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利用RACE技术克隆获得大菱鲆(Scophthalmus maximus)膜结合型免疫球蛋白D(Membrane-bounded Ig,mIgD)重链基因的cDNA序列全长,该基因全长3 357bp,包含1个2 997bp的开放阅读框,编码999个氨基酸,基因结构为VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM。重链恒定区δ1~δ7氨基酸序列同源比对结果显示,其与庸鲽(78%)、鳜(71%)、牙鲆(68%)具有较高的同源性;多序列比对结果显示,大菱鲆mIgD的每个δ恒定区内均存在色氨酸与半胱氨酸保守位点。利用邻位相连法构建硬骨鱼类免疫球蛋白系统发育树,结果显示,鱼类IgD聚为一大支,大菱鲆IgD与庸鲽及牙鲆聚为一支。利用RT-PCR分析了大菱鲆IgD重链基因的组织差异表达,结果显示,其在外周血白细胞、脾脏、前肾及中肾组织中具有较高的表达。将大菱鲆腹腔注射福尔马林灭活鳗弧菌后,实时荧光定量PCR检测其前肾组织中mIgD重链基因应答表达量,结果显示,mIgD重链基因在注射鳗弧菌后呈显著下调趋势,在注射后8h时降至最低值,其相对表达量约为对照组的1/28,然后呈逐渐恢复上调趋势,在48h时mIgD相对表达量与对照组无显著差异。
Abstract：A 3 357bp full-length cDNA sequence of membrane-bounded IgD(mIgD)heavy chain gene of turbot Scophthalmus maximus was obtained by rapid amplication of cDNA ends(RACE).The cDNA contained a 2 997bp open reading frame which encoded 999animo acids.The gene structure of turbot mIgD was VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM.BLAST result based on the mIgD heavy chain amino sequence of δ1~δ7domain showed that turbot mIgD shared high similarities with that of Hippoglossus hippoglossus(78%),Siniperca chuatsi(71%)and Paralichthys olivaceus(68%).The result of multiple sequence alignment showed that the deduced amino acid sequence possessed two highly conserved tryptophans and cysteines resieues in eachδconstant domain.Phylogenetic tree based on immunoglobulin heavy chain amino acid sequences of different teleosts was constructed using the neighbor-joining method,which exhibited that all the teleost IgDs clustered into one branch,and the turbot mIgD merged with that of H.hippoglossus and P.olivaceus.The result of RT-PCR showed that IgD gene of turbot was mainly expressed in peripheral blood leucocytes,spleen,pronephros and mesonephros.The expression profile of mIgD heary chain gene in pronephros was investigated after intraperitoneal injection with formalin inactivated Vibrio anguillarum by quantitative real-time PCR.The result showed that the expression level of mIgD heary chain gene was significantly down-regulated after injection and decreased to the lowest level at 8hwith approximate 1/28of control value,and then gradually returned to the control level at 48hpost inJection.
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基本信息:
DOI：10.16441/j.cnki.hdxb.2014.03.004
中图分类号:S917.4
引用信息:
[1]陈孔茂,唐小千,绳秀珍,等.大菱鲆mIgD重链基因的克隆与表达分析[J],2014,44(03):26-33.DOI:10.16441/j.cnki.hdxb.2014.03.004.
基金信息:
国家自然科学基金项目(31302216;31172429);; 山东省自然科学基金项目(ZR2011CQ022);; 中央高校基本科研业务费专项(201213013);; 国家重点基础研究发展规划项目(2012CB114406)资助