重组朊病毒的体外制备及动物模型研究

发布时间：2018-08-13 11:00

【摘要】：朊蛋白疾病,也被称作传染性海绵状脑病,是一类在哺乳动物中广泛存在的致死性神经退行性疾病。根据“朊病毒”假说,朊蛋白疾病的致病因子是朊病毒,其主要致病因子是由正常型的朊蛋白发生了构象转变形成的致病型朊蛋白,这种构象的朊蛋白可以诱导更多的正常朊蛋白发生构象转变,进而引发神经毒性和神经退行性病变。在朊蛋白发生构象转变的过程中,目前认为有某些辅助因子参与、促进了朊蛋白构象转变的进行,脂质就是一种有效的辅助因子。研究表明带负电荷的脂质可以和朊蛋白相互作用并诱导其发生构象转变,并形成具有PK抗性的构象体。此外RNA作为一种长链阴离子复合结构的物质,对朊蛋白构象转变也有加速的作用。在本论文中,我们在体外条件下用蛋白质错误折叠循环扩增的技术,在体系中加入了大肠杆菌表达的重组朊蛋白、带有负电荷的脂质POPG和小鼠肝脏提取的总RNA进行朊蛋白的构象转变,从无到有地制备出了重组朊病毒。经鉴定该朊病毒具有聚集化、PK抗性且抗性肽段在C端的特点,与生理状况下的朊病毒相似。在体外制备出重组朊病毒之后,我们通过颅内注射的方式感染野生型CD-1小鼠,在5个月的潜伏期后小鼠开始出现朊蛋白疾病的相关症状。在病程末期处死小数进行生化和组化检测,其具有脑部存在大量PrPsc(朊蛋白的一种错误折叠构象,具有传染性和致病性的特点)、脑组织海绵状空泡、异常朊蛋白聚集、胶质细胞增生等典型的朊蛋白疾病病理学特点,表明该重组朊病毒确实可以引发小鼠朊蛋白疾病。总的来说,用重组朊蛋白可以在脂质作为辅助因子以及RNA的帮助下,在体外用PMCA技术从无到有地制备出朊病毒,并且该朊病毒具有很强的传染性和致病性,这就强有力的支持了“朊病毒”假说,并且为进一步研究朊蛋白的构象转变提供了良好的实验平台。
[Abstract]:Prion disease, also known as infectious spongiform encephalopathy, is a widespread fatal neurodegenerative disease in mammals. According to the prion hypothesis, prion disease is caused by prion, and its main pathogenic factor is the conformational transition from normal prion protein to pathogenetic prion protein. This conformational prion protein can induce more normal prion proteins to undergo conformational transformation, which leads to neurotoxicity and neurodegenerative lesions. In the process of conformation transformation of prion proteins, it is considered that there are some auxiliary factors involved in the conformation transition of prion proteins, and lipid is an effective auxiliary factor. It is shown that negatively charged lipids can interact with prion proteins and induce conformation transformation and form conformation bodies with competitive resistance. In addition, as a long chain anion compound structure, RNA also accelerates the conformation transition of prion protein. In this paper, we added the recombinant prion protein expressed by E. coli into the system by using the technique of protein misfolding cycle amplification in vitro. The recombinant prions were prepared by conformational transformation of prion protein with negative charge lipid POPG and total RNA extracted from mouse liver. It was identified that the prion had the characteristics of agglomeration PK resistance and the resistance peptide was at the C-terminal, which was similar to that of the prion in physiological condition. After the recombinant prion was prepared in vitro, we infected the wild type CD-1 mice by intracranial injection. After the incubation period of 5 months, the mice began to develop the related symptoms of prion disease. At the end of the course of the disease, the small number was killed for biochemical and histochemical examination. It had a large number of PrPsc in the brain (a misfolded conformation of prion protein, with infectious and pathogenicity), cavernous vacuole in brain tissue, abnormal aggregation of prion protein. The pathological features of some typical prion diseases such as glial cell proliferation indicate that the recombinant prion can indeed cause prion disease in mice. In general, the recombinant prion protein can be used to prepare prions from scratch in vitro with the help of lipids and RNA, and the prions are highly infectious and pathogenicity. This strongly supports the prion hypothesis and provides a good experimental platform for the further study of the conformation transition of prion proteins.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373;R-332

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