【摘要】：本論文主要包括兩方面的內(nèi)容：奇異變形桿菌鞭毛蛋白質(zhì)FliD和人染色體移動(dòng)復(fù)合物的初步晶體學(xué)研究。 鞭毛是細(xì)菌的重要組成部分，同時(shí)也參與致病菌與宿主間的相互作用。細(xì)菌鞭毛的組裝與調(diào)控是由多種蛋白質(zhì)相互協(xié)調(diào)的一個(gè)復(fù)雜過程。然而鞭毛的數(shù)量及其長(zhǎng)度與鞭毛帽子蛋白質(zhì)FliD有著密切的關(guān)聯(lián)，但是對(duì)于鞭毛的組裝機(jī)制并不清楚。本研究通過克隆、表達(dá)及純化的方法在體外表達(dá)純化了大量的奇異變形桿菌鞭毛帽子蛋白質(zhì)FliD；并得到了蛋白質(zhì)的晶體，通過限制性蛋白酶水解實(shí)驗(yàn)及脫水等方法使得其衍射能力從無到3.5；同時(shí)也構(gòu)建了FliD與分子伴侶FliT的復(fù)合物，晶體學(xué)研究正在進(jìn)行中。 染色體移動(dòng)復(fù)合物（Chromosomal Passenger Complex, CPC)由蛋白激酶Aurora B、內(nèi)層著絲粒蛋白、存活蛋白及蛋白Borealin四部分組成，在細(xì)胞有絲分裂和減數(shù)分裂的各個(gè)階段CPC定位于相應(yīng)的位置發(fā)揮重要的調(diào)節(jié)功能。有研究表明Survivin、borealin和INCENP（1-58aa）組成的三元復(fù)合物在體外可以與H3T3ph結(jié)合，同時(shí)通過核磁共振波譜(NMR)驗(yàn)證了Survivin可以單獨(dú)與H3T3ph結(jié)合，表明Survivin是H3T3ph的受體。本研究通過克隆、表達(dá)及純化的方法表達(dá)純化了一系列的子復(fù)合物，最終發(fā)現(xiàn)Survivin單獨(dú)表達(dá)時(shí)穩(wěn)定性不好，存在降解條帶；但是與Borealin(10-109aa)構(gòu)建復(fù)合物時(shí)降解條帶消失。最后選擇克隆、表達(dá)純化Survivin-Borealin（10-109aa）后與生物合成的H3T3ph在體外通過共結(jié)晶的方法得到了衍射能力較好的晶體。解析結(jié)構(gòu)未發(fā)現(xiàn)有H3T3ph結(jié)合，，后續(xù)再繼續(xù)進(jìn)行相關(guān)復(fù)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R3411

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 林思祖;曹光球;丁國(guó)昌;劉寶;;杉木葉片蛋白質(zhì)提取方法的改良[J];安徽農(nóng)學(xué)通報(bào)(上半月刊);2009年17期

2 崔林開;葉華智;;3種ELISA法檢測(cè)Bt殺蟲蛋白的比較研究[J];安徽農(nóng)業(yè)科學(xué);2005年11期

3 王自良;張海棠;王艷榮;張改平;;氯霉素人工免疫原的合成與鑒定[J];安徽農(nóng)業(yè)科學(xué);2006年09期

4 張麗霞;張國(guó)強(qiáng);;大蒜蛋白酶抑制劑的分離純化與部分性質(zhì)研究[J];安徽農(nóng)業(yè)科學(xué);2006年18期

5 孫新城;司艷紅;張莉;王云龍;;抗煙草青枯病拮抗菌的篩選及抑菌活性研究[J];安徽農(nóng)業(yè)科學(xué);2008年14期

6 張曉晴;蔡宇杰;廖祥儒;張大兵;;香樟(Cinnamomum camphora)果實(shí)酪氨酸酶的分離純化及特性分析[J];安徽農(nóng)業(yè)科學(xué);2008年15期

7 柏玉潔;朱啟忠;陳晚華;王旭;任延剛;;毛栓菌木聚糖酶的純化[J];安徽農(nóng)業(yè)科學(xué);2008年19期

8 彌春霞;孫豐賓;李金龍;張妍;曹振嶺;;東北小油菜葉蛋白提取工藝的研究[J];安徽農(nóng)業(yè)科學(xué);2008年20期

9 謝焱;顧國(guó)賢;李崎;;重組β-1,3-1,4-葡聚糖酶的純化及部分酶學(xué)性質(zhì)研究[J];安徽農(nóng)業(yè)科學(xué);2008年20期

10 張靠穩(wěn);馬愛瑛;葉東平;楊婧;;DFRKN-1堿性磷酸酶的提取純化和主要理化性質(zhì)研究[J];安徽農(nóng)業(yè)科學(xué);2008年26期

相關(guān)會(huì)議論文 前10條

1 王在貴;何瑞國(guó);錢世鈞;劉朝良;;特異腐質(zhì)霉纖維素酶的酶譜分析[A];現(xiàn)代農(nóng)業(yè)理論與實(shí)踐——安徽現(xiàn)代農(nóng)業(yè)博士科技論壇論文集[C];2007年

2 張新妙;馬潤(rùn)宇;;用膜結(jié)晶技術(shù)制備蛋白質(zhì)晶體的研究[A];第二屆全國(guó)膜分離技術(shù)在食品工業(yè)中應(yīng)用研討會(huì)論文集[C];2006年

3 高娟;唐明森;王艷玲;蔣再學(xué);竺薇;羅滿林;;重組豬α干擾素發(fā)酵工藝的研究[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)獸醫(yī)公共衛(wèi)生學(xué)分會(huì)第三次學(xué)術(shù)研討會(huì)論文集[C];2012年

4 鄧?yán)?胡小芳;盧明s

本文編號(hào)：2291064