Expression and functional characterization of CK-1: A putative rainbow trout (Onchorynchus mykiss) CC chemokine.

Previous experiments by Dixon et al. (1997) succeeded in isolating a cDNA clone from rainbow trout (Onchorynchus mykiss) head kidney leukocytes that encodes an 8 kDa protein designated CK-1. This protein had structural similarities to molecules called chemokines identified in mammals and avians that attract leukocytes to inflammatory sites. It was similar to a certain family called CC chemokines and in particular the C6-beta subfamily because it had 6 cysteine residues. We have set a study to determine whether CK-1 was indeed a rainbow trout chemokine by testing for its function. We expressed CK-1 protein in prokaryotic expression vectors and purified it to homogeneity. When this protein was tested using seven different experiments of chemotaxis assay; which is the most conventional method to assay for chemotactic activity; it was chemotactic to rainbow trout leukocytes and in particular to lymphocytes. Another protein that we expressed under exactly identical conditions of CK-1 production called beta2 microglobulin (beta2m) was not chemotactic to trout leukocytes. Southern blot analysis of rainbow trout genomic DNA with CK-1 genomic fragments indicated that CK-1 was a single copy gene within rainbow trout genome. Tissue distribution and expression pattern of CK-1 transcript investigation revealed that CK-1 was an inducible gene like all other known chemokine genes. When rainbow trout was stimulated with a mitogen for 24 hours, CK-1 message was detected in the blood leukocytes, liver, head kidney and spleen upon northern blotting. A one hour stimulation failed to induce CK-1 transcription, however. CK-1 protein is the first teleost chemokine whose activity has been verified. The use of this chemokine could be helpful in aquaculture if used as a vaccine adjuvant against common fish pathogens.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis2001 .A43. Source: Masters Abstracts International, Volume: 40-03, page: 0704. Adviser: Brian Dixon. Thesis (M.Sc.)--University of Windsor (Canada), 2001