Abstract
Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.
Highlights
Riemerella anatipestifer (R. anatipestifer) is a Gram-negative, nonmotile, non-spore-forming, rod-shaped bacterium, belongs to the Flavobacteriaceae family in rRNA superfamily V [1]
Mass spectroscopic identification and analysis of proteins Based on the higher ratio ($2.5) of spot density and molecular weight (MW) range of 17–72 Kd, 12 (Fe01, Fe02, Fe05, Fe08, Fe09, Fe10, Fe17, Fe18, Fe19, Fe21, Fe22 and Fe23) of 23 above proteins from the iron restriction medium were selected for mass spectroscopic analysis
Twelve of 23 proteins were analyzed by mass spectrography
Summary
Riemerella anatipestifer (R. anatipestifer) is a Gram-negative, nonmotile, non-spore-forming, rod-shaped bacterium, belongs to the Flavobacteriaceae family in rRNA superfamily V [1]. Riemerellosis can cause significant economic losses in duck farmers [2,4], especially in China and Southeastern Asia [5,6,7]. In some Gram-positive, siderophore is one of these gene products produced in the environment of iron deficiency and forms the strongest soluble ferric-siderophore complexes that can be taken up by active transport mechanisms [10]. Because of this property, siderophores have attracted high attentions in the prevention and treatments of bacterial infectious diseases [10,12]
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