坚强芽胞杆菌主要分泌蛋白的鉴定及其分泌性序列

Mass spectrometry identification of secreted proteins from Bacillus firmus and analysis of its secretive sequences

摘要:

摘要:【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( chitin

Abstract:

Abstract:［Objective］Bacillus firmus was a common probiotics bacteria in nature and widely used in the shrimp aquaculture．In order to construct expression vectors for secretive proteins，we identified several major secretive proteins of Bacillus firmus by mass spectrometry and analyzed their gene sequences to find signal peptide sequences．［Methods］The secreted proteins of Bacillus firmus were extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)．The 3 highly expressed protein bands in the SDS-PAGE were identified by mass spectrometry Matrix Assisted Laser Desorption Ionization (MALDI)-Time of Flight (TOF)/Time of Flight (TOF) and the sequences were downloaded from National Center for Biotechnology Information (NCBI) database．Then Polymerase Chain Reaction (PCR) primers were designed according to the downloaded sequences and specific DNA bands were amplified sequenced and bioinformatics analyzed．［Results］The proteins were identified as the putative chitinase，enterotoxin A and protein BCG9842 of Bacillus firmus．Three signal peptides were conformed by using the online software SignaIP 3. 0，namely bf-43，bf-37 and bf-16. The cellular localization of the secreted sequences were analyzed by PSORT． And we found that bf-43 located in the outer membrane of cells，bf-37 and bf-16 located in the extracellular cell．［Conclusion］3 major secreted proteins of Bacillus firmus have been identified．3 possible signal peptides were obtained and will be useful for the construction of expression vectors for secretive proteins．