本文關(guān)鍵詞：基于CMV啟動子構(gòu)建CVB3感染性克隆的初步研究　出處：《安徽大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

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【摘要】： RNA病毒基因組較DNA病毒難以操作,對其進(jìn)行研究必須先通過反向遺傳學(xué)技術(shù)構(gòu)建其感染性克隆。隨著各類研究方法和實驗技術(shù)手段的不斷更新,反向遺傳學(xué)技術(shù)也在與時俱進(jìn),各種新型的回復(fù)系統(tǒng)不斷建立。 傳統(tǒng)的基于原核啟動子的回復(fù)系統(tǒng)的局限都是感染哺乳細(xì)胞時需要通過煩瑣的步驟來產(chǎn)生RNA聚合酶,增加了實驗操作的難度,而且一些產(chǎn)生聚合酶的手段,如使用輔助病毒等,本身就會對實驗有所干擾甚至由于輔助病毒對細(xì)胞的病變效應(yīng)(CPE)會影響負(fù)載細(xì)胞的生存而導(dǎo)致病毒回復(fù)無法進(jìn)行。真核細(xì)胞都可以合成自身的RNA聚合酶,而且這些RNA聚合酶是真核細(xì)胞的內(nèi)源蛋白,不需要通過輔助手段(如上述的輔助病毒,輔助質(zhì)粒,構(gòu)建細(xì)胞系等)去產(chǎn)生就可以啟動真核啟動子的表達(dá),十分方便,也從根本上杜絕了原核RNA聚合酶回復(fù)系統(tǒng)利用輔助病毒等對細(xì)胞CPE的發(fā)生；另外真核RNA聚合酶有的定位在細(xì)胞核,有的定位于細(xì)胞質(zhì),為回復(fù)各種類型的病毒都提供了可能。目前,真核RNA聚合酶回復(fù)系統(tǒng)的優(yōu)越性已經(jīng)越來越明顯,成為RNA病毒回復(fù)系統(tǒng)的研究熱點。 柯薩奇病毒(Coxsackie virus, CV)是一類常見的經(jīng)呼吸道和消化道感染人體的病毒,屬于微小RNA病毒科(Picornaviridae)腸道病毒屬(Enterovirus genus)。柯薩奇病毒血清型分A,B兩組,A組有24個血清型,B組有6個血清型,能起一系列的臨床疾病。 科薩奇病毒B組3型(CVB3)基因組為單股正鏈RNA,編碼一個大的開放讀碼框包括2207個氨基酸,兩側(cè)為5’非編碼區(qū)(5’-NTR)和3’多聚腺苷非編碼區(qū)。5’-NTR包括一個Ⅰ型核糖體進(jìn)入位點(IRE)使5’帽子能夠獨立翻譯成病毒多聚蛋白的起始。由于單股正鏈結(jié)構(gòu),病毒基因組可以被核糖體立即翻譯來產(chǎn)生進(jìn)行病毒生活周期下一步所需要的多肽。 CVB3的復(fù)制不經(jīng)過DNA的中間體,研究其結(jié)構(gòu)和功能比較困難,必須依賴反向遺傳學(xué)操作技術(shù)得到該病毒的感染性克隆。在感染性克隆的構(gòu)建過程中,設(shè)計一個有效的回復(fù)載體是至關(guān)重要的。鑒于傳統(tǒng)回復(fù)載體的缺陷,本研究設(shè)計了一個RNA聚合酶Ⅱ驅(qū)動的病毒回復(fù)載體。利用CMV啟動子構(gòu)建CVB3感染性克隆載體：先將質(zhì)粒pcDNA3.1(+)的CMV啟動子下游區(qū)域通過PCR技術(shù)替換成設(shè)計好的多克隆位點區(qū),分別插入PCR合成的HdvRz核心序列和polyA尾片段,形成框架載體pc-H-A,通過瞬時轉(zhuǎn)染Hela細(xì)胞初步鑒定pc-H-A的可用性；同時,為了得到CVB3病毒的全長序列,本研究用我室CVB3 (nancy株)感染來Hela細(xì)胞,取感染液上清提取病毒RNA,利用長鏈RT-PCR和長鏈PCR技術(shù)反復(fù)優(yōu)化條件擴增病毒基因組全長片段；最后,將CVB3全長基因組序列克隆至設(shè)計好的框架載體pc-H-A中,篩選陽性克隆,并對篩選到的CVB3重組全長克隆其進(jìn)行測序鑒定和初步回復(fù)功能評價。 綜上所述,本研究構(gòu)建了帶有CMV啟動子的回復(fù)框架載體pc-H-A并初步鑒定了該載體可用,利用此框架載體構(gòu)建了與基因庫中序列相似性為98%的CVB3全長cDNA克隆pc-CVB3-H-A并在轉(zhuǎn)錄水平進(jìn)行分析,同時進(jìn)行了轉(zhuǎn)染和盲傳實驗檢測有無子代病毒。雖然該系統(tǒng)回復(fù)正鏈RNA病毒的有效性仍需進(jìn)一步研究探討,但該研究為RNA聚合酶Ⅱ型回復(fù)系統(tǒng)的研究奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

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相關(guān)期刊論文 前2條

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本文編號：1405981