本文選題：牛I型干擾素 + 生物學(xué)特性　； 參考：《東北農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：干擾素(interferons,IFN)是見(jiàn)于所有有頜類(lèi)脊椎動(dòng)物的一大類(lèi)多效應(yīng)細(xì)胞因子,主要擔(dān)負(fù)宿主抗病毒感染免疫應(yīng)答功能,不同的病原感染往往傾向于誘導(dǎo)表達(dá)出特定的干擾素成員,發(fā)現(xiàn)并系統(tǒng)鑒定干擾素新成員對(duì)抗病毒感染免疫應(yīng)答具有重要的意義。固有免疫是機(jī)體抵御病原入侵的第一道防線,抗病毒固有免疫反應(yīng)起始于細(xì)胞內(nèi)模式識(shí)別受體(PRRs)。PRRs識(shí)別的病原相關(guān)分子模式(PAMPs)通過(guò)轉(zhuǎn)錄因子NF-κB和干擾素調(diào)節(jié)因子(IRF)(尤其是IRF3和IRF7)來(lái)進(jìn)一步誘導(dǎo)I型干擾素的產(chǎn)生,建立抗病毒狀態(tài)以促進(jìn)適應(yīng)性免疫應(yīng)答,最終從機(jī)體內(nèi)清除病毒感染,所以I型干擾素在建立抗病毒感染狀態(tài)和宿主細(xì)胞抵抗病毒復(fù)制方面具有重要的作用。本研究首先系統(tǒng)分析了牛I型干擾素基因座位,定位到牛的2個(gè)干擾素新亞型(IFN-ε、IFN-κ)以及1個(gè)包含有2個(gè)成員的新亞族(IFN-χ1、IFN-χ3),隨后圍繞這四種干擾素的抗病毒信號(hào)轉(zhuǎn)導(dǎo)與表達(dá)調(diào)控進(jìn)行了一系列的研究,具體研究?jī)?nèi)容如下:1.牛I型干擾素基因座圖譜的構(gòu)建與分析。綜合運(yùn)用多種在線BLAST方法(BLAST,Basic Local Alignment Search Tool,基本的基于局部比對(duì)的搜索工具)對(duì)牛全基因組篩選,全面分析各型干擾素的信息,發(fā)現(xiàn)牛I型干擾素基因排布于8號(hào)染色體上,并進(jìn)化出了具有鮮明特征的干擾素新家族,其中最為引人注目當(dāng)屬I(mǎi)FN-χ亞族。IFN-χ亞族具有4個(gè)成員,具備全新干擾素的特征,組成了1個(gè)新干擾素亞族。牛I型干擾素基因座按轉(zhuǎn)錄方向分成2個(gè)大的亞座位,長(zhǎng)度(746kp)分別是人(403kp)和鼠(323kb)的1.85和2.31倍,并且干擾素基因數(shù)目(64個(gè))也遠(yuǎn)超人(17個(gè))與鼠(19個(gè))。在座位末端5個(gè)IFN-δ假基因與4個(gè)IFN-χ樣分子混雜在12個(gè)IFN-β樣基因之中,并且這些基因的轉(zhuǎn)錄方向均相同;基因座其它區(qū)域則被數(shù)量龐大的IFN-α(13個(gè))和IFN-ω(24個(gè))家族所占據(jù)。IFN-κ和IFN-ε僅存在1個(gè)拷貝序列,位于基因組的起始端,且IFN-κ的轉(zhuǎn)錄方向與IFN-β相反,其與最近的干擾素基因間隔6.561Mb。2.牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3的特性和抗病毒信號(hào)轉(zhuǎn)導(dǎo)通路。本研究首先從牛肝基因組中克隆得到了牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3基因,并分析了這些序列的特征。隨后在大腸桿菌表達(dá)系統(tǒng)表達(dá)了重組牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3的成熟肽序列,并制備了抗牛IFN-ε、IFN-κ、IFN-χ多克隆抗體,在不同的系統(tǒng)上測(cè)定了其抗病毒活性。牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3均對(duì)胰酶敏感,一定程度上耐熱(42℃和60℃)耐酸堿(p H2.0和p H10.0),其抗病毒活性可被特異性多克隆抗體中和,也可被I型干擾素受體IFNAR1和IFNAR2抗體阻斷。進(jìn)一步研究發(fā)現(xiàn)牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3不僅可誘導(dǎo)干擾素刺激基因(ISGs,如Mx1,ISG15,ISG56)的轉(zhuǎn)錄,也可誘導(dǎo)Mx1,STAT1,NF-κB p65的表達(dá),這些分析表明目前已知的I型干擾素成員均依賴(lài)JAK-STAT信號(hào)通路轉(zhuǎn)導(dǎo)抗病毒信號(hào)。3.牛IFN-ε、IFN-κ、IFN-χ1、IFN-χ3對(duì)干擾素信號(hào)通路的影響。本試驗(yàn)采用轉(zhuǎn)錄組測(cè)序的方式分析了在牛睪丸原代細(xì)胞上牛IFN-αA和IFN-χ1的調(diào)控基因并比較了這些基因的表達(dá)差異,結(jié)果發(fā)現(xiàn)牛Bo IFN-χ1樣品處理組有40個(gè)差異基因表達(dá)上調(diào);Bo IFN-αA樣品處理組有80個(gè)差異基因表達(dá)上調(diào),1個(gè)基因表達(dá)下調(diào)。隨后采用熒光定量PCR進(jìn)一步驗(yàn)證了這些調(diào)控基因的在牛源細(xì)胞上的表達(dá)調(diào)控,結(jié)果顯示上調(diào)基因大部分屬于ISGs基因,隨機(jī)選取OAS、Mx1,STAT1、LGP2、GBP4、BST2、PML基因在MDBK、BT、BL細(xì)胞上進(jìn)行驗(yàn)證,證實(shí)這些基因在不同細(xì)胞上均有不同程度的上調(diào)。此外,牛I型干擾素可在BL和BT細(xì)胞上啟動(dòng)商品化的人NF-κB-Luc和ISRE-Luc報(bào)告載體的熒光素酶活性,并可激活本研究構(gòu)建的Bo IFNβ-Luc和Bo IFNχ-Luc啟動(dòng)子報(bào)告載體的熒光素酶活性,為進(jìn)一步開(kāi)展牛固有免疫信號(hào)通路的研究奠定了基礎(chǔ)。4.牛干擾素信號(hào)通路相關(guān)分子的研究。維甲酸誘導(dǎo)基因(RIG-I)、黑素瘤分化相關(guān)因子5(MDA5)是胞內(nèi)識(shí)別dsRNA的重要模式識(shí)別受體,IRF3和IRF7是參與固有免疫應(yīng)答的重要轉(zhuǎn)錄因子,轉(zhuǎn)錄因子IRF9參與誘導(dǎo)I型干擾素的產(chǎn)生,ELF4、MAVS、MITA作為信號(hào)通路中的接頭分子參與誘導(dǎo)I型干擾素的產(chǎn)生。本研究運(yùn)用RT-PCR的方法從犢牛原代腎細(xì)胞中克隆獲得了牛接頭分子ELF4、MAVS、MITA的c DNA,并分析了其序列特征,組織表達(dá)和細(xì)胞器定位,重點(diǎn)研究了其對(duì)干擾素信號(hào)通路的影響。研究表明在不同的細(xì)胞上接頭分子ELF4、MAVS、MITA能誘導(dǎo)產(chǎn)生不同的I型干擾素,促進(jìn)信號(hào)通路相關(guān)分子的轉(zhuǎn)錄與表達(dá),還能刺激ISRE、NF-κB、Bo IFN-β和Bo IFN-χ啟動(dòng)子激活的熒光素酶活性,為今后進(jìn)一步探討接頭分子ELF4、MAVS、MITA在牛感染性疾病中的作用奠定了基礎(chǔ)。除了接頭分子ELF4、MAVS、MITA基因外,本研究還相繼克隆得到了牛干擾素信號(hào)通路中的重要分子MDA5、IRF3、IRF7、IRF9、DAI,這為今后研究牛固有免疫奠定基礎(chǔ),同時(shí)也為研究牛病原與宿主相互作用提供便利工具。5.3C~(pro)、Lbpro在牛源細(xì)胞上對(duì)干擾素信號(hào)通路的影響。首先分析了不同刺激物在不同細(xì)胞上對(duì)I型干擾素及信號(hào)通路相關(guān)分子轉(zhuǎn)錄的影響,結(jié)合已有的研究證實(shí)FMDV可以拮抗宿主I型干擾素的產(chǎn)生,FMDV的3C~(pro)和L~(pro)可以作用在不同的靶分子上來(lái)逃避宿主的固有免疫應(yīng)答,拮抗I型干擾素的產(chǎn)生,本研究進(jìn)一步發(fā)現(xiàn)3C~(pro)和L~(pro)不但能抑制I型干擾素的產(chǎn)生,抑制接頭分子ELF4、MAVS,RIG-I樣受體RIG-I、MDA5,轉(zhuǎn)錄因子IRF3、IRF7的轉(zhuǎn)錄,還能抑制I型干擾素和接頭分子ELF4、MAVS、MITA激活的NF-κB、ISRE、Bo IFN-β、Bo IFN-χ啟動(dòng)子活性。Western Blot結(jié)果證實(shí)在BT和MDBK細(xì)胞上3C~(pro)和L~(pro)可以抑制poly(I:C)和Bo IFN-β誘導(dǎo)的NF-κB p65、Mx1、IRF3、IRF7、ELF4、MAVS的表達(dá)�？傊�,本研究系統(tǒng)分析了牛I型干擾素新成員IFN-ε、IFN-κ、IFN-χ的分子特征和生物學(xué)功能,初步建立了牛固有免疫信號(hào)通路研究的技術(shù)平臺(tái),利用該平臺(tái)重點(diǎn)分析了接頭分子ELF4、MAVS、MITA對(duì)干擾素信號(hào)通路的影響,系統(tǒng)地分析了口蹄疫病毒3C~(pro)、Lbpro逃避固有免疫應(yīng)答的機(jī)制,為病毒免疫預(yù)防和抗病毒藥物的研制提供理論依據(jù)和物質(zhì)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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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