大菱鲆(Scophthalmus maximus)MHCⅡA基因全长cDNA的克隆与组织表达分析

FULL LENGTH cDNA CLONING AND TISSUE EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX(MHC)ⅡA FROM TURBOT SCOPHTHALMUS MAXIMUS

采用同源克隆和末端快速扩增(RACE)方法,得到1131bp的大菱鲆MHCⅡA全长cDNA片段。该序列包括720bp的开放阅读框(0RF),75bp的5′末端非编码区(UTR)以及336bp的3′末端非编码区。利用CLASTAL W1.8软件进行MHCⅡA基因的氨基酸序列比对和同源性比较,结果表明,大菱鲆MHCⅡA与牙鲆MHCⅡA的相似性最高,为69.8%,与真鲷、鲈鱼和丽鱼的相似性次之,分别为65.5%、69.6%和61.4%,与人、鼠和鲨鱼MHCⅡA的相似性仅为19.8%、31.6%和26.9%。应用RT-PCR分析其组织表达发现MHCⅡA基因在正常大菱鲆组织中均表达,但表达量在各个组织中不同,其中在鳃、脾、头肾、心脏、小肠和皮肤中有较强的转录本,中等程度表达于肝和血,在性腺和肌肉中表达最弱。

The full-length cDNA encoding turbot MHC class Ⅱ A was cloned from turbot using homology cloning and RACE PCR approach. The MHC class Ⅱ A was 1131bp in length, including 75bp 5＇ terminal UTR, 720bp encoding region and 336bp 3＇ terminal UTR with a AU motif （ATTTA） that associated with mRNA instability and three canonical polyadenylation signals （AATAAA）, followed by an additional 26bp poly （A） tail. The 720bp ORF was found to code a protein with 239 amino acid residues, and its secondary structure was analyzed using PHDsec Program. One N-glycosylation site （N-X-S/T） was observed in alpha 1 domain region. Two protein kinase C phosphorylation sites （S/T-X-R/K） were identified in alpha 2 domain region and five casein kinase Ⅱ phosphorylation sites （S/T-X-X-D/E） were identified in alpha 1 and alpha 2. Four N-myristoylation sites （G-E/D/R/K/H/P/F/Y/W-X-X-S/T/A/G/C/N-G/I/T） were found in alpha 1, alpha 2 and transmembrane region. In addition, signature （F/Y-X-C-X-V/A-X-H） of immunoglobulins and MHC proteins occurred in alpha 2 domain region （residues 182-188）. Alignment of deduced amino acid sequences of the MHC class Ⅱ A and those of other vertebrates demonstrated that they shared many protein features common in those of other fish and mammalians. Phylogenetic analysis using CLASTAL W software showed that the putative MHC Ⅱ A and that of Japanese flounder were close to each other in the first sister group in similarity of 69.8%. For other animals, the similarity was 65.5%, 69.6%, 44.4%, 61.4%, 47.6%, 46.3%, 43.6%, 19.8%, 31.6% and 26.9% from red sea bream, striped sea bass, carp, cichlid, rainbow trout, Atlantic salmon, zebrafish, human, mouse and nurse shark, respectively. Semi-quantitative RT-PCR analysis demonstrated that MHC class Ⅱ A was ubiquitously expressed in tissues, but the expression level was locally unique. The MHCⅡ A transcript was detected at a high level in head kidney, spleen, intestine, gill and heart, moderate in liver and blood, and low in gonad and muscle.