金黄色葡萄球菌肽聚糖水解酶Ami的异源表达与纯化研究

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中图分类号：R978.1 文献标志码：A

Abstract Objective To screen the peptidoglycan hydrolase （PGH） which can response to carbazole amphiphilicantimicrobial agent CB29，express arecombinantPGH in Escherichiacoli，obtainpurifiedrecombinant PGH and determine its antimicrobial activity. MethodsFirstly，zymogram profiling was used to screen PGH that responsive to CB29 and the His-tag fusion expression sequence was designed.The recombinant expression plasmid was constructed using pET28a （+） as the starting plasmid through enzyme digestion and ligation and induced expression in E☉ . coli. Then， the cels were lysed by freeze-thaw method and ultrasonication，and the recombinant PGH was purified through anickel-Sepharose column，ultrafiltration tube，and dialysis bag.SDS-PAGEand highperformance liquid chromatography-massspectrometry were used to detect and identify the target PGH.Finally， the minimum inhibitory concentration （MIC）of recombinant PGH was determined by the broth micro-dilution method.ResultsThree kinds of PGH with molecular weightsof 51， 62，and 85kDa were identified in S. aureus. They revealed hydrolase bands on polyacrylamide gels as the concentration of antibacterial agent increased.After mass spectrometry identification，they were identifiedas glycosidase （Glc），amidase （Ami），and cell wallbinding protein SpA. The 62kDa band was identified as Ami. The core functional sequence of31l amino acids was determined by bioinformatic analysis，and the expression vector pET28a-rAmi was constructed accordingly. Freeze-thaw once， followed by ultrasonication achieved adequate celldisruption. After purification with nickel-Sepharose column， a 34kDa band of rAmi was obtained. The protein concentration was 578μg/mL after renatured with dialysis.The purified rAmi at a concentration of 50μg/mL had an inhibitory rate of 95% against S. aureus and 93% against methicillin-resistant S. aureus.ConclusionThe rAmi could be effciently expressed by a recombinant amidase-type PGH prokaryotic expressionand purified，and its invitro antibacterial activity was successfully validated，laying the foundation for further research on its role in antibacterial therapy and the development of new antibacterial agents.

Key WordsStaphylococcus aureus; Peptidoglycan hydrolase; Antimicrobial peptide; Recombinant amidase

金黄色葡萄球菌（Staphylococcusaureus）是主要临床分离菌种中检出率最高的革兰阳性菌。（剩余13708字）