Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella
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Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.
KeywordsCoccidiosis Sporulated Oocyst Serpin Gene Reactive Center Loop Invasion Inhibition
This work was supported by the Shanghai Science Foundation of China (Grant No. 09ZR1438700). We would like to thank Prof. Chan Ding (CAAS) for generously providing the DF-1 cell line used in this study. We also thank Zhixin Zhao and Beimin Zhang for their excellent technical assistance.
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