【摘要】 为了确定苦荞主要过敏原蛋白TB2 2的抗原决定簇 ,揭示其致敏机制 ,为以后的分子改造及育种改良打下基础 ,需要在体外微生物体系中获得大量的纯化蛋白。以pQE 31为表达载体 ,M1 5为表达菌株 ,使用IPTG诱导 ,在 37℃获得了以包涵体形式存在的表达产物。经WesternBlotting检测 ,证明表达条带N端带有Histidinetag。使用 8mol L尿素初步纯化后 ,目的蛋白含量达到 40 %以上。进一步HitrapChelatingHP亲和纯化 ,表达蛋白的纯度达到 90 %以上。建立了适合重组TB2 2纯化的基本方法 ,得到了纯化的包涵体 ,为抗TB2 2抗体的制备及其抗原决定簇的研究奠定了基础。更多还原

【Abstract】 Locating the antigenic determinant(epitope)of allergenic protein TB22 of tartary buckwheat,to elucidate the relative allergenic mechanism and lay the foundation for molecular modification and breeding improvement, it is essential to obtain large amount of purified protein in microbes.The TB22 gene was cloned into vector pQE\|31 and expressed as inclusion body in host strain M15 after induced by IPTG at 37℃.Western Blotting test proved that the Histidine tag has been attached to the N\|terminal of the expressed protein. The target protein accounted for over 40% of total precipitated protein after preliminarily purified by 8mol/L urea and finally reached to above 90% by Hitrap Chelating HP affinity chromatography. An effective purification method of recombinant TB22 was built and the purified inclusion body was obtained also, which laid the foundation for the following preparation of anti\|TB22 antibody and antigenic determinant research.更多还原