Characterization of flavivirus recombinant proteins
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...
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| Format: | Thesis |
| Language: | English |
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Universiti Malaysia Sarawak (UNIMAS)
2012
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| Online Access: | http://ir.unimas.my/id/eprint/14355/ http://ir.unimas.my/id/eprint/14355/4/Fatimah%20%20ft.pdf |
| _version_ | 1848837630669619200 |
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| author | Fatimah, binti Elie |
| author_facet | Fatimah, binti Elie |
| author_sort | Fatimah, binti Elie |
| building | UNIMAS Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-15T06:42:43Z |
| format | Thesis |
| id | unimas-14355 |
| institution | Universiti Malaysia Sarawak |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T06:42:43Z |
| publishDate | 2012 |
| publisher | Universiti Malaysia Sarawak (UNIMAS) |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | unimas-143552025-05-05T03:58:39Z http://ir.unimas.my/id/eprint/14355/ Characterization of flavivirus recombinant proteins Fatimah, binti Elie Q Science (General) 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. Universiti Malaysia Sarawak (UNIMAS) 2012 Thesis NonPeerReviewed text en http://ir.unimas.my/id/eprint/14355/4/Fatimah%20%20ft.pdf Fatimah, binti Elie (2012) Characterization of flavivirus recombinant proteins. Masters thesis, Universiti Malaysia Sarawak. |
| spellingShingle | Q Science (General) Fatimah, binti Elie Characterization of flavivirus recombinant proteins |
| title | Characterization of flavivirus recombinant proteins |
| title_full | Characterization of flavivirus recombinant proteins |
| title_fullStr | Characterization of flavivirus recombinant proteins |
| title_full_unstemmed | Characterization of flavivirus recombinant proteins |
| title_short | Characterization of flavivirus recombinant proteins |
| title_sort | characterization of flavivirus recombinant proteins |
| topic | Q Science (General) |
| url | http://ir.unimas.my/id/eprint/14355/ http://ir.unimas.my/id/eprint/14355/4/Fatimah%20%20ft.pdf |