大鳍鳠免疫球蛋白M重链基因的克隆及表达分析

Molecular cloning and expression analysis of the IgM heavy chain gene in largefin longbarbel catfish (Mystus macropterus)

用RACE-PCR和RT-PCR方法获得大鳍鳠分泌型免疫球蛋白M(sIgM)重链基因的全长cDNA序列。大鳍鳠sIgM的cDNA全长为1 992 bp,包含5'非编码区53 bp,3'非编码区226bp,开放阅读框1 713,编码570个氨基酸。推测的大鳍鳠sIgM蛋白质序列可变区含有4个骨架区(FR)和3个互补决定区(CDR),恒定区(CH)含有CH1、CH2、CH3和CH4 4个部分。与其它6种硬骨鱼类IgM氨基酸序列比对分析表明,大鳍鳠sIgM存在半胱氨酸和色氨酸保守位点;其氨基酸序列与斑点叉尾IgM的相似性最高,为54.3%;与石斑鱼的相似性最低,为24.6%。进化树分析表明,大鳍鳠sIgM与斑点叉尾聚为一支。RT-PCR显示,大鳍鳠IgM基因主要在血细胞、脾脏、头肾和中肾中转录表达;注射嗜水气单胞菌后,血细胞、脾脏和头肾IgM重链基因转录表达量在1～10 d内有显著上升。

Full cDNA sequence of a secretory IgM（sIgM）gene in largefin longbarbel catfish（Mystus macropterus Bleeker）was cloned using RACE-PCR and RT-PCR methods.It has 1 992 nucleotides,including 5′UTR of 53 nucleotides,3′UTR of 226 nucleotides and an open reading frame with 1 713 nucleotides encoding a 570 amino acid peptide.The deduced amino acid sequence in M.macropterus contains 4 constant regions（CH）and a variable domain（VH）which consists of 4 frame regions（FRs）and 3 complementary determining regions（CDRs）.The IgM comparison in seven teleost species showed that IgM in M.macropterus shared the highest identity（54.3%）with that in Ictalurus punctatus,and the lowest identity（24.6%）with that in Epinephelus coioides,and that the conserved cysteines and tryptophans existed in all fishes involved.Phylogenetic tree based on some teleost Ig heavy chain amino acids suggested IgM in M.macropterus was clustered closely with that of I.punctatus.Fluorescent real-time PCR showed that IgM mRNA expression of M.macropterus was mainly detected in blood cells,spleen,kidney and head kidney,and increased significantly in blood cells,spleen,head kidney between day 1 and day 10 after injection of Aeromonas hydrophila.