Identification of protective epitopes against dengue virus using single chain fragment of variable region technique

• Main Authors: Cian-DaiCiou, 邱千玳
• Other Authors: Trai-Ming Yeh
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/50399956880185742717

碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 101 === Dengue virus (DENV) is mainly found in tropical and subtropical regions, and divided into four serotypes. Dengue virus infection causes mild dengue fever and potentially fatal dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS), which is transmitted by the mosquito Aedes aegypti. Currently, there are no approved vaccines or antiviral therapies against four serotypes of DENV infections, and only fluid replacement is available for clinical treatment. Antibody-dependent enhancement (ADE) has been proposed as a mechanism for increased disease severity during secondary DENV infection. According to ADE, antibodies induced in previous infection can enhance DENV infection in monocytes/macrophages through Fc receptor. To develop vaccine candidates against DENV, we immunized BALB/c mice with different DENV proteins, such as PEG-precipitated-DENV particle, DENV RNA, recombinant DENV envelope protein domain III or recombinant DENV NS1, and compared the neutralizing ability of immune serum. Sera from PEG-precipitated-DENV particle immunized mice showed the best neutralizing ability against DENV infection than sera from other DENV proteins-immunized mice. To avoid ADE effect, we used phage display to generate single chain fragment of variable region (scFv) from PEG-precipitated DENV-immunized mice to further study their protective effects against DENV. After five times of panning, anti-DENV scFv phages were selected by ELISA to test their binding ability against DENV. In addition, anti-DENV scFv proteins were expressed from E.coli strain HB2151 and purified by nickel column. Some of them showed significantly neutralizing abilities against DENV infection by flow cytometry. Three potential DENV neutralizing scFv proteins 25, 37 and 39 were selected. The epitope of scFv recognized were identified by phage display random peptide library. We found that dengue patients’ sera can recognize the epitope of scFv 25. Furthur more, we immunized BALB/c mice with peptide of scFv 25 epitope.The results showed that mice sera can recogniz DENV as well as the epitope of scFv 25. Besides, immunized mice sera significantly inhibited DENV replication. Our data suggested that anti-DENV scFv 25 could be used as an anti-viral drug or diagnostic tool.