摘要
目的对细粒棘球蚴抗原B(EgAgB)的3个亚单位(EgAgB1、EgAgB2和EgAgB4)的反应性表位的结构进行预测分析并进行基因重组,鉴定构建的多表位重组抗原的反应性。方法用Bioedit和Discovery Studio Visualizer分析软件和I-TASSER在线服务器对EgAgB1、EgAgB2和EgAgB4等3个亚单位抗原序列及其不同组合方式的组合序列进行分析和结构预测。选择结构预测评分较高的表位或亚单位组合方式进行基因重组。设计特异性重叠引物,用重叠延伸PCR技术扩增目标序列。将目标序列克隆至pET32a(+)载体中构建表达质粒表达重组蛋白,表达产物经纯化后即为多表位重组抗原。采用蛋白质印迹(Western blotting)分析鉴定多表位重组抗原的反应性。结果结构预测显示,EgAgB1、EgAgB2和EgAgB4等3个亚单位的主要表位均呈"Z"字型结构,且主要表位区域亦均位于序列的中部;对拟进行组合的3个EgAgB亚单位和4个主要表位(KK36、RK30、B4-2和B4-3)的57种不同的组合方式进行了结构预测,选择6种组合方式进行重组表达。对6个多表位重组抗原(MEA-8、MEA-20、MEA-26、MEA-36、MEA-49和MEA-52)进行的Western blotting分析显示,多表位重组抗原与细粒棘球蚴病患者血清的反应条带明显强于AgB亚单位抗原。结论构建的6个多表位重组抗原与细粒棘球蚴病患者血清的反应性明显强于EgAgB亚单位抗原。
Objective To improve the reactivity of Echinococcus granulosus antigen B （EgAgB） by constructing muhi-epitope antigens using the gene fragment from 3 subunits, EgAgB1, EgAgB2, and EgAgB4. Methods Bioedit, Discovery Studio Visualizer software and 1-TASSER on-line server were used to predict protein structure and analyze the gene sequence of the 3 subunits and their combinations in different way. The epitope or subunit combination which had a higher prediction scores was selected for gene recombination. The target sequence was amplified with specific overlap primers and by using overlap extension PCR technology. The target sequence was then cloned to pET32a （＋） vector for constructing expression plasmid and expressing recombinant proteins. The expressed products were served as multi-epitope recombinant antigens after purification. The immuno-response of the recombinant multi-epitope antigens were explored by Western blotting analysis. Results Structure prediction showed that all the three subunits EgAgB1, EgAgB2 and EgAgB4 are in a ＂Z＂ word structure. The epitope region is located in the central part of the sequence. For combinations from the three subunits and four reactive epitopes （KK36, RK30, B4-2, and B4-3）, 57 different combinations were tried for structure prediction. Six of them were selected for recombination and expression. Western blotting analysis on the six multi-epitope antigens （MEA-8, MEA-20, MEA-26, MEA-36, MEA--49, and MEA-52） suggested that the band reactivity of muhi-epitope antigen was much stronger than AgB subunit antigens when the positive serum of cysticechinococcosis were used. Conclusion By using protein tertiary structural prediction and screening the higher prediction score of combinations, six multi-epitope recombinant antigens were constructed. Western blotting shows that the band reactivity of multi-epitope antigen is much stronger than that of AgB subunit antigens.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2012年第4期279-283,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.30872211)~~
关键词
细粒棘球绦虫
抗原B亚单位
结构预测
多表位抗原
Echinococcus granulosus
Antigen B subunit
Structure prediction
Multi-epitope antigen
