| Summary: | 碩士 === 國立臺灣大學 === 病理學研究所 === 91 === Epitope mapping is important for understanding the pathogenesis of viral infectious diseases as well as for developing vaccines and diagnostic reagents. Here we used phage display, a selection technique for protein-protein interaction, to find out and detect dengue virus (DEN) serotype-specific B-cell epitopes. 4G2 monoclonal antibody which reacted with E-protein of all 4 kinds of dengue serotypes was used to purify whole virus from culture supernatant. The virues were then used as antigens to immunize mice (BALB/c) to produce dengue hyperimmunized serum. We used dengue immunized serum as targets to select the immunopositive phage clones from random phage peptide library. Then we used these phage derived peptide motifs to identify dengue specific epitope, and synthesize peptides mimic dengue epitope as a detection agent. At first we found out two consensus motifs on DEN-2, TPQS and DxExRx.
When these two motifs were compared with four dengue serotypes, TPQS motif will react with all four dengue serotypes, and all phage clones belong to TPQS motif group had a structure aSxRM (a=Y/F) which can not pile up to dengue genome. To understand the function of this motif, we had designed new peptide P33M (AMYSNRMA). ELISA assay showed low affinity of P33M with all four dengue serotypes, and high titer anti-P33M sera could be collected when immunized mice with P33M. P33M will be examined in the future.
The motif DxExRx had been shown to mimic the sequence DLEKRH, corresponding to amino acid residues 341-346 on E-protein and motif TPQS corresponding to amino acid residues 165~168 on E-protein. E-protein is the major virion surface protein and the most important antigen for the virus. To find out the B-cell epitope according to the E-protein and further develop epitope-based antigen for serologic test reagents will be helpful in both vaccine development and prevention of dengue viral disease.
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