炭疽芽孢杆菌A16R株无菌培养滤液的蛋白质组学分析

Proteomic analysis of Bacillus anthracis A16R strain sterile culture filtrate

目的:初步分析由我国炭疽疫苗株A16R所生产的无菌培养滤液中的主要成分。方法:运用免疫蛋白质组学的手段,从炭疽A16R疫苗株无菌培养滤液的二维凝胶电泳中选择与保护性抗体结合和不与该血清结合但丰度高的蛋白质点,进行质谱鉴定和信号肽分析。结果:共选择73个点,从炭疽芽孢杆菌数据库中鉴定出66个。在66个点中,有43个点与保护性血清结合;23个为高丰度蛋白,不与该血清结合。在与保护性血清结合的43个点中,13个点为不同大小的PA分子,其中相对分子质量83×103和63×103占主导,相对分子质量较小的片段仅4个。其余成分包括炭疽表面蛋白EA1、Sap、胶原黏附蛋白、伴侣分子DnaK等。结论:我们制备的炭疽无菌培养滤液中含有PA成分,PA是炭疽疫苗的重要保护性抗原。但PA以外的其他成分在抗炭疽免疫中起何种作用,是否参与诱发机体免疫应答有待进一步研究。

Objective：To analyze the protein profile of sterile culture filtrate from a Bacillus anthracis vaccine A16R （pXO1^＋ , pXO2^- ） strain. Methods ：Two-dimensional gel electrophoresis （2-DGE） and immunoblot using protective rabbit polyclonal antisera were used to select cross-reactive and high-abundance spots, which were identified by MALDI-TOF- MS, then signal peptide of identified proteins was analyzed. Results ：A total of 66 spots identified from 73 spots selected represented 28 protein entries, including 43 spots reacting with antisera and 23 high-abundance non-cross-reactive spots. Of the 43 cross-reactive spots, 13 were PA molecules of different sizes, with the 83×10^3 and 63×10^3 species being dominant. Only four protein spots were low relative molecular mass fragments such as 20×10^3 and 50×10^3. The rest of the cross-reactive proteins included protein entries related to protein fate （chaperone protein DnaK and collagen adhesive protein）, protein entries of cell envelope （S-layer protein EA1 precursor, S-layer protein Sap precursor）. Conclusion： Our findings indicate that PA is present in B. anthracis vaccine A16R strain sterile culture filtrate and PA is the important protective component of anthrax vaccine. Further work will be required to find ont whether other components such as S-layer proteins, DnaK, collagen adhesive protein also play important roles in inducing immune response against anthrax.