Analysis Of β-lactoglobulin Epitopes And Recombinant Expression And Identification Of Key Epitopes Concatemer

Objective:Serum specific IgE is a common means of diagnosis of food allergy,Standardization of known antigen is the important precondition for development of specific IgE in vitro diagnostic reagents and the development of food components of antigen epitope recombinant is an important measure to solve the standardization of antigen. In this study, the most important milk allergen "β-lactoglobulin" as an example, starting from the analysis of its epitopes, and analyze the important antigen epitope and the patients serum sIgE response frequency, thus to select key epitopes and build key epitopes recombinant and try the possibility of using epitope recombinant protein instead of natural protein.Methods:(1) β-lactoglobulin key epitopes screening and serum heterogeneity analysis:First, in the NCBI database searched milk allergens β- lactoglobulin amino acid sequence(162 amino acids), then entrusted GL(Shanghai) Biochem Ltd synthesis antigen epitopes by the end of the overlap. 20 amino acids of a polypeptide, two adjacent overlapping peptides of 5 amino acids; The polypeptide pointed on nitrocellulose membrane and the specific IgE in milk allergy patients serum as a "probe". To observe polypeptide response frequency difference between patients with milk allergy and peptide identification by any differences between different individuals.(2) β-lactoglobulin key antigen epitope recombinant tandem of building and its preliminary application research: The highest response frequency of three polypeptide expressed in series, between adjacent two peptides do joint with a glycine. First using DNAstar bioinformatics software to forecast three tables of 6 kinds of combination and selecting the best combination; Then entrust Shanghai sangon biological co., LTD synthesis of the DNA sequence, Connection to DNA sequence with pET carrier-42a(+),cloned host and amplification recombinant plasmid. The constructed plasmid wastransformed into E. coli BL21(DE3), and expression was induced with IPTG inducer,and finally the use of nickel column purified single component key epitopes series body recombinant proteins, and finally using western-blot, dot blot methods and immunoassay methods luminol oxygen pathways identified immunologically active recombinant protein.Results:(1) Heterogeneity analysis of patients serum specific IgE to identify antigen epitope: In 20 patients with milk allergy spots a portion of the imprinting results serum antigen epitope identification by the same, such as the 1st, 5th, 6th, 11 th milk allergy serum identified peptide epitopes are on the 1st, Some milk allergy patients serum recognition by the table were not identical. Depending on the reaction conditions of peptide and serum IgE, an amino acid sequence selected β-lactoglobulin main epitopes for AA76-95, AA1-20, AA106-125, the amino acid sequence of the major epitopes for AA91-110, AA61-81, AA121-140, the amino acid sequence for a secondary epitopes for AA 46-65, AA 31-50, AA 151-162.(2) β-lactoglobulin expression and identification of key epitopes of recombinant proteins: By theoretically six kinds of DNAStar software combinations were analyzed to obtain the best combination, after successfully constructed an E. coli vector, IPTG induction of recombinant protein expression, access to the size of about 36 kDa recombinant protein and purified recombinant protein 36 kDa size of the individual components. And by Western blot, dot blot immunoreactivity was identified, and the use of recombinant proteins as antigens to detect oxygen established a luminol way immunoassay technology to detect serum sIgE.Conclusion:This study on the milk allergens mainly beta globulin antigen epitopes parsing,different milk allergy patients serum antigen epitopes identification by have heterogeneity. Successfully expressed the beta globulin key protein antigen epitope sseries weight group, and the combination of recombinant protein with milk allergy patients serum sIgE ability was better than that of natural milk allergens, in detectionof milk allergy disease could replace natural antigen, lay a foundation for the preparation of a new generation of diagnostic reagents.