【作者】 徐江；

【导师】 李柱一； 徐志凯；

【作者基本信息】 第四军医大学 ， 神经病学， 2009， 博士

【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。更多还原

【Abstract】 The purpose of the present study is to produce enough amount of soluble DAF for in vivo studies, i.e. the metabolization of soluble DAF in the blood and its inhibitive effect to the complement system. In order to achieve this goal, the 4 short concensus repeats (SCR1-4) of rat DAF (rDAF) and human DAF (hDAF) were successfully overexpressed in E. coli. The inclusion body was purified and refolded to obtain soluble and active DAF. Then, the recombinant rDAF was used as an immunongen to produce monoclonal antibodies against rDAF. Finally, an ELISA system was established in which as little as 10 ng/ml of rDAF could be detected. A monoclonal antibody which could detect as little as 40 ng of rDAF was isolated. The results of the in vivo experiment showed that rDAF was metabolized rapidly in the blood. Within 90 min after injection, it could not be detected. Even at the highest concentration, the negative effect of soluble DAF against the complement system was not detected. This result showed that soluble DAF is not a good candidate of complement inhibitor. In the mean time, two other studies were done. First, an efficienct and economic way of refolding DAF was established. Second, the possibility of soluble expression of DAF was explored and a possible reason was given for the soluble inclusion bodies which were formed by the NusA-DAF fusion protein.更多还原