【作者】 贺晓静；

【导师】 梁爱华； 饶子和；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 微管组织中心（MTOC）组织真核生物细胞中微管的数量,方向和极性。MTOC复制缺陷将阻断双极纺锤体的形成,使细胞分裂终止在G2/M期,相反,过量的MTOC又会导致癌症的产生。MTOC中含有大约150-200种蛋白,中心蛋白（centrin）是其中之一。中心蛋白是一种约19.5 kDa的酸性钙结合蛋白,属于钙调蛋白超家族。中心蛋白广泛存在于真核生物中,如藻类、酵母、原生动物、高等植物、哺乳动物等。这种蛋白的高保守性和在生物界的普遍存在表明它对于正常细胞的功能是必不可少的。中心蛋白参与了MTOC的复制和分裂,它的突变和缺失会导致细胞的异常分裂。中心蛋白还在依赖于它的纤维系统的收缩中起重要作用。 原生动物八肋游仆虫（Euplotes octocarinatus）是一种进化上处于特殊地位的单细胞真核生物,这类生物中无典型的中心粒和中心体存在,细胞分裂时纺锤体的微管由核液（Karyolymph）中的微管组成中心形成。对这类生物的中心蛋白的研究,有助于阐明低等真核生物的细胞分裂、细胞运动等重要生命活动过程的本质。本研究以八肋游仆虫为材料,克隆了该生物的中心蛋白基因（EoCen）。序列分析表明,该基因全长690 bp,两端为端粒序列C₄A₄C₄A₄C₄A₄C₄,开放阅读框有507bp,编码168个氨基酸,不含内含子。将全长和N端截短型中心蛋白基因连入表达载体,构建了两个重组表达质粒pGEX-6p-EoCen和pGEX-6p-EoCenN,在大肠杆菌中获得了高效可溶性表达,经过GST亲和层析和Mono Q离子交换层析两步纯化后,获得了纯度达95%以上的重组游仆虫中心蛋白,每升LB培养液可以得到纯蛋白约10mg左右。 通过凝胶排阻层析分析了游仆虫中心蛋白的聚合特性,发现盐离子对它结构的变化起很重要的作用。当有盐离子存在时,中心蛋白大部分以拉长的纤维状的单体结构存在,不含盐离子的蛋白随着pH的增高,聚合程度逐步增强。加钙离子与否对有盐离子存在的蛋白没有很大的影响,但没有盐离子的蛋白中如果加入钙离子,会引起蛋白的急剧沉降。N端缺失23个氨基酸的截短体则没有这一现象发生,表明N端对中心蛋白的聚合起重要作用。在中心蛋白的紫外、荧光光谱性质分析中,发现Ca²⁺与apoEoCen结合时引起了蛋白构象的变化,且优先占据中心蛋白的第Ⅲ更多还原

【Abstract】 The number, direction and polarity of microtubules in eukaryotic cells are usually organized by microtubule-organizing centre （MTOC）. Alteration of the MTOC duplication could severely perturb the bipolar spindle formation, and this blocks the cell division in the G2/M phase. In contrast, hyper amplification of the centrosome is associated with cellular transformation and cancer. About 150-200 proteins are permanently or temporarily part of this matrix and among them are several regulatory proteins such as centrin. Centrin is an acidic protein of 19.5 kDa, which belongs to the highly conserved EF-hand CaM super family of Ca²⁺-binding proteins. It is a ubiquitous highly conserved protein in diverse evolutionary lineages, including algal, higher plants, invertebrate and mammalian cells. The high degree of conservation and the ubiquity of the protein in all species investigated so far suggest that centrin is essential for proper cell function. It has been shown to be required for the normal duplication and separation of the microtubule-organizing center. Mutations and complete deletions of the centrin gene have shown that the protein is required for proper cell division. It also plays fundamental roles in contraction of centrin-based fiber systems in eukaryotic cells.Euplotes octocarinatus, a unicellular protozoa, locate in a special phylogenic degree showing a number of exceptional features. MTOC of the unicellular protozoa ciliate is not structurally defined. In Euplotes, the microtubules were organized by the microtubule-organizing center of karyolymph. In this study, the centrin gene was cloned from Euplotes octocarinatus using degenerated primers and telomeric primers. The results of sequence analysis indicated that the full-length gene contains 690 bp including one open reading frame （507 bp） without intron and two telomeric sequences C4A4C4A4C4A4C4 on the termini of the gene. A full length and a N-terminal truncated fragment of the centrin gene were ligated into the expression plasmid pGEX-6P. The fusion proteins GST-EoCen andGST-EoCenN were over-expressed in an E.coli strain B121（DE3）. The proteins were purified to above 95% of purity by GST affinity chromatography and ion exchange chromatography Mono Q and pure protein about 10 mg per liter LB medium were obtained.The aggregation characterization of EoCen was examined by size exclusion in vitro. We found that ion strength had an important impact on the protein’s aggregation and most EoCen showed elongated monomer when salt was presence. The protein without NaCl had the tendency to produce more aggregation with increased basic concentration. The aggregation of the protein in NaCl solution did not change greatly when Ca2+ was added. If put the Ca2+ into the protein solution without NaCl, the protein would precipitate until the NaCl was added and this process was reversible. A truncated form of EoCen, lacking the first 23 residues, shows no turbidity.The results from UV and fluorescence spectra showed that all four EF-hand motives could bind Ca2+ and the III and IV sites at C-terminal lobe were preferably occupied. The CD spectrum did not change much when the temperature and pH changed and this indicated that EoCen was a stable protein.Good quality, well diffracting crystals could be obtained from a condition containing 0.2M NaCl, 20%PEG 3350 and the protein was dissolved in 10 mM CaCl2- EoCen crystals belong to space group $2\2{l\ and diffract to 2.01 A. The unit-cell parameters are a=34.4 A, b=48.9A, c=72.6 A （Fig.2）. Assuming the presence of one molecule in the asymmetric unit, the solvent content is estimated to be about 19.4% and the Matthews coefficient （VM） is about 1.5 A3 Da"1 o All the results indicated that the NaCl and Ca2+ were important for the stability of the EoCen.Wistar rats were immunolized by the purified EoCen with the injection dosage of 0.4ug antigen/g body weight. The polyclonal antibody was prepared. The antibody titer of 1:2560 was detected by indirect ELISA. The antiserum was purified using DEAE-dextran and PVDF membrane. The total proteins from Euplotes was detected by Western blotting with anti-EoCen更多还原