新猪源干扰素刺激基因LOC100623143对日本乙型脑炎的抗病毒作用研究
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- 英文论文题名：Research on the Antiviral Effect of New Porcine Interferon-stimulated gene(ISG) LOC100623143 to Japanese Encephalitis
- 论文作者：王婧霞 ; 顾金燕 ; 刘珂 ; 马志永
- 英文论文作者：WANG Jing-xia ; GU jing-yan ; LIU Ke ; MA Zhi-yong ; Shanghai Veterinary Research Institute ; CAAS ; Swine respiratory disease Innovation Team
- 年：2016
- 作者机构：中国农业科学院上海兽医研究所猪呼吸道传染病创新团队;
- 论文关键词：干扰素刺激基因 ; 抗病毒因子 ; 日本乙型脑炎病毒
- 英文论文关键词：ISG ; Antiviral Factor ; JEV
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.65
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：8
- 文件大小：770k
- 原文格式：D
- 会议级别：全国
摘要
干扰素是一种具有广谱抗病毒、抗肿瘤、促进细胞生长分化和免疫调节等多种生物活性的蛋白质。在对干扰素的研究中发现,干扰素并不直接作用于病毒,而是诱导机体细胞产生众多的效应分子,通过这些效应分子发挥抗病毒作用,这些效应分子被称为干扰素刺激基因(ISG)。在对人源干扰素刺激基因的研究中发现,不同病原对不同的干扰素刺激基因有着不同的敏感性,某一个ISG基因在病原感染宿主的某个过程中发挥特定作用,干扰素通过不同ISG的协同机制发挥抗病毒作用。目前对人源的干扰素及干扰素刺激基因的研究较为全面,但猪源ISG的各方面研究较少,仍有大量的ISGs生物学功能未被阐明。为了分析不同ISGs在病原感染过程中发挥的作用,我们首先检测了日本乙型脑炎病毒(JEV)感染的PK-15细胞,并对感染后0h,6h,12h,24h及48h的PK-15细胞中ISGs变化进行检查,发现LOC100623143分子在JEV感染过程中呈上调变化趋势。然后,我们通过BALST比对以及生物信息学软件分析不同品种猪的ISGs基因序列的同源性和保守序列,Primer 5.0设计针对LOC100623143的引物,用RT-PCR方法扩增出目的基因LOC100623143。将目的基因与p Flag7.1载体连接,构建真核表达载体用于过表达实验。LOC100623143的过表达的实验中,LOC100623143基因过表达的细胞中JEV m RNA水平显著下调,这初步说明LOC100623143对JEV发挥抑制作用。随后的RNA干扰实验中,LOC100623143基因敲低的细胞中JEV m RNA水平显著上调,这进一步验证了LOC100623143对JEV的抑制作用。本研究对猪源ISG基因LOC100623143进行了分析,构建并初步鉴定了LOC100623143的生物学功能,为进一步探索干扰素刺激基因的作用机制和开发抗病毒药物奠定基础。
Interferon(IFN) is a kind of protein with broad-spectrum anti-viral,anti-tumor,promoting cell growth and differentiation,immune regulation and other biologically functions.IFN doesn't act on the virus but induce the cells produce a large number of effector molecules,these molecules exert antiviral effects and they are called interferon stimulated genes(ISG).It's found in the study of human-derived ISG that different pathogens have distinct sensitivities with different ISGs,one ISG plays a specific role in a pathogen infection,ISGs play antiviral function by cooperative mechanisms.Research on human-derived ISG is more comprehensive while less on swine ISG.There are still a large number of biological functions of ISGs have not been elucidated.To analyze the role of different ISGs during pathogen infection,we examined the PK-15 cells infected by JEV,check the change of ISGs in PK-15 cells 0h,6h,12 h,24 h and 48 h after infection,we found that LOC100623143 showed increase trends in the course of JEV infection.Then compare the gene homology of ISGs from different breeds of pigs byBALST and other bioinformatics software,design the primers for LOC100623143 by Primer 5.0 and amplify the target gene LOC100623143 through RT-PCR.Connect the target gene with p Flag7.1 to construct eukaryotic expression vector for over-expression experiment.Verify the role played by LOC100623143 of JEV through gene over-expression and RNAi.The result showed that ISG-LOC100623143 has inhibitory effect to JEV.This study analyzed porcine ISG gene LOC100623143,constructed and preliminary identify the biological functions of LOC100623143,lay the foundation for further exploration and development of interferon-stimulated genes antiviral drugs.
Interferon(IFN) is a kind of protein with broad-spectrum anti-viral,anti-tumor,promoting cell growth and differentiation,immune regulation and other biologically functions.IFN doesn't act on the virus but induce the cells produce a large number of effector molecules,these molecules exert antiviral effects and they are called interferon stimulated genes(ISG).It's found in the study of human-derived ISG that different pathogens have distinct sensitivities with different ISGs,one ISG plays a specific role in a pathogen infection,ISGs play antiviral function by cooperative mechanisms.Research on human-derived ISG is more comprehensive while less on swine ISG.There are still a large number of biological functions of ISGs have not been elucidated.To analyze the role of different ISGs during pathogen infection,we examined the PK-15 cells infected by JEV,check the change of ISGs in PK-15 cells 0h,6h,12 h,24 h and 48 h after infection,we found that LOC100623143 showed increase trends in the course of JEV infection.Then compare the gene homology of ISGs from different breeds of pigs byBALST and other bioinformatics software,design the primers for LOC100623143 by Primer 5.0 and amplify the target gene LOC100623143 through RT-PCR.Connect the target gene with p Flag7.1 to construct eukaryotic expression vector for over-expression experiment.Verify the role played by LOC100623143 of JEV through gene over-expression and RNAi.The result showed that ISG-LOC100623143 has inhibitory effect to JEV.This study analyzed porcine ISG gene LOC100623143,constructed and preliminary identify the biological functions of LOC100623143,lay the foundation for further exploration and development of interferon-stimulated genes antiviral drugs.
引文
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[2]李洲,范启修.干扰素刺激基因(ISGs)的研究进展[J].生物化学与生物物理进展,1994,05:414-417+473-474.
[3]蔡应木,焦晓阳,叶向群.干扰素抗病毒应答途径新基因--干扰素激活基因20结构和功能研究进展[J].国际检验医学杂志,2007,28(1):41-42.
[4]孙静静,邵军军,常惠芸.干扰素作用机制及其在抗口蹄疫病毒中的应用研究进展[J].中国畜牧兽医,2012,39(1):206-209.
[5]Nakajima,E.,Morozumi,T.,Tsukamoto,K.,Watanabe,T.,Plastow,G.,Mitsuhashi,T.,2007.A naturally occurring variant of porcine Mx1 associated with increased susceptibility to influenza virus in vitro.Biochem.Genet.45,11-24.
[6]李燕.猪干扰素刺激基因ISG20的克隆、真核表达及其对PRRSV增殖的影响[D].华中农业大学,2010.
[7]朱紫祥,魏建超,史子学,吴开宝,马志永.泛素样蛋白ISG15抗病毒机制研究进展[J].病毒学报,2012,01:78-83.
[8]刘维婷.重组猪源OAS1抗日本脑炎病毒的特性研究[D].南京农业大学,2013.
[9]Chu H,Wang J J,Qi M,et al.Tetherin/BST-2 Is Essential for the Formation of the Intracellular Virus-Containing Compartment in HIV-Infected Macrophages[J].Cell Host&Microbe,2012,12(3):360-372.
[10]Frias-Staheli N,Giannakopoulos N V,Kikkert M,et al.Ovarian Tumor Domain-Containing Viral Proteases Evade Ubiquitin-and ISG15-Dependent Innate Immune Responses[J].Cell Host&Microbe,2007,2(6):404-16.
[11]Pitha-Rowe I F,Pitha P M.Viral defense,carcinogenesis and ISG15:Novel roles for an old ISG[J].Cytokine&Growth Factor Reviews,2007,18(5-6):409-417.
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