欧洲鳗鲡免疫球蛋白轻链基因的克隆及表达分析
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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)


Molecular cloning and expression analysis of immunoglobulin light chain gene in Anguilla anguilla
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    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1 016 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。

    Abstract:

    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.

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冯建军,郭松林,林鹏.欧洲鳗鲡免疫球蛋白轻链基因的克隆及表达分析[J].华中农业大学学报,2015,34(5):81-89

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  • 收稿日期:2014-12-09
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  • 在线发布日期: 2015-07-22
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