金黄色葡萄球菌噬菌体裂解酶LysGH15的抗原性及其代谢动力学

Lysin is a kind of hydrolytic enzyme which is encoded by phage and able to lyse bacteria specifically and efficiently. The bactericidal mechanism of lysin is different from that of antibiotics. Bacteria that are resistant to antibiotics also exhibit sensitive to lysin. Hence, lysin belongs to a novel antibacterial agent. In the previous studies supported by the National Natural Science Foundation of China, we obtained a lysin named LysGH15 (encoded by phage GH15) which shows broad host-range and strong lytic activity against multi-drug resistant Staphylococcal aureus in vitro and in vivo. LysGH15 shows great potential as alternative treatment strategy for infections caused by multi-drug resistant S. aureus. The three-dimensional structure and molecular function mechanism of LysGH15 have been further revealed. All these researches laid solid foundation for the development of LysGH15 as a novel antibacterial. However, the antigenicity and the pharmacokinetic of LysGH15 have not been revealed. It was the scientific question that must be resolve for the application of LysGH15. Thus, in the following work, the antigenicity and the application formulation of LysGH15 will be studied. More importantly, the pharmacokinetic features of LysGH15, including the characterizations of adsorption, distribution, metabolism and excretion in vivo, will be revealed systematically by methods of isotope-labeling, ELISA, chromatography- and mass- spectrography. These studies will provide theoretical and experimental basis for utilization of LysGH15 as novel polypeptide drug in the treatment of infections caused by S. aureus, especially by drug resistant S. aureus.

噬菌体裂解酶可以特异、高效的裂解细菌，有着与抗生素完全不同的作用机制，是极具潜力的新概念抗菌物质。前期在国家自然基金重点课题的资助下获得了对耐药性金黄色葡萄球菌具有广谱、高效裂解活性的噬菌体裂解酶（LysGH15），并进一步揭示了LysGH15的三维结构与分子作用机制，其在体外和体内均表现出良好的治疗效果。但作为一种新型多肽药物，其进入体内后的抗原性、与组织细胞的相互作用及其体内代谢规律尚不清楚，而这是其作为药用必须回答的科学问题。因此，本项目将深入研究该裂解酶进入体内的抗原性及其对机体的影响；重点通过经典的同位素标记示踪法、免疫学鉴定法、色谱-质谱串联检测等方法，深入研究该裂解酶在体内吸收、分布、代谢和排泄等药代动力学特性，为将LysGH15作为治疗耐药金葡菌感染的新型抗菌多肽药物提供坚实的理论与实验基础。

噬菌体裂解酶可以特异性的杀灭细菌，但对植物、动物和人没有危害，由于抗菌作用的特异性，使得其也不会破坏机体的正常菌群，是新概念的“精准型”抗菌物质。课题组前期通过基因工程方法获得了裂解酶LysGH15，在体外和体内均对金黄色葡萄球菌表现出广谱、高效的裂解活性，显示出了很大的研究与应用潜力，且该裂解酶活性片段的三维空间结构及其裂解作用的分子机制也已经得到解析和揭示。但作为一种新型多肽药物，其进入体内后的抗原性、与机体组织细胞的相互作用及其体内代谢规律是作为其药用必须要回答的科学问题。围绕这一科学问题，我们开展了本项目的研究。首先研究了噬菌体裂解酶LysGH15的抗原性及对其裂解活性的影响，表明LysGH15不会诱导菌株产生抗性；其可以诱导机体产生特异性抗体，但抗体不会影响LysGH15体内外最终的抗菌活性；建立了裂解酶LysGH15在血液及各组织药代动力学的ELISA检测方法，该方法的准确度、精密度、回收率和稳定性良好；进一步进行了裂解酶LysGH15在大鼠体内药代动力学研究，检测了大鼠单次静脉注射给药后裂解酶在血浆中的浓度、血药浓度-时间曲线、大鼠单次静脉给药LysGH15的药代动力学参数及在组织的分布情况；对LysGH15进行了慢性毒性实验，给大鼠注射LsyGH15之后，其一般状况良好，体重没有发生明显的变化，其血液学指标和生化指标以及主要组织器官均正常；最后研究了裂解酶LysGH15对表皮葡萄球菌的抗菌活性及对生物被膜的抑制和清除能力，表明裂解酶LysGH15对表皮葡萄球菌和金黄色葡萄球菌均表现出广谱的抗菌活性，同时可以有效清除葡萄球菌形成的生物被膜，且裂解酶LysGH15对体内表皮葡萄球菌感染具有良好的治疗效果。通过本项目的研究，表明LysGH15在抗葡萄球菌和清除葡萄球菌生物被膜方面均表现出巨大的应用潜力，也为将LysGH15作为治疗耐药金葡菌感染的新型抗菌多肽药物提供了坚实的理论与实验基础。