牛Ⅰ型干扰素家族新成员（IFN-ε、IFN-κ、IFN-χ）抗病毒信号转导与表达调控研究

发布时间：2018-05-18 20:07

本文选题：牛I型干扰素 + 生物学特性　；参考：《东北农业大学》2017年博士论文

【摘要】：干扰素(interferons,IFN)是见于所有有颌类脊椎动物的一大类多效应细胞因子,主要担负宿主抗病毒感染免疫应答功能,不同的病原感染往往倾向于诱导表达出特定的干扰素成员,发现并系统鉴定干扰素新成员对抗病毒感染免疫应答具有重要的意义。固有免疫是机体抵御病原入侵的第一道防线,抗病毒固有免疫反应起始于细胞内模式识别受体(PRRs)。PRRs识别的病原相关分子模式(PAMPs)通过转录因子NF-κB和干扰素调节因子(IRF)(尤其是IRF3和IRF7)来进一步诱导I型干扰素的产生,建立抗病毒状态以促进适应性免疫应答,最终从机体内清除病毒感染,所以I型干扰素在建立抗病毒感染状态和宿主细胞抵抗病毒复制方面具有重要的作用。本研究首先系统分析了牛I型干扰素基因座位,定位到牛的2个干扰素新亚型(IFN-ε、IFN-κ)以及1个包含有2个成员的新亚族(IFN-χ1、IFN-χ3),随后围绕这四种干扰素的抗病毒信号转导与表达调控进行了一系列的研究,具体研究内容如下:1.牛I型干扰素基因座图谱的构建与分析。综合运用多种在线BLAST方法(BLAST,Basic Local Alignment Search Tool,基本的基于局部比对的搜索工具)对牛全基因组筛选,全面分析各型干扰素的信息,发现牛I型干扰素基因排布于8号染色体上,并进化出了具有鲜明特征的干扰素新家族,其中最为引人注目当属IFN-χ亚族。IFN-χ亚族具有4个成员,具备全新干扰素的特征,组成了1个新干扰素亚族。牛I型干扰素基因座按转录方向分成2个大的亚座位,长度(746kp)分别是人(403kp)和鼠(323kb)的1.85和2.31倍,并且干扰素基因数目(64个)也远超人(17个)与鼠(19个)。在座位末端5个IFN-δ假基因与4个IFN-χ样分子混杂在12个IFN-β样基因之中,并且这些基因的转录方向均相同;基因座其它区域则被数量庞大的IFN-α(13个)和IFN-ω(24个)家族所占据。IFN-κ和IFN-ε仅存在1个拷贝序列,位于基因组的起始端,且IFN-κ的转录方向与IFN-β相反,其与最近的干扰素基因间隔6.561Mb。2.牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3的特性和抗病毒信号转导通路。本研究首先从牛肝基因组中克隆得到了牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3基因,并分析了这些序列的特征。随后在大肠杆菌表达系统表达了重组牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3的成熟肽序列,并制备了抗牛IFN-ε、IFN-κ、IFN-χ多克隆抗体,在不同的系统上测定了其抗病毒活性。牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3均对胰酶敏感,一定程度上耐热(42℃和60℃)耐酸碱(p H2.0和p H10.0),其抗病毒活性可被特异性多克隆抗体中和,也可被I型干扰素受体IFNAR1和IFNAR2抗体阻断。进一步研究发现牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3不仅可诱导干扰素刺激基因(ISGs,如Mx1,ISG15,ISG56)的转录,也可诱导Mx1,STAT1,NF-κB p65的表达,这些分析表明目前已知的I型干扰素成员均依赖JAK-STAT信号通路转导抗病毒信号。3.牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3对干扰素信号通路的影响。本试验采用转录组测序的方式分析了在牛睾丸原代细胞上牛IFN-αA和IFN-χ1的调控基因并比较了这些基因的表达差异,结果发现牛Bo IFN-χ1样品处理组有40个差异基因表达上调;Bo IFN-αA样品处理组有80个差异基因表达上调,1个基因表达下调。随后采用荧光定量PCR进一步验证了这些调控基因的在牛源细胞上的表达调控,结果显示上调基因大部分属于ISGs基因,随机选取OAS、Mx1,STAT1、LGP2、GBP4、BST2、PML基因在MDBK、BT、BL细胞上进行验证,证实这些基因在不同细胞上均有不同程度的上调。此外,牛I型干扰素可在BL和BT细胞上启动商品化的人NF-κB-Luc和ISRE-Luc报告载体的荧光素酶活性,并可激活本研究构建的Bo IFNβ-Luc和Bo IFNχ-Luc启动子报告载体的荧光素酶活性,为进一步开展牛固有免疫信号通路的研究奠定了基础。4.牛干扰素信号通路相关分子的研究。维甲酸诱导基因(RIG-I)、黑素瘤分化相关因子5(MDA5)是胞内识别dsRNA的重要模式识别受体,IRF3和IRF7是参与固有免疫应答的重要转录因子,转录因子IRF9参与诱导I型干扰素的产生,ELF4、MAVS、MITA作为信号通路中的接头分子参与诱导I型干扰素的产生。本研究运用RT-PCR的方法从犊牛原代肾细胞中克隆获得了牛接头分子ELF4、MAVS、MITA的c DNA,并分析了其序列特征,组织表达和细胞器定位,重点研究了其对干扰素信号通路的影响。研究表明在不同的细胞上接头分子ELF4、MAVS、MITA能诱导产生不同的I型干扰素,促进信号通路相关分子的转录与表达,还能刺激ISRE、NF-κB、Bo IFN-β和Bo IFN-χ启动子激活的荧光素酶活性,为今后进一步探讨接头分子ELF4、MAVS、MITA在牛感染性疾病中的作用奠定了基础。除了接头分子ELF4、MAVS、MITA基因外,本研究还相继克隆得到了牛干扰素信号通路中的重要分子MDA5、IRF3、IRF7、IRF9、DAI,这为今后研究牛固有免疫奠定基础,同时也为研究牛病原与宿主相互作用提供便利工具。5.3C~(pro)、Lbpro在牛源细胞上对干扰素信号通路的影响。首先分析了不同刺激物在不同细胞上对I型干扰素及信号通路相关分子转录的影响,结合已有的研究证实FMDV可以拮抗宿主I型干扰素的产生,FMDV的3C~(pro)和L~(pro)可以作用在不同的靶分子上来逃避宿主的固有免疫应答,拮抗I型干扰素的产生,本研究进一步发现3C~(pro)和L~(pro)不但能抑制I型干扰素的产生,抑制接头分子ELF4、MAVS,RIG-I样受体RIG-I、MDA5,转录因子IRF3、IRF7的转录,还能抑制I型干扰素和接头分子ELF4、MAVS、MITA激活的NF-κB、ISRE、Bo IFN-β、Bo IFN-χ启动子活性。Western Blot结果证实在BT和MDBK细胞上3C~(pro)和L~(pro)可以抑制poly(I:C)和Bo IFN-β诱导的NF-κB p65、Mx1、IRF3、IRF7、ELF4、MAVS的表达。总之,本研究系统分析了牛I型干扰素新成员IFN-ε、IFN-κ、IFN-χ的分子特征和生物学功能,初步建立了牛固有免疫信号通路研究的技术平台,利用该平台重点分析了接头分子ELF4、MAVS、MITA对干扰素信号通路的影响,系统地分析了口蹄疫病毒3C~(pro)、Lbpro逃避固有免疫应答的机制,为病毒免疫预防和抗病毒药物的研制提供理论依据和物质支持。
[Abstract]:Interferons (IFN) is a large type of multiple effect cytokine, which is seen in all vertebrate vertebrates, mainly responsible for the immune response function of the host virus infection. Different pathogenic infections tend to induce specific interferon members, and identify and systematically identify the immune response of the new members of interferon against the virus infection. Important significance. Inherent immunity is the first line of defense against pathogen invasion, and the antiviral inherent immune response begins with the pathogen associated molecular pattern identified by the intracellular pattern recognition receptor (PRRs).PRRs (PAMPs) to further induce I interferon through the transcription factor NF- kappa B and interferon regulatory factor (IRF) (especially IRF3 and IRF7). To create an antiviral state in order to promote the adaptive immune response and eventually to remove the virus infection from the body, the I type interferon plays an important role in the establishment of antiviral infection and host cell resistance to virus replication. Type (IFN-, IFN- kappa) and 1 new subfamilies containing 2 members (IFN- CHI 1, IFN- x 3), followed by a series of studies on the anti viral signal transduction and expression regulation of these four interferons. The specific research contents are as follows: 1. the construction and analysis of the gene pedestal of bovine I type interferon (I). A variety of on-line BLAST methods (BLAST, Basic) are used. Local Alignment Search Tool, a basic local alignment search tool) to sift the whole genome of cattle and comprehensively analyze the information of various interferons. It is found that bovine I type interferon gene is distributed on chromosome 8 and has evolved a new family of interferon with distinct characteristics, most notably the IFN- Chi subfamily of the IFN- Chi subfamily of the Chi subfamily. 4 members, with the characteristics of a new interferon, composed of 1 new interferon subgroups. The bovine type I interferon locus is divided into 2 large subloci in the direction of transcription. The length (746kp) is 1.85 and 2.31 times of the human (403kp) and mouse (323kb), and the interferon gene number (64) is also far superhuman (17) and rats (19). At the end of the seat 5 IFN- The delta pseudogenes and 4 IFN- x like molecules are mixed in 12 IFN- beta like genes, and the transcriptional direction of these genes are the same; the other regions of the loci are only 1 copies of the large number of IFN- a (13) and IFN- omega (24) families, which are located at the beginning of the genome, and the direction of the IFN- kappa and IFN- On the contrary, the characteristics of bovine IFN- e, IFN- kappa, IFN- CHI 1, IFN- x 3 and the antiviral signal transduction pathway were interspaced with the nearest interferon gene. First of all, bovine IFN- e, IFN- kappa, IFN- CHI 1, IFN- Chi 3 gene were cloned from the bovine liver genome, and the characteristics of these sequences were analyzed. Subsequently, the recombinant expression system of Escherichia coli was expressed in the Escherichia coli expression system. Bovine IFN- epsilon, IFN- kappa, IFN- CHI 1, IFN- Chi 3, and the anti bovine IFN- e, IFN- kappa, IFN- x polyclonal antibody were prepared on different systems. Bovine IFN- e, IFN- kappa, IFN- CHI 1, IFN- Chi 3 were sensitive to trypsin, to a certain degree heat resistance (42 and 60), and its antiviral activity could be specific. The neutralization of sexual polyclonal antibodies can also be blocked by the IFNAR1 and IFNAR2 antibodies of type I interferon receptor. Further studies have found that bovine IFN- e, IFN-, IFN- CHI 1, IFN- x 3 not only induce the transcription of interferon stimulating genes (such as ISGs, Mx1, ISG15, ISG56), but also can induce the expression of Mx1. These analyses indicate the present known members of the interferon type interferon. The effect of JAK-STAT signaling pathway on the transduction of antiviral signal.3. bovine IFN- e, IFN- kappa, IFN- 1, IFN- x 3 on the interferon signaling pathway. This experiment was carried out to analyze the regulation genes of bovine IFN- alpha A and IFN- CHI 1 on bovine testicular primary cells and compare the differences in the expression of these genes. The results showed that the bovine Bo IFN- CHI 1 samples were found. The expression of 40 differentially expressed genes was up-regulated in the treatment group; 80 differentially expressed genes were up-regulated and 1 genes were down regulated in the Bo IFN- alpha A sample treatment group. Then fluorescence quantitative PCR was used to further verify the regulation of these regulatory genes on bovine cells. The results showed that most of the up-regulated genes were ISGs genes, and OAS, Mx1, STA were selected randomly. T1, LGP2, GBP4, BST2, and PML genes were tested on MDBK, BT, BL cells to verify that these genes were up to varying degrees on different cells. The luciferase activity of the Chi -Luc promoter, which lays the foundation for the study of the related molecules of.4. bovine interferon signaling pathway for further development of bovine inherent immune signaling pathway. Retinoic acid inducible gene (RIG-I), melanoma differentiation related factor 5 (MDA5) is an important pattern recognition receptor for intracellular identification of dsRNA, and IRF3 and IRF7 are involved in the study. The important transcription factor of the inherent immune response, the transcription factor IRF9 participates in the induction of the production of I type interferon. ELF4, MAVS, and MITA are involved in the induction of I type interferon. In this study, the RT-PCR method was used to clone bovine joint molecule ELF4, MAVS, MITA C DNA, and analyzed. Its sequence characteristics, tissue expression and organelle localization, focus on its effect on interferon signaling pathway. The study shows that the ELF4, MAVS, MITA can induce the production of different I type interferons, promote the transcription and expression of signal pathway related molecules, and stimulate ISRE, NF- kappa B, Bo IFN- beta and Bo IFN- X promoter. Activated luciferase activity lays the foundation for further exploration of the role of ELF4, MAVS and MITA in bovine infectious diseases. Besides the joint molecules ELF4, MAVS, and MITA genes, this study has also cloned important components of the bovine interferon signal pathway, MDA5, IRF3, IRF7, IRF9, DAI, for the future study of bovine inherent immunity. The pestilence lays the foundation, and also provides a convenient tool for the study of the interaction of bovine pathogens and hosts,.5.3C~ (pro), and the effect of Lbpro on the interferon signaling pathway on bovine cells. First, the effects of different stimuli on I type interferon and signal transduction related molecules on different cells are analyzed. Combined with previous studies, it has been proved that FMDV can be antagonized. The production of anti host type I interferon, 3C~ (pro) and L~ (pro) of FMDV can act on different target molecules to escape the inherent immune response of the host and antagonize the production of I type interferon. This study further found that 3C~ (pro) and L~ (pro) can not only inhibit the generation of I type interferon, but also inhibit the receptor, transcription, transcription, transcription, transcription, and transcription. The transcription of factor IRF3, IRF7 can also inhibit the I type interferon and the joint molecule ELF4, MAVS, MITA activated NF- kappa B, ISRE, Bo IFN- beta. The molecular and biological functions of IFN- - E, IFN- - kappa, IFN- Chi were analyzed systematically, and a technical platform for the study of bovine inherent immune signal pathway was established. The influence of ELF4, MAVS, MITA on the interferon signal pathway was analyzed with the platform, and the 3C~ (pro) and Lbpro of foot and mouth disease virus (pro) and Lbpro were systematically analyzed. The mechanism of evasion of innate immune response provides theoretical basis and material support for the prevention of virus and the development of antiviral drugs.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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