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Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC.
KTH, School of Biotechnology (BIO), Proteomics. (Proteomik)
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2008 (English)In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, 539-549 p.Article in journal (Refereed) Published
Abstract [en]

Variable surface protein Vmm and five Vmm-type proteins from Mycoplasma mycoides subsp. mycoides SC were analysed to determine whether these proteins are expressed in vivo in animals affected by contagious bovine pleuropneumonia (CBPP) and in vitro. Recombinant versions of these proteins were constructed and expressed in Escherichia coli after mutation of the TGA Trp codons to TGG. These proteins were then analysed by dot and Western blotting with sera from CBPP-affected cattle. Furthermore, affinity-purified polyclonal antibodies to the recombinant proteins were used in Western and colony blotting to look for expression of the putative Vmm-type proteins in cultured M. mycoides SC. This study demonstrates that immunoglobulins in CBPP sera recognize all putative Vmm-type proteins tested, indicating that these proteins or their homologues are expressed by mycoplasmas during natural infections. Vmm and one of the putative Vmm-type proteins showed variable expression in vitro.

Place, publisher, year, edition, pages
Reading: Society for General Microbiology , 2008. Vol. 154, 539-549 p.
Keyword [en]
CONTAGIOUS BOVINE PLEUROPNEUMONIA, ALBUMIN-BINDING REGION, SMALL COLONY TYPE, GENETIC-CHARACTERIZATION, AFFINITY-CHROMATOGRAPHY, GEL-ELECTROPHORESIS, SIGNAL PEPTIDES, LIPOPROTEIN, ERADICATION, AFRICA
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11088DOI: 10.1099/mic.0.2007/010694-0ISI: 000253927800020OAI: oai:DiVA.org:kth-11088DiVA: diva2:235612
Note
QC 20100719Available from: 2009-09-17 Created: 2009-09-17 Last updated: 2010-09-14Bibliographically approved
In thesis
1. Protein based approaches to understand and prevent contagious bovine pleuropneumonia
Open this publication in new window or tab >>Protein based approaches to understand and prevent contagious bovine pleuropneumonia
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 77 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:17
Keyword
CBPP, Mycoplasma mycoides subsp. mycoides small colony type, humoral immune responses, recombinant surface proteins, ELISA, subunit vaccine, suspension bead array
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11108 (URN)978-91-7415-417-7 (ISBN)
Public defence
2009-10-16, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
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Note
QC 20100719Available from: 2009-09-25 Created: 2009-09-18 Last updated: 2010-07-19Bibliographically approved

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