摘要
目的通过基因原核表达技术获取金黄色葡萄球菌(ATCC25923)自溶素片段AtlG重组蛋白,初步探讨其体外抗菌效应。方法根据GenBank中的atlG基因序列设计合成特异引物,采用PCR技术以金黄色葡萄球菌的基因组为模板扩增相应atlG基因序列;分别构建克隆载体PMD19-T-atlG和表达载体pET-32а(+)/atlG;构建的表达载体经由IPTG诱导后通过等电点洗脱技术获取纯化的重组蛋白AtlG;采用微量肉汤稀释法测定AtlG对金黄色葡萄球菌及其苯唑西林耐药株的最低抑菌浓度(MIC),动态测定金黄色葡萄球菌及其耐药株对AtlG的药物敏感性,初步探讨重组蛋白AtlG的体外抗菌活性。结果成功构建了原核表达载体pET-32а(+)/atlG,表达的重组蛋白AtlG对金黄色葡萄球菌标准株和苯唑西林耐药株的MIC分别为16μg/ml和64μg/ml;相比于未加药物的对照组,rAtlG对标准株作用1h后细菌数显著降低(P=0.045),抑菌效果持续到5h测定点(P=0.006);对苯唑西林耐药株作用3h后显示出抑菌效果(P=0.003),5h后与对照组相比差异无统计学意义。结论 50μg/ml的rAtlG对金黄色葡萄球菌标准株抑菌作用发生快、持续时间长,对苯唑西林耐药株也有一定的抑菌作用,具有作为抗菌药物的可能性。
OBJECTIVE To obtain the glucosaminidase fragment of autolysin （AtlG） from Staphylococcus aureus by prokaryotic expression system and investigate its antibacterial activity in vitro so as to provide basis for the exploration of in vitro antibacterial activity of autolysin fragment. METHODS The specific primers were designed according to atlG of S. aureus gene sequence, and atlG was amplified by PCR from the S. aureus genome. The recombiant plasmid PMD19-T-atlG and pET-32a（＋）/atlG were constructed, respectively. After the induction to pET-32a（＋）/atlG by IPTG, the recombinant protein AtlG（rAtlG ）was purified by electroeluting of bag filter. The MIC of rAtlG for ATCC25923 and resistant S. aureus were determined by the broth mierodilution method. In time-kill experiments, S. aureus and resistant S. aureus were exposured to AtlG（50μg/ml） in PBS and colonies were counted at different time. RESULTS The recombinant plasmid pET-32a（＋）/atlG was constructed success-fully. The MIC of ATCC25923 and resistant S. aureus for rAtlG were 16μg/ml and 64vg/ml, respectively. Time-kill experiments showed that rAtlG（50μg/ml）had bactericidal effect on both S. aureus and resistant S. aureus. At lh, rAtlG began to take effect to S. aureus（P=0. 045）, and the effect lasted to the 5 h detection point（P=0. 006）. To resistant S. aureus, rAtlG took effect at 3h（P=0. 003）, there was no statistical difference at 5 h as compared with the control group. CONCLUSION 50 μg/ml rAtlG has rapid and long-lasting antibacterial activity against the S. aureus, with certain antibacterial activity against the oxacillin-resistant strains, which can be served as the antirnicrobial agents.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2013年第11期2521-2523,共3页
Chinese Journal of Nosocomiology
基金
辽宁省科技厅自然科技基金资助项目(201202053)
