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Chemotactic activity of channel catfish, Ictalurus punctatus (Rafinesque), recombinant cyclophilin A

Author:
Yeh, H. Y., Shoemaker, C. A., Klesius, P. H.
Source:
Journal of fish diseases 2013 v.36 pp. 1041
ISSN:
0140-7775
Subject:
Escherichia coli, Ictalurus punctatus, affinity chromatography, chemotaxis, cyclophilins, fish, immunity, macrophages, mass spectrometry, peptide mapping, polyacrylamide gel electrophoresis, polyclonal antibodies
Abstract:
Cyclophilin A (CyPA), a member of the highly conserved immunophilin superfamily, is important to physiological and pathological processes, including immune cell signaling. Recently, several teleost fish CyPA have been cloned and characterized. However, CyPA function in fish immunity has not been fully determined. In order to provide a framework for post-genomic studies on the role(s) of CyPA in teleost fish immunity, we first expressed and purified the channel catfish, Ictalurus puntatus (Rafinesque), CyPA protein from an Escherichia coli expression system. The recombinant CyPA protein, analyzed by SDS-PAGE , was highly expressed as a major protein band. This protein was further confirmed by mass spectrometric peptide mapping and sequencing analysis. The recombinant CyPA (rCyPA) contained a six-His tag and was purified by nickel-iminodiacetic acid affinity chromatography. SDS-PAGE analysis indicated the protein was purified almost to homogeneity. The rCyPA protein was able to induce migration of peritoneal macrophages from channel catfish in vitro, suggesting that CyPA is involved in fish innate immunity. It is anticipated that this rCyPA will be very useful for future development of monoclonal and polyclonal antibodies to investigate this protein in the teleost immune and pathogenic processes.
Agid:
58450
Handle:
10113/58450