美洲大蠊变应原CrPI的表达、纯化与免疫学特性鉴定

Expression, purification and characterization of the American cockroach Cr PI allergen

以阳性噬菌体克隆为模板 ,通过PCR扩增出目的基因片段并克隆入T载体 ,经测序证实为美洲大蠊Periplanetaamericana变应原CrPI后 ,将该基因亚克隆入表达载体pGEX 5X 1。美洲大蠊变应原CrPI在大肠杆菌中得到高效表达 ,但主要以包涵体形式存在于沉淀中。目的蛋白溶于 6mol L盐酸胍并经稀释复性后 ,经GlutathioneSepharoseTM 4B亲和层析 ,纯度达 90 %以上。以蟑螂过敏病人血清进行免疫印迹检测 ,结果显示重组变应原具有良好的IgE结合活性。

Using the Cr PI clone from the λEXcell library as a template, the cDNA fragments were first generated by PCR techniques and then ligated into T vector. After being confirmed by DNA sequencing, the cDNA encoding the American cockroach Cr PI allergen was subcloned into pGEX-5X-1 and expressed as GST-fusion protein in the form of inclusion bodies. After being dissolved in 6 mol/L guanidine hydrochloride and renatured with a simple dilution method, the proteins of target were purified to above 90% purity by affinity chromatography with Glutathione Sepharose 4B. Tested with sera from subjects allergic to cockroach, the recombinant allergen was shown to possess good IgE-binding activity as determined by Western blotting.