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Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum
Dissertation (MSc (Microbiology))--University of Pretoria, 2006.
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University of Pretoria
2013
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| _version_ | 1867613663398985728 |
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| access_status_str | Open Access |
| author2 | Nel, Louis Hendrik |
| author_browse | Nel, Louis Hendrik |
| author_facet | Nel, Louis Hendrik |
| collection | Thesis |
| dc_rights_str_mv | © 2001 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 (Microbiology))--University of Pretoria, 2006. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/28901 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:39:43.676Z |
| 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:2263/28901 Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum Nel, Louis Hendrik upetd@up.ac.za Theron, Jacques Rossouw, Jennifer Livestock botulism vaccines Clostridium botulinum vaccines research UCTD Dissertation (MSc (Microbiology))--University of Pretoria, 2006. The neuroparalytic syndrome called botulism is caused by the neurotoxins produced by bacteria in the genus Clostridium. There are seven toxigenic types of C. botulinum (A to G) based on antigenically distinct toxins produced by different strains of the organism. Animal botulism is caused by C. botulinum type C and D neurotoxins and has a severe economic impact on cattle farming in South Africa and neighbouring countries. Current treatment regimes include the use of acetylcholine for symptomatic treatment, but this is unfortunately very seldom successful. All indications are that there is no cure for this disease and that effective control can only be achieved through development of efficacious vaccines. The botulinum vaccine currently in use in South Africa contains an adjuvanted toxoid form of the type C and o neurotoxins. However, this bivalent vaccine relies on problematic anaerobic cultivation of the Clostridium bacterium followed by isolation, purification and inactivation of the toxin by treatment with formalin. Apart from the fastidious growth requirements of this organism, it has been reported that the production of toxin by these cells declines rapidly and eventually ceases, following laboratory passaging of the bacterial cultures. In addition, improper inactivation of the toxins may also lead to the demise of animals following vaccination. Thus, there exists a great need for a safe, effective and inexpensive vaccine against botulism. To investigate the potential of types C and D botulinum neurotoxins as efficacious recombinant vaccine candidates against botulism, full-length copies of the genes were obtained by polymerase chain reaction (PCR) amplification from bacteriophage DNA isolated from Clostridium botulinum type C (Stockholm) and D (South Africa) cultures. The full-length genes were cloned and subsequently sequenced to verify their integrity. By making use of PCR-based site-directed mutagenesis procedures, three amino acid mutations were introduced in the zinc-binding motif of the respective neurotoxins. Mutation of this domain has previously been reported to successfully detoxify type C neurotoxin. The wild-type and mutant genes were subsequently expressed in insect cells using the BAC-to-BAC™ baculovirus system. Although, unique protein bands corresponding to the size of the neurotoxins could not be seen in Coomassie brilliant blue-stained gels, Western blot analysis indicated immunoreactive material for wild-type and mutant type C corresponding to the size of the type C toxin light chain. However, there was no conclusive evidence to support the successful expression of the full-length wild-type and mutant type D genes. Microbiology and Plant Pathology unrestricted 2013-09-07T14:26:40Z 2006-02-20 2013-09-07T14:26:40Z 2001-04-01 2006-02-20 2006-02-15 Dissertation Rossouw, J 2001, Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/28901 > H997/ag http://hdl.handle.net/2263/28901 http://upetd.up.ac.za/thesis/available/etd-02152006-160530/ © 2001 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 | Livestock botulism vaccines Clostridium botulinum vaccines research UCTD Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title | Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title_full | Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title_fullStr | Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title_full_unstemmed | Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title_short | Molecular cloning and expression of type C and D neurotoxin genes of Clostridium botulinum |
| title_sort | molecular cloning and expression of type c and d neurotoxin genes of clostridium botulinum |
| topic | Livestock botulism vaccines Clostridium botulinum vaccines research UCTD |
| url | http://hdl.handle.net/2263/28901 http://upetd.up.ac.za/thesis/available/etd-02152006-160530/ |