Abstract：
The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.
摘要：
苏云金芽孢杆菌在产孢过程中产生的杀虫晶体蛋白是抗鳞翅目的活性成分，双翅目,和鞘翅目昆虫。已经报道了几种定量方法，例如晶体计数，ELISA,和SDS-PAGE/光密度测定。工业过程中的主要任务之一是分析原材料依赖性和成本。因此，晶体蛋白定量方法有望与复杂且廉价的培养基成分的存在相兼容。这项工作提出了一种重新验证的基于洗脱的方法，用于定量天然菌株苏云金芽孢杆菌RT产生的杀虫晶体蛋白。为了量化蛋白质，通过改变加载到SDS-PAGE凝胶中的BSA的量产生校准曲线。首先,进行SDS-PAGE用于生物杀虫剂的质量控制。然后，从10%聚丙烯酰胺凝胶上切下染色的蛋白质条带,在95℃下45分钟内,用SDS醇溶液(3%SDS在50%异丙醇中)洗脱蛋白质缔合的染料。该方案是定量2.0-10.0µg范围内的蛋白质的敏感程序。作为概念的证明，通过SDS-PAGE分离从复杂发酵液中获得的样品的蛋白质。然后，适当定量Cryl和Cry2蛋白。
