Prediction of B-Cell Epitopes and Overexpression of Truncated gl of Duck Enteritis Virus

摘要

At present, little has been known about the immunogenicity of DEV gI protein. In this study we predicted the B-cell epitopes of DEV gI protein by bioinformatics tools and expressed the truncated gI in prokaryotic expression system. The location of B-cell epitopes was predicted by screening the secondary structure of DEV gI protein, then analysis was on the basis of the prediction results of hydrophilicity, flexibility, accessibility, antigenicity followed by a verification using ABCpred program. The predicted B-cell epitopes were found to principally locate at the protein sequence of amino acids 217-233,198-208,258-270 and 181-194 for gl. We amplified the truncated gl gene(88-813bp, named gl M) which encoded the major antigen sites by PCR, then subcloned into clone vector pMD18-T, finally cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)/gI M. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)/gI M was transformed into E.coli BL21(DE3) competent cells for overexpression. Truncated gl gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). SDS-PAGE showed that the recombinant protein 6×His-tagged gI M molecular weight was about 47 kDa, consistent with the calculation. Subsequently, the purified protein was indentified by rabbit anti-DEV IgG in Western blot analysis, showed good immunogenicity. In conclusion, these findings would be helpful for estimate of the epitopes sites localized within DEV gI, and also provide foundation for the development of DEV subunit vaccine or diagnostic reagent.