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

【摘要】：嚴重急性呼吸道綜合征(SARS)是一種新現的主要經呼吸道傳播的嚴重傳染病,它的疾病特點很多還不為人知。我們利用分子生物學的方法,研究SARS冠狀病毒(SARS-CoV)對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,并逆轉錄為cDNA,然后用SARS-CoV不同基因區(qū)間的特定引物進行PCR擴增,對其中1例患者肺組織中病毒株的全序列進行了測定。結果顯示有2例患者僅在肺組織中擴增到病毒序列,另1例患者除在肺組織中檢測到病毒外,還在氣管、腎、淋巴結及肝組織中檢測到了病毒的存在。研究結果進一步說明SARS-CoV具有多組織嗜性,這對研究SARS-CoV感染機體細胞的受體提供線索。病毒全序列分析結果顯示,該序列全長為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四種結構蛋白基因外,還有另外8個蛋白編碼框。經與GenBank上登錄的其它SARS-CoV全序列進行比對,發(fā)現該序列除存在少量的SNP外,還多出一段29個核苷酸序列,這段序列的存在完全改變了ORF10和ORF11兩個蛋白編碼框,使得在此終止的蛋白翻譯能夠繼續(xù)進行下去,從而使ORF10和ORF11兩個讀框合成為一個讀框。這一結果可能提示該序列有可能是病毒從動物傳到人的原始序列。 為了充分認識SARS的本質,獲得更多關于SARS-CoV的免疫致病機理的信息,我室開始構建SARS-CoV主要結構蛋白原核表達載體,并探討各結構蛋白在大腸桿菌BL21(DE3)中的表達情況及其抗原性。從SARS病人組織中抽提出RNA,經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基因的兩個區(qū)段、N基因和M基因分別克隆至表達載體pET-32和pET-28上,轉化大腸桿菌BL21,利用IPTG進行誘導表達,SDS-PAGE檢測表達情況,并通過WB鑒定表達蛋白的抗原性。結果我們成功構建了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.
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R373.1

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本文編號：2020275