Proceedings of the 10th International Colloquium on Paratuberculosis

#118 Cloning, expression and purification <strong>of</strong> Mycobacterium avium subsp. paratuberculosis
stress-associated genes
Satoko Kawaji, Sanjeev Gumber, Richard Whittington, Faculty <strong>of</strong> Veterinary Science, University <strong>of</strong> Sydney, Australia
It is believed that Mycobacterium avium subsp. paratuberculosis (MAP) can survive in an unfavourable environment
for a long time in a dormant state. In a previous study, we developed an in vitro model <strong>of</strong> stressed/
dormant MAP under three different environmental stresses <strong>of</strong> temperature flux, nutrient starvation and oxygen
limitation, and identified proteins expressed under <strong>the</strong>se conditions by proteomics analysis. It was hypo<strong>the</strong>sised
that <strong>the</strong>se stress response proteins might also be expressed in vivo, and could be useful targets for early
diagnosis <strong>of</strong> JD. In this study, 28 MAP stress-associated proteins were cloned, expressed and purified, and <strong>the</strong>
immunogenicity <strong>of</strong> <strong>the</strong>se recombinant proteins was evaluated by ELISA.
Twenty eight MAP genes previously identified as differentially regulated under conditions <strong>of</strong> physiological
stress were randomly selected for cloning and expression. 15 <strong>of</strong> 28 were cloned using Invitrogen
Gateway ® Technology, while <strong>the</strong> o<strong>the</strong>r 13 clones were obtained using a traditional restriction enzymebased
cloning method. His-tagged proteins were expressed in BL21 Escherichia coli and purified using
affinity chromatography. Soluble proteins confirmed by Western-blotting analysis were purified under native
conditions, while insoluble proteins were solubilised with denaturing buffer and <strong>the</strong>n purified.
The immunogenicity <strong>of</strong> purified recombinant proteins was evaluated for detection <strong>of</strong> MAP infection by
ELISA using serum samples from 50 infected and 50 uninfected sheep. Some antigens showed significantly
higher OD values in <strong>the</strong> infected group compared to <strong>the</strong> uninfected group. When an arbitrary cut <strong>of</strong>f line was
set, <strong>the</strong>se antigens had similar sensitivity to a commercial Pourquier ELISA, but detected more infected sheep
that had no detectable histological lesions and fewer that had multibacillary lesions. The results suggest that
<strong>the</strong>se recombinant MAP stress-associated proteins might be expressed in MAP infected sheep at an early
stage <strong>of</strong> infection. This is consistent with <strong>the</strong> hypo<strong>the</strong>sis <strong>of</strong> dormancy.
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