Design,Construction And Bioactivity Analysis Of The Hypoallergenic Molecule Derived From Cow’s Milk Allergen Bos D 5

ObjectiveFood allergy is a common allergic reaction.Allergen-specific immunotherapy(AIT)uses the immune system of the patient to establish a counter immune response,antagonizing the allergic immune response by vaccination with the disease-causing allergens or derivatives.With the development of epitope recognition,molecular diagnosis and biotechnology,AIT has entered the stage of epitope-based AIT.Cow’ s milk is the most common allergic food and Bos d 5 is one of the major allergen in cow’ s milk.In this study,we designed and constructed a hypoallergenic allergen derivatives molecule B5 M.Through biological prediction,physical structure analysis and biological activity analysis,it was preliminarily proved that B5 M reduced the allergenicity and retained its immunogenicity.Thus,B5 M represents a suitable candidate molecule for allergen-specific immunotherapy(AIT)of Cow’ s milk allergy(CMA).Methods1.The design of hypoallergenic molecule-B5MThe amino acid sequence and the key epitopes of Bos d 5 were obtained through literature and the webcite NCBI.Based on the principle of destroying the B-cell epitopes and retaining the T-cell epitopes,several amino acid sequences of alternative hypoallergenic molecule B5 M were designed.Hydrophilicity,secondary structure and antigenicity was analyzed using bioinformatics software.The amino acid sequence of B5 M was optimized and determined.2.The purification and expression of B5MBased on the amino acid sequence of the B5 M,through synthesis of gene,construction of plasmid,expression by using E.coli,treatment of inclusion body and affinity purification,B5 M protein was finally obtained.3.The bioacitivity analyse of B5MThe allergenicity analysis of B5 M was divided into two parts.Firstly,the binding ability of B5 M to free Bos d-sIgE in serum was determined by Dot blot and ELISA.Second,basophilic activation assay(BAT)was used to verify the binding ability ofB5 M to the sIgE on the surface receptor of basophils.Newzland white rabbits were immunized with B5 M and rBos d 5 and antiserum was obtained.ELISA inhibition assay verified the ability of B5M-antiserum to inhibit the binding of rBos d 5 to free sIgE in serum.BAT neutralization assay verified the ability of B5 M antiserum to inhibit the binding of r Bos d 5 to sIgE on the cell surface receptor.T cell proliferation assay verified whether B5 M retained the T cell epitopes of Bos d 5.MTT method was used to carry out this part of experiment.Results1.The design of hypoall ergenic molecule B5MThe hydrophilicity,antigenicity and secondary structure of Several candidate molecules were analyzed by bioinformatics,and The amino acid sequence of B5 M was screened out.2.The purification and expression of B5MBy using the E.coli expression systemBL21(DE3),B5 M was expressed.After the treatment of inclusion body and nickel column affinity purification,B5 M with a molecular weight of 52 KDa was obtained.Circular dichroism(CD)analysis showed that the disulfide bond of Bos d 5 disappeared,the secondary structure of B5 M was significantly changed compared with Bos d 5.3.The bioactivity analysis of B5MThe allergenicity analyse of B5M:Dot-blot and ELISA showed that the binding ability of B5 M to free sIgE in serum of patients with CMA was decreased compared with rBos d 5.The results of ELISA showed that the binding capacity of B5 M to free sIgE in serum of 20 patients with CMA decreased to 67.84-45.48%,with an average of 58.22%.BAT showed that compared with the stimulation index(SI)obtained from stimulated the basophils presensitized by sera from 4 patients with rBo s d 5 of different concentrations,the stimulation index(SI)was significantly decreased due to B5M(0.001-10 ng/ml).This experiment proved that the allergenicity of B5 M was decreased than that of rBos d 5.The immunogenicity analysis of B5M:The rabbite antiserum was obtained by immunizing rabbits with rBos d 5 and B5 M,respectively.The titer of sIgG antibody was analyzed.ELISA inhibition assay showed that the antiserum of rabbit immunizedwith B5 M was able to inhibit the binding of serum sIgE to rBos d 5.The results showed that the inhibition rate of B5 M to inhibit the binding of free-sIgE to the allergen was between 51.53% and 75.43%,and the inhibition rate of rBos d 5 was between 42.24% and 66.67%.BAT inhibition assay showed that the anti-serum of rabbits immunized with B5 M was able to inhibit the binding of Bos d 5 to sIgE on the surface receptors of basophils in peripheral blood of allergic patients.The T-cell epitope integrity analysis of B5M: The results of MTT assay showed that compared with the rBos d 5,B5 M retained the T cell epitope of the allergen,providing a prerequisite for the production of protective antibodies in vivo.ConclusionIn this study,we design and construct a hypoallergenic molecule B5 M for the study of AIT of cow’s milk allergy.After design and predicted by bioinformatics software,the amino acid sequence of B5 M was determined.After expression and purification,this hypoallergenic molecule was collected.By biological activity analysis,it was proved that the allergenicity of B5 M was decreased,but its immunogenicity was retained.Therefore,B5 M could be used as a candidate molecule for AIT of CMA and provided theoretical support for AIT of food allergy.