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
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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。
Objective:To Establish and optimize the methods of proteomics of adult worm soluble antigen of Schistosoma japonicum with mass spectrum identification, search for specific adult worm antigen inducing immunologic response from soluble protein componant. Methods:The adult worm soluble antigen of S. japonicum was purified and subjected to the two-dimensional electrophoresis and immunoblotting assay. The electrophoregram and immunoblotting pattern was obtained. Some bioinformation was obtained including molecular weight and isoionic point of the proteins with the PDQuest two-dimensional electrophoresis image analysis software and the spots of specific antigens were identified and among these 30 protein spots were picked up. LC-MS/MS was used for sequencing following trypsase digestion of the polypeptides in gel. Non-redundant protein sequence library of Schistosoma was searched with SEQUEST software. The peptide mass fingerprints (PMF) and sequencing data of 24 protein spots were successfully obtained, which belong to 18 proteins, for structural and functional analyses of these proteins by bioinformatics. Results:The soluble antigen of the S.japomicum adult worm were treated by two-dimensional electrophoresis,stainned by coomassie R250 .The electrophoretogram demonstrates about 152 major protein spots.The molecular weight is 14 kDa~134 kDa.And the isoelectric point mainly concentrate upon 4.94～9.50. assayed by Western blotting ,on the experimental group,the number of the blot which embody the reaction between the antigen and antibodies is about 57.While on the control group,the number of blot is zero. 30 protein spots were incised from coomassie staining gel and digested in gel by trypisin. 24 maps of PMF. were obtained and 18 proteins were identified. The PMF has high definition and powerful peak signal.It has more fragment peaks between m/z=200～2000. Analyze the sequence information of the polypeptide according to the acquried data and use the Data Base Retrieval(DBK) to determine the protein.By Internet on-line analysis and the soft of bioinformatics.At last,we got the information on the struture and function of protein. The result of the Data Base Retrieval demonstrate that the protein is correlation with cell movement or energy metabolism or signal transduction..It also has the functional correlation with the overlap,modification and synthesis of the protein.The C4 protein has the important biologic functional locus and domain similar to glyceraldehyde-3-phosphate dehydrogenase. Take further essay of the code geues、stucture and fuction of the C4 protein.The result of the sequence analysis show that the cDNA contain a open reading frame of 816 bp.It can encode 271 amino acid,the mole cule weight of the encode protein is 29.34 kDa,pI 9.12. The protein has the active site similar to the dehydrogenase of the glyceraldehyde phosphate,calmodulin binding dotain on N teminal、the catalytic domain retate to the transportation and metabolism of sugar and the potential epotope domain.Conclusion: Using the technique of two demengional electrophoress,mass sprctrum assay and bioinformatics to analyse the soluble antigen of the S.japomicun adult worm.Sucessfully assay several protein antigens of Sj AWA and demonstrate their structure and function.It provide new clue and ider for study on Sj AWA and setablish the base of the study on proteomics of the S.japonicum.It also provide the possiblity of getting the vaccines of the infection by schistosoma and the Diagnostic antigen of immunodignosis.
Objective:To Establish and optimize the methods of proteomics of adult worm soluble antigen of Schistosoma japonicum with mass spectrum identification, search for specific adult worm antigen inducing immunologic response from soluble protein componant. Methods:The adult worm soluble antigen of S. japonicum was purified and subjected to the two-dimensional electrophoresis and immunoblotting assay. The electrophoregram and immunoblotting pattern was obtained. Some bioinformation was obtained including molecular weight and isoionic point of the proteins with the PDQuest two-dimensional electrophoresis image analysis software and the spots of specific antigens were identified and among these 30 protein spots were picked up. LC-MS/MS was used for sequencing following trypsase digestion of the polypeptides in gel. Non-redundant protein sequence library of Schistosoma was searched with SEQUEST software. The peptide mass fingerprints (PMF) and sequencing data of 24 protein spots were successfully obtained, which belong to 18 proteins, for structural and functional analyses of these proteins by bioinformatics. Results:The soluble antigen of the S.japomicum adult worm were treated by two-dimensional electrophoresis,stainned by coomassie R250 .The electrophoretogram demonstrates about 152 major protein spots.The molecular weight is 14 kDa~134 kDa.And the isoelectric point mainly concentrate upon 4.94～9.50. assayed by Western blotting ,on the experimental group,the number of the blot which embody the reaction between the antigen and antibodies is about 57.While on the control group,the number of blot is zero. 30 protein spots were incised from coomassie staining gel and digested in gel by trypisin. 24 maps of PMF. were obtained and 18 proteins were identified. The PMF has high definition and powerful peak signal.It has more fragment peaks between m/z=200～2000. Analyze the sequence information of the polypeptide according to the acquried data and use the Data Base Retrieval(DBK) to determine the protein.By Internet on-line analysis and the soft of bioinformatics.At last,we got the information on the struture and function of protein. The result of the Data Base Retrieval demonstrate that the protein is correlation with cell movement or energy metabolism or signal transduction..It also has the functional correlation with the overlap,modification and synthesis of the protein.The C4 protein has the important biologic functional locus and domain similar to glyceraldehyde-3-phosphate dehydrogenase. Take further essay of the code geues、stucture and fuction of the C4 protein.The result of the sequence analysis show that the cDNA contain a open reading frame of 816 bp.It can encode 271 amino acid,the mole cule weight of the encode protein is 29.34 kDa,pI 9.12. The protein has the active site similar to the dehydrogenase of the glyceraldehyde phosphate,calmodulin binding dotain on N teminal、the catalytic domain retate to the transportation and metabolism of sugar and the potential epotope domain.Conclusion: Using the technique of two demengional electrophoress,mass sprctrum assay and bioinformatics to analyse the soluble antigen of the S.japomicun adult worm.Sucessfully assay several protein antigens of Sj AWA and demonstrate their structure and function.It provide new clue and ider for study on Sj AWA and setablish the base of the study on proteomics of the S.japonicum.It also provide the possiblity of getting the vaccines of the infection by schistosoma and the Diagnostic antigen of immunodignosis.
引文
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