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  本文關(guān)鍵詞：多粘類芽孢桿菌中抗菌物質(zhì)的分離純化及沙門氏菌毒性因子啟動子庫的構(gòu)建與應(yīng)用　出處：《西北大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 多粘類芽孢桿菌 金黃色葡萄球菌 沙門氏菌 啟動子庫 毒性因子 抗生素

【摘要】：金黃色葡萄球菌是一種常見的病原菌,目前已對多種抗生素產(chǎn)生了抗藥性。尋找新的抗金黃色葡萄球菌的物質(zhì),已成為醫(yī)學(xué)和微生物學(xué)的一個重要課題。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn)多粘類芽胞桿菌分泌物對金黃色葡萄球菌有抑制作用。多粘類芽孢桿菌(Paenibacillus polymyxa)是類芽孢桿菌屬中的一種革蘭氏陽性菌,由于其產(chǎn)生的多種次生代謝產(chǎn)物具有抑菌、防腐作用,近年在植物病蟲害防治和抗生素分離研究方面受到了很大關(guān)注。 本實(shí)驗(yàn)從多粘類芽孢桿菌J株培養(yǎng)物中,通過柱層析、高效液相等方法分離純化出了一種活性物質(zhì),其對耐藥性的金黃色葡萄球菌(MRSA)有明顯的抑殺作用。再通過后續(xù)的質(zhì)譜、核磁共振等實(shí)驗(yàn)確定出了該抑菌物質(zhì)主要是兩種分子量為414.5的非多肽類、容易相互轉(zhuǎn)化的類似物。 另一方面,在抗生素的后期分離純化中,由于抗菌物質(zhì)在溶液中含量低,對其抗菌活性跟蹤極其困難。為了解決該問題,構(gòu)建了一個沙門氏菌的毒性因子啟動子-LuxCDABE報告子庫。構(gòu)建好的報告子菌株庫,通過藥物刺激其Lux發(fā)光情況的變化,可以用來檢測低濃度的藥物存在或者研究藥物對啟動子對應(yīng)的毒性因子的影響。這種檢測具有高靈敏、高通量、實(shí)時快速、重復(fù)性好的特點(diǎn)。 研究挑選了沙門氏菌(Salmonella typhimurium LT2)的47個毒性因子,并擴(kuò)展出了它們各自的啟動子區(qū)域,將其連接到了一個質(zhì)粒載體pCS26上,該質(zhì)粒含有未帶啟動子的發(fā)光報告基因LuxCDABE。連接后的啟動子-LuxCDABE報告子質(zhì)粒,經(jīng)多種方法驗(yàn)證后最終轉(zhuǎn)回到了沙門氏菌中。 構(gòu)建好的啟動子-LuxCDABE報告子可以用來檢測濃度很低的多粘類芽孢桿菌抗性提取物：經(jīng)柱層析純化后的抗菌物稀釋1000倍左右,對構(gòu)建好的四株菌的發(fā)光有明顯的減弱作用,但卻不影響沙門氏菌的生長。由于多粘類芽孢桿菌抗性物質(zhì)在高效液相單峰回收液中含量極低,常規(guī)的抑菌圈檢測法很難滿足需求,操作復(fù)雜。高靈敏度的啟動子-LuxCDABE發(fā)光檢測法可以很靈敏地檢測出該物質(zhì)的存在。此外,該毒性因子啟動子庫可以用來研究沙門氏菌的致病因子,而致病因子又是新的抗菌靶位。
[Abstract]:Staphylococcus aureus is a common pathogen, has a variety of antibiotic resistant Staphylococcus aureus. Looking for new material, has become an important topic in medicine and microbiology. Our previous studies showed that polymexa Bacillus secretion has inhibitory effect on Staphylococcus aureus. Paenibacilluspolymyxa (Paenibacillus polymyxa) is a kind of gram positive bacteria Paenibacillus species, due to a variety of secondary metabolites which has antibacterial, antiseptic effect, in recent years in plant pest control and antibiotic separation research has attracted much attention.
This experiment from Paenibacillus polymyxa strain J in culture, through column chromatography, HPLC separation purification method of a kind of active substance, the drug resistance of Staphylococcus aureus (MRSA) has obvious anti-tumor effects. Then through the subsequent mass spectrometry, nuclear magnetic resonance experiment determined the antibacterial substances are mainly two kinds of molecular weight of 414.5 non peptide analogues, easily transformed into each other.
On the other hand, in the separation of antibiotics in purification of antibacterial substances in solution, because the content is low, extremely difficult for antibacterial activity tracking. In order to solve the problem, constructs a virulence factor of Salmonella -LuxCDABE promoter report libraries. The report sub strains library, through the changes of light stimulation of Lux, can be used to detect low concentrations of drug effects of drugs on promoter or corresponding virulence factors. This detection with high sensitivity, high throughput, fast real-time, good reproducibility.
Study on selection of Salmonella (Salmonella typhimurium LT2) 47 virulence factors, and expand their promoter region, connect it to a plasmid pCS26, the plasmid containing luminescent reporter gene LuxCDABE. not with promoter after connecting -LuxCDABE promoter report plasmids by many method validation after the final turn back to the Salmonella.
Construction of promoter -LuxCDABE can be used to report good paenibacilluspolymyxa resistance detection of very low concentration of extract by column chromatography purification of antibacterial substances after diluted 1000 times, the constructed four strains light weaken obviously, but it did not affect the growth of Salmonella. The content of polymyxa resistant substance in HPLC peak recovery liquid is very low, the antibacterial circle detection routine is difficult to meet the demand, complex operation and high sensitivity. The promoter -LuxCDABE luminescence assay can sensitively detect the existence of matter. In addition, the pathogenic factor of virulence factor promoter library can be used to study on the Salmonella bacteria, and pathogenic factors and antimicrobial new target sites.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

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本文編號：1399211