大菱鲆多聚免疫球蛋白受体基因的克隆及表达分析

Molecular cloning and expression analysis of the pIgR gene in Scophthalmus maximus

鱼类多聚免疫球蛋白受体(polymeric immunoglobulin receptor,pIgR)是黏膜免疫系统的关键因子,能够介导多聚免疫球蛋白向黏液中的转运和分泌。本研究利用同源克隆和cDNA末端快速扩增技术(rapid amplification ofcDNA ends,RACE),首次得到了大菱鲆(Scophthalmus maximus)pIgR完整的cDNA序列,全长1 584 bp,该序列包含一个1 005 bp的开放阅读框及5’和3’UTR,编码334个氨基酸。同源性比较发现,大菱鲆pIgR氨基酸序列与牙鲆(Paralichthys olivaceus)、斜带石斑鱼(Epinephelus coioides)鲀、红鳍东方(Takifugu rubripes)的序列相似性分别是74%、74%和59%,表现出较高的保守性。多序列比对显示,硬骨鱼pIgR包含两个ILD(Ig-like domain),分别对应哺乳类的ILD1和ILD5区域。系统进化树分析表明,大菱鲆pIgR和牙鲆的聚为一支。半定量RT-PCR显示,pIgR在健康大菱鲆各组织中均有表达,但表达水平不同,其中在黏膜相关淋巴组织中表达最强。经灭活的鳗弧菌菌液浸泡处理后,实时荧光定量PCR结果显示,大菱鲆各组织中pIgR的相对表达量在72 h内均呈现先增加后减少的趋势,在48 h内均有一个最高值出现,且黏膜相关淋巴组织中峰值出现较早,说明pIgR在硬骨鱼类黏膜免疫中发挥了重要作用。

Polymeric immunoglobulin receptor （plgR） is a key immune factor, medicating the transport of polymeric immunoglobulins （pig） through the epithelial cells into the mucosal secretions to protect the organisms from pathogens. In this study, the pIgR of Scophthalmus maximus was first cloned and sequenced by rapid amplification of cDNA ends approaches （RACE）. The plgR contained 1 584 nucleotides, 3＇UTR, 5＇UTR, and an open reading frame （ORF） of 1005 nucleotides that encoded for a polypeptide of 334 amino acid. Gene homology comparisons showed that S. maximus shared 74% amino acid identity with Paralichthys olivaceus and Epinephelus coioides, and 59% with Takifugu rubripes, indicating a high conservation ofplgR gene. Multiple sequence alignment demonstrated the plgR of teleosts was composed of two Ig-like domains （ILDs）, corresponding to the ILD 1 and ILD5 of Mammal＇s plgR. The phylogenetic tree was constructed from the amino acid sequences of plgR of 18 vertebrates and indicated that plgR amino acids in S. maximus were clustered together with P. olivaceus. Expression analysis revealed that plgR gene was expressed in almost all tissues of healthy S. maximuss, with higher levels in the mucosa-associated lymphoid tissues. The dynamics changes ofplgR expression in S. maximu were evaluated following bath immunization with inactivated Vibrio anguillarum by real-time PCR, and the results showed that the relative expression amount increased firstly and then decreased within 72 h in all the tested tissues, and reached its maximum expression within 48 h and the peak appeared earlier in mucosa-associated lymphoid tissues. These results suggested pIgR played a critical role in the mucosal immunity of teleost.