Acquired Immunodeficiency Syndromes (AIDS), a disease caused by the infection of human immunodeficiency virus (HIV), is still incurable to date. Various types of anti-viral drugs have been developed and most of these drugs are targeted on HIV reverse transcriptase and protease. Highly active antiretroviral therapy (HAART) has been used on AIDS treatment recently. However, new drugs are required to delay the resistance onset and to maximize the effectiveness of combination therapy by inhibiting a variety of targets simultaneously. / In the second part, the possibility of using the vector-based approach of RNA interference (RNAi) to reduce the expression of HIV-1 integrase and HIV replication in mammalian cells was examined. RNAi suppressed protein synthesis through the induction of sequence-specific gene silencing of 21-25 nucleotides (nt) double stranded RNA fragments, termed small interfering RNA (siRNA). pSilencer series vectors with different promoters (p Silencer 1.0-U6, pSilencer 2.0-U6, pSilencer 3.0-H1) were used on shRNA expression inside HeLa cells. Four different hairpin constructs containing the 19-nt corresponding to the nucleotide sequence of HIV integrase at positions 19-27, 79-96, 158-176 and 495-513 were generated for RNAi study. (Abstract shortened by UMI.) / Integrase is one of the important enzymes on HIV infection. It acts by integrating viral RNA to host DNA and this is one of the ideal targets for therapeutic intervention. Previous results in our laboratory demonstrated that luffin, a type-I ribosome inactivating protein (RIP), had high potency on integrase inhibition. In the first part of this thesis, alpha-luffin cDNA was cloned from the seed of Luffa cylindrica. Three different sets of expression vectors were used to produce recombinant luffin. Different deletion mutants of luffin were also generated for structural analysis on integrase inhibition. Recombinant alpha-luffin and its various deletion mutants were expressed exclusively in the form of inclusion bodies despite different expression conditions had been attempted. Various refolding strategies and conditions were carried out but the problem of insolubility was consistently found after removal of the denaturing reagents. The problem of insolubility was improved by using the maltose binding protein (MBP) luffin fusion construct. However, there is evidence that this soluble MBP-luffin formed a multimeric fusion protein complex rather than monomer and removal of MBP tag resulted in the precipitation of luffin. / Lau Tat San. / "August 2005." / Adviser: C. C. Wan. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 202-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343669 |
| Date | January 2005 |
| Contributors | Lau, Tat San., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (vi, 225 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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