金黄色葡萄球菌rAtlM、rAtlG的原核表达及体内外抗菌效应初步探讨

发布时间：2018-06-17 07:59

  本文选题：金黄色葡萄球菌 + 自溶素　； 参考：《大连医科大学》2012年硕士论文

【摘要】：目的金黄色葡萄球菌是引起人和动物化脓性感染的重要病原体，可引起局部化脓感染，也可引起败血症、脓毒血症等全身感染。抗生素的频繁使用加剧了金黄色葡萄球菌耐药现象尤其是多重耐药，现有抗生素已经不足以治愈金黄色葡萄球菌引起的感染，寻找新型抗菌药物成为研究热点。 金黄色葡萄球菌的自溶素(AtlA)是一个双功能蛋白，，经过蛋白酶作用可分解为51kD的氨基葡萄糖苷酶（glucosaminidase，AtlG）和62kD的酰胺酶（amidase，AtlM）。本研究通过基因工程技术获取金黄色葡萄球菌（ATCC25923）自溶素（autolysin，Atl）的两个功能片段AtlM和AtlG重组蛋白，并初步探讨其在体内、体外的抗菌作用，为自溶素作为抗菌药物的深入研究奠定基础。 方法1.获取rAtlM和rAtlG：根据GenBank中金黄色葡萄球菌自溶素的基因序列（AC:D17366）设计合成两对特异的引物，采用PCR技术以ATCC25923的基因组为模板扩增相应的atlM、atlG序列，分别构建克隆质粒及表达质粒；经由IPTG诱导表达重组蛋白rAtlM、rAtlG，通过透析袋电洗脱的方法纯化并测定其浓度。 2.体外抑菌试验：采用微量肉汤稀释法测定rAtlM和rAtlG对标准菌株/耐药菌株的MIC。用0.01MPBS（pH7.0）混悬金黄色葡萄球菌（终浓度为5×105CFU/ml）,并加入rAtlM/rAtlG(终浓度为50μg/ml)，测定1h、3h、5h时间点rAtlM及rAtlG对细菌生长的抑制作用及差异。 3.体内（小鼠）抑菌试验：以金黄色葡萄球菌标准株及苯唑西林耐药株分别对小鼠行腹腔接种（菌液浓度107CFU/ml，接种0.5ml），1h后再依次给每组小鼠尾静脉注射rAtlM或rAtlG0.2ml(蛋白量1mg/只)，对照组接种0.2ml无菌N.S。2h和4h后每只小鼠眼球采血10μl加入血培养瓶，最终以平板菌落计数测定金黄色葡萄球菌及其耐药株对rAtlM及rAtlG的药物敏感性，判断比较重组蛋白AtlM和AtlG的抗菌活性。 结果1.成功构建了原核表达载体pET-32а(+)/atlM和pET-32а(+)/atlG，表达的重组蛋白AtlM、AtlG经过SDS-PAGE电泳鉴定符合预期结果，约为80kD和66kD，经微量紫外分光光度计测得浓度为1.25mg/ml和1.63mg/ml。 2.rAtlM对金黄色葡萄球菌及其耐药株的MIC分别为16μg/ml和64μg/ml；rAtlG对金黄色葡萄球菌及其耐药株的MIC分别为8μg/ml和64μg/ml。体外抑菌试验显示，rAtlM、rAtlG对金黄色葡萄球菌及其苯唑西林耐药株均有一定的抑菌作用（3h测定点rAtlM作用于标准株和耐药株的p值分别为0.004，0.001；3h测定点rAtlG作用于标准株和耐药株的p值分别为0.004、0.003）；3h和5h测定点，rAtlM对耐药株的抑菌效果优于rAtlG（p值分别为0.001、0.000）。 3.体内溶菌试验初步表明，注射rAtlM或rAtlG的小鼠体内金黄色葡萄球菌及其耐药株数量显著低于对照组(2h时rAtlM作用于标准株和耐药株的p值分别为0.008，0.014；2h时rAtlG作用于标准株和耐药株的p值分别为0.011、0.026)。 结论rAtlM和rAtlG对金黄色葡萄球菌及其苯唑西林耐药株均有一定的抑菌作用，具有作为抗菌药物的可能性。
[Abstract]:Objective Staphylococcus aureus is an important pathogen causing suppurative infection in human and animal. It can cause partial pyogenic infection, sepsis, sepsis and other systemic infections. The frequent use of antibiotics has aggravated the phenomenon of drug resistance of Staphylococcus aureus, especially multidrug resistance. The existing antibiotics are not enough to cure the infection caused by Staphylococcus aureus. The autolysin AtlA of Staphylococcus aureus is a bifunctional protein, which can be decomposed into 51kD glucosaminidase (AtlG) and 62kD amidase (AtlMN) by protease. In this study, two functional fragments, AtlM and AtlG recombinant proteins of S.aureus ATCC25923) were obtained by genetic engineering, and their antibacterial activities in vitro and in vivo were studied. It lays a foundation for the further study of self-lysins as antimicrobial agents. Method 1. To obtain rAtlM and rAtlG: two pairs of specific primers were designed and synthesized according to the gene sequence of Staphylococcus aureus autolysin in GenBank (AC1: D17366). Using the genome of ATCC25923 as template, two pairs of primers were designed and amplified by PCR, and the cloned plasmids and expression plasmids were constructed. The recombinant protein was induced by IPTG to express the recombinant protein rAtlG. The recombinant protein was purified and determined by electroelution with dialysis bag. In vitro bacteriostatic test: the MICM and rAtlG of rAtlM and rAtlG against standard / resistant strains were determined by broth dilution method. Staphylococcus aureus (final concentration 5 脳 10 5 CFU / ml) was mixed with 0.01MPBS0 pH 7.0, and rAtlM / rAtlG (final concentration was 50 渭 g / ml) was added to determine the inhibitory effect of rAtlM and rAtlG on bacterial growth at 1 h and 3 h / 5 h. In vivo (mouse) bacteriostasis test: mice were inoculated intraperitoneally with staphylococcus aureus standard strain and oxacillin resistant strain respectively (the concentration of bacterial fluid was 107 CFU / ml, 1 h after inoculation with 0.5 ml / 1 h), and then each group was injected with rAtlM or rAtlG 0.2 ml (protein content 1mg/). The control group was inoculated with 0.2ml aseptic N.S. 2 h and 4 h later, 10 渭 l blood was collected from each mouse eyeball and added to the blood culture bottle. Finally, the drug sensitivity of Staphylococcus aureus and its resistant strains to rAtlM and rAtlG were determined by plate colony count, and the antibacterial activities of the recombinant protein AtlM and AtlG were compared. Result 1. The prokaryotic expression vectors pET-32 (pET-32 M and pET-32 G) were successfully constructed, and the recombinant protein AtlMN AtlG was identified by SDS-PAGE electrophoresis to meet the expected results. The mics of 1.25mg/ml and 1.63 mg / ml 路rAtlM against Staphylococcus aureus and its resistant strains were 16 渭 g/ml and 64 渭 g / ml, respectively, and the mics of microultraviolet spectrophotometer were 8 渭 g/ml and 64 渭 g / ml for Staphylococcus aureus and Staphylococcus aureus resistant strains, respectively. In vitro bacteriostasis test showed that rAtlM rAtlG had a certain bacteriostatic effect on Staphylococcus aureus and its oxacillin resistant strain. The p value of rAtlM on standard strain and drug resistant strain was 0.004 and 0.001, respectively. The p values of rAtlG acting on standard strain and resistant strain were 0.004 ~ 0.003 ~ 3 h and 5 h respectively. The bacteriostatic effect of rAtlG on resistant strain was better than that of rAtlG _ (P) 0.001 ~ 0.000 ~ 0.000 ~ (-3), respectively. The bacteriolytic test in vivo showed that the number of staphylococcus aureus and its resistant strains in mice injected with rAtlM or rAtlG was significantly lower than that in control group at 2h after treatment with rAtlM (p = 0.0080.14). At 2h, the p value of rAtlG on standard strain and resistant strain was 0.011 ~ 0.026, respectively. Conclusion both rAtlM and rAtlG have certain bacteriostatic effects on Staphylococcus aureus and its oxacillin resistant strains, and have the possibility of being used as antimicrobial agents.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.11

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