摘要
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 016bp,开放阅读框为714bp,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27ku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 AaIgLis 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 AaIgLin 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 7dand 14 d,respectively.The peak expression level of AaIgL was much higher in the spleen and the expression recovered to normal level at 21 din 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 AaIgLexpression after stimulation.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2015年第5期81-89,共9页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(31272685)
福建省教育厅项目(JA11150)
福建省海洋与渔业厅项目(201212140006)
集美大学科研基金项目(C60819)
关键词
欧洲鳗鲡
免疫球蛋白轻链
原核表达
荧光定量PCR
Anguilla anguilla
immunoglobulin light chain（IgL）
prokaryotic expression
realtime PCR
