Prokaryotic Expression,Purification And Identification Of Immune Activity Of Allergenic Protein TB24kDa From Tartary Buckwheat

Buckwheat is a kind of valuable plant with great advantages in nutrition and medicine. Its high content of bioactive substances has showed significant effects in preventing currently common cardiovascular diseases such as diabetes, hypertention etc. and some other diseases including rheumatic arthritis. However, another unfavorable reaction, buckwheat allergy, which was aroused by touching or eating buckwheat food, has brought people much trouble. Many scientists have embarked on the research of the reasonable allergen in common buckwheat (CB). Considering comparatively few research at present on the allergen in tartary buckwheat (TB), it is necessary for some attention and concentration in this field.A protein with molecular mass of 24kDa has identified as the major allergen in tartary buckwheat. Further study to locate the antigenic determinant (epitope) of the allergen and to find it's effect in immunology and molecular genetics will help to understand allergenic mechanism and provide scientific ground for immunotherapy of allergy to buckwheat and the breeding of low-allergenic species. The target of this study is to express and purify the recombinant allergenic protein and identify it's immune activity, which will lay a foundation for the following study to locate the epitope.TB24kDa gene was cloned into vector pQE-31 and expressed as inclusion body in host strain Ml5 after induced by IPTG at 37 C.Western Blotting test proved that the Histidine tag has been attached to the N-terminal of the expressed protein. After preliminarily purified by 8mol/L urea and further purified by Hitrap Chelating HP affinity chromatography, the purity of the target protein reached above 90%. Part of the inclusion body became soluble after refolded by dialysis and fixed refolding by Ni2+ affinity column.In order to prepare antiserum which will be used in following identification of recombinant protein, the natural TB24kDa was extracted , purified and injected into mice to produce special antiserum. The antiserum of a mouse which had special IgE was selected and applied in the following study to identify the immunol activity of recombinant protein. The binding ability to IgE of recombinant protein was founded through the experiment of competitive ELISA.