重组苦荞麦过敏蛋白TBa的原核表达及其免疫活性鉴定

TBa ［tartary buckwheat allergen］是苦荞麦中的一种主要过敏蛋白.根据长度为585 bp 的TBacDNA序列，以pET-28a为表达载体并选择合适的酶切位点合成上、下游引物，采用基因克隆技术构建重组表达载体pET-28a-TBa.进一步将重组质粒转入大肠杆菌BL21(DE3) 中进行表达.从而获得以包涵体形式存在的TBa目的蛋白.该目的蛋白经Ni ²⁺ -NTA琼脂糖柱亲和纯化及SDS-PAGE分析显示, 纯度达到95% 以上.用透析复性的方法将目的蛋白重折叠，其复性产率可达到约68%.Western印迹证实，目的蛋白N端带有6个组氨酸标签.ELISA检测表明，通过基因重组及表达获得的重组苦荞麦过敏蛋白，与天然苦荞种子中的该蛋白具有相似的免疫学活性，与荞麦过敏病人血清中的IgE有特异性的结合.

Prokaryotic Expression and Immunological Identification of Tartary Buckwheat Allergenic Protein (TBa)

TBa is a major allergen of the tartary buckwheat.The structure gene had been obtained in our laboratory, and 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.coli BL21 (DE3) host cells.The protein product was expressed in inclusion bodies mostly.After purified by Ni~ 2 -NTA agarose affinity chromatography column and analyzed by SDS-PAGE, the purity of the target protein reached above 95%, and renatured by gradient dialysis, about 68% soluble TBa protein was obtained.The six histidine residues were confirmed by Western blotting.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 immunology activity.