SARS-CoV结构蛋白的表达、纯化和鉴定及中国家养果子狸类SARS-CoV病毒的血清学检测
发布时间：2018-07-25 06:15
【摘要】：本论文在原核表达系统中对SARS-CoV 的主要结构蛋白基因进行了克隆、表达与多克隆抗体制备,同时对中国家养果子狸类SARS-CoV 病毒流行情况在血清学检测方面做了初步研究,论文包括四个章节。 第一章对SARS-CoV 近年的研究进展作了简要综述,并对本研究的背景和目的意义作了简要介绍。 第二章对SARS-CoV 的主要结构蛋白基因基因进行了克隆、原核表达、纯化与多克隆抗体制备。根据SARS-CoV 基因组全序列,设计相应的引物,用RT-PCR 的方法扩增得到S, S_1, S_2, S_△,M, E, N,ORF03,ORF08 基因。将这些结构蛋白基因分别克隆至原核表达载体pGEX-KG,经IPTG 诱导,S_1,S_2, S_△,M,E, N,ORF08 基因在E.coli BL21 (DE3)菌株中得到了高效表达。表达的目的蛋白大小分别为98 kDa,86 kDa, 58 kDa,52 kDa,34 kDa,74 kDa,38 kDa,我们纯化了其中的5 种蛋白GST-S2, GST-S△, GST-E, GST-N , GST-08,并用纯化了的蛋白免疫新西兰大白兔制备多克隆抗体,1:2000 倍稀释后用于Western Blot 分析,获得特异性显色信号,所得重组蛋白和抗体将用于SARS-CoV 的诊断、亚单位疫苗的研究。另外我们对所纯化蛋白做免疫原性的初步分析,发现GST-N 蛋白免疫小鼠可以产生较强的免疫应答,其抗体与灭活SARS-CoV 病毒产生特异性的反应,表明该蛋白具有较强的免疫原性,适用于SARS 的早期检测。 第三章主要内容为家养果子狸类SARS-CoV病毒的血清学检测。我们在2003年和2004 年从不同的养殖场收集了110 份家养果子狸的血清,这些血清来自于中国四个省五个地区不同的养殖场,这些养殖场都在较为偏远的山区且没有受2003 年SARS 流行的影响。我们用两种不同的方法(Western blot 和ELISA)检测中国家养果子狸血清中的抗体,结果显示84%的样品含有抗SARS-CoV 的抗体,ELISA 检测还发现来自于不同地区的家养果子狸血清样品含有抗SARS-CoV
[Abstract]:In this paper, the major structural protein genes of SARS-CoV were cloned in prokaryotic expression system, expressed with polyclonal antibody, and the serological detection of SARS-CoV virus in Chinese house-bred civets was preliminarily studied. The paper includes four chapters. In the first chapter, the research progress of SARS-CoV in recent years is briefly reviewed, and the background and purpose significance of this study are briefly introduced. In chapter 2, the main structural protein genes of SARS-CoV were cloned, expressed in prokaryotic, purified and prepared polyclonal antibody. According to the whole sequence of SARS-CoV genome, the corresponding primers were designed and amplified by RT-PCR. These structural protein genes were cloned into the prokaryotic expression vector pGEX-KG and were highly expressed in E.coli BL21 (DE3) strain by IPTG induction. The expressed target proteins were 98 kDa-86 kDa, 58 kDa-52-kDa-34 kDa-74 kDa-38kDa. five proteins, GST-S2, GST-S, GST-E, GST-N, GST-08, were purified. The purified proteins were used to immunize New Zealand white rabbits to prepare polyclonal antibodies, which were diluted by 12: 2000 times and then analyzed by Western Blot. Specific chromogenic signals were obtained and recombinant proteins and antibodies were obtained for the diagnosis of SARS-CoV and the study of subunit vaccines. In addition, we made a preliminary analysis of the immunogenicity of the purified protein, and found that the GST-N protein immunized mice could produce a strong immune response, and its antibody had a specific reaction with inactivated SARS-CoV virus, which indicated that the protein had strong immunogenicity. Suitable for early detection of SARS. The third chapter focuses on the serological detection of SARS-CoV virus in the family of civets. We collected 110 sera from different farms in 2003 and 2004 from four provinces and five regions in China. These farms are in remote mountain areas and are not affected by the 2003 SARS epidemic. Two different methods, (Western blot and ELISA, were used to detect the antibodies in the sera of Chinese house-grown civets. The results showed that 84% of the samples contained antibodies against SARS-CoV and the samples from different regions contained anti-SARS-CoV.
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
本文编号：2142883
[Abstract]:In this paper, the major structural protein genes of SARS-CoV were cloned in prokaryotic expression system, expressed with polyclonal antibody, and the serological detection of SARS-CoV virus in Chinese house-bred civets was preliminarily studied. The paper includes four chapters. In the first chapter, the research progress of SARS-CoV in recent years is briefly reviewed, and the background and purpose significance of this study are briefly introduced. In chapter 2, the main structural protein genes of SARS-CoV were cloned, expressed in prokaryotic, purified and prepared polyclonal antibody. According to the whole sequence of SARS-CoV genome, the corresponding primers were designed and amplified by RT-PCR. These structural protein genes were cloned into the prokaryotic expression vector pGEX-KG and were highly expressed in E.coli BL21 (DE3) strain by IPTG induction. The expressed target proteins were 98 kDa-86 kDa, 58 kDa-52-kDa-34 kDa-74 kDa-38kDa. five proteins, GST-S2, GST-S, GST-E, GST-N, GST-08, were purified. The purified proteins were used to immunize New Zealand white rabbits to prepare polyclonal antibodies, which were diluted by 12: 2000 times and then analyzed by Western Blot. Specific chromogenic signals were obtained and recombinant proteins and antibodies were obtained for the diagnosis of SARS-CoV and the study of subunit vaccines. In addition, we made a preliminary analysis of the immunogenicity of the purified protein, and found that the GST-N protein immunized mice could produce a strong immune response, and its antibody had a specific reaction with inactivated SARS-CoV virus, which indicated that the protein had strong immunogenicity. Suitable for early detection of SARS. The third chapter focuses on the serological detection of SARS-CoV virus in the family of civets. We collected 110 sera from different farms in 2003 and 2004 from four provinces and five regions in China. These farms are in remote mountain areas and are not affected by the 2003 SARS epidemic. Two different methods, (Western blot and ELISA, were used to detect the antibodies in the sera of Chinese house-grown civets. The results showed that 84% of the samples contained antibodies against SARS-CoV and the samples from different regions contained anti-SARS-CoV.
【学位授予单位】：中国科学院研究生院（武汉病毒研究所）
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373
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