金黄色葡萄球菌噬菌体穿孔素及其与裂解酶CHAP片段重组蛋白的构建与作用机制

穿孔素（holin）和裂解酶（lysin）共同构成噬菌体的二元裂解系统，分别可以特异性地破坏细菌的细胞膜和细胞壁，具有与抗生素完全不同的作用机制，是新概念的抗菌物质。前期本课题组获得了对金黄色葡萄球菌具有广谱、高效裂解活性的裂解酶LysGH15，揭示了其三维结构和分子作用机制，该裂解酶的CHAP片段发挥了主要的裂解活性。近期课题组成功表达了穿孔素HolGH15，它可以有效裂解金黄色葡萄球菌、单核增生李斯特杆菌、枯草芽孢杆菌、大肠杆菌和铜绿假单胞菌，对治疗耐药菌感染具有很大的应用潜力。本项目首先通过工程菌株的筛选和工艺的优化以解决HolGH15难以大量获得的问题，并进一步揭示其作用机制；重点将HolGH15与LysGH15的CHAP片段进行融合表达，深入研究二者的协同作用机制，以获得裂解活性更强、裂解谱更宽的重组蛋白。这将为耐药细菌、尤其是耐药性金黄色葡萄球菌感染的治疗提供有效的新型药物。

The lytic system of phage infecting bacterium consists of holin and lysin which destroy the cell membrane and cell wall respectively. The bactericidal mechanisms of holin and lysin are different from that of antibiotics. Bacteria that are resistant to antibiotics also exhibit sensitive to holin and lysin. Hence, holin and lysin belong to novel bactericidal agents. In the previous studies, we obtained a lysin named LysGH15 (encoded by phage GH15) which shows broad host-range and strong lytic activity against Staphylococcus aureus in vitro and in vivo, especially multi-drug resistant S. aureus. The three-dimensional structure and molecular function mechanism of LysGH15 have been further revealed. The CHAP domain of LysGH15 is responsible for the strong lytic activity. Recently, HolGH15, the holin protein of phage GH15, also has been expressed and shows efficient bactericidal activity against S. aureus, Listeria monocytogenes, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Thus, HolGH15 shows a great potential in the treatment of infections caused by drug-resistant bacteria. In the following work, the engineering strains will be screened and the expression system will be optimized for the sake of obtaining an enormous amount of HolGH15. The bactericidal mechanism of HolGH15 will be researched afterwards. In order to obtain the fusion protein which possesses much stronger and broader bactericidal activity, the recombinant protein CHAP-HolGH15 (or HolGH15-CHAP) will be constructed. Furthermore, the synergistic bactericidal mechanism of HolGH15 and CHAP will be studied comprehensively. Through these researches, the efficacious drugs which possess great potential in treating infectious caused by drug-resistant bacteria, especially by mult-drug resistant S. aureus, will be obtained.