猪瘟病毒新型反向遗传操作技术平台的建立和病毒复制调控研究

发布时间：2018-06-23 03:39

本文选题：猪瘟病毒 + 反向遗传操作　；参考：《武汉大学》2015年博士论文

【摘要】：猪瘟病毒(classical swine fever virus, CSFV)是猪的高致死性、烈性传染病——猪瘟(classical swine fever, CSF)的病原体。猪瘟的发生和流行对猪养殖业造成重大的经济损失。CSFV和牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)、羊边界病病毒(border disease virus, BDV)同属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)。CSFV的基因组为长度约12.3kb的单股正链RNA,包含两端的非编码区(untranslated region, UTR)和中间一个大的开放阅读框(open reading frame, ORF)。ORF编码一个大的多聚蛋白,该多聚蛋白经宿主细胞和病毒编码的蛋白酶水解加工产生具有功能的成熟蛋白,参与完成病毒的生命周期。作为RNA病毒研究的一个重要平台,反向遗传操作技术在CSFV基因组结构功能研究以及疫苗研发等方面具有重要作用。我们利用猪的RNA聚合酶Ⅰ(polⅠ)启动子驱动细胞内DNA转录产生RNA的特性,构建了一个新型的拯救CSFV反向遗传操作系统。将猪RNA pol Ⅰ启动子序列插入CSFV基因组cDNA的5'末端,鼠polⅠ终止子序列插入其3'末端,成功构建了CSFV石门株和猪瘟疫苗C株的全长cDNA感染性克隆pSPTI/SM和pSPTI/C。CSFV基因组cDNA在宿主细胞内利用细胞pol Ⅰ驱动猪pol Ⅰ启动子起始转录以及鼠的pol Ⅰ终止子终止转录,合成病毒基因组RNA,该vRNA没有5'端加帽修饰及3'poly(A)尾,具有精确的5'UTR和3'UTR末端；同时vRNA可作为mRNA直接翻译、加工得到病毒蛋白。分别将pSPTI/SM和pSPTI/C直接转染PK-15细胞,拯救出相应的CSFV强毒石门株和猪瘟疫苗C株病毒。这种基于polⅠ启动子的反向遗传操作系统能产生具有精确末端基因组的CSFV,且具有更高的拯救效率。以猪瘟疫苗C株的cDNA感染性克隆pSPTI/C为骨架,用强毒石门株UTR替换猪瘟疫苗C株的对应区域,构建获得了嵌合重组病毒株vC/SM 5UTR、 vC/SM3'UTR和vC/SMUTRs。特性研究表明,与猪瘟疫苗C株相比,石门株非编码区替换显著增强了重组嵌合病毒的复制能力,其复制效率由高至低依次为vC/SMUTRs、vC/SM3UTR和vC/SM 5'UTR;且重组病毒在PK-15细胞上形成蚀斑的能力也显示出与其病毒复制效率一致的趋势。嵌合重组病毒在PK-15细胞连续传代后其生长特性保持稳定。CSFV非结构蛋白NS2作为病毒编码的一种自切割半胱氨酸蛋白酶,对NS2-3前体蛋白之间的不完全加工而释放复制复合物关键因子NS3,NS2通过调节NS2-3的切割效率进而调节病毒基因组的复制。为了研究NS2蛋白在CSFV生命周期中的作用,利用反向遗传操作进行特异性位点突变分析,我们重点探讨了NS2 N端跨膜区的结构与功能。结果表明,NS2 N端NS2/D60A, NS2/D60K和NS2/D78K特异性位点突变完全妨碍了CSFV感染性病毒产生；NS2/R100A突变显著降低感染性病毒滴度；NS2/T37A、NS2/K52A、NS2/D78A及NS2/W85A突变不影响产生病毒的能力。通过构建单顺反子复制子研究证实,NS2/D60A、 NS2/D60K和NS2/D78K位点突变导致病毒基因组丧失复制能力,NS2/R100A使基因组复制效率显著降低,NS2/T37A、NS2/K52A、NS2/D78A和NS2/W85A基因组复制效率与野生型一致。表达Rluc活性重组病毒vA187-Rluc和vA187-Rluc/R100A特性分析与复制子结果显示,NS2通过调节病毒基因组RNA复制进而影响感染性病毒的产生。体外NS2-3前体蛋白生化分析结果显示,NS2 N端氨基酸点突变不影响NS2-3的切割效率和NS2-NS2自身的相互作用及蛋白稳定性；双顺反子复制子报告系统分析发现,NS2蛋白对病毒基因组的复制具有负调节作用；NS2氨基酸对基因组复制的调节作用不依赖NS2-3前体蛋白的加工效率。将致死突变体体外转录产物RNA转染细胞,经细胞连续传代获得感染性病毒。全基因组序列分析显示,病毒的恢复在于NS2/D60A和NS2/D60K位点的回复突变或NS2/D78K拟回复突变为NS2/K78E; NS2/R100A突变体的第二位点补偿突变NS2／I90L也导致病毒滴度达到野生型水平,暗示CSFV NS2 N端跨膜结构域之间存在相互作用。我们的研究成果为进一步理解CSFV基因组结构与功能、致病分子机制和发展基因工程疫苗奠定了坚实的基础。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.651

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