PDF(Pubmed)
Abstract：
Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and ΦJB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and ΦJB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and ΦJB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The ΦJB-EAD exhibited stronger lytic activity than ΦJB-lys, possibly due to ΦJB-CBD-mediated sequestration of ΦJB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and ΦJB-lys and their EADs are good candidates for LBP purity reagent development.
摘要：
噬菌体内溶素特异性结合特性和杀伤活性支持其在生物技术应用中的潜在用途。包括活的生物治疗产品（LBP）的效力和纯度测试。LBP含有活的生物体，如乳酸菌（LAB），并打算用作药物。我们的方法使用内溶素细胞壁结合域（CBD）进行LBP效力测定，并使用内溶素杀伤活性进行纯度测定。表征了以下五种乳杆菌噬菌体溶素的CBD：CL1，Jlb1，Lj965，LL-H，和ΦJB。它们对27个LAB菌株表现出不同的结合，并发现它们结合肽聚糖或表面聚合物。基于CBD结合的流式细胞术用于计算混合物中两种菌株的活菌计数。CL1-lys,jlb1-lys,和ΦJB-lys及其酶域（EAD）表现出针对LAB的细胞壁消化活性和裂解活性。Jlb1-EAD和ΦJB-EAD对缓冲pH和NaCl变化比各自的溶血素更敏感。ΦJB-EAD表现出比ΦJB-lys更强的裂解活性，可能是由于ΦJB-CBD介导的ΦJB-lys被细胞碎片隔离。CBD多重测定表明，这些蛋白质可能是有用的LBP效力试剂，裂解活性表明CL1-lys，jlb1-lys,和ΦJB-lys及其EAD是LBP纯度试剂开发的良好候选者。
