细粒棘球绦虫铜锌超氧化物歧化酶基因的表达与特征分析

Prokaryotic Expression and Characterization of Cu/Zn Superoxide Dismutase of Echinococcus granulosus

旨在探讨Eg-SOD基因的特征及其在细粒棘球绦虫上的抗氧化作用。通过原核表达得到重组Eg-SOD,对其进行酶活性测定、免疫印迹、ELISA以及免疫荧光定位分析。经原核表达出的重组Eg-SOD蛋白为可溶性蛋白,以羟胺法测定其酶学活性,可达(464.55±19.99)U·mg-1。免疫印迹结果显示,该蛋白质能够被实验室人工感染细粒棘球蚴的小鼠阳性血清所识别,具有较强的反应原性;ELISA分析显示,重组Eg-SOD对人工感染细粒棘球蚴的小鼠血清和自然感染包虫病的绵羊血清检出率均为100%,而对细颈囊尾蚴的交叉反应为37.5%,提示该蛋白质可以作为潜在的诊断候选分子。免疫荧光定位显示该蛋白质广泛分布于成虫和原头蚴的组织间隙,以及少量分布于表皮。本研究对Eg-SOD的特点进行了初步的探讨,并揭示该蛋白质与虫体的抗氧化损伤有着密切的联系。

The aim of this study was to characterise the Cu/Zn superoxide dismutase of Echinococcus granulosus（Eg-SOD）,and analyze its role in the parasite antioxidant system.The recombinant Eg-SOD was expressed in E.coli,and the enzymatic activity was measured,and analyses including western blotting,ELISA and immunofluorescence localization were also performed.The recombinant Eg-SOD was expressed as soluble protein,and its activity was up to（464.55 ±19.99）U·mg-1 measured by hydroxylamine method.As showed by the result of western blot,the recombinant Eg-SOD could be recognized by the serum of mice secondary experimentally infected with E.granulosus protoscolexes,and showed a high immunogenicity;ELISA tests showed that recombinant Eg-SOD could recognize all the sera from mice experimentally infected with protoscolexs and from sheep naturally infected with E.granulosus,and have 37.5% cross-reactions with sera from sheep infected with Cysticercus tenuicollis,which indicates Eg-SOD might be a potential diagnose antigen.The immunolocalization of Eg-SOD showed a wide extent of distribution,mostly in the tissue clearance of adult worms and protoscolexes,and fewer on the tegument.These results provide the fundamental understanding of Eg-SOD,and revealed its capable of antioxidation effect.