日本血吸虫成虫可溶性抗原蛋白质谱鉴定与生物信息学分析
发布时间：2018-11-11 10:59
【摘要】： 目的:建立及优化日本血吸虫成虫的蛋白质组学分析方法,寻找可溶性蛋白组分中与免疫应答相关的特异性成虫抗原。方法:纯化日本血吸虫成虫可溶性蛋白,应用双向电泳(2D-E)结合免疫印迹技术,获得相应的电泳图谱和免疫印迹图谱,应用PDQuest双向电泳图像分析软件对图像进行分析比对,分析获得蛋白的理论分子量(Mr)和等电点(pI)等信息,标定特异性的抗原蛋白点;从中选取了30个蛋白质点,胶内胰蛋白酶酶切成多肽,利用LC-MS/MS质谱分析技术进行分析,并用SEQUEST软件搜索Schistosoma的非冗余蛋白质序列数据库。成功获得了24个蛋白斑点的肽指纹图谱(PMF)及肽序列检测数据,分属18种蛋白,运用生物信息学等技术在线分析这些蛋白的结构和功能。结果:日本血吸虫成虫可溶性蛋白经双向电泳后,考马斯亮蓝R250染色,电泳图谱显示了约152个主要蛋白斑点。其分子量分布约为14 kDa～134 kDa,pI主要集中在4.94～9.50。进一步用Western blotting鉴定,结果显示实验组图像可见的抗原抗体反应点数目约为57个,对照组为0个;对选定的蛋白质点进行脱色和胶内原位消化、酶解。酶解后的肽混合物经LC-MS/MS分析,获得肽质量指纹图,该图质量较高,峰信号较强,以m/z=200～2000间的片段峰较多,从获得的数据中解析出多肽序列信息,再通过数据库检索确定相应的蛋白质;通过Internet在线分析和生物信息学软件分析,获得这些蛋白的结构、功能相关信息,数据库检索结果表明这些蛋白主要与细胞运动、能量代谢、信号转导、蛋白折叠、修饰、合成等功能相关。对C4蛋白的编码基因、蛋白结构和功能做进一步的分析,序列分析结果提示该cDNA序列含有一个816 bp的开放阅读框序列,编码271个氨基酸,其编码蛋白的理论分子量为29.34 kDa ,pI为9.12。蛋白含有磷酸甘油醛脱氢酶样活性位点、N端NAD(P)结合区域、C端糖的运输和代谢的催化区域等保守结构功能域,具有潜在的抗原表位区域。结论:利用双向电泳分析技术、质谱鉴定技术和生物信息学分析了日本血吸虫成虫可溶性抗原,成功鉴定了多个日本血吸虫成虫抗原蛋白质,并展示其结构和功能。给研究日本血吸虫成虫抗原机制提供新的线索和思路,为今后日本血吸虫的蛋白质组学深入系统研究打下了基础,为开发抗血吸虫感染疫苗提供新的候选分子以及筛选新的免疫诊断抗原提供了可能。
[Abstract]:Aim: to establish and optimize the proteomic analysis method of adult Schistosoma japonicum, and to find out the specific adult antigen in soluble protein components related to immune response. Methods: the soluble protein of adult Schistosoma japonicum was purified, and the corresponding electrophoresis and Western blotting patterns were obtained by 2D-E and Western blotting. The image was analyzed and compared by PDQuest two-dimensional electrophoresis image analysis software. The theoretical molecular weight (Mr) and isoelectric point (pI) of the protein were obtained and the specific antigen protein spots were calibrated. Thirty protein spots were selected, which were digested into polypeptides by trypsin. The polypeptides were analyzed by LC-MS/MS mass spectrometry, and the non-redundant protein sequence database of Schistosoma was searched by SEQUEST software. The peptide fingerprint (PMF) and peptide sequence detection data of 24 protein spots belonging to 18 kinds of proteins were obtained successfully. The structure and function of these proteins were analyzed online by bioinformatics and other techniques. Results: the soluble protein of adult Schistosoma japonicum was stained with Coomassie brilliant blue R250 after two dimensional electrophoresis. Its molecular weight distribution of about 14 kDa~134 kDa,pI was mainly concentrated in 4.94 ~ 9.50. The results showed that the number of antigen-antibody reaction points was about 57 in the experimental group and 0 in the control group. The selected protein spots were decolorized and digested in situ and hydrolyzed by enzyme. The peptide mixture was analyzed by LC-MS/MS, and the peptide mass fingerprint was obtained, which was characterized by high quality and strong peak signal. The polypeptide sequence information was analyzed from the obtained data. Then the corresponding protein was identified by database retrieval. The structural and functional information of these proteins were obtained by Internet online analysis and bioinformatics software analysis. The results of database retrieval showed that these proteins were mainly associated with cell movement, energy metabolism, signal transduction, protein folding, modification. Synthesis and other functions related. Further analysis of the encoding gene, protein structure and function of C4 protein showed that the cDNA sequence contained an open reading frame sequence of 816 bp, encoding 271 amino acids, and the theoretical molecular weight of the encoded protein was 29.34 kDa. PI is 9. 12. The protein contains some conserved functional domains, such as glyceraldehyde phosphate dehydrogenase-like sites, N-terminal NAD (P) binding region, C-terminal carbohydrate transport and metabolic catalytic region, and has potential antigen epitopes. Conclusion: the soluble antigens of adult Schistosoma japonicum were analyzed by two-dimensional electrophoresis, mass spectrometry and bioinformatics. Several proteins of adult Schistosoma japonicum were successfully identified, and their structures and functions were demonstrated. It provides new clues and ideas for studying the antigenic mechanism of adult Schistosoma japonicum and lays a foundation for further and systematic study of the proteomics of Schistosoma japonicum in the future. It is possible to develop a new vaccine against Schistosoma japonicum infection and to screen new immunological diagnostic antigens.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2324626
[Abstract]:Aim: to establish and optimize the proteomic analysis method of adult Schistosoma japonicum, and to find out the specific adult antigen in soluble protein components related to immune response. Methods: the soluble protein of adult Schistosoma japonicum was purified, and the corresponding electrophoresis and Western blotting patterns were obtained by 2D-E and Western blotting. The image was analyzed and compared by PDQuest two-dimensional electrophoresis image analysis software. The theoretical molecular weight (Mr) and isoelectric point (pI) of the protein were obtained and the specific antigen protein spots were calibrated. Thirty protein spots were selected, which were digested into polypeptides by trypsin. The polypeptides were analyzed by LC-MS/MS mass spectrometry, and the non-redundant protein sequence database of Schistosoma was searched by SEQUEST software. The peptide fingerprint (PMF) and peptide sequence detection data of 24 protein spots belonging to 18 kinds of proteins were obtained successfully. The structure and function of these proteins were analyzed online by bioinformatics and other techniques. Results: the soluble protein of adult Schistosoma japonicum was stained with Coomassie brilliant blue R250 after two dimensional electrophoresis. Its molecular weight distribution of about 14 kDa~134 kDa,pI was mainly concentrated in 4.94 ~ 9.50. The results showed that the number of antigen-antibody reaction points was about 57 in the experimental group and 0 in the control group. The selected protein spots were decolorized and digested in situ and hydrolyzed by enzyme. The peptide mixture was analyzed by LC-MS/MS, and the peptide mass fingerprint was obtained, which was characterized by high quality and strong peak signal. The polypeptide sequence information was analyzed from the obtained data. Then the corresponding protein was identified by database retrieval. The structural and functional information of these proteins were obtained by Internet online analysis and bioinformatics software analysis. The results of database retrieval showed that these proteins were mainly associated with cell movement, energy metabolism, signal transduction, protein folding, modification. Synthesis and other functions related. Further analysis of the encoding gene, protein structure and function of C4 protein showed that the cDNA sequence contained an open reading frame sequence of 816 bp, encoding 271 amino acids, and the theoretical molecular weight of the encoded protein was 29.34 kDa. PI is 9. 12. The protein contains some conserved functional domains, such as glyceraldehyde phosphate dehydrogenase-like sites, N-terminal NAD (P) binding region, C-terminal carbohydrate transport and metabolic catalytic region, and has potential antigen epitopes. Conclusion: the soluble antigens of adult Schistosoma japonicum were analyzed by two-dimensional electrophoresis, mass spectrometry and bioinformatics. Several proteins of adult Schistosoma japonicum were successfully identified, and their structures and functions were demonstrated. It provides new clues and ideas for studying the antigenic mechanism of adult Schistosoma japonicum and lays a foundation for further and systematic study of the proteomics of Schistosoma japonicum in the future. It is possible to develop a new vaccine against Schistosoma japonicum infection and to screen new immunological diagnostic antigens.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
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1 胡梅梅;钟政荣;罗庆礼;方婷娜;沈继龙;;基于2-DE和蛋白质组技术筛选的日本血吸虫鸟氨酸氨基转移酶的表达及其诊断应用[J];中国人兽共患病学报;2011年06期
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1 胡梅梅;基于蛋白质组技术筛选的日本血吸虫鸟氨酸氨基转移酶和亲免素的克隆表达及其诊断应用[D];安徽医科大学;2011年
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