Expression and functional characterization of CK-1, a putative rainbow trout, Onchorynchus mykiss, CC chemokine
Abstract (Summary)Previous expenments by Dixon et al. (1997) succeeded in isolating a cDNA clone from rainbow trout (Onchorynchus mykiSS) head kidney Ieukocytes that encodes an 8 D a protein designated CK-1. This protein had structural similarities to molecules called chemokines identified in rnammals and avians that attract leukocytes to inflammatory sites. It was similar to a certain family called CC chemokines and in particular the C6-P 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 fbnction. We expressed CK-1 protein in prokaryotic expression vectors and purified it to hornogeneity. When this protein was tested using seven different experiments of chernotaxis 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 P2 microglobulin (P2m) was not chemotactic to trout Ieukocytes. Southem 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 reveaIed that CK-1 was an inducible gene like al1 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.
Source Type:Master's Thesis
Date of Publication:01/01/2001