SARS病人组织中SARS冠状病毒全基因组克

发布时间：2018-01-10 18:14

本文关键词：SARS病人组织中SARS冠状病毒全基因组克隆、序列分析及病毒样颗粒装配　出处：《南京农业大学》2005年博士论文　论文类型：学位论文

【摘要】：2003年春季在我国广东、北京等地人群中爆发了以高热、寒战、肌肉痛、干咳、呼吸困难、高度接触性传染为主要特点的传染性疾病，随后该疾病被正式命名为“严重急性呼吸综合症(简称SARS)”。病因学研究表明，SARS的病因是一种新型冠状病毒，世界卫生组织将其命名为SARS相关冠状病毒(SARS-associated coronavirus，，SARS-CoV)。作为一种新发现的病毒，目前对其遗传学和复制、装配机制等还所知甚少，因此开展此方面研究具有重要意义。笔者参与了SARS病原的调查工作。为深入阐明北京地区流行的SARA-CoV的病原学特征，首先从SARS病人病料标本中进行病毒分离试验。将SARS病人临床标本，如咽拭子、痰液和肺组织标本接种Vero细胞，连续传代后，可观察到显著的细胞病理改变(CPE)，表明可能标本中可能存在病毒。电镜观察分离物培养上清负染样本，，可见到形态与典型冠状病毒外观相似的病毒样颗粒；用SARS病人康复期血清进行的间接免疫荧光试验，显示分离物接种培养的细胞胞浆内存在特异性荧光，证实培养物中存在与SARS-CoV相关的病毒抗原；提取分离物总RNA进行RT-PCR检测，可用冠状病毒基因组序列特异性引物扩增特异性片段，克隆扩增片段后进行DNA序列分析，结果显示所扩增的片段为冠状病毒保守的复制酶基因区序列。因此综合以上结果，提示分离到的病毒为SASR冠状病毒为比较来源于SARS病人人体组织和已经分离、培养的SARS-CoV基因组之间的差异，阐明病毒的遗传和变异特点并深入探讨其致病机制，利用RT-PCR从北京地区一例SARS死亡病人(#5)肺组织总RNA中对SARS-CoV组织毒的基因组全长cDNA进行了克隆和序列分析。参考已经发表的SARS-CoV全序列，将待测定的SARS-CoV全长基因组分成26个片段，设计引物进行扩增。扩增并克隆的26个目的片段覆盖SARS-CoV全长基因组。测序结果表明本次实验中SARS-CoV-5#毒株基因组全长29707个核苷酸。同源性比对结果表明，SARS-CoV组织毒5#基因组序列与BJ01-BJ04地方分离株为同一流行株型。本次试验测定的组织毒的基因组序列在基因组水平上与18株全序列已知人源毒株的碱基差异不明显，而与10株动物来源的SARS-CoV分离株全序列在92个核酸位点上存在明显差异，这说明人源SARS-CoV毒株的全序列与动物源SARS-CoV毒株的全序列存在基因组水平的碱基差异，而细胞培养和人体组织来源的SARS毒株不存在基因组水平的碱基差异。对各种来源的SARS病毒的S蛋白质基因分析同样也显示了类似的碱基差异
[Abstract]:In 2003 in Guangdong, Beijing and other places in China, outbreaks of infectious diseases were characterized by high fever, shivering, muscle pain, dry cough, dyspnea, and highly contagious infections. The disease was later officially named "severe Acute Respiratory Syndrome (SARS)". Etiological studies have shown that SARS is a new type of coronavirus. The World Health Organization named it SARS associated coronavirus SARS-associated coronavirus. As a newly discovered virus, SARS-CoV has little knowledge of its genetics, replication and assembly mechanism, so it is of great significance to study this aspect. In order to elucidate the etiological characteristics of SARA-CoV epidemic in Beijing, the authors participated in the investigation of the pathogen of SARS. Firstly, virus isolation test was carried out from the samples of SARS patients. The clinical specimens of SARS patients, such as pharyngeal swabs, sputum and lung tissues, were inoculated with Vero cells and then passed on continuously. Significant cytopathological changes were observed, indicating the possible presence of viruses in the specimens. The negative staining samples of the culture supernatants of the isolates were observed by electron microscopy. The appearance of virus like particles was similar to that of typical coronavirus. The indirect immunofluorescence test in the convalescent serum of SARS patients showed that specific fluorescence existed in the cytoplasm of the cells inoculated with the isolate. It was confirmed that there were virus antigens related to SARS-CoV in the culture. Total RNA was extracted for RT-PCR detection. Specific fragments of coronavirus genome sequence were amplified by specific primers, and then amplified by DNA sequence analysis. The results showed that the amplified fragment was conserved by the sequence of the replicase gene of coronavirus. Therefore, the above results suggest that the isolated virus is SASR coronavirus. In order to compare the differences between the human tissues from SARS patients and the isolated and cultured SARS-CoV genomes, the genetic and variation characteristics of the virus were elucidated and the pathogenetic mechanism of the virus was discussed. #5, a case of SARS death in Beijing using RT-PCR. The genomic full-length cDNA of SARS-CoV tissue virus was cloned and sequenced from the total RNA of lung tissue. Reference was made to the published full SARS-CoV sequence. The full-length SARS-CoV genome was divided into 26 fragments. The primers were designed to amplify. The 26 target fragments were amplified and cloned to cover the full-length SARS-CoV genome. The sequencing results showed that the total length of the genome of the SARS-CoV-5# strain in this experiment was 297. The homology analysis of 07 nucleotides showed that. The genomic sequence of SARS-CoV tissue virus is the same as that of the local isolate of BJ01-BJ04. The genomic sequence of the tissue virus determined in this experiment is totally sequenced with 18 strains at the genomic level. There was no significant difference in the bases of known human strains. However, there were significant differences in 92 nucleic acid loci between the whole sequence of SARS-CoV isolates and 10 animal isolates. This indicated that there was a genomic difference between the whole sequence of human SARS-CoV strain and that of animal SARS-CoV strain. However, SARS strains derived from cell culture and human tissues did not have genome-level differences. The S-protein gene analysis of SARS viruses from various sources also showed similar base differences.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R511.9;R373

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