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

作　　者：高波[1] 刘志刚[1] 邢苗[1] 徐宏[2] 罗时文[2] 赖仞[2] 

机构地区：[1]深圳大学生命科学学院,深圳518060 [2]深圳市微生物基因工程重点实验室,深圳518060

出　　处：《昆虫学报》2005年第1期13-17,共5页Acta Entomologica Sinica

基　　金：国家自然科学基金项目 (3 9660 0 73 );广东省科技重点项目 (2 0 0 3A3 0 80 5 0 2 );深圳市科技计划资助项目

摘　　要：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.

关 键 词：美洲大蠊 重组变应原Cr PI 蛋白表达 亲和层析 免疫印迹 

分 类 号：Q966[生物学—昆虫学]