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Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics
Dissertation (MSc)--University of Pretoria, 2012.
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University of Pretoria
2013
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| _version_ | 1867613448911716352 |
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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:2263/23688 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:36:19.085Z |
| 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:2263/23688 Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics Beukes, Mervyn Jhn.Scho@gmail.com Schoombie, Johannes Loubser Single chain variable fragments Transversion Transition Tuberculosis (TB) Mycolic acids Error prone polymerase chain reaction Protein engineering UCTD Dissertation (MSc)--University of Pretoria, 2012. Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs. Biochemistry unrestricted 2013-09-06T15:44:29Z 2013-04-24 2013-09-06T15:44:29Z 2013-01-17 2012 2013-01-17 Dissertation Schoombie, JL 2012, Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/23688 > C13/4/65/gm http://hdl.handle.net/2263/23688 http://upetd.up.ac.za/thesis/available/etd-01172013-111741/ © 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 | Single chain variable fragments Transversion Transition Tuberculosis (TB) Mycolic acids Error prone polymerase chain reaction Protein engineering UCTD Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title | Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title_full | Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title_fullStr | Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title_full_unstemmed | Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title_short | Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics |
| title_sort | genetic engineering of recombinant anti mycolic acid antibody fragments for use in tuberculosis diagnostics |
| topic | Single chain variable fragments Transversion Transition Tuberculosis (TB) Mycolic acids Error prone polymerase chain reaction Protein engineering UCTD |
| url | http://hdl.handle.net/2263/23688 http://upetd.up.ac.za/thesis/available/etd-01172013-111741/ |