVW, WVDL docking beta-lactoglobulin reduce sensitization effect is better. The test mice serum IgG showed that QWVW and WVDL, these two groups of aptamer should cover up the beta-lactoglobulin sensitization loci, reduced beta lactoglobulin sensitization. The aptamer QWVW corresponding to the VTQT, as well as aptamer WVDL corresponds to the TQLE beta lactoglobulin on sensitization loci.