本文選題：嚴(yán)重急性呼吸道綜合征 + 冠狀病毒　； 參考：《第一軍醫(yī)大學(xué)》2005年碩士論文

【摘要】：嚴(yán)重急性呼吸道綜合征(SARS)是一種新現(xiàn)的主要經(jīng)呼吸道傳播的嚴(yán)重傳染病,它的疾病特點(diǎn)很多還不為人知。我們利用分子生物學(xué)的方法,研究SARS冠狀病毒(SARS-CoV)對(duì)不同組織的感染情況,并了解SARS流行初期病毒株的基因組序列。從感染SARS病毒而死亡的3例早期患者的不同組織中,用TRIzol RNA抽提試劑提取總RNA,并逆轉(zhuǎn)錄為cDNA,然后用SARS-CoV不同基因區(qū)間的特定引物進(jìn)行PCR擴(kuò)增,對(duì)其中1例患者肺組織中病毒株的全序列進(jìn)行了測(cè)定。結(jié)果顯示有2例患者僅在肺組織中擴(kuò)增到病毒序列,另1例患者除在肺組織中檢測(cè)到病毒外,還在氣管、腎、淋巴結(jié)及肝組織中檢測(cè)到了病毒的存在。研究結(jié)果進(jìn)一步說(shuō)明SARS-CoV具有多組織嗜性,這對(duì)研究SARS-CoV感染機(jī)體細(xì)胞的受體提供線索。病毒全序列分析結(jié)果顯示,該序列全長(zhǎng)為29 760堿基,具有典型的冠狀病毒序列特征,除具有RNA聚合酶基因和S、E、M和N四種結(jié)構(gòu)蛋白基因外,還有另外8個(gè)蛋白編碼框。經(jīng)與GenBank上登錄的其它SARS-CoV全序列進(jìn)行比對(duì),發(fā)現(xiàn)該序列除存在少量的SNP外,還多出一段29個(gè)核苷酸序列,這段序列的存在完全改變了ORF10和ORF11兩個(gè)蛋白編碼框,使得在此終止的蛋白翻譯能夠繼續(xù)進(jìn)行下去,從而使ORF10和ORF11兩個(gè)讀框合成為一個(gè)讀框。這一結(jié)果可能提示該序列有可能是病毒從動(dòng)物傳到人的原始序列。 為了充分認(rèn)識(shí)SARS的本質(zhì),獲得更多關(guān)于SARS-CoV的免疫致病機(jī)理的信息,我室開始構(gòu)建SARS-CoV主要結(jié)構(gòu)蛋白原核表達(dá)載體,并探討各結(jié)構(gòu)蛋白在大腸桿菌BL21(DE3)中的表達(dá)情況及其抗原性。從SARS病人組織中抽提出RNA,經(jīng)RT-PCR獲得了SARS冠狀病毒刺突蛋白(S)、核衣殼蛋白(N)和膜蛋白(M)基因,將S基因的兩個(gè)區(qū)段、N基因和M基因分別克隆至表達(dá)載體pET-32和pET-28上,轉(zhuǎn)化大腸桿菌BL21,利用IPTG進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE檢測(cè)表達(dá)情況,并通過(guò)WB鑒定表達(dá)蛋白的抗原性。結(jié)果我們成功構(gòu)建了SARS-CoV重組
[Abstract]:Severe Acute Respiratory Syndrome (SARS) is a new severe infectious disease mainly transmitted by respiratory tract, the characteristics of which are still unknown. We used molecular biology to study the infection of SARS coronavirus SARS-CoV in different tissues, and to understand the genome sequence of SARS coronavirus strain in the early stage of SARS epidemic. Total RNAs were extracted by TRIzol RNA extraction reagent from different tissues of three early patients who died of SARS virus infection, and then reverse transcripted to cDNA.Then PCR amplification was carried out with specific primers from different regions of SARS-CoV gene. The whole sequence of the virus strain in the lung tissue of one patient was determined. The results showed that the virus sequence was only amplified in the lung tissue in 2 cases, and the virus was detected in the trachea, kidney, lymph nodes and liver tissues in the other case in addition to the virus detected in the lung tissue. The results further indicate that SARS-CoV has multi-tissue tropism, which provides clues for studying the receptor of SARS-CoV infection cells. The results of the whole sequence analysis showed that the total length of the virus was 29 760 base, which had the characteristic of typical coronavirus sequence. Besides the RNA polymerase gene and the four structural protein genes of Sequen M and N, there were 8 other protein coding frames. After alignment with other SARS-CoV sequences recorded in GenBank, it was found that there were 29 nucleotide sequences in addition to a small number of SNP in the sequence. The existence of this sequence completely changed the ORF10 and ORF11 protein coding frames. So that the protein translation that terminates here can continue so that the ORF10 and ORF11 reading frames are synthesized into one reading frame. This result may suggest that the sequence may be the original sequence of the virus from animals to humans. In order to fully understand the nature of SARS and obtain more information about the immune pathogenesis of SARS-CoV, we began to construct the prokaryotic expression vector of SARS-CoV main structural protein, and to investigate the expression and antigenicity of each structural protein in Escherichia coli BL21DDE3. The RNAs were extracted from the tissues of SARS patients and the nucleocapsid protein (NN) and membrane protein (MN) genes of SARS coronavirus (SARS-CoV) were obtained by RT-PCR. The N gene and M gene of S gene were cloned into the expression vectors pET-32 and pET-28, respectively. The expression was detected by SDS-PAGE induced by IPTG and the antigenicity of the expressed protein was identified by WB. As a result, we successfully constructed the SARS-CoV recombinant.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373.1

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本文編號(hào)：2020275