【作者】 王玉霞；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2006， 博士

【摘要】 本文尝试运用“shotgun”蛋白质组学研究技术和策略解决生物工程中的问题。首先将蛋白质组学应用于抗生素代谢途径的研究;其次将蛋白质组学应用于肿瘤生物标志物和潜在药物靶标的寻找;在此基础上,引入18O同位素标记技术,定量地考察K562细胞调亡过程中蛋白质组的变化。结果如下:运用2D LC-MS/MS分析了藤黄灰链霉菌抗生素生产期菌株103和cnn1的总蛋白,分别得到726和809个蛋白。运用生物信息学手段,结合实验室研究结果,将所有可能的代谢途径关键酶在所构建的数据库中进行搜索,两个菌株中均发现有聚酮合成途径的关键酶聚酮合成酶和其它几个与该途径相关的酶,说明麦拓莱霉素通过聚酮合成途径合成。结合β-酮酯酰合酶的抑制剂实验结果,说明以上结论的正确性。展示了蛋白组学在寻找生物合成途径方面的价值。基于“shotgun”、一维凝胶电泳预分离策略,分离鉴定了K562细胞总蛋白提取物,得到1707个蛋白。运用生物信息学手段,对肿瘤生物标志物和药物靶标进行了预测,表明MLL3、SET、DEK、Stathmin、Nucleophosmin等蛋白可能为潜在的生物标志物或治疗肿瘤和白血病的药物作用靶点。通过亚细胞分级、一维凝胶电泳、串联质谱分析了K562细胞核蛋白质,共鉴定了334个蛋白。其中检测到了与肿瘤发生、发展、诊断相关的重要基因产物,包括NF45 protein、PIBF1、DDX48、IKAP等蛋白,提供了可深入研究的肿瘤相关分子靶标。采用18O同位素标记法定量比较了多烯紫杉醇诱导k562细胞凋亡过程中可溶性蛋白质组的变化,在所分析的86个蛋白中,46个蛋白表达量没有发生变化,15个蛋白表达量下调,包括SET蛋白、延长因子EEF1A1等;20个蛋白表达量上调,包括α-烯醇酶、过氧化物还原酶等。这些被抑制和被激活的蛋白可能为肿瘤生物标志物和药物作用靶点,为肿瘤的诊断和治疗提供了新的候选蛋白。更多还原

【Abstract】 The“shotgun”proteomic technologies and strategies were introduced to solving the bioengineering problems. Firstly, it was used to investigate the antibiotic biosynthesis pathways. Secondly, it was used to seek candidate tumor biomarkers and developing of new drug targets. Thirdly, 18O stable isotope labeling technology was introduced to quantificationally investigate the proteome changes in process of apoptosis of k562 induced by docetaxel.For strain 103 and cnn1, a total of 726 and 809 proteins were identified respectively using 2D LC-MS/MS. By exploring the key enzymes of several probable antibiotic biosynthesis pathways in the identified proteins of strain 103, only polyketide synthase and other proteins associated with the polyketide pathways were found, indicating that Maituolaimysin might be synthesized through the polyketide pathway. Combining with the results of the inhibition experiments of beta-ketoacyl ACP synthase, the above conclusion was proved.SDS-PAGE and sub-cellular pre-fractionation were added to achieve a more comprehensive protein profile of k562 cell line. A total of 1707 non-redundant proteins were identified. Proteins like to be associated with cell cycle, apoptosis, tumor and leukemia were investigated. Many proteins that have the potential to be biomarkers or drug targets of leukemia therapy, including MLL3, SET, DEK, Stathmin and nucleophosmin were identified unambiguously to exist in k562 cell line. For the nuclear proteins of k562 cell line, a total of 334 proteins were identified. Proteins probably associated with tumor such as NF45 protein、PIBF1、DDX48、IKAP were found.A total of 86 proteins were analyzed in the experiment of 18O labeled quantitative proteomics. Forty-six proteins were equal-expressed. Fifteen proteins were down-expressed including SET, EEF1A1, etc. Twenty proteins were up-expressed including alpha enolase, Peroxiredoxin-2, etc. These repressed or activated proteins were the potential tumor biomarkers and drug targets, which would offer the candidate proteins for tumor diagnosis and treatments.更多还原