Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter
Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter
复制标题
新型干扰素诱导基因Gig2及其启动子的表达调控和功能表征
DOI：
10.1016/j.molimm.2009.05.183
复制
发表时间：
2009-09-01
影响因子：
3.6
通讯作者：
Gui, Jian-Fang
中科院分区：
文献类型：
--
作者：
Jiang, Jun;Zhang, Yi-Bing;Gui, Jian-Fang
草鱼出血病毒（Grasscarp hemorrhagic virus，GCHV）诱导基因2（Gig 2）是从紫外线灭活的鲫鱼囊胚胚胎细胞（Carassius auratus blastulae embryonic，CAB）中发现的一个新基因，它可能在干扰素（IFN）抗病毒反应中起关键作用。在这项研究中，产生了多克隆抗Gig 2抗血清，并用于通过Western印迹分析研究诱导表达模式，显示在正常CAB细胞中没有基础表达，但在UV灭活的GCHV、聚肌胞苷酸（Poly I：Q）和重组IFN（rIFN）后显著上调。然而，在草鱼（Ctenopharyngodon idellus）的所有测试组织中观察到Gig 2的组成型表达，并且Poly I：C注射增加了皮肤、脾脏、干肾、鳃、后肠和胸腺中Gig 2蛋白的相对量。此外，覆盖整个Gig 2 ORF和上游启动子区域的基因组序列通过基因组步移扩增。重要的是，Gig 2启动子包含3个IFN刺激的应答元件（ISRE）、9个GAAA/TfTC基序和5个γ-IFN激活位点（GAS），这是对I型IFN和11型IFN都应答的基因的特征。随后，将完整的Gig 2启动子序列克隆到pGL 3-Basic载体中，并通过转染的CAB细胞中的荧光素酶测定来测量其活性。Gig 2启动子驱动的构建体在用Poly I：C或rIFN处理后在CAB细胞中被高度诱导，并且功能能力依赖于IFN调节因子7（IRF 7），因为其活性可以被IRF 7刺激。总的来说，这些数据提供了强有力的证据，Gig 2确实是一种新的IFN诱导基因，其表达可能依赖于IRF 7对Poly I：C或IFN。(C)2009爱思唯尔有限公司保留所有权利。
Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.