【摘要】：流感病毒反向遺傳系統(tǒng)能夠在體外對流感病毒的基因組進(jìn)行操作,可以極大的方便對流感病毒的研究。流感病毒最小的復(fù)制單位由核糖核蛋白復(fù)合物(ribonucleoprotein complex,RNP)組成。在流感病毒反向系統(tǒng)中,PB1、PB2、PA、NP蛋白可以識別由Ⅰ型RNA聚合酶啟動(dòng)子驅(qū)動(dòng)生成的負(fù)鏈病毒RNA,并以此為模板轉(zhuǎn)錄生成正鏈互補(bǔ)RNA及m RNA。真核生物Ⅰ型RNA聚合酶啟動(dòng)子具有種屬特異性。一種生物的Ⅰ型RNA聚合酶不能識別另一種生物的Ⅰ型RNA聚合酶啟動(dòng)子。研究者已經(jīng)克隆了人源、犬源、雞源、鼠源Ⅰ型RNA聚合酶啟動(dòng)子,并將其用于流感病毒反向遺傳系統(tǒng)的構(gòu)建。但是,目前為止,還沒有馬源Ⅰ型RNA聚合酶啟動(dòng)子的研究報(bào)道。因此,對于流感病毒的很多研究不能在馬源細(xì)胞上進(jìn)行�；诖�,在本研究中,利用真核生物Ⅰ型RNA聚合酶啟動(dòng)子的位置特點(diǎn),在Gen Bank數(shù)據(jù)庫和ensembl數(shù)據(jù)庫中的馬基因組中,檢索并確定了馬Ⅰ型RNA聚合酶啟動(dòng)子轉(zhuǎn)錄起始位點(diǎn)。以馬肺細(xì)胞基因組DNA為模板,通過PCR,擴(kuò)增得到了馬Ⅰ型RNA聚合酶啟動(dòng)子,并構(gòu)建了不同長度的馬Ⅰ型RNA聚合酶啟動(dòng)子驅(qū)動(dòng)的馬流感病毒小基因組復(fù)制系統(tǒng)。結(jié)果表明馬Ⅰ型RNA聚合酶啟動(dòng)子為500bp時(shí),可以發(fā)揮最大轉(zhuǎn)錄活性。用建立的馬流感病毒小基因組復(fù)制系統(tǒng),對比了兩株馬流感病毒聚合酶活性的差異,發(fā)現(xiàn)PA蛋白可能是決定兩者聚合酶活性差異的關(guān)鍵蛋白。在本研究中,還使用該系統(tǒng)證實(shí)了馬Mx A蛋白會影響流感病毒聚合酶活性。最后,利用馬Ⅰ型RNA聚合酶啟動(dòng)子構(gòu)建了馬流感病毒反向遺傳系統(tǒng),成功拯救出一株野生型馬流感病毒和一株重組型馬流感病毒。馬Ⅰ型RNA聚合酶啟動(dòng)子的成功克隆及應(yīng)用,豐富了我們對真核生物Ⅰ型RNA聚合酶啟動(dòng)子的認(rèn)識。利用馬Ⅰ型RNA聚合酶啟動(dòng)子構(gòu)建的流感病毒小基因組復(fù)制系統(tǒng)是在馬源細(xì)胞上研究流感病毒聚合酶活性的有效工具,而在此基礎(chǔ)上構(gòu)建的馬流感病毒反向系統(tǒng)將方便研究者在馬源細(xì)胞上對流感病毒進(jìn)行操作。最近的研究表明,人源、豬源、雞源、鴨源、鼠源IFITM蛋白都具有抗流感病毒活性。馬屬動(dòng)物是流感病毒的重要宿主,但對于馬源IFITM蛋白是否也具有抗流感病毒作用卻未見報(bào)道。在本研究中,通過在Gen Bank數(shù)據(jù)庫中檢索馬IFITM基因,設(shè)計(jì)了靶向馬IFITM基因的特異性引物。通過RT-PCR,擴(kuò)增得到了6個(gè)馬IFITM基因。首先,我們描述了馬IFITM m RNA的表達(dá)特性:通過熒光定量PCR,檢測了馬源細(xì)胞及組織中的IFITM m RNA表達(dá)水平,發(fā)現(xiàn)不同IFITM的m RNA表達(dá)水平有所差異;經(jīng)干擾素處理和流感病毒感染后,馬源細(xì)胞中IFITM m RNA表達(dá)水平會上調(diào);但是感染馬皰疹病毒后,馬源細(xì)胞中IFITM m RNA表達(dá)水平未發(fā)生變化。然后,為了明確馬IFITM蛋白的細(xì)胞定位,構(gòu)建了表達(dá)馬IFITM蛋白的真核表達(dá)載體,間接免疫熒光和Western blot實(shí)驗(yàn)都表明馬IFITM蛋白成功表達(dá);激光共聚焦實(shí)驗(yàn)的結(jié)果表明不同馬IFITM蛋白的細(xì)胞定位并不相同;其中,馬IFITM3蛋白與LAMP1蛋白呈現(xiàn)共定位現(xiàn)象,而其它馬IFITM蛋白不與LAMP1蛋白共定位。最后,通過瞬時(shí)轉(zhuǎn)染外源表達(dá)質(zhì)粒和構(gòu)建穩(wěn)定表達(dá)IFITM蛋白細(xì)胞系這兩種策略,利于基于Ⅰ型RNA聚合酶啟動(dòng)子的雙熒光素酶報(bào)告系統(tǒng),檢測了馬IFITM蛋白的抗流感活性:通過慢病毒感染實(shí)驗(yàn),建立了穩(wěn)定表達(dá)馬IFITM蛋白的MDCK細(xì)胞系;間接免疫熒光試驗(yàn)的結(jié)果表明,在MDCK細(xì)胞系中,馬IFITM蛋白都成功表達(dá);利用基于馬或犬Ⅰ型RNA聚合酶啟動(dòng)子的雙熒光素酶報(bào)告系統(tǒng),證實(shí)在細(xì)胞中過表達(dá)或者穩(wěn)定表達(dá)馬IFITM蛋白都會限制流感病毒復(fù)制。對馬源IFITM蛋白的抗流感病毒活性研究,將加深我們對IFITM蛋白抗流感病毒作用的理解,也為相應(yīng)的抗流感藥物的研發(fā)提供了理論基礎(chǔ)。2012年以來,在馬群中發(fā)現(xiàn)了三種新的黃病毒科病毒:馬丙型肝炎病毒(equine hepatitis C virus,EHCV)、EPg V(equine pegivirus)、泰勒病相關(guān)病毒(Theiler’s disease-associated virus,TDAV)。目前,在世界范圍內(nèi),關(guān)于這三種新發(fā)馬病病毒的報(bào)道較少。在國內(nèi)的馬群中,也沒有這三種病毒的流行病學(xué)報(bào)道。在2014年和2015年,我們從國內(nèi)的馬群中采集了177份血清樣品。通過PCR,在血清樣品中,檢測到了6份EHCV陽性樣品和2份EPg V陽性樣品。在檢測過程中,沒有發(fā)現(xiàn)這兩種病毒共感染的證據(jù)。在所有樣品中沒有檢測到TDAV核酸。對EHCV的NS5B基因、NS3基因、5’非轉(zhuǎn)錄區(qū)序列以及EPg V的NS3基因序列進(jìn)行進(jìn)化樹分析。結(jié)果表明,EHCV和EPg V已經(jīng)進(jìn)化為兩個(gè)分支,而且這兩支病毒都在國內(nèi)馬群中流行。本研究豐富了我們對這三種新發(fā)黃病毒科馬病病毒的流行地區(qū)以及基因進(jìn)化的認(rèn)識。
[Abstract]:Influenza virus reverse genetic system can manipulate the genome of influenza virus in vitro, so it is very convenient to study influenza virus. The smallest replication unit of influenza virus is composed of ribonucleoprotein complex (RNP). In influenza virus reverse system, PB1, PB2, PA, NP proteins can be recognized by I. Eukaryotic type I RNA polymerase promoters are species specific. One organism's type I RNA polymerase does not recognize another organism's type I RNA polymerase promoter. Chicken-derived, mouse-derived RNA polymerase promoter and its application in the construction of reverse genetic system of influenza viruses have not been reported so far. Therefore, many studies on influenza viruses can not be carried out on horse-derived cells. Location characteristics of polymerase promoter were studied. The transcription initiation sites of horse type I RNA polymerase promoter were searched and identified in the horse genome of Gen Bank and Ensembl databases. The horse type I RNA polymerase promoter was amplified by PCR using the genomic DNA of horse lung cells as template, and the horse type I RNA polymerase promoter with different lengths was constructed. The results showed that the promoter of equine influenza virus type I RNA polymerase could maximize the transcriptional activity when it was 500 bp. The difference of polymerase activity between the two equine influenza viruses was compared with the established system of equine influenza virus small genome replication. In this study, we also used this system to confirm that horse Mx A protein can affect the polymerase activity of influenza virus. Finally, a reverse genetic system of equine influenza virus was constructed using horse type I RNA polymerase promoter, and a wild equine influenza virus and a recombinant equine influenza virus were successfully rescued. The successful cloning and application of the promoter of NA polymerase enriched our understanding of the promoter of Eukaryotic type I RNA polymerase.The replication system of influenza virus small genome constructed by the promoter of horse type I RNA polymerase is an effective tool for studying the polymerase activity of influenza virus in horse-derived cells. The virus reverse system will facilitate researchers to operate on influenza virus on horse origin cells. Recent studies have shown that IFITM protein from human, pig, chicken, duck * and mouse sources has anti influenza activity. Equine is an important host of influenza virus, but it has not reported whether IFITM protein has anti influenza effect. In this study, six horse IFITM genes were amplified by RT-PCR. Firstly, we described the expression characteristics of horse IFITM RNA: IFITM RNA expression levels in horse-derived cells and tissues were detected by fluorescence quantitative PCR. It was found that the expression level of IFITM m RNA was different in different IFITM cells; IFITM m RNA expression level was up-regulated in horse-derived cells after treatment with interferon and infection with influenza virus; however, the expression level of IFITM m RNA in horse-derived cells did not change after infection with equine herpes virus. Eukaryotic expression vectors, indirect immunofluorescence and Western blot assays showed that equine IFITM protein was successfully expressed; confocal laser scanning showed that different equine IFITM proteins had different cellular localization; among them, equine IFITM3 protein and LAMP1 protein were co-localized, while other equine IFITM proteins were not co-localized with LAMP1 protein. Then, two strategies, transient transfection of exogenous expression plasmids and construction of stable IFITM expression cell lines, were used to detect the anti-influenza activity of equine IFITM proteins by using a dual luciferase reporting system based on type I RNA polymerase promoter. The results of epidemic fluorescence assay showed that equine IFITM protein was successfully expressed in MDCK cell lines, and the double luciferase reporting system based on horse or dog type I RNA polymerase promoter was used to confirm that overexpression or stable expression of equine IFITM protein in cells would restrict influenza virus replication. Since 2012, three new Flaviviridae viruses have been found in horses: equine hepatitis C virus (EHCV), EPg virus (equine pegivirus), and Theiler disease-related virus (Theiler). At present, there are few reports about these three new equine disease s viruses in the world. There is no epidemiological report of these three viruses in domestic horses. In 2014 and 2015, 177 serum samples were collected from domestic horses. TDAV nucleic acid was not detected in all samples. Evolutionary tree analysis was performed on the NS5B gene, NS3 gene, 5'non-transcriptional region sequence and NS3 gene sequence of EHCV. This study enriches our understanding of the epidemic areas and genetic evolution of the three new flavivirus equine diseases viruses.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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