【摘要】 【目的】建立广州管圆线虫特异性抗原中间纤维(IF)基因的体外表达系统,研究IF蛋白的结构和功能关系。【方法】将IF基因亚克隆入原核表达载体pET-30a-c(+)并在原核中诱导表达,Westernblot鉴定融合表达产物的免疫特性,用组氨酸(His)亲合层析柱分离纯化表达产物,鉴定表达体系效率。生物信息学分析目的蛋白结构和功能关系。【结果】抗原基因IF在原核中与6个组氨酸进行了融合表达,以可溶性表达为主,并能在该系统中大量、稳定表达,其相对分子质量Mr=48×103,该表达产物具有较好的抗原性,可被大鼠感染血清识别,抗原IF具有典型的中间纤维家族特征,该蛋白具有多个抗原表位存在。【结论】建立了IF的有效原核表达系统,IF抗原蛋白的结构决定了它的属性和功能。更多还原

【Abstract】 Objective To construct the expression system in vitro of specific antigen cDNA,the intermediate filament gene(IF)of Angiostrongylus cantonensis,and study IF protein structure correlating its property and function.Methods The IF cDNA was subcloned into the prokaryotic expressing vector pET-30a-c(+)and expressed in vitro.The immuno-characteristics of expressed products were identified by western blot,the expressed products His-tagged were isolated and purified by affinity chromatography resins and the expression ratio was analyzed.The correlation between structure and function of IF proteins was analyzed by bioinformatics.Results The antigen IF was expressed as recombinant protein fused with 6 histidine,it could be expressed effectively as soluble protein.The molecular mass of antigen IF was Mr=48×103,it had effective antigenicity and could be recognized by infected rat antiserum.IF protein had the characteristics of intermediate filament protein family and several hydrophilic domain as well.Conclusion An effective prokaryotic expression system of antigen IF of Angiostrongylus cantonensis was constructed,the study showed that the structure of IF protein determine its property and function.更多还原