Abstract: Objective   To improve the reactivity of Echinococcus granulosus antigen B （EgAgB） by constructing multi-epitope antigens using the gene fragment from 3 subunits， EgAgB1， EgAgB2， and EgAgB4.  Methods   Bioedit， Discovery Studio Visualizer software and I-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 multi-epitope antigen was much stronger than AgB subunit antigens when the positive serum of cystic echinococcosis 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.

Key words: Echinococcus granulosus, Antigen B subunit, Structure prediction, Multi-epitope antigen