本文選題：金黃色葡萄球菌 + 自溶素��； 參考：《大連醫(yī)科大學》2012年碩士論文

【摘要】：目的金黃色葡萄球菌是引起人和動物化膿性感染的重要病原體，可引起局部化膿感染，也可引起敗血癥、膿毒血癥等全身感染�？股氐念l繁使用加劇了金黃色葡萄球菌耐藥現(xiàn)象尤其是多重耐藥，現(xiàn)有抗生素已經(jīng)不足以治愈金黃色葡萄球菌引起的感染，尋找新型抗菌藥物成為研究熱點。 金黃色葡萄球菌的自溶素(AtlA)是一個雙功能蛋白，，經(jīng)過蛋白酶作用可分解為51kD的氨基葡萄糖苷酶（glucosaminidase，AtlG）和62kD的酰胺酶（amidase，AtlM）。本研究通過基因工程技術(shù)獲取金黃色葡萄球菌（ATCC25923）自溶素（autolysin，Atl）的兩個功能片段AtlM和AtlG重組蛋白，并初步探討其在體內(nèi)、體外的抗菌作用，為自溶素作為抗菌藥物的深入研究奠定基礎(chǔ)。 方法1.獲取rAtlM和rAtlG：根據(jù)GenBank中金黃色葡萄球菌自溶素的基因序列（AC:D17366）設計合成兩對特異的引物，采用PCR技術(shù)以ATCC25923的基因組為模板擴增相應的atlM、atlG序列，分別構(gòu)建克隆質(zhì)粒及表達質(zhì)粒；經(jīng)由IPTG誘導表達重組蛋白rAtlM、rAtlG，通過透析袋電洗脫的方法純化并測定其濃度。 2.體外抑菌試驗：采用微量肉湯稀釋法測定rAtlM和rAtlG對標準菌株/耐藥菌株的MIC。用0.01MPBS（pH7.0）混懸金黃色葡萄球菌（終濃度為5×105CFU/ml）,并加入rAtlM/rAtlG(終濃度為50μg/ml)，測定1h、3h、5h時間點rAtlM及rAtlG對細菌生長的抑制作用及差異。 3.體內(nèi)（小鼠）抑菌試驗：以金黃色葡萄球菌標準株及苯唑西林耐藥株分別對小鼠行腹腔接種（菌液濃度107CFU/ml，接種0.5ml），1h后再依次給每組小鼠尾靜脈注射rAtlM或rAtlG0.2ml(蛋白量1mg/只)，對照組接種0.2ml無菌N.S。2h和4h后每只小鼠眼球采血10μl加入血培養(yǎng)瓶，最終以平板菌落計數(shù)測定金黃色葡萄球菌及其耐藥株對rAtlM及rAtlG的藥物敏感性，判斷比較重組蛋白AtlM和AtlG的抗菌活性。 結(jié)果1.成功構(gòu)建了原核表達載體pET-32а(+)/atlM和pET-32а(+)/atlG，表達的重組蛋白AtlM、AtlG經(jīng)過SDS-PAGE電泳鑒定符合預期結(jié)果，約為80kD和66kD，經(jīng)微量紫外分光光度計測得濃度為1.25mg/ml和1.63mg/ml。 2.rAtlM對金黃色葡萄球菌及其耐藥株的MIC分別為16μg/ml和64μg/ml；rAtlG對金黃色葡萄球菌及其耐藥株的MIC分別為8μg/ml和64μg/ml。體外抑菌試驗顯示，rAtlM、rAtlG對金黃色葡萄球菌及其苯唑西林耐藥株均有一定的抑菌作用（3h測定點rAtlM作用于標準株和耐藥株的p值分別為0.004，0.001；3h測定點rAtlG作用于標準株和耐藥株的p值分別為0.004、0.003）；3h和5h測定點，rAtlM對耐藥株的抑菌效果優(yōu)于rAtlG（p值分別為0.001、0.000）。 3.體內(nèi)溶菌試驗初步表明，注射rAtlM或rAtlG的小鼠體內(nèi)金黃色葡萄球菌及其耐藥株數(shù)量顯著低于對照組(2h時rAtlM作用于標準株和耐藥株的p值分別為0.008，0.014；2h時rAtlG作用于標準株和耐藥株的p值分別為0.011、0.026)。 結(jié)論rAtlM和rAtlG對金黃色葡萄球菌及其苯唑西林耐藥株均有一定的抑菌作用，具有作為抗菌藥物的可能性。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378.11

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