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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。
In order to determine the antigenic determinants of TB2 2, a major allergen of tartary buckwheat, the mechanism of its sensitization was revealed, which laid the foundation for subsequent molecular transformation and breeding improvement. It was necessary to obtain a large amount of purified protein in vitro microbial system. Using pQE 31 as the expression vector and M1 5 as the expression strain, induced by IPTG, the expression product in the form of inclusion bodies was obtained at 37 ° C. Western Blotting detection, the expression of N-terminal band with Histidinetag. After the initial purification with 8mol L urea, the target protein content reached more than 40%. Further HitrapChelatingHP affinity purification, expression of protein purity of more than 90%. The basic method for purification of recombinant TB2 2 was established and the purified inclusion bodies were obtained, which laid the foundation for the preparation of anti-TB2 2 antibody and its antigenic determinants.