Domain Ⅲ of Dengue Virus Serotype 2 Envelope: Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry
Abstract Obejective The domain Ⅲ of dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the recombinant protein inhibited virus effect was tested. Methods In this study, the domain Ⅲ (DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was purified using enzymatic cleavage and affinity purification. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢ protein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a specific soluble 29 kD protein was obtained, and the expression product accounted for 68.87% of the total protein of the cell lysate. Western blotting demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was purified using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢ protein. At a 1:16 dilution, the antibodies produced at least 90% neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢ antibody titer reached 1:1 024. Conclusions DENV-2 EDⅢ was cloned and expressed successfully. DENV-2 EDⅢ protein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.