Xu Xiaowen, Lai Qinan, Gu Meihui, Liu Dan, Hou Qunhao, Liu Xiancheng, Mi Yichuan, Sun Zhicheng, Wang Haizhou, Lin Gang, Hu Chengyu
Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China; Infectious Diseases Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China.
Fish Shellfish Immunol. 2015 Dec;47(2):978-85. doi: 10.1016/j.fsi.2015.10.039. Epub 2015 Nov 3.
Interferon Regulatory Factors (IRFs) belong to a family of transcription factor involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF3 full-length cDNA (termed CiIRF3, JX999055) and its promoter sequence were cloned by homology cloning strategy and genome walking from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiIRF3 is comprised of a 5'UTR (195 bp), a 3'UTR (269 bp) and a largest open reading frame (ORF) of 1377 bp encoding a polypeptide of 458 amino acids. CiIRF3 has a conservative DNA-binding domain (DBD) at N-terminal and a relatively conserved interferon regulatory factors association domain (IAD). Phylogenetic tree analysis indicated that CiIRF3 gathers together with other IRF-3 from higher vertebrates in the same branch. The promoter sequence of CiIRF3 (596 bp) consists of three IRF-E, a C/EBP beta, a NF-kappa B and a TATA-BOX. CiIRF3 was constitutively expressed at low level in different grass carp tissues but was rapidly up-regulated with Poly I:C stimulation. To study the molecular mechanism of CiIRF3 regulating the transcription of IRFs, CiIRF3 was expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assays revealed the affinity of CiIRF3 protein with promoters of CiIRF1, CiIRF2, CiIRF3 and CiIRF7 respectively. Then, CIK cells were co-transfected with pcDNA3.1-CiIRF3, pGL3-promotor (pGL3-CiIRF1, pGL3-CiIRF2, pGL3-CiIRF3, pGL3-CiIRF7) and luciferase reporter vector respectively. The cotransfection experiment showed that CiIRF3 increased the promoter activity of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. Furthermore, overexpression of CiIRF3 in CIK cells also up-regulated the expressions of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. So, CiIRF3 can improve the transcriptional level of CiIRF1, CiIRF2, CiIRF3 and CiIRF7.
干扰素调节因子（IRFs）属于一类转录因子家族，参与细胞中I型干扰素和干扰素刺激基因（ISG）的转录调控。在本研究中，通过同源克隆策略和基因组步移技术，从草鱼（Ctenopharyngodon idella）中克隆了IRF3全长cDNA（命名为CiIRF3，JX999055）及其启动子序列。CiIRF3的全长cDNA序列由一个5'UTR（195 bp）、一个3'UTR（269 bp）和一个1377 bp的最大开放阅读框（ORF）组成，编码一个458个氨基酸的多肽。CiIRF3在N端有一个保守的DNA结合结构域（DBD）和一个相对保守的干扰素调节因子关联结构域（IAD）。系统发育树分析表明，CiIRF3与其他高等脊椎动物的IRF-3聚集在同一分支。CiIRF3的启动子序列（596 bp）由三个IRF-E、一个C/EBPβ、一个NF-κB和一个TATA盒组成。CiIRF3在草鱼不同组织中组成性低水平表达，但在Poly I:C刺激下迅速上调。为了研究CiIRF3调节IRFs转录的分子机制，将CiIRF3在大肠杆菌BL21中表达，并用Ni-NTA His-Bind树脂通过亲和层析进行纯化。凝胶迁移率变动分析分别揭示了CiIRF3蛋白与CiIRF1、CiIRF2、CiIRF3和CiIRF7启动子的亲和力。然后，将CIK细胞分别与pcDNA3.1-CiIRF3、pGL3-启动子（pGL3-CiIRF1、pGL3-CiIRF2、pGL3-CiIRF3、pGL3-CiIRF7）和荧光素酶报告载体共转染。共转染实验表明，CiIRF3增加了CiIRF1、CiIRF2、CiIRF3和CiIRF7的启动子活性。此外，CiIRF3在CIK细胞中的过表达也上调了CiIRF1、CiIRF2、CiIRF3和CiIRF7的表达。因此，CiIRF3可以提高CiIRF1、CiIRF2、CiIRF3和CiIRF7的转录水平。
