Chen Huarong, Sun Changgui, Liu Wenqun, Gu Meihui, Lin Gang, Liu Yong, Mi Yichuan, Fan Lihua, Wang Binhua, 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.
Fish Shellfish Immunol. 2015 Feb;42(2):249-55. doi: 10.1016/j.fsi.2014.11.008. Epub 2014 Nov 15.
Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (+). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were co-transfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3.
草鱼呼肠孤病毒（GCRV）诱导基因2（Gig2）被认为是一种参与病毒感染应答的新型抗病毒因子。然而，关于病毒感染时Gig2转录调控背后的机制知之甚少。在本研究中，通过同源克隆策略鉴定了草鱼（Ctenopharyngodon idella）Gig2基因（CiGig2）的上游启动子区域。发现CiGig2启动子序列长度为859 bp，包含三个分散的干扰素刺激反应元件（ISRE）。此外，将一些草鱼IRF（CiIRF1、CiIRF2和CiIRF3）的开放阅读框序列亚克隆到表达质粒pET-32a中，并在大肠杆菌BL21中表达，然后用Ni-NTA His-Bind树脂通过亲和层析纯化表达的蛋白。采用凝胶迁移率变动分析筛选CiGig2的转录调控因子。结果显示，CiIRF1、CiIRF2和CiIRF3的重组多肽与CiGig2启动子具有高亲和力结合；表明IRF1、IRF2和IRF3可能是Gig2潜在的转录调控因子。随后，将CiGig2启动子序列克隆到pGL3-Basic载体中，将CiIRF1、CiIRF2和CiIRF3的开放阅读框克隆到表达质粒pcDNA3.1(+)中。然后，将pGL3-CiGig2启动子序列和pcDNA3.1-CiIRFs共转染到草鱼肾（CIK）细胞中。通过双荧光素酶分析在转染的CIK细胞中测定CiIRFs对CiGig2启动子的体内作用。我们的结果表明，CiIRFs在调节CiGig2转录中作用多样，例如，CiIRF3在此过程中起积极作用；相反，CiIRF1起抑制作用；然而CiIRF2对CiGig2转录的影响不明显。为了进一步研究三个ISRE在CiGig2转录中的作用，我们分别克隆了三个突变的CiGig2启动子，即ISRE1mut-luc（缺失ISRE1）、ISRE2mut-luc（缺失ISRE2）和ISRE3mut-luc（缺失ISRE3）。在体外，凝胶迁移率变动分析表明所有三个突变启动子也与CiIRFs结合。将CiGig2启动子突变体（分别为ISRE1mut-luc、ISRE2mut-luc或ISRE3mut-luc）与pcDNA3.1-IRFs共转染CIK细胞。结果表明不同的ISRE发挥不同的作用。ISRE2对CiIRF1诱导的CiGig2转录比ISRE1和ISRE3更重要。ISRE1和ISRE3对CiIRF2和CiIRF3诱导的CiGig2转录很重要。
