【摘要】 核糖体是抗生素的主要靶点,而获得足量高纯度的核糖体是进行结构和药物研究的基础。结核分枝杆菌壁厚且生长缓慢,制备足量高纯度的核糖体具有挑战性。本研究改进并优化了核糖体纯化制备方法,通过大量培养和安全处理致病菌,应用高效破碎厚壁革兰阳性菌的技术,结合传统的蔗糖密度离心分离和蛋白液相色谱纯化技术,经多步纯化和分离,获得了高纯度和较高产率的耻垢分枝杆菌与结核分枝杆菌的核糖体样品,为后续生化实验和结构生物学研究提供了保证。该分枝杆菌核糖体制备方法也可应用于其他革兰阳性致病菌复合物样品的直接提纯,以及复合物特异性的进一步研究,特别是利用晶体学与冷冻电镜结合的高精度复合物结构研究,有助于揭示细菌耐药性机制及用于新型抗生素的研发。更多还原

【Abstract】 Ribosomes of pathogenic bacteria are major targets for antibiotics,and how to prepare ribosome samples with quality and quantity is a key step for structure and antibiotic studies.In particular,it is a challenge to purify enough ribosome from slow-growing Mycobacterium such as Mycobacterium tuberculosis due to slow growth rate and thick cell wall.With the optimized steps,including growing and processing bulk culture with biosafety measures,breaking the thick cell wall of Gram-positive bacteria with effective techniques,and purification methods to combine the fast protein liquid chromatography(FPLC)and sucrose gradient separation,enough ribosome samples with high purity were obtained from Mycobacterium.The prepared samples could be subjected to complex specificity studies,such as high-resolution structure studies with X-ray crystallography and cryo-electron microscopy(cryo-EM),and shed light on the mechanism of antibiotics.This improved method is applicable in other Gram-positive bacteria.更多还原