Molecular cloning and expression of interleukin Ibeta (IL-1β) from red seabream (Pagrus major)

摘要

The interleukin 1(3 (IL-1β) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known I L-1β sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1β (RS IL-1β) , The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp a nd an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28 6 kD and putative isoelectric point pI of 5 29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the cleat ICE cut site, which were common in other nonmammalian IL-1β genes The RS IL-1β had the highest homology with piscine IL-1β according to phylogenetic tree anal ysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RT-PCR Results showed that the RS IL-1β mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.