本文選題：豬瘟病毒 + 反向遺傳操作　； 參考：《武漢大學(xué)》2015年博士論文

【摘要】：豬瘟病毒(classical swine fever virus, CSFV)是豬的高致死性、烈性傳染病——豬瘟(classical swine fever, CSF)的病原體。豬瘟的發(fā)生和流行對(duì)豬養(yǎng)殖業(yè)造成重大的經(jīng)濟(jì)損失。CSFV和牛病毒性腹瀉病毒(bovine viral diarrhea virus, BVDV)、羊邊界病病毒(border disease virus, BDV)同屬于黃病毒科(Flaviviridae)瘟病毒屬(Pestivirus)。CSFV的基因組為長(zhǎng)度約12.3kb的單股正鏈RNA,包含兩端的非編碼區(qū)(untranslated region, UTR)和中間一個(gè)大的開放閱讀框(open reading frame, ORF)。ORF編碼一個(gè)大的多聚蛋白,該多聚蛋白經(jīng)宿主細(xì)胞和病毒編碼的蛋白酶水解加工產(chǎn)生具有功能的成熟蛋白,參與完成病毒的生命周期。作為RNA病毒研究的一個(gè)重要平臺(tái),反向遺傳操作技術(shù)在CSFV基因組結(jié)構(gòu)功能研究以及疫苗研發(fā)等方面具有重要作用。我們利用豬的RNA聚合酶Ⅰ(polⅠ)啟動(dòng)子驅(qū)動(dòng)細(xì)胞內(nèi)DNA轉(zhuǎn)錄產(chǎn)生RNA的特性,構(gòu)建了一個(gè)新型的拯救CSFV反向遺傳操作系統(tǒng)。將豬RNA pol Ⅰ啟動(dòng)子序列插入CSFV基因組cDNA的5'末端,鼠polⅠ終止子序列插入其3'末端,成功構(gòu)建了CSFV石門株和豬瘟疫苗C株的全長(zhǎng)cDNA感染性克隆pSPTI/SM和pSPTI/C。CSFV基因組cDNA在宿主細(xì)胞內(nèi)利用細(xì)胞pol Ⅰ驅(qū)動(dòng)豬pol Ⅰ啟動(dòng)子起始轉(zhuǎn)錄以及鼠的pol Ⅰ終止子終止轉(zhuǎn)錄,合成病毒基因組RNA,該vRNA沒(méi)有5'端加帽修飾及3'poly(A)尾,具有精確的5'UTR和3'UTR末端；同時(shí)vRNA可作為mRNA直接翻譯、加工得到病毒蛋白。分別將pSPTI/SM和pSPTI/C直接轉(zhuǎn)染PK-15細(xì)胞,拯救出相應(yīng)的CSFV強(qiáng)毒石門株和豬瘟疫苗C株病毒。這種基于polⅠ啟動(dòng)子的反向遺傳操作系統(tǒng)能產(chǎn)生具有精確末端基因組的CSFV,且具有更高的拯救效率。以豬瘟疫苗C株的cDNA感染性克隆pSPTI/C為骨架,用強(qiáng)毒石門株UTR替換豬瘟疫苗C株的對(duì)應(yīng)區(qū)域,構(gòu)建獲得了嵌合重組病毒株vC/SM 5UTR、 vC/SM3'UTR和vC/SMUTRs。特性研究表明,與豬瘟疫苗C株相比,石門株非編碼區(qū)替換顯著增強(qiáng)了重組嵌合病毒的復(fù)制能力,其復(fù)制效率由高至低依次為vC/SMUTRs、vC/SM3UTR和vC/SM 5'UTR;且重組病毒在PK-15細(xì)胞上形成蝕斑的能力也顯示出與其病毒復(fù)制效率一致的趨勢(shì)。嵌合重組病毒在PK-15細(xì)胞連續(xù)傳代后其生長(zhǎng)特性保持穩(wěn)定。CSFV非結(jié)構(gòu)蛋白NS2作為病毒編碼的一種自切割半胱氨酸蛋白酶,對(duì)NS2-3前體蛋白之間的不完全加工而釋放復(fù)制復(fù)合物關(guān)鍵因子NS3,NS2通過(guò)調(diào)節(jié)NS2-3的切割效率進(jìn)而調(diào)節(jié)病毒基因組的復(fù)制。為了研究NS2蛋白在CSFV生命周期中的作用,利用反向遺傳操作進(jìn)行特異性位點(diǎn)突變分析,我們重點(diǎn)探討了NS2 N端跨膜區(qū)的結(jié)構(gòu)與功能。結(jié)果表明,NS2 N端NS2/D60A, NS2/D60K和NS2/D78K特異性位點(diǎn)突變完全妨礙了CSFV感染性病毒產(chǎn)生；NS2/R100A突變顯著降低感染性病毒滴度；NS2/T37A、NS2/K52A、NS2/D78A及NS2/W85A突變不影響產(chǎn)生病毒的能力。通過(guò)構(gòu)建單順?lè)醋訌?fù)制子研究證實(shí),NS2/D60A、 NS2/D60K和NS2/D78K位點(diǎn)突變導(dǎo)致病毒基因組喪失復(fù)制能力,NS2/R100A使基因組復(fù)制效率顯著降低,NS2/T37A、NS2/K52A、NS2/D78A和NS2/W85A基因組復(fù)制效率與野生型一致。表達(dá)Rluc活性重組病毒vA187-Rluc和vA187-Rluc/R100A特性分析與復(fù)制子結(jié)果顯示,NS2通過(guò)調(diào)節(jié)病毒基因組RNA復(fù)制進(jìn)而影響感染性病毒的產(chǎn)生。體外NS2-3前體蛋白生化分析結(jié)果顯示,NS2 N端氨基酸點(diǎn)突變不影響NS2-3的切割效率和NS2-NS2自身的相互作用及蛋白穩(wěn)定性；雙順?lè)醋訌?fù)制子報(bào)告系統(tǒng)分析發(fā)現(xiàn),NS2蛋白對(duì)病毒基因組的復(fù)制具有負(fù)調(diào)節(jié)作用；NS2氨基酸對(duì)基因組復(fù)制的調(diào)節(jié)作用不依賴NS2-3前體蛋白的加工效率。將致死突變體體外轉(zhuǎn)錄產(chǎn)物RNA轉(zhuǎn)染細(xì)胞,經(jīng)細(xì)胞連續(xù)傳代獲得感染性病毒。全基因組序列分析顯示,病毒的恢復(fù)在于NS2/D60A和NS2/D60K位點(diǎn)的回復(fù)突變或NS2/D78K擬回復(fù)突變?yōu)镹S2/K78E; NS2/R100A突變體的第二位點(diǎn)補(bǔ)償突變NS2／I90L也導(dǎo)致病毒滴度達(dá)到野生型水平,暗示CSFV NS2 N端跨膜結(jié)構(gòu)域之間存在相互作用。我們的研究成果為進(jìn)一步理解CSFV基因組結(jié)構(gòu)與功能、致病分子機(jī)制和發(fā)展基因工程疫苗奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Classical swine fever virus (CSFV) is the pathogen of pig's high death, severe infectious disease, classical swine fever (CSF). The occurrence and epidemic of swine fever have caused major economic losses to the pig breeding industry, such as.CSFV and bovine viral diarrhea virus (bovine viral), sheep border disease virus. Sease virus, BDV) with the genome of the genus Flaviviridae (Pestivirus).CSFV, the genome of the genus Pestivirus.CSFV is a single strand RNA with a length of approximately 12.3kb, containing a non coding region at both ends (untranslated region, UTR) and a large open reading frame (open), which encodes a large polyprotein, the polyprotein. The hydrolytic protease encoded by host cells and viruses produce functional mature proteins and participate in the completion of the life cycle of the virus. As an important platform for RNA virus research, reverse genetic manipulation plays an important role in the research of CSFV genome structure function and vaccine development. We use the RNA polymerase I of pig I. (pol i) the characteristics of DNA transcription produced by the promoter in the cell to produce RNA, a new reverse CSFV operation system for saving the RNA Pol I was inserted into the 5'terminal of the CSFV genome cDNA, and the mouse Pol I terminator sequence was inserted into the 3' terminal, and the full-length infection of the CSFV Shimen strain and the swine fever vaccine was successfully constructed. The clone pSPTI/SM and pSPTI/C.CSFV genome cDNA use cell Pol I to drive the starting transcription of porcine Pol I promoter and the termination transcription of Pol I terminator of rat Pol I, to synthesize the genomic RNA of the virus, which has no 5'end cap modification and 3'poly (A) tail. PSPTI/SM and pSPTI/C were directly transfected into PK-15 cells to save the corresponding CSFV virulent Shimen strain and the swine fever vaccine C strain. This reverse genetic operating system based on Pol I promoter could produce CSFV with precise terminal genome and have higher saving efficiency. CDNA vaccine C strain cDNA The infectious clone pSPTI/C was the skeleton, and the corresponding region of the swine fever vaccine C strain was replaced with the strong virus Shimen strain UTR. The characterization of vC/SM 5UTR, vC/SM3'UTR and vC/SMUTRs. of the chimeric recombinant virus strain showed that the replication ability of the recombinant chimeric virus was significantly increased by the non coding region substitution of the swine fever vaccine C strain, and the replication efficiency of the recombinant chimeric virus was significantly increased. From high to low, vC/SMUTRs, vC/SM3UTR and vC/SM 5'UTR are in turn, and the ability of recombinant virus to form plaque on PK-15 cells also shows a tendency to be consistent with the replication efficiency of the virus. The chimeric recombinant virus maintains stable.CSFV non structural egg white NS2 as a self cut cysteine encoded by the virus after continuous passage of PK-15 cells. The release of replication complex key factor, NS3, is released from the incomplete processing of NS2-3 precursor proteins. NS2 regulates the replication of the virus genome by regulating the cutting efficiency of NS2-3. In order to study the role of the NS2 protein in the life cycle of the CSFV, the specific site mutation analysis is carried out by reverse genetic manipulation. We focus on the analysis of the specific site mutation. The structure and function of the NS2 N end transmembrane region were discussed. The results showed that the NS2/D60A, NS2/D60K and NS2/D78K specific site mutations at the NS2 N end completely hindered the production of CSFV infected virus; NS2/R100A mutation significantly reduced the titer of the infected virus; NS2/T37A, NS2/K52A, NS2/D78A and mutation did not affect the ability to produce the virus. The anti subclone studies confirmed that NS2/D60A, NS2/D60K and NS2/D78K mutations cause the virus genome to lose replication ability, and NS2/R100A makes the genome replication efficiency significantly reduced. The genomic replication efficiency of NS2/T37A, NS2/K52A, NS2/D78A and NS2/W85A is in accordance with the wild type. The vA187-Rluc and vA187-Rluc/R100A characteristics of the Rluc active recombinant virus are expressed. The analysis and replicator showed that NS2 could affect the production of infectious virus by regulating viral genome RNA replication. The biochemical analysis of NS2-3 precursor protein in vitro showed that the mutation of NS2 N terminal amino acid point did not affect the cutting efficiency of NS2-3 and the interaction of NS2-NS2 itself and protein stability; the analysis of the BIS CIS counter replicon system analysis It was found that NS2 protein had a negative regulating effect on the replication of the virus genome, and the regulation of NS2 amino acid on genome replication was not dependent on the processing efficiency of NS2-3 precursor protein. The transfected cells of the lethal mutant in vitro RNA were transfected to the cells and the infected virus was obtained through the cell continuous passage. The whole genome sequence analysis showed that the virus was recovered in the genome sequence. The response mutation or NS2/D78K pseudo recovery mutation at the NS2/D60A and NS2/D60K sites is NS2/K78E, and the second locus compensation mutation NS2 / I90L of the NS2/R100A mutant also causes the virus titer to reach the wild type, suggesting the existence of the interaction between the CSFV NS2 N end transmembrane domains. Our research results are the further understanding of the CSFV genome structure. It laid a solid foundation for function, pathogenic molecular mechanism and development of genetic engineering vaccine.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.651

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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本文編號(hào)：2055594