Cry1Ah蛋白标准物质候选物的制备与纯化

Preparation and purification of Cry1Ah protein candidate reference material

摘要:

随着转基因技术的迅猛发展，转基因产品的安全性受到了广泛关注。转基因检测用有证标准物质在确保转基因产品定性、定量检测结果的可比性和可追溯性方面发挥着重要作用。但转基因蛋白质标准物质的开发相对缓慢，其中一个难点是制备高纯度的转基因蛋白质候选物。苏云金芽胞杆菌Bacillus thuringiensis cry1Ah1基因因其对亚洲玉米螟等鳞翅目害虫有很好的杀虫活性，已用于转基因抗虫作物的研制，并获得具有较好抗虫性状的转基因株系。为了研发Cry1Ah蛋白有证标准物质，亟需建立其制备及纯化体系。文中优化了利用Bt表达系统制备Cry1Ah蛋白的体系，利用离子交换色谱法和排阻色谱法逐级纯化的方法，获得了高纯度的Cry1Ah蛋白 (排阻色谱纯度：99.6%)。生物活性测定结果表明，纯化的Cry1Ah蛋白与原毒素对小菜蛾Plutella xylostella的杀虫活性没有显著差异。最后使用Edman降解法和质谱法确定了Cry1Ah蛋白活化后的氨基酸序列。综上所述，获得的Cry1Ah纯蛋白可用于蛋白质标准物质的研制。

Abstract:

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.