SARS病毒刺突蛋白片段的表达纯化及单克隆抗体的制备

发布时间：2018-05-15 06:00

本文选题：冠状病毒 + 刺突蛋白　；参考：《吉林大学》2005年硕士论文

【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/S_2,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。
[Abstract]:The outbreak of severe acute respiratory syndrome (acute respiratory syndrome), which began in southern China and affected the whole world, has seriously affected human health and life. Who has confirmed that SARS pathogen is a new coronavirus named SARS coronavirus SARS-CoV. It was found that the carboxyl terminal of S2 segment of SARS virus spike protein had obvious homology with that of mouse and bovine coronavirus spines protein S2 segment. There are neutralizing antibody epitopes in S2 segment of bovine coronavirus, so the S2 segment of SARS virus may also have an epitope that can activate neutralizing antibody. First of all, we selected a fragment of the antigen epitope and cloned it into the prokaryotic expression vector pET-28. The expression vector pET-28a / SSP _ 2 containing SARS coronavirus stinging protein fragment was constructed, and the highly expressed protein was obtained in Escherichia coli. DEAE-Sepharose FF ion exchange chromatography was selected to purify the protein and the purity of the protein was over 90%. Then, the purified SARS antigen was immunized to prepare anti-SARS monoclonal antibody. A stable monoclonal antibody against SARS spike protein was obtained by ELISA detection and screening. The antibody can recognize the binding site of SARS antigen. Using the monoclonal antibody against SARS as the first antibody and the goat anti-mouse anti-antibody as the second antibody, a method for the detection of SARS stinging antigen was established.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【共引文献】