基于CMV启动子构建CVB3感染性克隆的初步研究

发布时间：2018-01-10 16:35

本文关键词：基于CMV启动子构建CVB3感染性克隆的初步研究　出处：《安徽大学》2010年硕士论文　论文类型：学位论文

【摘要】： RNA病毒基因组较DNA病毒难以操作,对其进行研究必须先通过反向遗传学技术构建其感染性克隆。随着各类研究方法和实验技术手段的不断更新,反向遗传学技术也在与时俱进,各种新型的回复系统不断建立。传统的基于原核启动子的回复系统的局限都是感染哺乳细胞时需要通过烦琐的步骤来产生RNA聚合酶,增加了实验操作的难度,而且一些产生聚合酶的手段,如使用辅助病毒等,本身就会对实验有所干扰甚至由于辅助病毒对细胞的病变效应(CPE)会影响负载细胞的生存而导致病毒回复无法进行。真核细胞都可以合成自身的RNA聚合酶,而且这些RNA聚合酶是真核细胞的内源蛋白,不需要通过辅助手段(如上述的辅助病毒,辅助质粒,构建细胞系等)去产生就可以启动真核启动子的表达,十分方便,也从根本上杜绝了原核RNA聚合酶回复系统利用辅助病毒等对细胞CPE的发生；另外真核RNA聚合酶有的定位在细胞核,有的定位于细胞质,为回复各种类型的病毒都提供了可能。目前,真核RNA聚合酶回复系统的优越性已经越来越明显,成为RNA病毒回复系统的研究热点。柯萨奇病毒(Coxsackie virus, CV)是一类常见的经呼吸道和消化道感染人体的病毒,属于微小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus genus)。柯萨奇病毒血清型分A,B两组,A组有24个血清型,B组有6个血清型,能起一系列的临床疾病。科萨奇病毒B组3型(CVB3)基因组为单股正链RNA,编码一个大的开放读码框包括2207个氨基酸,两侧为5’非编码区(5’-NTR)和3’多聚腺苷非编码区。5’-NTR包括一个Ⅰ型核糖体进入位点(IRE)使5’帽子能够独立翻译成病毒多聚蛋白的起始。由于单股正链结构,病毒基因组可以被核糖体立即翻译来产生进行病毒生活周期下一步所需要的多肽。 CVB3的复制不经过DNA的中间体,研究其结构和功能比较困难,必须依赖反向遗传学操作技术得到该病毒的感染性克隆。在感染性克隆的构建过程中,设计一个有效的回复载体是至关重要的。鉴于传统回复载体的缺陷,本研究设计了一个RNA聚合酶Ⅱ驱动的病毒回复载体。利用CMV启动子构建CVB3感染性克隆载体：先将质粒pcDNA3.1(+)的CMV启动子下游区域通过PCR技术替换成设计好的多克隆位点区,分别插入PCR合成的HdvRz核心序列和polyA尾片段,形成框架载体pc-H-A,通过瞬时转染Hela细胞初步鉴定pc-H-A的可用性；同时,为了得到CVB3病毒的全长序列,本研究用我室CVB3 (nancy株)感染来Hela细胞,取感染液上清提取病毒RNA,利用长链RT-PCR和长链PCR技术反复优化条件扩增病毒基因组全长片段；最后,将CVB3全长基因组序列克隆至设计好的框架载体pc-H-A中,筛选阳性克隆,并对筛选到的CVB3重组全长克隆其进行测序鉴定和初步回复功能评价。综上所述,本研究构建了带有CMV启动子的回复框架载体pc-H-A并初步鉴定了该载体可用,利用此框架载体构建了与基因库中序列相似性为98%的CVB3全长cDNA克隆pc-CVB3-H-A并在转录水平进行分析,同时进行了转染和盲传实验检测有无子代病毒。虽然该系统回复正链RNA病毒的有效性仍需进一步研究探讨,但该研究为RNA聚合酶Ⅱ型回复系统的研究奠定了基础。
[Abstract]:The genome of RNA DNA virus is difficult to operate, its study must through the construction of its infectious clone reverse genetics technology. With all kinds of research methods and experimental techniques of reverse genetics technology constantly updated, in the times, all kinds of new reply system continue to be build.
The traditional reply prokaryotic promoter system based on the limitation of all infected mammalian cells when the tedious steps to produce RNA polymerase, increase the difficulty of the experimental operation, and some means to generate polymerase, such as the use of helper virus, itself has the interference even due to the cytopathic effect on cells of experimental helper virus (CPE) will affect the load cell survival and cause the virus cannot reply. Eukaryotic cells can synthesize the RNA polymerase and the RNA polymerase is endogenous protein in eukaryotic cells, does not require the auxiliary means (such as helper virus, the helper plasmid construction, cell lines, etc.) to produce can express start, eukaryotic promoter is very convenient, but also fundamentally eliminate the prokaryotic RNA polymerase system using helper virus to reply CPE cells; other eukaryotic RNA polymerase Some of them are located in the nucleus, and some are located in cytoplasm. It is possible for all kinds of viruses to recover. Currently, the superiority of eukaryotic RNA polymerase recovery system has become more and more obvious, and has become the research focus of RNA virus recovery system.
Coxsackie virus (Coxsackie virus CV) is a kind of common human virus respiratory and digestive tract infection, belonging to the small RNA virus (Picornaviridae) enterovirus (Enterovirus genus). Coxsackie virus serotype A, B two groups, A group has 24 serotypes, B group of 6 a serum type, can play a series of clinical disease.
Kosach virus group B type 3 (CVB3) genome is a single strand RNA, encoding a large open reading frame consists of 2207 amino acids, on both sides of 5 'non encoding region (5' -NTR 3 ') and polyadenosine non encoding region of.5' -NTR contains a type 1 ribosome entry site (IRE) 5 "to cap independent translation initiation of the viral polyprotein. The positive strand virus genome structure, can be immediately translated polypeptide ribosome generated viral life cycle of the next step needed.
CVB3 without DNA replication intermediates, study its structure and function is difficult, must rely on the reverse genetics technology of infectious clone of the virus. In the process of the construction of infectious clone, design an effective response vector is crucial. In view of the shortcomings of the traditional response vector, this study designed a RNA polymerase II virus vector driven by CMV promoter. To construct CVB3 infectious clone carrier: the plasmid pcDNA3.1 (+) CMV promoter region replaced multiple cloning sites designed by PCR technology, were inserted into the PCR synthesis of HdvRz core sequence and polyA tail fragment, forming the framework of vector pc-H-A. The availability of identification of transfected Hela cells pc-H-A; at the same time, in order to get the full-length sequence of CVB3 virus, this study in my room CVB3 (Nancy strain) infection to Hela cells after infection The supernatant extract virus RNA, optimize the conditions for amplification of viral genome was amplified by long RT-PCR and long chain PCR; finally, the full-length CVB3 genome sequence was cloned into the vector pc-H-A design framework, the positive clones were screened and the screening of the recombinant CVB3 cloning sequencing and its preliminary reply function evaluation.
In summary, this study constructed with CMV promoter vector pc-H-A response framework and identification of the carrier can be constructed, and the gene pool of sequence similarity of CVB3 full-length cDNA clone of pc-CVB3-H-A 98% and at the transcriptional level analysis using the frame vector, were transfected and blind transfer experimental detection with offspring virus. Although the effectiveness of the system response of positive strand RNA virus still needs further study, but the study laid the foundation for the study of RNA polymerase II recovery system.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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