【摘要】：根據(jù)WHO公布的資料,至2003年7月8日,全球29個(gè)國家和地區(qū)共發(fā)生SARS 8096例,死亡774例;中國內(nèi)地發(fā)病5329例,死亡349例。面對這一場突如其來的瘟疫,全球多個(gè)國家和地區(qū)的實(shí)驗(yàn)室參加了SARS病原體的協(xié)作研究,并很快取得重大突破。中國香港、加拿大、美國、德國、新加坡和我國內(nèi)地等國家和地區(qū)先后從SARS患者標(biāo)本中成功分離到新型冠狀病毒,經(jīng)科赫法則(Koch’s postulate)確認(rèn)新型冠狀病毒是本次SARS全球流行的病原體。 SARS 病毒是人類面臨的新挑戰(zhàn),許多性狀是通過與其它 3 組冠狀病毒類推、或者是通過一些理論模型推導(dǎo)出來的。但 SARS 病毒畢竟不是其它 3 組冠狀病毒,還需要做大量的實(shí)驗(yàn)研究來明確它的來源、演變、復(fù)制、轉(zhuǎn)錄、基因變異、致病機(jī)理、免疫學(xué)應(yīng)答等科學(xué)問題。而這一切,則是指導(dǎo)預(yù)防和治療的基礎(chǔ)。 本實(shí)驗(yàn)將通過對 SARS-CoV 基因組中 5’UTR 末端非編碼區(qū)的序列分析和功能研究,初步探討該段序列在病毒復(fù)制過程中的作用。 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 的序列構(gòu)成特點(diǎn)和差異;了解其空間結(jié)構(gòu),從而為進(jìn)一步研究其功能打下基礎(chǔ)。 方 法:先從 GenBank 中下載現(xiàn)有 SARS-CoV 全基因組 5’UTR和 5’UTR 片段,采用分子生物學(xué)軟件 DNAStar-MegAlign 和 BLAST進(jìn)行序列同源性分析;取其代表株 RNA 序列,經(jīng) RNADraw3.0 進(jìn)行二級結(jié)構(gòu)預(yù)測。 結(jié) 果:⑴序列同源性:101 個(gè) SARS-CoV 5’UTR 序列中,全長264nt 者 83 株,占 82.2%;18 株有缺失突變,所有缺失位于 5’端。5個(gè)點(diǎn)突變位置,點(diǎn)突變率為 1.92×19-4。⑵ SARS-CoV 5’UTR RNA 二級結(jié)構(gòu)預(yù)測:RNADraw3.0 分析表明,在 37℃、最小自由能為-79.5kcal時(shí),SARS-CoV 5’UTR RNA 可折疊,形成比較穩(wěn)定的二級結(jié)構(gòu)。包含4 個(gè)莖環(huán)結(jié)構(gòu)(stem-loop):Stem-loop-Ⅰ由第 7~31 位核苷酸構(gòu)成;Stem-loop-Ⅱ由第 42~221 位核苷酸構(gòu)成,是 4 個(gè)莖環(huán)結(jié)構(gòu)中最大的,含多個(gè)莖環(huán)結(jié)構(gòu),形成復(fù)雜的假結(jié);Stem-loop-Ⅲ由第 227~251 位核苷酸構(gòu)成,Stem-loop-Ⅳ第 252~261 位核苷酸構(gòu)成。 結(jié) 論:⑴ SARS-CoV 5’UTR 序列在各分離株之間同源性比較高,堿基構(gòu)成保守。⑵ SARS-CoV 5’UTR RNA 的二級結(jié)構(gòu)可形成 4 個(gè)莖環(huán)結(jié)構(gòu)域,推測可能具有啟動(dòng)子樣活性。 第二部分:SARS-CoV 5’UTR cDNA 啟動(dòng)子活性研究 目 的: 研究 SARS-CoV 5’-UTR cDNA 序列在真核細(xì)胞中的啟動(dòng)子活性,初步探討 SARS 病毒(SARS-CoV)復(fù)制調(diào)控機(jī)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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