SARS-CoV 5’UTR启动子活性研究

发布时间：2018-11-01 14:16

【摘要】：根据WHO公布的资料,至2003年7月8日,全球29个国家和地区共发生SARS 8096例,死亡774例;中国内地发病5329例,死亡349例。面对这一场突如其来的瘟疫,全球多个国家和地区的实验室参加了SARS病原体的协作研究,并很快取得重大突破。中国香港、加拿大、美国、德国、新加坡和我国内地等国家和地区先后从SARS患者标本中成功分离到新型冠状病毒,经科赫法则(Koch’s postulate)确认新型冠状病毒是本次SARS全球流行的病原体。 SARS 病毒是人类面临的新挑战,许多性状是通过与其它 3 组冠状病毒类推、或者是通过一些理论模型推导出来的。但 SARS 病毒毕竟不是其它 3 组冠状病毒,还需要做大量的实验研究来明确它的来源、演变、复制、转录、基因变异、致病机理、免疫学应答等科学问题。而这一切,则是指导预防和治疗的基础。 本实验将通过对 SARS-CoV 基因组中 5’UTR 末端非编码区的序列分析和功能研究,初步探讨该段序列在病毒复制过程中的作用。 第一部分:SARS-CoV 5’UTR RNA 序列分析 目 的:了解 SARS-CoV 不同毒株 5’UTR RNA 的序列构成特点和差异;了解其空间结构,从而为进一步研究其功能打下基础。 方 法:先从 GenBank 中下载现有 SARS-CoV 全基因组 5’UTR和 5’UTR 片段,采用分子生物学软件 DNAStar-MegAlign 和 BLAST进行序列同源性分析;取其代表株 RNA 序列,经 RNADraw3.0 进行二级结构预测。 结 果:⑴序列同源性:101 个 SARS-CoV 5’UTR 序列中,全长264nt 者 83 株,占 82.2%;18 株有缺失突变,所有缺失位于 5’端。5个点突变位置,点突变率为 1.92×19-4。⑵ SARS-CoV 5’UTR RNA 二级结构预测:RNADraw3.0 分析表明,在 37℃、最小自由能为-79.5kcal时,SARS-CoV 5’UTR RNA 可折叠,形成比较稳定的二级结构。包含4 个茎环结构(stem-loop):Stem-loop-Ⅰ由第 7~31 位核苷酸构成;Stem-loop-Ⅱ由第 42~221 位核苷酸构成,是 4 个茎环结构中最大的,含多个茎环结构,形成复杂的假结;Stem-loop-Ⅲ由第 227~251 位核苷酸构成,Stem-loop-Ⅳ第 252~261 位核苷酸构成。 结 论:⑴ SARS-CoV 5’UTR 序列在各分离株之间同源性比较高,碱基构成保守。⑵ SARS-CoV 5’UTR RNA 的二级结构可形成 4 个茎环结构域,推测可能具有启动子样活性。 第二部分:SARS-CoV 5’UTR cDNA 启动子活性研究 目 的: 研究 SARS-CoV 5’-UTR cDNA 序列在真核细胞中的启动子活性,初步探讨 SARS 病毒(SARS-CoV)复制调控机制。
[Abstract]:According to the data released by WHO, from July 8, 2003, there were 8096 cases of SARS in 29 countries and regions, and 5329 cases of SARS and 349 cases of death in mainland China. In the face of this sudden plague, laboratories from many countries and regions around the world participated in the collaborative research of SARS pathogens, and soon made a major breakthrough. Hong Kong, China, Canada, the United States, Germany, Singapore and the mainland of China have successfully isolated a novel coronavirus from SARS patient samples. The new coronavirus has been confirmed by Koch Rule (Koch's postulate) as the global pathogen of SARS. SARS virus is a new challenge for human beings. Many traits are derived by analogy with other three groups of coronaviruses or by some theoretical models. But SARS virus is not the other three groups of coronaviruses after all, and a large number of experimental studies are needed to identify its origin, evolution, replication, transcription, gene variation, pathogenesis, immunological response and other scientific problems. All this is the basis for guiding prevention and treatment. The purpose of this study was to analyze the sequence and function of the non-coding region of 5'UTR terminal in the SARS-CoV genome, and to explore the role of the sequence in the replication process of the virus. Part I: sequence analysis of SARS-CoV 5'UTR RNA: to understand the characteristics and differences of 5'UTR RNA sequences of different strains of SARS-CoV; Understand its spatial structure, thus lay a foundation for further study of its function. Square method: firstly, the existing SARS-CoV genomic 5'UTR and 5'UTR fragments were downloaded from GenBank, and the sequence homology was analyzed by molecular biology software DNAStar-MegAlign and BLAST, and the RNA sequence of its representative strain was predicted by RNADraw3.0. Fruit formation: 1 sequence homology: out of 101 SARS-CoV 5'UTR sequences, 83 strains (82.2%) had full-length 264nt; 18 strains had deletion mutations, all of which were located at the 5 'end and 5 point mutation sites. The point mutation rate was 1.92 脳 19-4.2 SARS-CoV 5'UTR RNA secondary structure prediction. RNADraw3.0 analysis showed that at 37 鈩，

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