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United States Department of Agriculture

Agricultural Research Service

Research Project: Integrated Aquatic Animal Health Strategies

Location: Aquatic Animal Health Research

Title: Chemotactic activity of channel catfish, Ictalurus punctatus (Rafinesque), recombinant Cyclophilin A

Authors
item Yeh, Hung-Yueh
item Shoemaker, Craig
item Klesius, Phillip

Submitted to: Journal of Fish Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 16, 2013
Publication Date: November 6, 2013
Repository URL: http://handle.nal.usda.gov/10113/58450
Citation: Yeh, H., Shoemaker, C.A., Klesius, P.H. 2013. Chemotactic activity of channel catfish, Ictalurus punctatus (Rafinesque), recombinant Cyclophilin A. Journal of Fish Diseases. 36:1041-1046.

Interpretive Summary: Cyclophilin A (CyPA) is an enzyme, which is found in the cytosol of vertebrates and invertebrates. This protein is highly conserved among living organisms and plays many important roles in physiological and pathological processes. Recently, several economic fish CyPA have been cloned and characterized. However, the 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 fish immunity, we first produced and purified the channel catfish CyPA protein from an Escherichia coli expression system. The recombinant CyPA protein (rCyPA) was highly expressed as a major protein band in SDS-PAGE gels. This protein was further confirmed by mass spectrometry and sequencing analysis. The rCyPA contained a six-His tag and was purified by affinity chromatography. The protein was purified almost to homogeneity judged by SDS-PAGE analysis. The rCyPA was able to induce movement of peritoneal macrophages from channel catfish in vitro, suggesting that CyPA is involved in fish immunity. It is anticipated that this rCyPA will be very useful for future development of antibodies to investigate this protein in the fish immune and pathogenic processes. This study is related to the goal of ARS National Program 106-Aquaculture, Component 4C-Prevention of Diseases.

Technical 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 punctatus (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.

Last Modified: 11/22/2014
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