增生型胸腺组织重症肌无力相关蛋白的鉴定研究  

Identification of the related proteins in myasthenia gravis hyperplastic thymus tissues

作　　者：高峰[1] 程豪为 崔新征[2] 郭亮[2] 杜英[3] 张清勇[2] 

机构地区：[1]河南省医学科学研究所,郑州450052 [2]郑州大学第二附属医院胸外科 [3]郑州大学基础医学院微生物与免疫学教研室,郑州45005

出　　处：《中国实用神经疾病杂志》2006年第6期11-14,共4页Chinese Journal of Practical Nervous Diseases

基　　金：国家自然科学基金资助项目30570625;河南省科研院专项基金资助项目0441130303

摘　　要：目的筛选出增生型胸腺组织重症肌无力(myasthenia gravis,MG)相关蛋白,并对其作出鉴定。方法分别利用异体MG患者血清、正常人血清和自身免疫病患者血清为第一抗体,与电泳转移到硝酸纤维素膜上的蛋白点孵育,做Western Blot-ting试验;然后在利用双向电泳技术获得的参考凝胶上挖取MG组特异显色点所对应的蛋白质点,经胰蛋白酶胶内酶切,对所得多肽混合物进行基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF-MS)分析,得到肽质量指纹图谱(peptide mass fingerprinting,PMF)数据,登陆http://prospector.ucsf.edu/ucs fhtml4.0/msfit.htm网站,用MS Fit程序在NCBInr.10.13.2004数据库检索,鉴定蛋白质。结果Western Blotting试验共获得5个MG特异显色点,编号为2、4、6、7、9;经质谱分析得到5个蛋白质PMF图谱,数据库检索这些蛋白分别为:2号蛋白点isopeptidaseT、4号蛋白点importin beta subunit和6号蛋白点SOUL protein。7号和9号蛋白点未能得到确认,需要更换检索条件或进行蛋白质测序才能进一步认定。结论运用蛋白质组学和Western Blotting研究方法,共获得5个MG相关蛋白,得到鉴定的三个蛋白分别为2号蛋白点isopeptidaseT、4号蛋白点importin beta subunit和6号蛋白点SOUL protein。Objective With techniques of 2-DE（Two-dimensional gel electrophoresis）,the thesis analyzes the proteome of myasthenia gravis hyperplastic thymus tissue and screens the MG related proteins,which then identified by matrix assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF-MS）. Methods Compared with the control（normal serum）and AID（autoimmune disease serum）, MG serums were incubated with protein spots on the NCM. After western blotting, the colored corresponding protein spots in 2-DE gel were cut and done ingel digestion,and the mixture of peptides was send to MALDI-TOF-MS. Proteinidentification was finished by searching the PMF（peptide mass fingerprinting） data in NCB Int. 10. 13. 2004 protein data bank using MS Fit program at http：//prospector, ucsf. edu/ucsfhtml4.0/msfit, htm. Results （1）5 MG specificity colored protein spots are obtained and marked NO. 2, NO. 4, NO. 6, NO. 7 and NO. 9;（2） Searching the NCBInr. 10.13. 2004 protein data bank by inputting the PMF data,NO. 2,NO. 4 and NO. 6 protein are considered as isopeptidase T,importin beta subunit and SOUL protein, respectively. NO. 7 and NO. 9 are unidentified. Conclusion Utilizing 2-DE techniques and western blotting assay,5 MG related proteins are obtained. NO. 2,NO. 4 and NO. 6 protein are considered as isopeptidase T,importin beta subunit and SOUL protein, respectively.

关 键 词：重症肌无力 胸腺组织 双向电泳 基质辅助激光解析电离飞行时间质谱 蛋白质组 

分 类 号：R746.1[医药卫生—神经病学与精神病学]