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【目的】坚强芽胞杆菌是一种在自然界普遍存在的益生菌,在对虾养殖中应用较为广泛.为了研究其分泌性蛋白从而为分泌性载体的构建提供理论依据,本文对坚强芽胞杆菌的主要分泌蛋白进行质谱鉴定及分泌性序列的分析。【方法】从本实验室分离保存的1株来自对虾肠道的坚强芽胞杆菌(Bacillus firmus)培养液中获取了分泌性蛋白,进行SDS-PAGE,并对表达量较高的3条蛋白区带进行MALDI-TOF/TOF质谱鉴定及克隆测序,进行生物信息学分析。【结果】鉴定出的蛋白分别是坚强芽胞杆菌几丁质酶(chitinase)、坚强芽胞杆菌肠毒素A(enterotoxin A)和坚强芽胞杆菌BCG9842蛋白(hypothetical protein BCG9842)。经在线软件SignaIP 3.0分析,确定chitinase、enterotoxin和BCG9842均存在不同的分泌性信号肽序列bf-43、bf-37和bf-16,通过在线软件PSORT分析表明,bf-43定位于细胞的外膜上,bf-37和bf-16定位于细胞的胞外。【结论】本研究鉴定出了坚强芽胞杆菌的3个主要分泌性蛋白,分析筛选出了3条分泌性序列,为分泌性载体的构建提供理论依据。
【Objective】 Bacillus subtilis is a kind of probiotics which is ubiquitous in nature and is widely used in shrimp culture.In order to study its secreted protein and provide a theoretical basis for the construction of secreted vector, Protein mass spectrometry identification and secretion sequence analysis. 【Method】 Secreted proteins were obtained from a culture of Bacillus firmus isolated from the intestinal tract of shrimp and analyzed by SDS-PAGE. Three protein bands with high expression level MALDI-TOF / TOF mass spectrometry identification and cloning and sequencing, bioinformatics analysis. 【Result】 The identified proteins were respectively Chlamydophila sp., Enterotoxin A and Hypothetical protein BCG9842. The bf-43, bf-37 and bf-16 were found to be differentially expressed in chitinase, enterotoxin and BCG9842 by online software SignaIP 3.0. The results of online software PSORT showed that bf-43 was located on the outer membrane of cells On the bf-37 and bf-16 located in the extracellular. 【Conclusion】 In this study, three major secreted proteins of Bacillus persica were identified. Three secreted proteins were screened out to provide a theoretical basis for the construction of secreted vector.