拉曼镊子分析单个苏云金芽孢杆菌伴孢晶体蛋白

应用拉曼镊子研究了来自同一亚种的两个苏云金芽孢杆菌(Bacillus thuringiensis,Bt)菌株的单个伴孢晶体蛋白的拉曼光谱。一束30mW、785nm的近红外激光导入倒置显微镜,形成光镊,俘获水溶液中的单个伴孢晶体蛋白,同时收集被俘伴孢晶体蛋白的拉曼信号。结果表明,单个晶体的拉曼光谱反映了晶体蛋白的分子结构和蛋白组成,得到的拉曼光谱信号更清晰、更灵敏。平均光谱和主成分分析均显示,同一亚种的H7、D4菌株的晶体蛋白的拉曼光谱比较接近。但在同一菌株内有两类不同的伴孢晶体存在,得到了群体分析方法难以得到的信息。本方法不需要复杂的纯化伴孢晶体过程,直接俘获单个伴孢晶体并收集其拉曼光谱,既可以得到群体伴孢晶体的信息,更可以得到群体内每个晶体蛋白之间的信息。

Analysis of Single Parasporal Crystal from Bacillus Thuringiensis by Raman Tweezers

Parasporal crystal, an intracellular protein crystal produced by Bacillus thuringiensis (Bt) during sporulation, are toxic to insects, and used as a successful bio-pesticide wildly. To investigate the biochemical components and structure in single parasporal crystal from Bt, a Raman tweezers setup was used to record the Raman spectrum of single crystal. A NIR laser beam (785 nm) was introduced into an inverted microscope to form a tweezers for trapping the parasporal crystal suspended in water, and the Raman scatter was excited by the same beam. Strains H7 and D4 from the same subspecies Kurstaki of B. thuringiensis were tested. The Raman peaks derived from single crystal were more clear and sharp than from mass crystals. Raman spectrum of single crystal detailed the structural information and the protein components. Mean spectrum and principal component analysis (PCA) revealed the difference in protein components between strain H7 and D4. PCA plots also indicated the two strains produced two kinds of parasporal crystals. The convenience of trapping and scattering of single parasporal crystal suspended in water directly, without the complex preparation of purifying, predict Raman tweezers as a powerful tool on insight into individual crystal of Bt.