奇异变形杆菌鞭毛蛋白质FliD和人染色体移动复合物的初步晶体学研究

发布时间：2018-10-24 10:02

【摘要】：本论文主要包括两方面的内容：奇异变形杆菌鞭毛蛋白质FliD和人染色体移动复合物的初步晶体学研究。鞭毛是细菌的重要组成部分，同时也参与致病菌与宿主间的相互作用。细菌鞭毛的组装与调控是由多种蛋白质相互协调的一个复杂过程。然而鞭毛的数量及其长度与鞭毛帽子蛋白质FliD有着密切的关联，但是对于鞭毛的组装机制并不清楚。本研究通过克隆、表达及纯化的方法在体外表达纯化了大量的奇异变形杆菌鞭毛帽子蛋白质FliD；并得到了蛋白质的晶体，通过限制性蛋白酶水解实验及脱水等方法使得其衍射能力从无到3.5；同时也构建了FliD与分子伴侣FliT的复合物，晶体学研究正在进行中。染色体移动复合物（Chromosomal Passenger Complex, CPC)由蛋白激酶Aurora B、内层着丝粒蛋白、存活蛋白及蛋白Borealin四部分组成，在细胞有丝分裂和减数分裂的各个阶段CPC定位于相应的位置发挥重要的调节功能。有研究表明Survivin、borealin和INCENP（1-58aa）组成的三元复合物在体外可以与H3T3ph结合，同时通过核磁共振波谱(NMR)验证了Survivin可以单独与H3T3ph结合，表明Survivin是H3T3ph的受体。本研究通过克隆、表达及纯化的方法表达纯化了一系列的子复合物，最终发现Survivin单独表达时稳定性不好，存在降解条带；但是与Borealin(10-109aa)构建复合物时降解条带消失。最后选择克隆、表达纯化Survivin-Borealin（10-109aa）后与生物合成的H3T3ph在体外通过共结晶的方法得到了衍射能力较好的晶体。解析结构未发现有H3T3ph结合，，后续再继续进行相关复合物的研究。
[Abstract]:This paper mainly includes two aspects: Proteus mirabilis flagellum protein FliD and the preliminary crystallographic study of human chromosome moving complex. Flagella is an important component of bacteria and also participates in the interaction between pathogenic bacteria and hosts. Bacterial flagellum assembly and regulation is a complex process coordinated by a variety of proteins. However, the amount and length of flagella are closely related to the protein FliD of flagella cap, but the mechanism of flagellum assembly is not clear. In this study, a large number of Proteus mirabilis flagella protein FliD; was expressed and purified in vitro by cloning, expression and purification, and the crystal of the protein was obtained. The diffraction ability of FliD was increased from zero to 3.5 by means of restriction protease hydrolysis and dehydration. The complex of FliD and molecular chaperone FliT was also constructed, and crystallographic studies were in progress. Chromosome moving complex (Chromosomal Passenger Complex, CPC) consists of four parts: protein kinase Aurora B, inner centromere protein, survival protein and protein Borealin, which play an important role in regulating the location of CPC in various stages of cell mitosis and meiosis. Some studies have shown that the ternary complexes composed of Survivin,borealin and INCENP (1-58aa) can bind to H3T3ph in vitro, and Survivin can bind to H3T3ph alone by NMR (NMR), indicating that Survivin is the receptor of H3T3ph. In this study, a series of subcomplexes were expressed and purified by cloning, expression and purification. Finally, it was found that the Survivin expressed alone had poor stability and degradation bands, but the degradation bands disappeared when it was constructed with Borealin (10-109aa). Finally, the crystal with good diffraction ability was obtained by cocrystallization of Survivin-Borealin (10-109aa) and biosynthetic H3T3ph in vitro. No H3T3ph binding was found in the analytical structure, and further studies on the related complexes were carried out.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3411

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