DAF的原核表达，重折叠，ELISA检测系统的建立，及其在大鼠体内的代谢和生物学活性的测定

发布时间：2018-07-18 20:29

【摘要】： 本研究的主要目的是表达足量的可溶性促衰变因子(DAF)蛋白用于动物试验,测定DAF在血清中的代谢规律和对补体系统的抑制效果。在本研究中,充分采用了如PCR、载体构建、转化、蛋白诱导表达和液相色谱等技术得到了高度纯化的蛋白质。进而采用单克隆抗体技术,制备了20株抗-大鼠DAF(rDAF)的单克隆抗体,建立了检测可溶性rDAF的ELISA系统(最低浓度下限可达10 ng/ml)和Western blot用单抗株(可检出40 ng rDAF蛋白)。通过大鼠的在体试验证明rDAF在大鼠体内代谢非常快且对大鼠血浆补体抑制程度很小,其本身并不适合作为一种补体抑制剂使用。除此以外,还完成了以下两个工作:建立了新的高效折叠复性DAF蛋白的方案;探索了可溶性表达DAF的可能性并揭示了NusA-DAF融合蛋白形成可溶性包涵体的原因。
[Abstract]:The main purpose of this study was to express sufficient soluble decay factor (DAF) protein in animal experiments and to determine the metabolism of DAF in serum and its inhibitory effect on complement system. In this study, highly purified proteins were obtained by using techniques such as PCR, vector construction, transformation, protein induction and expression, and liquid chromatography. Furthermore, 20 strains of monoclonal antibodies against rat DAF (rDAF) were prepared by using monoclonal antibody technique. An Elisa system for detecting soluble rDAF (the minimum concentration was up to 10 ng/ml) and a monoclonal antibody strain for Western blot (40 ng rDAF protein could be detected) were established. In vivo experiments in rats showed that rDAF was metabolized very quickly and had little inhibition on plasma complement in rats, so it was not suitable for use as a complement inhibitor. In addition, the following two works were accomplished: a new highly folded and refolded DAF protein was established, and the possibility of soluble expression of DAF was explored and the cause of the formation of soluble inclusion body of NusA-DAF fusion protein was revealed.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392;R446.6

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