细粒棘球绦虫原头蚴、囊壁蛋白质组学的初步研究

发布时间：2018-08-01 08:28

【摘要】：目的1.寻找出适用于研究细粒棘球蚴的蛋白组学实验方法,获得较为理想的细粒棘球绦虫原头蚴、囊壁组织的双向电泳图谱。2、运用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)获取各蛋白点的肽指纹图谱(PMF)。3、利用生物信息学技术对原头蚴、囊壁的蛋白质组分进行配比分析,了解细粒棘球蚴蛋白质的构成和种类,并将这些蛋白质进行功能划分。最终,为抗包虫病疫苗研究、早期诊断方法的研究和包虫病新型药物作用靶蛋白的筛选提供研究资料。方法采用研磨法处理各样本组织,并用Bradford法对其进行蛋白定量。按照样品中各蛋白质等电点和分子量大小的不同,将蛋白样品进行双向电泳分离。然后利用凝胶成像扫描仪采集并保存双向电泳图像,并使用PDQuest软件分析各蛋白点的相关数据。最后,将凝胶置于自动切胶仪中,通过软件选取切割的目标蛋白,切胶仪将自动切取、收集胶粒。胶粒需经胰蛋白酶酶解处理后,使得蛋白裂解为多个肽段。再通过MALDI-TOF的检测,获得各蛋白的肽指纹图谱(PMF)数据。将该PMF数据与各数据库中的蛋白数据相配比,并初步鉴定各蛋白组分的性质。结果1.在原头蚴、囊壁组织的双向电泳图中,分别分离出233个和107个蛋白点。2.各蛋白点通过MALDI-TOF-MS检测,原头蚴和囊壁分别有106个蛋白点和42个蛋白点成功获得肽指纹图谱(PMF)。3.原头蚴蛋白点中共有23个蛋白点得以初步鉴定,包括有:①细胞骨架蛋白:肌动蛋白、原肌球蛋白、肌球蛋白、副肌球蛋白、动力蛋白激活蛋白。②代谢相关酶类:过氧化物还原酶、转酮醇酶、磷脂酶A2、糖原合成酶。③信号转导蛋白:钙结合蛋白、酪氨酸蛋白激酶、丝/苏氨酸蛋白磷酸酶。④物质转运相关蛋白:细胞色素C、ADP/ATP运输蛋白、脂肪酸结合蛋白;囊壁蛋白点中共有14个蛋白点得到初步鉴定,它们是:①信号转导蛋白:钙结合蛋白、酪氨酸蛋白激酶、丝/苏氨酸蛋白磷酸酶。②代谢相关酶类:过氧化物还原酶、转酮醇酶、磷脂酶A2。③物质转运相关蛋白:细胞色素P450、脂肪酸结合蛋白、载脂蛋白A-I以及白蛋白。结论1.已总结出一套适用于研究细粒棘球蚴蛋白组学的实验方案,获得质量较好的原头蚴、囊壁的双向电泳图谱,分别分离到233个及107个蛋白点。2.获取原头蚴、囊壁共计106个和42个蛋白点的肽指纹图谱以及相关数据。3.初步鉴定了细粒棘球蚴原头蚴、囊壁组织中的部分蛋白点,其中,原头蚴中共有23个蛋白点,囊壁中有14个蛋白点。这些蛋白可能与细粒棘球蚴运动、生长、发育、代谢调控等方面有关。
[Abstract]:Objective 1. To find out the method of proteomics experiment suitable for studying Echinococcus granulosus, and obtain the more ideal echinococci of Echinococcus granulosus (Echinococcus granulosus). Two-dimensional electrophoretic map of cystic wall tissue. (PMF). 3 peptide fingerprinting of each protein point was obtained by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The protein components of protocercaria and cyst wall were analyzed by bioinformatics. To understand the composition and species of echinococcus granulosus proteins and to divide these proteins into functions. Finally, the research data are provided for the study of anti-hydatid vaccine, early diagnosis and screening of target proteins for new drug action of hydatid disease. Methods the samples were treated by grinding and the protein was quantified by Bradford. Protein samples were separated by two-dimensional electrophoresis according to their isoelectric point and molecular weight. Then the gel imaging scanner was used to collect and save the two-dimensional electrophoresis images, and the PDQuest software was used to analyze the relevant data of each protein point. Finally, the gel was placed in the automatic gel cutter, and the target protein was selected by software. After enzymatic hydrolysis of gelatin, the protein was split into multiple peptide segments. The peptide fingerprint (PMF) data of each protein were obtained by MALDI-TOF detection. The PMF data were matched with the protein data in each database, and the properties of each protein component were preliminarily identified. Result 1. 233 protein spots and 107 protein spots were isolated from the two dimensional electrophoretogram of the metacercariae and cystic wall tissues, respectively. The peptide fingerprinting (PMF). 3) was successfully obtained from 106 protein spots and 42 protein spots of procaria and cyst wall by MALDI-TOF-MS detection. A total of 23 protein spots were preliminarily identified, including 1: 1 cytoskeleton proteins: actin, promyosin, accessory myosin, Dynamic protein Activator protein 2 Metabolism related enzymes: peroxidase reductase, transketonase, phospholipase A2, glycogen synthase .3 signal transduction protein: calcium binding protein, tyrosine protein kinase, Silk / threonine protein phosphatase .4 substance transporter associated protein: cytochrome Con ADP / ATP transport protein, fatty acid binding protein; Cystoid protein site, 14 protein spots were preliminarily identified, they are the signal transduction protein of 1: calcium binding protein. Tyrosine protein kinase, silk / threonine protein phosphatase .2 metabolization-related enzymes: peroxidase, transketonase, phospholipase A2.3 substance transporter associated protein: cytochrome P450, fatty acid binding protein, Apolipoprotein A-I and albumin. Conclusion 1. A set of experimental schemes suitable for studying the proteomics of Echinococcus granulosus have been summarized, and a two-dimensional electrophoresis map of the hydatid and cyst wall has been obtained, and 233 and 107 protein spots, respectively, have been isolated. The peptide fingerprints of 106 and 42 protein spots were obtained and the related data. 3. 3. Some protein spots in the cyst wall of Echinococcus granulosus were preliminarily identified. Among them, there were 23 protein spots in the echinococcus and 14 protein spots in the wall of the cyst. These proteins may be related to the movement, growth, development and metabolic regulation of echinococcus granulosus.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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