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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 the in vitro Giardia lamblia trophozoites by mass spectrometry. Methods The trophozoites of Giardia lamblia were cultured in modified TYI-S-33 medium, and the total protein was extracted. The protein content was determined by Bradford method. The protein was stained with silver nitrate by 2D-electrophoresis and the gel electrophoresis was detected by scanning The number of protein spots was used to obtain the protein spot matching information. Then the spots with high matching were selected and analyzed by MALDI-TOF-MS to identify the species. Results The total protein content of parasites (4.8241 ± 8) μg / ml. Protein two-dimensional electrophoresis showed that the protein spots were densely packed at about 1 253 points, which covered the entire gel and showed high protein expression. The protein spots were dark, clear and bright. A total of 80 well-matched protein spots were randomly selected from the gel map and 46 proteins of 5 types were identified by mass spectrometry. Among them, 6 (14 points) of cytoskeletal proteins, namely α-Jardine, β- , Giardia, δ-Giardin, voxel, flagellin-1, 18 kinds of metabolic enzymes or related proteins (28 points), 5 kinds of transcribed proteins (5 points), 3 kinds of other proteins (3 points), 14 unknown proteins (14 points). Another 6 points were not detected. Conclusion Mass spectrometry can be used to identify the cytoskeletal proteins of Giardia lamblia in vitro.