大鳍鳠免疫球蛋白轻链3型基因cDNA的克隆及表达

摘要

The technique of homologous cloning and Rapid Amplification of cDNA Ends(RACE) was used to amplify full length cDNA of immunoglobulin light chain 3 isotype (IgL3) gene from largefin longbarbel catfish (Mystus macropterus Bleeker). IgL3 in M. macropterus has 947 nucleotides, including 5'-UTR of 58 nucleotides, 3'-UTR of 184 nucleotides and an open reading frame with 705 nucleotides encoding a peptide of 234 amino acids. The deduced amino acid sequence contains an constant region(CL) and a variable domain(VL) consisting of 4 frame regions(FRs) and 3 complementary determining regions(CDRs). The IgL3 comparison in seven teieost species showed that IgL3 in M macropterus shared the highest identity (68.8%) with that(F) in lctalurus punctatus, and the lowest identity(47.2%) with that in Salmo salar. Phylogenetic tree based on some teleost IgL amino acids showed that IgL3 in M. macropterus was clustered closely with that (F) of I. punctalus and grouped with IgL3 in other teleost, and was far away from L1 and L2 of teleost. Real-time PCR showed that lgL3 mRNA expression of M. macropterus was mainly detected in head kidney, spleen and blood cells and increased significantly in these tissues after injection of Aeromonas hydrophila. The result indicated that head kidney, spleen and blood cell are main organs for IgL3 expression after stimulation, and play a critical role in immunity interaction in M. macropterus.%应用同源克隆和RACE-PCR方法获得大鳍鳠免疫球蛋白轻链3型(IgL3)基因的全长cDNA序列,并分析了该基因在组织中的表达.大鳍鳠IgL3的cDNA全长为947 bp,包含5 '非编码区58 bp,3′非编码区184 bp,开放阅读框705 bp,编码234个氨基酸.推测的蛋白质序列分为可变区(VL)和恒定区(CL).VL被进一步划分为4个骨架区(FR)和3个互补决定区(CDR).大鳍鳠与其它6种硬骨鱼类免疫球蛋白轻链L3氨基酸序列比对分析表明,大鳍鳠氨基酸序列与斑点叉尾鲴IgL3型(F型)的相似性最高,为68.8％,与南极鱼IgL3型的相似性最低,为47.2％.进化树分析表明,大鳍鳠IgL与斑点叉尾鲴IgL3型(F型)聚为一支并与其它硬骨鱼类IgL3型聚为一簇,明显与L1和L2进化支不同.实时荧光定量PCR显示,大鳍鳠IgL3基因主要在头肾、脾脏和血细胞中转录表达;注射嗜水气单胞菌后,头肾、脾脏和血细胞IgL3基因转录表达量有显著上升.研究表明,头肾、脾脏和血液是大鳍鳠IgL3型基因主要的表达器官,在免疫反应中起重要作用.