苦荞TBW16重组表达及靶向结合蛋白的初步分析

苦荞16kD过敏蛋白(Tartary buckwheat 16kD allergen,TBW16)是定位于种子胚中的过敏蛋白,其生物学功能未知。该研究以苦荞种子灌浆期cDNA文库中获得苦荞过敏原TBW16基因的序列为基础,构建TBW16成熟蛋白的原核表达载体pET47b-TBW16,实现了其在大肠杆菌BL21Star(DE3)中的高效表达。结果表明:该过敏原以包涵体的形式表达,经包涵体复性及金属离子螯合层析纯化了目标蛋白;以1,4丁二醇二缩水甘油醚环氧基介导的蛋白偶联技术固定化TBW16于Sepharose CL 6B上,采用亲和层析分离与TBW16靶向结合的蛋白,MALDI-TOF质谱鉴定显示,苦荞TBW16过敏原靶向结合蛋白与细菌膜孔蛋白高度同源,该研究结果为分析苦荞TBW16生物学功能奠定了基础。

Recombinant Expression of TBW16 Allergen in Tartary Buckwheat and Preliminary Analysis of Its Targeting Binding Protein

Tartary buckwheat 16 kD allergen (TBW16) is located in seed embryo and its biological function is still unknown.Based on the TBW16 sequence obtained from the seed-filling period cDNA library of tartary buckwheat,prokaryotic expression vector pET47b-TBW16 was constructed and TBW16 was successfully overexpressed in E.coli BL21 Star(DE3) in form of inclusion bodies.The TBW16 was renatured by dialysis against gradually decreasing urea solution and further purified by cobalt chelating chromatography.TBW16 was then coupled to Sepharose CL 6B activated by 1,4 butyl glycol two glycidyl ether,and the TBW16 interacting protein was obtained by affinity chromatography protocols.Result of MALDI-TOF mass spectrometry showed that the TBW16 interacting protein has high homology to bacteria porin.This result laid the basis for further revealing the biological functions of TBW16 in tartary buckwheat.