Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the
pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in
cattle, sheep and many other domestic and wild animals. It is considered the most
important Clostridium producing economic losses in livestock. Typically, animals
infected with blackleg die rapidly without any signs of illness. Animals quickly die
within 12 to 48 hours after contracting the disease. Therefore, the control of this
disease is done by commercial vaccines consisting of whole formolized cultures.
Immunity against C. chauvoei is associated with whole cell, including its somatic and
flagellar antigens while in other clostridial diseases, protective immunity is obtained
by the use of vaccines containing toxoids. Moreover, it is essential to obtain new
information about the somatic antigens of C. chauvoei.
Proteomics is the study of the proteome, the protein complement of the genome. The
proteome has been defined as the entire complement of proteins expressed by a cell,
organism, or tissue type, and accordingly, proteomics is the study of this complement
expressed at a given time or under certain environmental conditions. 2-DE with
Immobilized pH Gradients (IPGs) combined with protein identification by Mass
Spectrometry (MS) is currently the workhorse for proteomics. Much of information
about immunogenic component can be derived from proteomics coupled to Western
blotting, namely immunoproteomics.
Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify
candidate immunogenic antigens for development of new vaccines. Analyses were
performed by Western blot and dot blot techniques against the whole cell extract
proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two
different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After
mice immunization studies with experimental vaccines prepared, sera were obtained
for evaluation of the immunoglobulin G antibody level by ELISA. After high level of
antibody response determination, 1-DE, 2-DE and immunoblot studies were
performed for the characterization of immunogenic proteins.
In the study, a total of 460 protein spots could be detected on the 2-DE gels by the
help of Delta2D image analysis software and 30 of them were reacted with polyclonal
antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and
8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots
revealed four different gene products (distinct ORFs). Ornithine decarboxylase,
methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin
protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has
been identified as an immunogenic protein for a pathogenic microbe and in C.
chauvoei for the first time. Methionine adenosyltransferase and ornithine
decarboxylation were identified as immunogenic for C. chauvoei for the first time.
The last defined protein is the flagellin protein FliB(C) which is known to be major
immunogenic protein of C. chauvoei.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12611360/index.pdf |
| Date | 01 December 2009 |
| Creators | Coral, Didem |
| Contributors | Ozcengiz, Gulay |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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