乌鳢免疫球蛋白M重链和免疫球蛋白轻链的克隆与特征分析

Molecular Cloning and Characterization of IgM Heavy Chain and IgLight Chain from Snakehead(Channa argus)

应用RACE-PCR方法克隆并鉴定了乌鳢免疫球蛋白M(i mmunoglobulin M,Ig M)重链(H)和免疫球蛋白轻链(L)全长cDNA。乌鳢IgH cDNA全长1 912个核苷酸(nt),包含1 797 nt的开放阅读框,编码598个氨基酸(aa)。IgH恒定区(CH)均包含1对保守的半胱氨酸残基形成链内二硫键,其中CH1区氨基末端存在保守的半胱氨酸残基与轻链形成链间二硫键,4个推测的N-糖基化位点(NXT和NXS)则分别位于CH1、CH2和CH4。序列比对发现乌鳢IgHCH1与CH2交界处氨基酸序列的插入使铰链区肽段较其他硬骨鱼IgH长,此外IgH CH4区羧基末端参与单体间聚合的半胱氨酸被赖氨酸替换,这反映乌鳢IgH分子结构的特异性。序列相似性比较显示乌鳢IgH与点带石斑鱼、黑鳍冰鱼、牙鲆、虹鳟的相似性依次为55%、50%、45%和36%,与斑马鱼类的相似性较低(为28%)。乌鳢IgL cDNA全长941 nt,包含729 nt的开放阅读框,编码242个aa。轻链可变区(VL)和恒定区(CL)均包含保守的半胱氨酸,形成链内和链间二硫键。序列比对分析表明乌鳢IgL与五条鰤和花狼IgL的相似性为78%和77%,而与斑点叉尾鮰和虹鳟的相似性仅为32%和31%。根据IgH和IgL恒定区氨基酸序列构建系统进化树,鱼类IgH形成硬骨鱼和软骨鱼两大分支,其中乌鳢IgH与河豚IgH的亲缘关系较近;硬骨鱼IgL系统树显示3个主要的分支:L1、L2和L3,乌鳢IgL位于L1分支的L1 A亚类。

By RACE-PCR,immunoglobulin M heavy chain （H） and immunoglobulin light （L） chain cDNAs have been cloned and characterized from snakehead Channa argus. The IgH cDNA was found to be 1 912 nucleotides （nt） and contains an open reading frame （ORF） of 1 797 nt that is translated into a putative peptide of 598 amino acids （aa）. The constant region （CH1-4） of IgH each contain a pair conserved Cys residues which may be responsible for the formation of intra-domain disulfide bridge. The Cys of CH1 may be concerned with inter-chain disulfide bridge, four N-linked glycosylation sites are located in CH1 ,CH2 and CH4 respectively. The multiple sequence alignment reveal the presence of a remarkable insertion at the CH1-CH2 boundary,result in a CH1,CH2 hinge peptide longer than other teleost IgH, respectively. Noteworthy,the Cys in CH4 is substituted by Lys in snakehead fish, that not find in other teleost IgH. Protein sequence of snakehead IgH has 55%, 50% ,45%, 36% and 28% identical to orangespotted grouper, blackfin icefish, bastard halibut, rainbow trout and zebrafish IgH, respectively. The IgL cDNA was found to be 941 nt and contains an ORF of 729 nt that translates into a putative peptide of 242 aa. The cysteine residues involved in the formation of disulfide bonds are completely conserved among fish IgL. Snakehead IgL exhibits 78 %, 77 %,32 % and 31 % overall identity to Japanese amberjack, spotted wolfish, channel catfish and rainbow trout IgL, respectively. The phylogenetic tree of fish IgH conserved region contains two major branches of teleost and elasmobranch, and snakehead IgH is most closely related to pufferfish. The phylogenetic tree of IgL conserved region shows three major branches, and snakehead IgL locate as a branch of IgL1A.