The Swimming Crab Allergens Analysis And Tropomyosin Key Epitope Recombinant Expression

Objectives:Food allergy is today’s major health issues, food allergen identification, purification, and standardization is the central link of food allergic disease diagnosis and treatment. The ultimate purpose of this study is to start from the two aspects of the analysis the allergens and analysis to be tested IgE polymorphism, and strive to solve the problems of the current serum specific IgE. First of all, the blue crab as the research object, and specific IgE antibodies in the serum of allergic patients were appointed as "probe" to analysis the blue crab major allergen component; Secondly, crab-allergic patients for the study, analysis of patients allergic to crab serum specific IgE against the allergen heterogeneity. In addition, a fusion peptide containing three epitopes was designed based on crustaceans common allergens-tropomyosin, and the following expression was employed by using a prokaryote protein expression system. Meanwhile, these experiments could provide the further technical support for the tandem expression from different epitopes of allergens.Methods:1. Swimming crab allergen component analysis:Total protein extraction swimming crab, By SDS-PAGE analysis of protein fractions, and serum specific IgE as a "probe" allergic to blue crab with specific IgE binding component technology by western-blot analysis.2. Heterogeneity analysis of crab allergic patients specific IgE against the allergen: Select50cases of crab allergy serum,and the specific IgE antibodies in the serum of patients with different polymorphism analysis by western-blot, and analysis of crab allergy serum specific IgE against the allergen individual heterogeneity.3.Tropomyosin of TM32a protein recombinant expression and immune activity identification:Select the section of tropomyosin containing the antigenic epitope of a polypeptide (abbreviated as TM32a). The DNA sequence of the synthetic TM32a, and select pET42a vector, then the EcoRI and XhoI double digestion construct a plasmid vector, and cloning host strain ToplO amplification of the recombinant plasmid, the most common use of prokaryotic expression host E. coli BL21(DE), IPTG-inducible protein expression, and carried on the carrier His-tagged protein was purified by Ni column purified single component TM32a protein. Western-blot were used for identification of the immune activity of the target protein.Results:1. The swimming crab allergen component analysis:swimming crab’s total crude protein were observed nine major protein bands. Positive pooled serum as a probe blot,94kD,74kD,70kD,48kD,43kD,40kD and36kD at both the positive bands, however, of protein both higher concentrations74kD and70kD protein were not present the stronger responeswith mixed serum.On the contrary, the strongest medium protein content94kD protein mixed serum response.2. Heterogeneity analysis of crab allergic patients specific IgE against the allergen:a singleton crab serum for the study of western-blot experiments, the presence of significant heterogeneity found in the different serum specific IgE,and the number and variety of different reactive protein are different, including:①one protein as the main allergen;②two or more proteins are major allergens;③there is no strong reaction of the major allergen, and only a fewlighter response allergens. Statistics found that the highest a36kD protein response rate up to80%.3.Recombinant expression of tropomyosin TM32a and immunocompetence identification:the DNA sequence of tropomyosin synthesis TM32a size96bp, and constructed by ligating the plasmid vector pET-42a, size of6030bp about.And build good plasmid was transformed into E. coli BL21(DE), and IPTG-induced expression.To obtain the size of the recombinant protein of about34kD TM32a,then The recombinant protein purified by nickel ion column purification column,and obtain the34kD size TM32a single component and their immune activity were analysed by Western-blot.Conclusion:1. There are seven major allergen components in the swimming crab, and the94kD allergen has showed the strongest reaction with mixed serum.2. Crab allergy serum of patients with specific IgE antibodies against allergens presence of significant heterogeneity.Identified a total of three different serum specific IgE against the allergen type, and analysis of the the nine major allergen reaction rate, wherein the highest reaction rate of allergen protein is36kD.3. The recombinant protein TM32a of tropomyosin,and the target protein molecular weight of about34kD,and the immune activity of protein were.observed through western-blotting.