【作者】 方勇；

【导师】 盛军；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2005， 硕士

【摘要】 始于中国南方并波及全球的重症急性呼吸综合征（severe acute respiratory syndrome ,SARS）的暴发性流行严重影响人类的健康和生命。WHO已经确认SARS病原体为一种新的冠状病毒,命名为SARS冠状病毒（SARS-CoV）。研究发现SARS病毒刺突蛋白S2段的羧基端与鼠、牛冠状病毒刺突蛋白S2段具有明显的同源性。鼠、牛冠状病毒S2段有中和抗体表位,因此SARS病毒的S2段也可能存在能够激发中和抗体的表位。首先我们选取抗原表位相对集中的一段基因克隆至原核表达载体pET-28上,构建了含有SARS冠状病毒刺突蛋白片段的表达载体pET-28a/S₂,并在大肠杆菌中获得了高表达（50%）,选择DEAE-Sepharose FF离子交换层析纯化,得到了纯度在90%以上的蛋白样品。然后,将纯化的SARS 抗原免疫小鼠,制备抗SARS 单克隆抗体。经ELISA 检测和筛选,获得一株稳定的抗SARS 刺突蛋白单克隆抗体株。该抗体能够识别SARS 抗原结合位点。以制备的抗SARS 单克隆抗体为第一抗体,HRP 标记山羊抗小鼠抗抗体为第二抗体,初步建立了SARS 刺突抗原检测方法。更多还原

【Abstract】 A novel coronavirus has been identified as the major cause of severe acute respiratory syndrome (SARS). Severe acute respiratory syndrome (SARS), referred to as atypical puenmoina, is an infectious disease of a kind of breath spread in the near future. An outbreak example appear in November of 2002 the region in Guangdong in the our country, and become the popular situation quickly, and become the popular situation quickly. Its contagion is strong, the infection rate is high, affecting the political economy development of the world-wide locations seriously. Have no special effect medicine and the treatment methods of cure SARS still currently, although after in time of the support treatment and to disease treatment, great majority the patient can recover from illness to recover from illness, its high infection rate, the high contagion and high possibility that patient relapse needs the medicine and the vaccine of the special effect urgently. But defend heavy in cure, the vaccine researches to manufacture to seem to be more important and urgent. According to the genome series of the virus of SARS research enunciation that measures the preface and former concerning the coronaviruses, the spike protein of the virus of SARS is the ideal antigen of target probably at keep virus from invade, moderating the virus aspect to rise the decisive function. The severe acute respiratory syndrome, (SARS) is main pathogenic body that causes SARS. The severe acute respiratory syndrome, (SARS) is a kind of coronaviruses. A sequence of SARS virus genes has already completed the measurement. The spike protein may be a second unit vaccine. The result of the SARS protein compared indicated that the N-terminal of spike protein has a evidence homology with the formerly MHV and BCV. S2 protein of MHV and BCV has counteracted antibody epitopes. So we presume that the S2 protein of SARS also has counteracted antibody epitopes. S2 protein gene was amplified fromthe genomic RNA of SARS-CoV by RT-PCR. The fragment was cloned into a prokaryotic expression vector pET-28a to construct a recombinant plasmid pET-28a/S2. The Escherichia coli cells were transformed with the recombinant plamid and the transformants were induced with IPTG to express the recombinant protein and the target protein was identified. To study the key influence factors when lactose, the inducer substitute to the IPTG, was used in the processes of the recombinant gene’s expression. The BL21(DE3)/S2 which expressed the fragment of the spike protein of the SARS virus was used as model Escherichia coli strain.Application of glucose/glycerol for carbon source and lactose/allolactose for induction of SARS/ S2 genes in BL21(DE3) was studied in shake flask. The result of the shake flask was verificated in 40 liters fermentor. Shake flask cultivations showed: 1. lactose induced gene expression is as efficiently as IPTG when concentration of the lactose was up to 0.5mM; 2. addition of glucose in the medium did inhibit the effect induced by the lactose but not the effect by allolactose; 3. addition of glycerol in the medium didn’t inhibit the effect by the lactose or allolactose. The studying in fermentor has confirmed the same results. The lactose can be used to induce the lacI-based expression system. The medium must contain the non-glucose carbon source when the Escherichia coli was induced by lactose. The next in order, we carry on the first step to the target protein to purified, suspending the engineering germ in the broken up liquid(10mM Tris-HCl ,1mMEDTA),break the germ with a broken up instrument under the condition that ice bathe. Broken up evenly, centrifuge, 10000 rpm,10min. Leave up pure. After precipitate was washed away dirt by foundation buffer liquid (20mM PB, 8MUrea , PH 7.4) and fused. Indoor temperature 5-6h, centrifuge, 10000rpm,10min. Discarded the precipitate. Pass further analysis to the S2 protein property, We更多还原