绿脓杆菌SecA ATPase抑制剂筛选模型的建立和应用

发布时间：2018-01-16 14:00

本文关键词：绿脓杆菌SecA ATPase抑制剂筛选模型的建立和应用　出处：《中国协和医科大学》2010年硕士论文　论文类型：学位论文

更多相关文章： 绿脓杆菌 SecA蛋白 抑制剂筛选 酶活

【摘要】： 绿脓杆菌是一种重要的院内感染条件致病菌。它可以引起多种类型的感染,并多为天然耐多种抗生素菌株,感染后不易被控制。近年来,由于广谱抗生素的大量使用以及药物的联合治疗,导致了多药耐药株以及泛耐药株的急速出现。因此,迫切需要发现新型低毒、高效抗绿脓杆菌药物。 Sec (secretion pathway)途径是细胞蛋白质转运中的主要途径,SecA是Sec途径中唯一已知的能量转化酶,可通过水解循环驱使蛋白质前体穿过细菌内膜,是细菌所独有且不可缺少的。因此,本研究选择SecA为药物的靶点并建立相应的药物筛选模型,以期发现新型高效低毒的抗绿脓杆菌药物。本研究首先克隆和表达了PaSecAN68(绿脓杆菌SecA蛋白N端609个氨基酸残基组成的片段,大小约68kDa)和PaSecAN75(绿脓杆菌SecA蛋白N端645个氨基酸残基组成的片段,大小约75kDa)蛋白并研究其酶学性质。选择酶活较高的PaSecAN75作为靶酶,建立和优化了两种绿脓杆菌SecA ATPase抑制剂酶水平筛选模型。采用孔雀绿比色法筛选了3220个化合物样品,得到4个阳性样品；采用NADH比色法筛选了7196个发酵液样品,得到66个阳性样品。随后,又运用细胞水平绿脓杆菌SecA ATPase筛选模型,对上述阳性样品进行了复筛,最终确定了3个阳性化合物样品和6个阳性发酵液样品,这些样品在酶水平和细胞水平对绿脓杆菌SecA ATPase均有较好的抑制作用,为进一步发展新型抗绿脓杆菌药物奠定了基础。
[Abstract]:Pseudomonas aeruginosa is an important opportunistic pathogen of nosocomial infection. It can cause many kinds of infections, and most of them are natural antibiotic resistant strains, which are difficult to control after infection. Due to the extensive use of broad-spectrum antibiotics and the combined treatment of drugs, multidrug resistant strains and pan-resistant strains appear rapidly. Therefore, there is an urgent need to find new low-toxic and high-effective anti-Pseudomonas aeruginosa drugs. The Sec section-pathway pathway is the main pathway in cell protein transport. Seca is the only known energy converting enzyme in the Sec pathway. It is unique and indispensable for bacteria to drive protein precursors through endobacterial membrane by hydrolysis cycle. Therefore, SecA was selected as the target of drug and the drug screening model was established. In order to find a new effective and low-toxic anti-Pseudomonas aeruginosa drug. In this study, we first cloned and expressed a fragment composed of 609 amino acid residues at the N-terminal of SecA protein of Pseudomonas aeruginosa. A fragment consisting of 645 amino acid residues at the N-terminal of SecA protein of Pseudomonas aeruginosa and PaSecAN75. the size of which is about 68 kDa. The size of the protein was about 75kDa and its enzymatic properties were studied. PaSecAN75 with high enzyme activity was selected as the target enzyme. Two SecA ATPase inhibitor enzyme level screening models were established and optimized. The samples of 321 compounds were screened by malachite green colorimetry and 4 positive samples were obtained. A total of 7196 fermentation broth samples were screened by NADH colorimetry, and 66 positive samples were obtained. Then, the cell-level SecA ATPase model was used to screen Pseudomonas aeruginosa. Three positive compound samples and six positive fermentation broth samples were determined. These samples can inhibit SecA ATPase of Pseudomonas aeruginosa at the enzyme level and cell level, which lays a foundation for the further development of new anti-Pseudomonas aeruginosa drugs.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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