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Expression of a Tandem of Epitopes on Bovine Beta-lactoglobulin Allergen

Author: HanZuo
Tutor: ChenHongBing
School: Nanchang University
Course: Of Food Science
Keywords: β-lactoglobulin Epitope series body Expression Milk allergy
CLC: TS252.1
Type: Master's thesis
Year: 2010
Downloads: 72
Quote: 0
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Abstract


Milk allergy is a common food allergies, including β-lactoglobulin is recognized as the main milk allergens, about 82% of milk allergy in patients allergic to β-lactoglobulin. Epitope to trigger the material basis of food allergy, food allergy original epitope exploration is an important part of the food allergy research. Therefore, to carry out the study of bovine p-lactoglobulin epitopes is important. In addition, the study will be to construct p-lactoglobulin epitope epitope series body, to optimize the combination of a series will have independent antigen, has potential applications in the research and development based on the food allergen epitopes detected. The work of the paper include of milk p-lactoglobulin linear epitopes series body molecular design, expression and purification of the epitope series prokaryotic systems, anti-epitope the series body polyclonal antibody preparation and epitope series of body antigenic and allergenic assessment. Methods, results and conclusions of the study are as follows: 1. 7 bovine p-lactoglobulin and human serum IgE binding linear epitopes of B cells (B1, B2, B3, B4, B5, B6, B7) and a T-cell epitope (T) is the object of study, select the appropriate interval sequence series. Theoretically 5040 combinations. Filter by the the DNAStar \u0026 SOMPA network server suitable combination, independent antigenic epitope build series body. The interval between the B-cell epitope sequence to the glycine (G), the sequence of the interval between the T cell and B cell epitopes of two lysine (KK). The combined structure of the final design of the molecule of the series body TKK-B1-G-B6-G-B5-G-B3-G-B7-G-B2-G-B4. 2. Designed series at both ends of the molecule, respectively with the BamHI and EcoRI restriction sites for gene synthesis, the synthetic gene was cloned into the vector pMD19-T. After double digestion, ligation, transformation steps, successfully constructed expression vector pGEX-4T-1-string (pGEX-4T-1-tan), and transformed into the expression strain E.coliBL21 (DE3) pLysS. Build a good carrier induced by IPTG, the expressed protein concatemer fusion protein, molecular weight about 39.5kD. After SDS-PAGE and Western blotting, the expression result is correct. By induction temperature, time and IPTG concentration optimization, the best conditions of the target protein soluble expression: 23 ℃ induced 4h IPTG concentration for 0.3mmo1 / L. The expression of the protein with GST tag, after purification, the protein concentration of 4mg/ml. Purified tandem fusion protein as an antigen, two Japanese white rabbits were immunized according to the routine immunization program, an indirect ELISA test results showed that the tandem protein purified antigen, Japanese white rabbits its having a good immune response. The resulting antibody titers were 1:128,000 and 1:64,000. By indirect ELISA and Western blot experiments, the results showed that the series body of this experiment, the purified protein and milk β-lactoglobulin strong immunological cross-reaction exists. 5 with the the allergenic milk allergy serum testing series fusion protein, the results showed that the tandem fusion protein has allergenic.

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