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Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1
Dissertation (MSc)--University of Pretoria, 2012.
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University of Pretoria
2013
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| _version_ | 1867613602571091968 |
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| access_status_str | Open Access |
| author2 | Beukes, Mervyn |
| author_browse | Beukes, Mervyn |
| author_facet | Beukes, Mervyn |
| collection | Thesis |
| dc_rights_str_mv | © 2012, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria |
| description | Dissertation (MSc)--University of Pretoria, 2012. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/29320 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:38:45.051Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/29320 Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 Beukes, Mervyn jiyageorge@yahoo.com George, Jiya Marian Human immunodeficiency virus (HIV) Molecules UCTD Dissertation (MSc)--University of Pretoria, 2012. Human immuno deficiency virus (HIV) incorporates host cellular protein, beta-2-microglobulin (β2m), into its surface envelope during budding. β2m is a cellular protein that belongs to the major histocompatibility complex (MHC) Class I molecules. Studies have shown anti- β2m monoclonal antibodies (mAbs) has the ability of to neutralize the virus. An epitope consisting of seven amino acids of the β2m protein designated as R7V produces antibodies that protect HIV infected people from progressing to AIDS. These protective antibodies, called anti-R7V antibodies, were able to neutralize different HIV isolates, despite their genomic variations, various cellular targets and geographic origin. Anti-R7V antibodies in the format of single chain variable fragments (scFvs) were produced in our laboratory using the M13 phage display technology. These scFv antibody fragments were used during in vitro studies for the detection and neutralization of the R7V antigen by enzyme linked immune sorbent assay (ELISA). The scFv fragments produced against the R7V epitope showed interaction, however the antibody-antigen affinity was too weak for the virus neutralization assay. Hence, this project focused on the affinity maturation of the anti-R7V scFv fragments through random mutagenesis using the error prone (EP) PCR method. The EP PCR method generated two mutated anti-R7V scFvs. The mutated clones were subcloned into the pAK400 expression vector. The computer-based models, created using the Swiss PDB Deep Viewer 4.02 software, were used to predict the antigen-binding site and affinity analysis of both parent and mutated scFv’s. Mutated clone 1 failed to bind to the R7V epitope whereas mutated clone 2 had similar binding pattern as the parent scFv. Mutated clone 2 was predicted to have a higher binding affinity compared to the parent scFv. The results obtained demonstrate the efficacy of EP PCR to generate high affinity antibodies. Future experiments using high affinity anti-R7V scFv’s may lead to its potential use in diagnostics, therapeutics or vaccine development. Copyright Biochemistry unrestricted 2013-09-07T15:25:08Z 2012-11-09 2013-09-07T15:25:08Z 2012-09-07 2012-11-09 2012-11-08 Dissertation George, JM 2012, Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/29320 > E12/9/78/gm http://hdl.handle.net/2263/29320 http://upetd.up.ac.za/thesis/available/etd-11082012-112904/ © 2012, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria application/pdf University of Pretoria |
| spellingShingle | Human immunodeficiency virus (HIV) Molecules UCTD Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title | Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title_full | Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title_fullStr | Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title_full_unstemmed | Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title_short | Generation and analysis of mutated clonal scFv antibody fragments against R7V epitope of HIV-1 |
| title_sort | generation and analysis of mutated clonal scfv antibody fragments against r7v epitope of hiv 1 |
| topic | Human immunodeficiency virus (HIV) Molecules UCTD |
| url | http://hdl.handle.net/2263/29320 http://upetd.up.ac.za/thesis/available/etd-11082012-112904/ |