首页 > 过刊浏览>2020年第44卷第11期 >1804-1812. DOI:10.11964/jfc.20200112119
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东北七鳃鳗NF-κB基因启动子的构建及活性
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教育部留学回国人员科研启动基金(D-8002-15-0042)；中美海洋研究中心基金(A1-0209-15-0806)；水产动物疾病与基因编辑育种的平台建设和前沿科学研究(A1-3201-19-3013)


Construction and activity of NF-κB promoter reporter in Lampetra morii
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  • ZHOU Zebin

    ZHOU Zebin

    Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China
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  • DING Shaoqing

    DING Shaoqing

    Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China
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  • CHENG Peiru

    CHENG Peiru

    Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China
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  • REN Jianfeng

    REN Jianfeng

    Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China
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  • LI Weiming

    LI Weiming

    Department of Fisheries and Wildlife, Michigan State University, East Lansing 48824, USA
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  • ZHANG Qinghua

    ZHANG Qinghua

    Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China;Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai 201306, China
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Fund Project:

Ministry of Education Overseas Returnees Research Fund (D-8002-15-0042); China-US Ocean Research Center Fund (A1-0209-15-0806); Construction platform of aquatic animal diseases and gene editing breeding and cutting-edge scientific research（A1-3201-19-3013）

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    摘要:

    核转录因子NF-κB (nuclear factor kappa B)对机体的免疫反应、炎症反应等起重要的调控作用。为了获得NF-κB基因启动子的活性报告基因，进一步研究原始脊椎动物免疫应答的机制，实验根据此前克隆得到的东北七鳃鳗NF-κB基因的编码序列(coding sequence，CDS)，以与东北七鳃鳗高度同源的日本七鳃鳗同源基因5′上游启动子特征序列为模板设计引物，克隆获得了东北七鳃鳗NF-κB启动子序列，该序列全长3 169 bp，提交至NCBI数据库GenBank获得登录号MN368861。经AliBaba 2.1软件预测该序列包含CREB (cgtgacttca)、Oct-1 (aatatgaatt)、GATA-1 (gaacctatct)、AP-1 (ggttgagtca)、NF-kappa B (ctttcctgtt)、IRF-1 (tttcctgttc)、Sp-1 (tgtgaggggt)、c-Jun (acgtgacttc)和c-Fos (ggttgagtca)等多个转录因子结合位点，并包含TATA box转录核心元件。通过MethPrimer软件预测在序列1 207~1 402 bp处存在CG含量大于50%，长196 bp的CpG岛。利用双酶切技术构建了pGL3-NF-κB-pro重组质粒，并将其转染至人胚肾细胞(human embryonal kidney, HEK293T)和鲤上皮瘤细胞(Epithelioma papulosum cyprinid, EPC)中。双荧光素酶报告基因系统检测结果显示，该片段序列在哺乳动物细胞和鱼类细胞中均具有启动子活性，同时在HEK293T中经过Toll样受体(Toll-like receptors，TLR)的配体LPS刺激后，启动子活性显著上升。本研究成功构建了东北七鳃鳗NF-κB启动子报告基因，并在不同细胞中证实了其活性。LPS刺激后的检测结果揭示东北七鳃鳗NF-κB参与了TLR信号通路的免疫应答。东北七鳃鳗NF-κB报告基因的构建为探究原始脊椎动物免疫系统信号传导机制奠定了基础。

    Abstract:

    Nuclear factor kappa B (NF-κB) plays an important role in regulating immune response and inflammation. In order to obtain the NF-κB promoter reporter gene and further study the mechanism of the immune response in primitive vertebrates, based on the CDS sequence of the NF-κB gene previously cloned from Lampetra morii, we obtained the L. morii NF-κB promoter sequence by using the 5′upstream promoter characteristic sequence of the NF-κB from L. japonica, which is highly homologous to L. morii, as a template to design primers. The cloned NF-κB promoter sequence from L. morii was 3 169 bp, which was submitted to NCBI Genbank to obtain accession number MN368861. The sequence is predicted by AliBaba2.1 software to include CREB (cgtgacttca), Oct-1 (aatatgaatt), GATA-1 (gaacctatct), AP-1 (ggttgagtca), NF-kappa B (ctttcctgtt), IRF-1 (tttcctgttc), Sp-1 (tgtgaggggt), c-Jun (acgtgacttc), c-Fos (ggttgagtca) and many other transcription factor binding sites, and contains TATA box transcription core elements. The MethPrimer software predicts that there are CpG islands with a CG content greater than 50% and a length of 196 bp at the 1 207-1 402 bp sequence. pGL3-NF-κB-pro recombinant plasmid was constructed by double digestion technology and transfected into human embryonal kidney (HEK293T) cell and epithelioma papulosum cyprinid (EPC) cell. The results of double luciferase reporter gene system assay showed that the fragment sequence had promoter activity in both mammalian cell lines and fish cell lines, while the promoter activity increased significantly after Toll-like receptors (TLR) ligands LPS stimulation in HEK293T cell. In this study, the NF-κB reporter gene of L. morii was successfully constructed, and its activity was confirmed in different cell lines. The results showed that NF-κB is involved in the immune response of TLR signaling pathway after LPS stimulation in L. morii. The construction of the NF-κB reporter gene of L. morii laid a foundation for exploring the signaling mechanism of the primitive vertebrate immune system.

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周泽斌,丁少青,程沛茹,任建峰,李伟明,张庆华.东北七鳃鳗NF-κB基因启动子的构建及活性[J].水产学报,2020,44(11):1804~1812

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  • 收稿日期:2020-01-02
  • 最后修改日期:2020-04-08
  • 录用日期:2020-04-14
  • 在线发布日期: 2020-11-09
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