發(fā)布時(shí)間：2018-06-17 07:59

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

【摘要】：目的金黃色葡萄球菌是引起人和動(dòng)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(diǎn)。 金黃色葡萄球菌的自溶素(AtlA)是一個(gè)雙功能蛋白，，經(jīng)過(guò)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(guò)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(gè)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(shè)計(jì)合成兩對(duì)特異的引物，采用PCR技術(shù)以ATCC25923的基因組為模板擴(kuò)增相應(yīng)的atlM、atlG序列，分別構(gòu)建克隆質(zhì)粒及表達(dá)質(zhì)粒；經(jīng)由IPTG誘導(dǎo)表達(dá)重組蛋白rAtlM、rAtlG，通過(guò)透析袋電洗脫的方法純化并測(cè)定其濃度。 2.體外抑菌試驗(yàn)：采用微量肉湯稀釋法測(cè)定rAtlM和rAtlG對(duì)標(biāo)準(zhǔn)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(cè)定1h、3h、5h時(shí)間點(diǎn)rAtlM及rAtlG對(duì)細(xì)菌生長(zhǎng)的抑制作用及差異。 3.體內(nèi)（小鼠）抑菌試驗(yàn)：以金黃色葡萄球菌標(biāo)準(zhǔn)株及苯唑西林耐藥株分別對(duì)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(duì)照組接種0.2ml無(wú)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(jì)數(shù)測(cè)定金黃色葡萄球菌及其耐藥株對(duì)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(dá)載體pET-32а(+)/atlM和pET-32а(+)/atlG，表達(dá)的重組蛋白AtlM、AtlG經(jīng)過(guò)SDS-PAGE電泳鑒定符合預(yù)期結(jié)果，約為80kD和66kD，經(jīng)微量紫外分光光度計(jì)測(cè)得濃度為1.25mg/ml和1.63mg/ml。 2.rAtlM對(duì)金黃色葡萄球菌及其耐藥株的MIC分別為16μg/ml和64μg/ml；rAtlG對(duì)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(yàn)顯示，rAtlM、rAtlG對(duì)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(cè)定點(diǎn)rAtlM作用于標(biāo)準(zhǔn)株和耐藥株的p值分別為0.004，0.001；3h測(cè)定點(diǎn)rAtlG作用于標(biāo)準(zhǔn)株和耐藥株的p值分別為0.004、0.003）；3h和5h測(cè)定點(diǎn)，rAtlM對(duì)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(yàn)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(duì)照組(2h時(shí)rAtlM作用于標(biāo)準(zhǔn)株和耐藥株的p值分別為0.008，0.014；2h時(shí)rAtlG作用于標(biāo)準(zhǔn)株和耐藥株的p值分別為0.011、0.026)。 結(jié)論rAtlM和rAtlG對(duì)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R378.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 韓耀倫;郝玉慶;;變異鏈球菌自溶素研究新進(jìn)展[J];國(guó)際口腔醫(yī)學(xué)雜志;2008年S1期

2 章銳鋒;金黃色葡萄球菌對(duì)糖肽類(lèi)抗生素耐藥機(jī)制研究進(jìn)展[J];國(guó)外醫(yī)藥(抗生素分冊(cè));2003年03期

3 朱德妹;;2005年中國(guó)CHINET葡萄球菌屬耐藥性分析[J];中國(guó)感染與化療雜志;2007年04期

4 王宇新;周曉巍;黨穎;黃培堂;;提高蛋白質(zhì)和多肽類(lèi)藥物代謝穩(wěn)定性的研究進(jìn)展[J];生物技術(shù)通訊;2005年06期

5 劉申;;細(xì)菌自溶素的研究進(jìn)展[J];中國(guó)實(shí)用醫(yī)藥;2009年12期

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