欧洲鳗鲡免疫球蛋白轻链基因的克隆及表达分析
Molecular cloning and expression analysis of immunoglobulin light chain gene in Anguilla anguilla

通过 RT-PCR 技术克隆欧洲鳗鲡（Anguilla anguilla）Ig轻链基因cDNA全长序列，命名为AaIgL，其全长为1 016 bp，开放阅读框为714 bp，编码238个氨基酸。将该基因片段与 pET-his 载体连接构建原核表达载体，在大肠杆菌 BL21 中诱导表达，其表达产物经 SDS-PAGE 和 Western blotting 分析，结果表明新增的 27 ku 蛋白条带与预期值相符，且与兔抗欧洲鳗鲡 IgM 血清发生特异性显色反应，证实了AaIgL基因能够在大肠杆菌中以包涵体形式高效表达；实时荧光定量PCR检测结果发现：AaIgL在欧洲鳗鲡各组织中均有表达，其中脾脏的表达量最高，肾脏和心脏中也有较高的表达水平，而肝脏、肌肉、鳃以及肠中的表达量较低；欧洲鳗鲡经山羊IgG肌肉注射后脾脏和肾脏的AaIgL表达水平明显上升，其峰值分别为第7天和第14天， AaIgL在脾脏的表达量显著高于肾脏，但是第21天后均恢复至正常水平。以上结果表明，AaIgL在欧洲鳗鲡机体免疫防御中发挥重要作用，脾脏是AaIgL基因的主要表达器官。
The cDNA sequence of the immunoglobulin light chain gene,AaIgL,in European eel (Anguilla anguilla) was cloned using reverse transcription PCR (RT-PCR).The full-length cDNA sequence of AaIgL is 1 016 bp,with a 714 bp open reading frame encoding 238 amino acids.The amplified DNA fragments were ligated into pET-his vector,transformed into E.coli BL21 and then induced by IPTG.The size of the expressed protein was 27 ku as estimated by SDS-PAGE electrophoresis and the recombinant proteins were strongly recognized by the serum anti-IgM of European eel detected by Western blotting,demonstrating that the fusion protein is expressed stably in E. coli BL21 with the form of inclusion bodies.Quantitative real-time RT-PCR analysis revealed a broad expression of AaIgL in a wide range of tissues,with the highest expression in spleen,followed by kidney and heart,and lowest in liver,muscles,gills,and intestine.After injection with goat IgG,AaIgL were obviously up-regulated in the spleen and the kidney,peaking at 7 d and 14 d,respectively.The peak expression level of AaIgL was much higher in the spleen and the expression recovered to normal level at 21 d in both spleen and kidney.Our results suggested that AaIgL might play an important role in fish’s immune defense system,and the spleen was the main organ for AaIgL expression after stimulation.