Characterization of flavivirus recombinant proteins

Fatimah, binti Elie (2012) Characterization of flavivirus recombinant proteins. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

The envelope protein of the flavivirus consists of three distinct domains named domain I, II, and ID. Secondary structure of envelope protein shows that this protein is folded in such a way that makes the domain III protein separate from domains I and II. This has made domain III protein possible to be cloned on its own because of continuity in gene sequence compared to domains I and II which overlaps with each other. Besides that, function of domain III as a receptor binding domain and its ability to elicit neutralizing antibodies when challenged has made this protein interesting to study. Here, we cloned and expressed the domain III of eight different flaviviruses which are of DENVI-4, WNV, lEV, KUNV and MVEV. The domain III fragment was cloned into pET-SUMO cloning vector and the expression was done in a bacterial expression system) All the domain III proteins were expressed as a fusion protein to histidine-tag protein. The expression of domain III was confirmed by probing a western blot with Ni-HRP which detects the presence of his-tag in the recombinant protein. The reactivity test done on these recombinant domain III proteins had shown that they were reactive when probed with high positive pooled dengue reference sera (HPR). These domain III proteins were then purified using nickel affinity chromatography before characterization work were performed. The purified products were tested in indirect IgO ELISA and the results were compared to the OACE which uses native antigens. The result shows that the sensitivity of domain III based assay is only 55.28%, however the specificity is 91.70%. The domain III proteins were also tested in latex beads agglutination assay, however results were worse in terms of sensitivity and specificity. The results obtained suggest that domain III is not a good candidate for use in diagnostic assay in place of authentic antigen. However, due to the simpler work involve in constructing the recombinant protein, its still can be used for other various functional studies. Interestingly, neutra1ization test using domain III positive pooled serum shows nearly 10 fold higher neutralizing antibodies titer compared to domain III negative pooled, indicating that this domain might be a good candidate in developing an antiviral agent or vaccine for preventing the infections by flavivirus.

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