质谱技术鉴定体外蓝氏贾第鞭毛虫的细胞骨架蛋白  

In vitro identification of the cytoskeleton proteins of Giardia lamblia using a MS technique

作　　者：何冰[1] 刘广伟[1] 曹蕾[1] 余源[1] 陈阳[1] 田喜凤[1] 杨志宏[2] 卢思奇[3] 赵永强[4] 

机构地区：[1]河北联合大学生物科学系,河北唐山063000 [2]唐山市职业技术学校 [3]首都医科大学病原生物学系,北京100069 [4]军事医学科学院国家生物医学分析中心,北京100850

出　　处：《中国病原生物学杂志》2010年第12期898-900,共3页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30970313);河北省科技厅项目(No.07276101D-68)

摘　　要：目的用质谱技术鉴定体外蓝氏贾第鞭毛虫滋养体的细胞骨架蛋白。方法用改良TYI-S-33培养基培养蓝氏贾第鞭毛虫滋养体,提取虫体总蛋白,Bradford法测定蛋白质含量,样品经蛋白质2D-电泳后用硝酸银染色,扫描检测凝胶电泳图蛋白点的数量,获取蛋白质点匹配信息,然后挑选匹配性高的蛋白点,进行质谱(MALDI-TOF-MS)分析,鉴定其种类。结果虫体总蛋白质含量(4.8241±8)μg/ml。蛋白质二维电泳显示,蛋白点密集,约1 253个点,布满整块凝胶,蛋白表达量高,蛋白点颜色深且清晰、明亮。在胶图中随机选取80个匹配良好的蛋白点,经质谱分析,鉴别出5类46种蛋白质,其中细胞骨架蛋白6种(14个点),即α-贾第素、β-贾第素、γ-贾第素、δ-贾第素、中体素、鞭毛蛋白-1,代谢酶或相关蛋白18种(28个点),翻译转录蛋白5种(5个点),其他蛋白3种(3个点)、未知蛋白14种(14个点)。另有6个点未测出。结论质谱分析技术可用于体外蓝氏贾第鞭毛虫细胞骨架蛋白的鉴定。Objective To identify the cytoskeletal proteins of Giardia lamblia trophozoites in vitro using mass spectrometry. Methods G.lamblia was cultured axenically with modified TYI-S-33 medium.Total protein from trophozoites was extracted and quantified using Bradford＇s method.Then,the samples were subjected to 2D-protein electrophoresis and electrophoresis gels were stained with silver nitrate.The number and types of protein spots scanned by image scanning were determined and analyzed using Image Master TM 2D 5.0 software.Information on matching protein spots was obtained and matching protein spots were selected for mass spectrometry（MALDI-TOF MS） analysis.Results The total protein content was（4.8241±8）μg/ml.Two-dimensional gel electrophoresis map analysis identified about 1 253 protein spots covering the entire gel.Protein spots were concentrated and expressed a high level of protein,and proteins were richly colored,bright,and distinct.From among 80 well-matched protein spots,mass spectrometry analysis identified 5 categories of 46 kinds of proteins.Six（14 points） were the cytoskeleton proteins α-giardin,β-giardin,γ-giardin,δ-giardin,centrin,and phase-1 flagellin.Eighteen were metabolic enzymes or associated proteins（28 spots）,5 were translated transcription proteins（5 spots）,3 were other proteins（3 spots）,and 6 spots could not be identified.Conclusion An MS technique can be used to identify the cytoskeleton proteins of Giardia lamblia trophozoites in vitro.

关 键 词：蓝氏贾第鞭毛虫 双向电泳 质谱 细胞骨架蛋白 

分 类 号：R382.2[医药卫生—医学寄生虫学]