【摘要】：金黃色葡萄球菌是最常見的社區(qū)和醫(yī)院感染相關(guān)病原菌。由于廣譜耐藥菌株的出現(xiàn),預(yù)防和治療由金葡菌所引起的疾病變得更加具有挑戰(zhàn)性,尋求新的治療策略已是勢在必行。噬菌體裂解酶是雙鏈DNA噬菌體在感染宿主周期期間產(chǎn)生的細(xì)菌細(xì)胞壁水解酶,其能夠通過水解細(xì)胞壁肽聚糖從而破壞細(xì)胞壁的完整性,最終導(dǎo)致細(xì)菌崩解死亡。由于具有高效、特異的殺菌活性,噬菌體裂解酶被認(rèn)為是控制細(xì)菌病原體的新型潛在藥物。中藥單體黃芩苷能靶向?qū)菇鹌暇玖σ蜃应?溶血素,可以有效治療金葡菌感染。前期,本實(shí)驗(yàn)室獲得了金葡菌的噬菌體裂解酶Lys GH15,其對金葡菌表現(xiàn)出廣譜、高效的殺菌活性。為進(jìn)一步探索裂解酶作用的分子機(jī)制及其與中藥聯(lián)合應(yīng)用的協(xié)同機(jī)理,本研究進(jìn)一步對其催化區(qū)與底物互作的關(guān)鍵位點(diǎn)進(jìn)行分析,并且將裂解酶Lys GH15與黃芩苷進(jìn)行聯(lián)合應(yīng)用,對金葡菌的肺炎感染模型的治療效果進(jìn)行評價。裂解酶作用的分子機(jī)制應(yīng)用Auto Dock 4.2軟件對Lys GH15的CHAP片段與多肽DGln NH2-LLys-DAla-5Gly進(jìn)行分子對接,預(yù)測Asp33、Ser35、Phe36、Tyr50、Arg71、Tyr72、Gln53和Asn75在CHAP與底物作用過程中應(yīng)該會起作用。分別將這7個位點(diǎn)突變?yōu)楸彼岷?通過誘導(dǎo)表達(dá)發(fā)現(xiàn)位點(diǎn)Asp33與Asn75突變?yōu)楸彼岷蟮鞍谉o法成功表達(dá),而Arg71、Tyr72、Ser35、Phe36和Tyr50這五個位點(diǎn)的突變體蛋白成功獲得了表達(dá)。對CHAP片段和Arg71、Tyr72、Ser35、Phe36和Tyr50這五個位點(diǎn)的突變體進(jìn)行了圓二色譜分析,CHAP片段的突變體在蛋白質(zhì)二級結(jié)構(gòu)組成上(α-螺旋和β-折疊的含量)沒有顯著的差異,表明這五個位點(diǎn)不影響CHAP的二級結(jié)構(gòu),也就不會影響CHAP片段的三級結(jié)構(gòu)。酶譜實(shí)驗(yàn)結(jié)果表明,只有F36A不能形成透明條帶,其余四個突變體均可以形成,這說明F36A突變體失去裂菌活性。天然的CHAP和S35A、Y50A、R71A、T72A這四個突變體均可以在60 min內(nèi)使菌液的OD600值從0.6下降到幾乎檢測不到的狀態(tài),對照組加入了緩沖液作為空白對照,在加入同樣的蛋白量的條件下(終濃度50μg/ml),只有F36A突變體的菌液OD600值變化趨勢與對照組相似,菌液濃度沒有下降,而同樣,在菌落計數(shù)的試驗(yàn)中也發(fā)現(xiàn)只有突變體F36A活性完全喪失,而S35A、Y50A、R71A、T72A這四個突變體的抗菌活性與天然的CHAP沒有顯著的差異。由此表明,位點(diǎn)F36在CHAP蛋白與底物互作過程中起關(guān)鍵作用,而Asp33與Asn75對蛋白的折疊可能起關(guān)鍵的作用。裂解酶與黃芩苷聯(lián)合應(yīng)用對Lys GH15與黃芩苷在體外活性的相互影響進(jìn)行了研究。單獨(dú)的Lys GH15處理(劑量為50μg/ml,USA300菌懸液的OD600值在10 min內(nèi)從0.785降至0.194)與Lys GH15(劑量50μg/ml)和黃芩苷(劑量16μg/ml)聯(lián)合處理(USA300菌懸液的OD600值在10 min內(nèi)從0.745降至0.124)相比,裂解活性沒有顯著差異(P0.05)。在溶血實(shí)驗(yàn)中,當(dāng)兔紅細(xì)胞與黃芩苷(16μg/ml)和Lys GH15(≥50μg/ml)聯(lián)合同時孵育時,沒有檢測到溶血。相比之下,用黃芩苷單獨(dú)處理時溶血率為13.1%,當(dāng)用終濃度為25、50或100μg/ml的Lys GH15單獨(dú)處理時,溶血率分別為34.5%、22.74%和20.7%。通過Western blot實(shí)驗(yàn)檢測黃芩苷和Lys GH15對α-溶血素表達(dá)量的影響,表明同時加入黃芩苷(16μg/ml)和Lys GH15(100μg/ml)時幾乎沒有α-溶血素產(chǎn)生。進(jìn)一步研究Lys GH15與黃芩苷聯(lián)合使用治療金葡菌肺炎的效果。結(jié)果表明,聯(lián)合治療組在提高小鼠的健康狀況、降低肺臟W/T比值以及BALF中細(xì)胞數(shù)、蛋白含量及細(xì)胞因子水平上優(yōu)于單獨(dú)治療組。在Lys GH15單獨(dú)治療和聯(lián)合治療的情況下,肺臟細(xì)菌菌落數(shù)降低至8.0×102CFU/mg和5.0×101CFU/mg,在黃芩苷單獨(dú)治療下,肺臟細(xì)菌菌落數(shù)達(dá)到1.0×104CFU/mg。作為對照,在沒有治療的情況下,24 h后的肺臟的細(xì)菌數(shù)量分別增加至3.3×1010CFU/mg,說明Lys GH15與黃芩苷聯(lián)合使用對金葡菌肺炎能產(chǎn)生良好的治療效果。
[Abstract]:Staphylococcus aureus is the most common pathogen associated with community and hospital infections. As a result of the emergence of broad-spectrum drug-resistant strains, prevention and treatment of diseases caused by Staphylococcus aureus have become more challenging. It is imperative to seek new treatment strategies. Phage lysozymes are produced by double-stranded DNA phages during the infection host cycle. Bacterial cell wall hydrolase, which can destroy the integrity of cell wall by hydrolyzing cell wall peptidoglycan, eventually leads to bacterial disintegration and death. Because of its high efficiency and specific bactericidal activity, bacteriophage lysozyme is considered as a new potential drug to control bacterial pathogens. Baicalin, a traditional Chinese medicine monomer, can target the virulence of Staphylococcus aureus. In the early stage, the phage lysozyme Lys GH15 of Staphylococcus aureus was obtained, which showed broad-spectrum and high-efficiency bactericidal activity against Staphylococcus aureus. The key sites of substrate interaction were analyzed and Lys GH15 and baicalin were combined to evaluate the therapeutic effect of Staphylococcus aureus pneumonia infection model. The molecular mechanism of Lys GH15 was docked with DGln NH2-LLys-DAla-5Gly by Auto Dock 4.2 software to predict Asp33. Ser35, Phe36, Tyr50, Arg71, Tyr72, Gln53 and Asn75 should play a role in the interaction between CHAP and substrates. After mutation of these seven sites into alanine, it was found that Asp33 and Asn75 could not be successfully expressed after mutation into alanine, but the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50 were formed. The results showed that there was no significant difference in protein secondary structure composition (alpha-helix and beta-folding content) between the mutants of CHAP fragments and the mutants of Arg71, Tyr72, Ser35, Phe36 and Tyr50, indicating that these five sites did not affect the secondary structure of CHAP, nor did they affect the secondary structure of C. The results of Enzymogram showed that only F36A could not form transparent bands and the other four mutants could form, which indicated that F36A mutants lost fission activity. In the control group, buffers were added as the blank control. Under the same protein content (final concentration 50 ug/ml), the change trend of OD600 value of F36A mutant was similar to that of the control group, and the concentration of F36A did not decrease. Similarly, only F36A mutant completely lost its activity, but S35A, Y50A, R71A mutant completely lost its activity in the colony count test. These results suggest that site F36 plays a key role in the interaction between CHAP protein and substrate, and Asp33 and Asn75 may play a key role in protein folding. Lys GH15 treatment alone (dosage 50 ug/ml, USA 300 suspension OD600 value decreased from 0.785 to 0.194 in 10 minutes) and Lys GH15 (dosage 50 ug/ml) and Baicalin (dosage 16 ug/ml) combined treatment (USA 300 suspension OD600 value decreased from 0.745 to 0.124 in 10 minutes) showed no significant difference in lysis activity (P 0.05). In hemolysis test In contrast, the hemolysis rate was 13.1% when treated with baicalin alone, and 34.5%, 22.74% and 20.7% when treated with baicalin 25, 50 or 100 ug/ml respectively. The effect of baicalin and Lys GH15 on the expression of alpha-hemolysin showed that there was almost no production of alpha-hemolysin when baicalin (16 ug/ml) and Lys GH15 (100 ug/ml) were added at the same time. Bacterial colonies in lung were reduced to 8.0 *102 CFU/mg and 5.0 *101 CFU/mg in Lys GH15 alone and in combination. Bacterial colonies in lung were up to 1.0 *10 CFU/mg in baicalin alone. After 24 hours, the number of bacteria in the lungs increased to 3.3 *1010 CFU/mg, indicating that Lys GH15 combined with baicalin can produce a good therapeutic effect on staphylococcal pneumonia.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R563.1

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1 胡麗媛;金葡菌噬菌體裂解酶LysGH15關(guān)鍵位點(diǎn)的確定及其與黃芩苷聯(lián)合治療小鼠肺炎的研究[D];吉林大學(xué);2017年

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