| Tartary buckwheat is a cultivated species, its medicinal and nutritional value has been long concerned, tartary buckwheat contain a lot of flavonoids, phenolic compounds, proteins and other substances, regular consumption can prevent and treatment of cardiovascular and cerebrovascular diseases. It’s known as the "ever-young health food". But there are many reports shown that edible buckwheat might cause allergic reactions, such as urticaria, asthma, severe shock or even death. In recent decades, scientists have identified that buckwheat allergy is an IgE-mediated immediate-type reaction. Some natural allergenic proteins from buchwheat with different molecular weights of 6,9,19,24, and 60-70 kDa have been identified, and among them, the 24 kDa protein was regarded as the main allergen. However, reports on the allergenic composition of TB are limited. Previously, we have obtained three main allergens with molecular weight of 56kDa,34kDa and 24kDa by cloning and expression. Detection of western blotting found that they could specific binding with allergenic serum to buckwheat, so study the low allergenic buckwheat food is very necessary. Usually, there are many methods to reduce the allergenic protein immune activity, such as heating, regulating pH, interaction metal ion and allergens, physical radiation, and so on. In this stuty, we investigated the effect of metal ions on the structure and activity of tartary buckwheat allergenic protein TBt.According to the previous method, TBt was separated and purified, the purity of purified protein was 95% and with a concentration of 1μmol/L. The different metal ion solution of 1 mol/L (Cu2+, Mg2+, Ca2+, Ba2+, Ni2+, AL3+) and TBt was mixed by different proportionma, and then the characteristics of spectroscopy was analysis. The results showed that among selected metal ion, Cu has obvious effect on the structure and activity of TBt. Analysis of fluorescence spectroscopy showed that Cu2+ could be bound to TBt at a molar ratio of 1:1. The result of CD spectroscopic analysis showed the secondary structure of TBt did not change, but the spatial structure of TBt was changed in native PAGE. Briefly, the spatial structures of TBt changed into hexamers after interaction with Cu2+. Inhibitory ELISA and indirect ELISA showed that TBt became a hexamer, and a part of epitopes were covered and the IgG binding capacity was decreased.The experimental results suggest that some metal, including to Cu2+, may as a main or crucial ion in allergenic proteins, they can influence the polymeric degree of proteins by interfere the secondary structure, further affect the immunologic activity. These results can provide theoretical reference and scientific basis for further research the regulation of metal ions on the storage protein in plant, as also as the molecular mechanism of allergen.In order to detect the trace amounts of buckwheat allergenic proteins in some foods, allergen TBt was prepared, and specific antibodies against the allergen were produced. A double-antibody sandwich enzyme linked immunosorbent assay (ELISA) based on rabbit polyclonal antibody and mouse polyclonal antibody was then established. Experimental results showed that the newly sandwich ELISA can detect the concentration of 0.16μg/mL of TBt, and the sensitivity and specificity of this method was higher than that of indirect ELISA. |
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