[Epitope structure prediction of antigen B of Echinococcus granulosus and construction of multi-epitope recombinant antigen].
OBJECTIVE
To improve the reactivity of Echinococcus granulosus antigen B (EgAgB) multi-epitope antigens using the gene fragment from 3 subunits, EgAgB1, EgAgB2, and EgAgB4.


METHODS
Discovery Studio Visualizer software and I-TASSER on-line server were used to predict protein structure gene sequence of the 3 subunits and their combinations in different way. The epitope or subunit combination higher prediction scores was selected for gene recombination. The target sequence was amplified with primers and by using overlap extension PCR technology. The target sequence was then cloned to pET32a constructing expression plasmid and expressing recombinant proteins. The expressed products were served recombinant antigens after purification. The immuno-response of the recombinant multi-epitope antigens were Western blotting analysis.


RESULTS
Structure prediction showed that all the three subunits EgAgB1, EgAgB4 are in a "Z" word structure. The epitope region is located in the central part of the sequence. For from the three subunits and four reactive epitopes (KK36, RK30, B4-2, and B4-3), 57 different combinations tried for structure prediction. Six of them were selected for recombination and expression. Western blotting six multi-epitope antigens (MEA-8, MEA-20, MEA-26, MEA-36, MEA-49, and MEA-52) suggested reactivity of multi-epitope antigen was much stronger than AgB subunit antigens when the positive echinococcosis were used.


CONCLUSIONS
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 hand reactivity of multi-epitope antigen is much stronger than that of AgB subunit antigens.
Shanghai Municipal Center for Disease Control and Prevention, Shanghai Institutes of Preventive Medicine, Shanghai 200336, China. jlsh_819@163.com
Li
L
Jiang
Yao-Guang
YG
Zhang
Shou-Fu
SF
Jiang
Zheng
Z
Feng
D000818:Animals; D017931:DNA Primers; D048209:Echinococcus granulosus / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D008074:Lipoproteins / Q000235:genetics* / Q000276:immunology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis*
D017931:DNA Primers; D000939:Epitopes; D008074:Lipoproteins; D011994:Recombinant Proteins; C514022:antigen B, Echinococcus granulosus