Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis

Proteome Science

Wojtek P Michalski²  Gary Beddome²  Jill A Vaughan²  Michelle L Colgrave¹  Brian J Shiell²  James W Wynne² 

[1] Queensland Bioscience Precinct, CSIRO Livestock Industries, St Lucia, Queensland 4067, Australia;Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, Victoria 3219, Australia

关键词: Mass spectrometry;    Diagnostic antigen;    Interferon gamma;    PPDj;    Johnin PPD;    Johne's disease;   

Others  :  817410
DOI  :  10.1186/1477-5956-10-22

 received in 2011-11-25, accepted in 2012-03-26,  发布年份 2012

【 摘 要 】

Background

Effective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production.

Results

Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa) and Mycobacterium bovis (PPDb). Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition.

Conclusions

This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.

【 授权许可】

   
2012 Wynne et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 2.

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Figure 4.

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