鲫抗病毒蛋白Viperin表达模式及启动子活性分析

Expression Pattern and Promoter Activity Analysis of Antiviral Protein Viperin in Crucian Carp

目的:研究鲫抗病毒蛋白Viperin(Ca Viperin)的表达特征,分析其启动子区主要转录因子结合位点并验证启动子活性。方法:采用半定量PCR法检测鲫Viperin在紫外灭活和活GCRV病毒、Poly(I:C)、LPS、重组干扰素处理鲫CAB细胞后不同时间点在RNA水平的表达量;采用Genome walking法从鲫基因组中扩增出鲫Viperin启动子片段-1832/+23,采用p GL3Basic荧光素酶报告基因系统对启动子活性进行分析;在NCBI中选代表性物种(从鱼类到哺乳类)Viperin启动子序列,分析其主要转录因子结合元件ISRE和GAS,预测其受上游信号调控的保守性。结果:分析了紫外灭活或活GCRV病毒、Poly(I:C)、LPS、重组干扰素刺激鲫CAB细胞后Viperin表达的时序特征;成功克隆鲫Viperin启动子片段并构建报告基因表达质粒;分析并证实鲫Viperin启动子活性;从进化角度预测分析了Viperin基因受上游信号调节的保守性。结论:除LPS外,灭活或活GCRV病毒、Poly(I:C)、重组干扰素皆能诱导鲫Viperin转录;Viperin启动子区富含IFN信号诱导元件ISRE和GAS,且在不同物种间相对保守,因此推测Viperin基因受IFN及其上游信号诱导经由相对保守的信号通路。

Objective： To investigate the expression pattern of crucian carp antiviral protein viperin（ Ca Viperin）,and to analyze the key transcription factor binding sites of its promoter and verify the promoter activity. Methods： In this study,semi-quantitative PCR was used to detect the Ca Viperin expression at different time points after treating CAB cells with activated or UV inactivated GCRV,Poly（ I： C）,LPS or r IFN. The-1832 / ＋ 23 fragment of Ca Viperin promoter was amplified by Genmome walking method from crucian carp genomic DNA. The promoter activity of Ca Viperin was analyzed via using the p GL3 Basic luciferase reporter system. The key transcription factor binding elements（ ISRE and GAS） of Viperin promoter were analyzed in different species（ from fish to mammalian） to predict the conservation of the regulation mechanism of Vipeirn. Results： We characterized the expression pattern of Ca Viperin after treating CAB cells with activated or UV inactivated GCRV,Poly（ I： C）,LPS or r IFN. We cloned the fragment of Ca Viperin promoter and constructed a reporter gene expression vectors. The promoter activity of Ca Viperin was analyzed and verified. And the conservation of Ca Viperin＇s regulagiotn mechanism were also predicted and analyzed from an evolutionary perspective. Conclusion： Ca Viperin can be induced by UV inactivated or activated GCRV,Poly（ I： C）,r IFN but not LPS; the IFN signal induced elements such as ISRE and GAS are enriched in Ca Viperin promoter; the VP3 and VP6 constructs showed the best activity; the ISRE and GAS elements existing in Viperin promoter are relatively conserved among different species; thus,it is speculated that the transcriptional regulation of Viperin was via a conserved signal pathway.