Molecular characterization and expression analysis of MHC class II alpha and beta genes in large yellow croaker...

MHC class II molecules play an important role in the activation of CD4(+) T cells, which are the central orchestrating cells of an immune response. Here, we report the cloning of MHC class II alpha and beta cDNAs from large yellow croaker (Pscr-DAAs and Pscr-DAB) by expressed sequence tags analysis and RACE-PCR techniques. Three different class II alpha and two class II beta sequences were obtained from spleens of two individual fish. Each of the three class II alpha sequences encodes a polypeptide of 239 amino acids while the two class II beta cDNA sequences encode for a protein of 249 aa. All the characteristic features of MHC class II chain structure could be identified in the deduced proteins of three class II alpha and two class II beta sequences, including the leader peptide, alpha1/beta1 and alpha2/beta2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. RT-PCR analysis showed that MHC class II alpha and beta mRNAs were broadly expressed in various tissues examined, although at different levels. Upon stimulation with inactivated trivalent bacterial vaccine or polyinosinic polycytidylic acid (poly(I:C)), the expression levels of both alpha and beta genes were obviously up-regulated in intestine, kidney and spleen. Real-time PCR analysis demonstrated that the expression levels of class II alpha and beta were quickly up-regulated in spleen, kidney, and intestine at 12 h after induction with poly(I:C), while their expression levels significantly increased at 48 h upon immunization with bacterial vaccine, indicating that the up-regulation of both class II alpha and beta expression was induced by bacterial vaccine or poly(I:C) at the early phase of induction, and that class II alpha and beta transcripts were quicker up-regulated by poly I:C than by bacterial vaccine.