SARS冠状病毒全基因测定、结构蛋白基因克

发布时间：2018-06-15 02:54

本文选题：严重急性呼吸道综合征 + 冠状病毒　；参考：《第一军医大学》2005年硕士论文

【摘要】：严重急性呼吸道综合征(SARS)是一种新现的主要经呼吸道传播的严重传染病,它的疾病特点很多还不为人知。我们利用分子生物学的方法,研究SARS冠状病毒(SARS-CoV)对不同组织的感染情况,并了解SARS流行初期病毒株的基因组序列。从感染SARS病毒而死亡的3例早期患者的不同组织中,用TRIzol RNA抽提试剂提取总RNA,并逆转录为cDNA,然后用SARS-CoV不同基因区间的特定引物进行PCR扩增,对其中1例患者肺组织中病毒株的全序列进行了测定。结果显示有2例患者仅在肺组织中扩增到病毒序列,另1例患者除在肺组织中检测到病毒外,还在气管、肾、淋巴结及肝组织中检测到了病毒的存在。研究结果进一步说明SARS-CoV具有多组织嗜性,这对研究SARS-CoV感染机体细胞的受体提供线索。病毒全序列分析结果显示,该序列全长为29 760碱基,具有典型的冠状病毒序列特征,除具有RNA聚合酶基因和S、E、M和N四种结构蛋白基因外,还有另外8个蛋白编码框。经与GenBank上登录的其它SARS-CoV全序列进行比对,发现该序列除存在少量的SNP外,还多出一段29个核苷酸序列,这段序列的存在完全改变了ORF10和ORF11两个蛋白编码框,使得在此终止的蛋白翻译能够继续进行下去,从而使ORF10和ORF11两个读框合成为一个读框。这一结果可能提示该序列有可能是病毒从动物传到人的原始序列。为了充分认识SARS的本质,获得更多关于SARS-CoV的免疫致病机理的信息,我室开始构建SARS-CoV主要结构蛋白原核表达载体,并探讨各结构蛋白在大肠杆菌BL21(DE3)中的表达情况及其抗原性。从SARS病人组织中抽提出RNA,经RT-PCR获得了SARS冠状病毒刺突蛋白(S)、核衣壳蛋白(N)和膜蛋白(M)基因,将S基因的两个区段、N基因和M基因分别克隆至表达载体pET-32和pET-28上,转化大肠杆菌BL21,利用IPTG进行诱导表达,SDS-PAGE检测表达情况,并通过WB鉴定表达蛋白的抗原性。结果我们成功构建了SARS-CoV重组
[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.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373.1

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