犬poll启动子双向转录表达载体的构建及流感病毒基因组克隆

发布时间：2018-04-08 07:10

本文选题：流感病毒　切入点：犬RNA多聚酶I　出处：《吉林大学》2012年硕士论文

【摘要】：流感病毒属于正黏病毒家族（Orthomyxoviridae），按照其NP和M蛋白的抗原性不同，可以将其分为A、B和C三个血清型，所有动物流感病毒都属于A型。依据流感病毒表面糖蛋白血凝素（HA）和神经氨酸酶（NA）的抗原性差异，又可将A型流感病毒分为16个HA亚型和9个NA亚型。在水禽中可以分离到A型流感病毒的所有亚型。从人和多种动物（如猪、马、海洋哺乳动物、猫、犬和禽类）体内已经分离到多株A型流感病毒。在过去十年，高致病性禽流感H5N1和甲型流感H1N1已经向全世界展示流感病毒在人与动物之间的快速扩散和传播能力。H3N8亚型流感病毒主要引起马的呼吸系统感染，，但在2004年的美国，马源H3N8亚型流感病毒已经扩散到犬，引起犬呼吸道疾病的广泛流行。利用反向遗传系统拯救A型流感病毒对于流感病毒致病机理研究以及疫苗开发均具有重要意义。通过将流感病毒基因组8个片段克隆到人的RNA多聚酶I启动子下游构建能够转录病毒基因组的8个重组质粒，再将其与编码NP和三个多聚酶基因表达质粒共转染适宜细胞系即可拯救出具有感染性的流感病毒颗粒。后来，Hoffman等对这一反向遗传系统进行了改进，目前的拯救系统只需8个基于pol I/II的双向表达质粒而不是原来的12个质粒即可完成病毒拯救。然而，目前的拯救系统存在一个缺陷，那就是它是基于人的pol I启动子，病毒拯救只能在人和灵长类细胞上完成，对其它种属的细胞系，如MDCK并不支持。为了开发能够在MDCK细胞上直接拯救流感病毒的反向遗传系统，本研究从MDCK细胞基因组中扩增了犬的pol I启动子。PCR产物克隆到pTA2载体后测序，序列经BLAST比对，与已知的犬RNA多聚酶I启动子区同源性达93%。接下来，我们构建了基于犬pol I启动子的双向转录表达载体pVW3000，这一载体系统支持以同一个模板同时转录病毒基因组RNA和mRNA。首先，通过重叠PCR方法将犬pol I启动子、两个并列的BsmB I位点和33nt的鼠pol I终止子序列进行拼接。而后，约300bp的犬pol I启动子表达盒被反向插入到pVAX1载体CMV启动子和poly（A）之间的BamHI/EcoR I位点。最后，双向转录表达载体pVW3000经酶切和测序鉴定均正确。为了鉴定本研究所克隆的犬pol I启动子在MDCK细胞上的转录效率，我们还构建了荧光报告质粒pVW-M-GFP。简言之，将含流感病毒M蛋白非编码区侧翼序列的增强型绿色荧光蛋白基因克隆至pVW3000的BsmB I位点，重组质粒经酶切和测序均正确。接下来，我们利用A型流感病毒通用引物RT-PCR方法克隆了H3N8亚型流感病毒全基因组8个片段，并将其克隆至自行构建的双向转录表达载体pVW3000，成功获得7个重组质粒pVW3000-PB1, pVW3000-PA, pVW3000-HA, pVW3000-NA,pVW3000-NP, pVW3000-M和pVW3000-NS，重组质粒经酶切和测序鉴定均正确。这些研究为在MDCK细胞上直接拯救A型流感病毒以及进一步研究病毒致病机制和实现疫苗的快速制备奠定了坚实的基础。
[Abstract]:Influenza virus belongs to Orthomyxoviridaeae family. According to the antigenicity of NP and M proteins, influenza virus can be divided into three serotypes: Agna B and C, and all animal influenza viruses belong to type A.According to the antigenicity of influenza virus surface glycoprotein hemagglutinin (HA) and neuraminidase (NAA), influenza A virus can be divided into 16 HA subtypes and 9 na subtypes.All subtypes of influenza A virus can be isolated from waterfowl.A number of influenza A viruses have been isolated from humans and various animals, such as pigs, horses, marine mammals, cats, dogs and birds.In the past decade, highly pathogenic avian influenza H5N1 and influenza A H1N1 have demonstrated to the world the rapid spread and transmission of influenza viruses between humans and animals. H3N8 subtype influenza viruses mainly cause respiratory infections in horses, but in the United States in 2004,Equine H3N8 subtype influenza virus has spread to dogs, causing widespread prevalence of respiratory diseases in dogs.Rescuing influenza A virus by reverse genetic system is of great significance for the study of the pathogenic mechanism of influenza virus and the development of vaccine.Eight recombinant plasmids capable of transcription of influenza virus genome were constructed by cloning 8 fragments of influenza virus genome into human RNA polymerase I promoter downstream.The influenza virus particles could be saved by co-transfection with NP and three plasmids encoding polymerase gene into suitable cell lines.The reverse genetic system was improved by Hoffman et al. The current rescue system only needs 8 bidirectional expression plasmids based on pol I/II instead of 12 original plasmids to complete virus rescue.However, the current rescue system has a defect, that is, it is based on human pol I promoter, virus rescue can only be done on human and primate cells, and it does not support other cell lines, such as MDCK.In order to develop a reverse genetic system capable of directly saving influenza virus from MDCK cells, the dog pol I promoter. PCR product was amplified from the genome of MDCK cells and cloned into pTA2 vector. The sequence was sequenced by BLAST alignment.The homology was 933 with the known promoter of RNA polymerase I in dogs.Next, we constructed a bidirectional transcriptional expression vector pVW3000based on canine pol I promoter, which supports the simultaneous transcription of virus genomic RNA and mRNA with the same template.Firstly, the dog pol I promoter, two parallel BsmB I loci and the 33nt pol I Terminator sequence were spliced by overlapping PCR method.Then, about 300bp's canine pol I promoter cassette was inserted into the BamHI/EcoR I site between the pVAX1 vector CMV promoter and Poly A.Finally, the bidirectional transcriptional expression vector pVW3000 was confirmed by restriction endonuclease digestion and sequencing.In order to identify the transcriptional efficiency of the cloned canine pol I promoter in MDCK cells, we also constructed the fluorescent reporter plasmid pVW-M-GFP.In short, the enhanced green fluorescent protein gene containing the flanking region of M protein of influenza virus was cloned to the BsmB I site of pVW3000, and the recombinant plasmid was digested and sequenced correctly.Next, we cloned the whole genome of H3N8 subtype influenza virus by using the universal primer RT-PCR method of influenza A virus.Seven recombinant plasmids pVW3000-PB1, pVW3000-PA, pVW3000-HA, pVW3000-NAVW3000-NPN, pVW3000-M and pVW3000-NSN were successfully cloned into the self-constructed bidirectional transcriptional expression vector pVW3000. the recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing.These studies have laid a solid foundation for the direct rescue of influenza A virus in MDCK cells and the further study of the pathogenic mechanism of the virus and the rapid preparation of vaccine.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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