Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) CC chemokine cDNAs by use of suppression subtractive hybridization

摘要

Suppression subtractive hybridization was carried out by using cDNAs of peripheral white blood cells (PWBCs) of banded dogfish (Triakis scyllia) after phorbol 12-myristate 13-acetate (PMA) stimulation. The Trsc-SCYA107, MIP3α1 and MIP3α2 clones contained an open reading frame encoding 97, 99 and 97 amino acids, respectively. Comparison of the deduced amino acids showed that the banded dogfish MIP3α1 and MIP3α2 sequences shared 42.3% and 40.0% identity with human SCYA20, respectively, while the Trsc-SCYA107 sequence shared 50.6, 44.2 and 42.0% identity with the catshark (Scyliorhinus canicula) Scca-SCYA107, rainbow trout (Oncorhynchus mykiss) CK4A and CK4B, respectively. The genomic sequences of banded dogfish Trsc-SCYA107, MIP3α1 and MIP3α2 contain four exons and three introns, and MIP3α1 and MIP3α2 shared the same intron/exon organization with that of human. The MIP3α1 and MIP3α2 genes of lipopolysaccharide (LPS)-unstimulated banded dogfish were expressed in gill, kidney and liver, while Trsc-SCYA107 mRNA was detected in various tissues except for brain. However, the constitutive expression of MIP3α2 gene was much lower than the Trsc-SCYA107 and MIP3α1 genes. RT-PCR analysis of the Trsc-SCYA107 expression in tissues of LPS-stimulated fish showed enhanced expression at 24 h poststimulation in the gill, heart, leydig, spleen and testes, while the expression of MIP3α1 and MIP3α2 was not influenced by LPS-stimulation in vivo. Furthermore, a relative increase in the expression of the Trsc-SCYA107 and MIP3α2 genes in PWBCs was observed at 1–12 h poststimulation with PMA and LPS, with maximal expression observed at 3 h, while MIP3α1 expression was observed at 3–12 h poststimulation only with PMA.