CAJ | 학술논문

目的通过基因工程技术获取金黄色葡萄球菌（ ATCC25923）自溶素片段AtlM重组蛋白（ rAtlM），初步探讨其体外抗菌效应。方法根据GenBank 中金黄色葡萄球菌atlM基因序列（ AC：D17366）设计合成特异引物，采用PCR技术扩增相应的序列并构建重组表达质粒pET-32а（＋）/atlM，经由IPTG诱导后通过等电点洗脱技术获取纯化的rAtlM；采用微量肉汤稀释法测定rAtlM对ATCC25923及其苯唑西林诱导耐药株的最低抑菌浓度（ minimal inhibitory concentration ，MIC）；体外试验测定rAtlM对ATCC25923菌株及其耐药株的抗菌活性。结果成功构建了原核表达载体pET-32а（＋）/atlM，表达的重组蛋白AtlM对ATCC25923标准株和耐药株的MIC分别为8μg/ml和64μg/ml。体外抑菌试验显示rAtlM作用于ATCC25923标准株和耐药株1 h后即对细菌生长具有抑制作用（ P值分别为0．004和0．026），对标准株作用持续到5 h测定点（P＝0．012），对苯唑西林耐药株作用持续到3 h测定点（P＝0．001），5 h后与未加药物的对照组相比差异无统计学意义（P＝0．102）。结论50μg/ml的rAtlM对金黄色葡萄球菌（ ATCC25923）及苯唑西林耐药株初步显示出一定的抑菌作用，具有作为抗菌药物的可能性。
목적통과기인공정기술획취금황색포도구균（ ATCC25923）자용소편단AtlM중조단백（ rAtlM），초보탐토기체외항균효응。방법근거GenBank 중금황색포도구균atlM기인서렬（ AC：D17366）설계합성특이인물，채용PCR기술확증상응적서렬병구건중조표체질립pET-32а（＋）/atlM，경유IPTG유도후통과등전점세탈기술획취순화적rAtlM；채용미량육탕희석법측정rAtlM대ATCC25923급기분서서림유도내약주적최저억균농도（ minimal inhibitory concentration ，MIC）；체외시험측정rAtlM대ATCC25923균주급기내약주적항균활성。결과성공구건료원핵표체재체pET-32а（＋）/atlM，표체적중조단백AtlM대ATCC25923표준주화내약주적MIC분별위8μg/ml화64μg/ml。체외억균시험현시rAtlM작용우ATCC25923표준주화내약주1 h후즉대세균생장구유억제작용（ P치분별위0．004화0．026），대표준주작용지속도5 h측정점（P＝0．012），대분서서림내약주작용지속도3 h측정점（P＝0．001），5 h후여미가약물적대조조상비차이무통계학의의（P＝0．102）。결론50μg/ml적rAtlM대금황색포도구균（ ATCC25923）급분서서림내약주초보현시출일정적억균작용，구유작위항균약물적가능성。
Objective To construct the recombinant protein AtlM ( rAtlM) of Staphylococcal au-reus through prokaryotic expression system and to investigate its antibacterial activity in vitro.Methods The specific primers were designed according to atlM gene sequence of Staphylococcal aureus recorded in Gen-Bank, and atlM gene was amplified by PCR from the Staphylococcal aureus strain (ATCC25923).The re-combinant plasmid pET-32а(+)/atlM was constructed and transformed into Transetta ( DE3 ) to express AtlM after induced by IPTG .The expressed protein AtlM was further analyzed by SDS-PAGE and purified by electroeluting of bag filter.The minimal inhibitory concentrations (MICs) of rAtlM to ATCC25923 and oxac-illin-resistant S.aureus strain were determined by the broth microdilution method .S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain (final concentration of 5×105 CFU/ml) were exposed to rAtlM (50 μg/ml) respectively to test its antibacterial activity in vitro.Results The recombinant protein AtlM was expressed and purified successfully with a relative molecular weight of 80 ×103 and a concentration of 1.25 mg/L.The MICs of rAtlM to ATCC25923 strain and oxacillin-resistant S.aureus strain were 8 μg/ml and 64 μg/ml, respectively.In vitro test showed that rAtlM had inhibitory effects on the growth of ATCC25923 strain and oxacillin-resistant S.aureus strain after 1 h of intervention (P=0.004 and P=0.026, respectively), which lasted to 5 h for ATCC25923 strain (P=0.012) and 3 h for oxacillin-resistant strain (P=0.001).Conclusion This study shows that rAtlM has a certain antibacterial effects on S.aureus ATCC25923 strain and oxacillin-resistant S.aureus strain, suggesting a possibility of serving as antimicrobial agent.