Prokaryotic Expression And Immunological Activity Identification Of C-terminal Domain Of The Allergenic Protein From Tartary Buckwheat

As a kind of functional food Buckwheat is attracting public's attention more and more, with great advantages in nutrition and medicine. Buckwheat protein is composed of some special amino acids. Its high content of bioactive substances such as flavone has showed significant effects in preventing currently common cardiovascular diseases such as diabetes, hypertension, high cholesterol etc. However, another unfavorable reaction, buckwheat allergy, which was aroused by touching or eating buckwheat food, has brought people much trouble. Researchers from Japan and Korea successively obtained allergenic proteins with molecular weight of 16 kDa, 22-24 kDa, 34-38 kDa and 69 kDa respectively from the common buckwheat (CB). Although there is not identical viewpoint on the major allergen in the buckwheat and the reports from every research group have discrepancy, the protein with molecular weight of 22-24 kDa is thought as the major allergen in the buckwheat. At present, reports in allergic composition research of tartary buckwheat (TB) are rare. By separation and purification, our laboratory have obtained one natural protein with molecular weight of 24 kDa named TBa (tartary buckwheat allergen) from the Yunnan tartary buckwheat weeds, and it was identified as one of the major allergens in the tartary buckwheat by immunoblot. The structure gene of TBa was obtained for the first time by gene cloning and was registered in the GenBank (No.AY044918).The objective of the experiment is to express the TBa in the prokaryotic system further. The TBa gene was cloned into the expression vector pET-28a, and expressed in E. coil BL21(DE3) host cells. The protein product was expressed in inclusion bodies mostly. After purified by Ni²⁺-NTA agarose affinity chromatography column, the purity of the target protein reached above 95 %, and renatured by gradient dialysis, and about 68 % soluble TBa protein was obtained. The six histidine residues were confirmed by Western blot. The recombinant TBa had a specific binding activity with IgE antibody. The result of ELISA indicated that the recombinant TBa could integrate with IgE peculiarly, which implied it had higher immunologyactivity.The amino acid sequence of TBa was deduced through the structure gene, according to which the antigen epitopes were doped out using the Antigenic programme. The result indicated that there were eight possible epitope regions, among of which two regions were chose to investigate. Primers were designed to clone the encoding sequences and then the two sequences were subcloned into the expression vector pET-32a. The recombinant plasmids were transformed into the E. coil BL21(DE3) host cells to express respectively. Then the two expression antigen epitopes were purified and the immunology activity was identified preliminary.In the experiment, by constructing the prokaryotic expression vector and optimizing the expression conditions, the high-yield expression of TBa in the prokaryotic cells was fulfilled. The result of the immunological activity identification indicated that the recombinant TBa had the similar immunological activity with the natural TBa and had the special integration with the IgE in the patient's sera, which provided the theoretical basises for the buckwheat food and production hypersensitivity. At the same time two regions with the higher possibility of having the antigen epitopes in the TBa structure gene were cloned, expressed and analyzed preliminarily, which laid a foundation for the research of the TBa's allergenic mechanism, the analysis of the relationship between it's structure and function and the following study to locate the epitope.