本文選題：普通變形桿菌　切入點：噬菌體　出處：《吉林大學》2011年碩士論文　論文類型：學位論文

【摘要】：變形桿菌在自然界中分布較廣泛是人體腸道的正常菌群。只有離開了正常寄居的腸道而進入人體其他部位時才引起感染。在醫(yī)院內,長期接受廣譜抗生素、激素、免疫抑制劑、抗腫瘤藥等治療的機體抵抗力下降的病人易感染,先進的檢查治療方法在某種程度上也為變形桿菌入侵人體提供了途徑,使變形桿菌在醫(yī)院內感染中占有重要地位。 噬菌體治療已表現(xiàn)出許多突出的優(yōu)越性,尤其是針對使用抗生素治療動物細菌感染時易產(chǎn)生細菌耐藥性及殘留等缺點。經(jīng)過大量的動物模型試驗及人類臨床治療的實踐,對于噬菌體治療中存在的缺點和不足,人們已經(jīng)開始在噬菌體的選擇、宿主譜、作用效力及其作用的動力學原理等方面進行探索性的改造,從而獲得較理想的治療效果,維護人類及動物的健康。 實驗中我們采用雙層瓊脂噬斑法分離、提純變形桿菌噬菌體,根據(jù)《分子克隆指南手冊》第二版λ噬菌體核酸提取方法提取核酸并進行酶切。測定該噬菌體的最佳感染復數(shù),繪制一步生長曲線。 分析實驗結果得出如下結論:本實驗利用10株臨床分離的變形桿菌為宿主菌,從居民污水中分離得到4株毒性噬菌體,分別命名為噬菌體Pr-v 01、噬菌體Pr-v 02、噬菌體Pr-v 03、噬菌體Pr-v 04,其形成噬斑為圓形、透明、邊界清晰。其中噬菌體Pr-v 04滴度最高為2.06×1012PFU/ml,形成直徑0.8～2.3mm的溶菌空斑。交叉實驗顯示噬菌體Pr-v 04裂解能力強、滴度高、較寬的裂解譜,故篩選為本實驗的研究對象。電鏡顯示,噬菌體Pr-v 04蝌蚪形,頭部呈多面體立體對稱,直徑約為40～50nm,有一短尾�；蚪MDNA經(jīng)EcoRⅠ、HindⅢ和NedⅠ酶切后、證實為雙鏈DNA,其基因組大小約為26kb,且有多個EcoRⅠ的酶切位點。噬菌體Pr-v 04感染宿主的一步生長曲線顯示潛伏期為15min作用,爆發(fā)時間為18min左右,裂解量170左右。噬菌體Pr-v 04感染其宿主普通變形桿菌的最佳感染復數(shù)為0.1。在4℃條件下,噬斑可保存10個月左右,而噬菌體Pr-v 04與其宿主菌的混懸液可保存至少6個月。
[Abstract]:Proteus is more widely distributed in nature is the normal flora of the human gut. Infection occurs only when it leaves the normal sojourn intestine and enters other parts of the human body. In hospitals, long-term exposure to broad-spectrum antibiotics, hormones, immunosuppressants, Patients with lower body resistance to antitumor drugs are susceptible to infection. Advanced examination and treatment methods also provide a way for Proteus to invade human body to some extent, which makes Proteus play an important role in nosocomial infection. Bacteriophage therapy has shown many outstanding advantages, especially in the treatment of bacterial infections in animals with antibiotics, which are easy to produce bacterial drug resistance and residues, etc. After a large number of animal model tests and human clinical treatment practice, For the shortcomings and shortcomings of bacteriophage therapy, people have begun to make exploratory modifications in the selection of phage, host spectrum, efficacy and the kinetic principle of its action, so as to obtain a better therapeutic effect. To maintain the health of human beings and animals. In the experiment, we isolated and purified Proteus phage by double layer Agar plaque method, extracted nucleic acid and digested it according to the second edition of 位 phage nucleic acid extraction method, and determined the best complex number of the phage infection. Draw a one step growth curve. The results are as follows: in this experiment, 10 strains of Proteus were used as host bacteria and 4 strains of toxic bacteriophages were isolated from the sewage. They were named phage Pr-v 01, phage Pr-v 02, phage Pr-v 03 and phage Pr-v 04, respectively. The boundary was clear. The titer of bacteriophage Pr-v 04 was 2.06 脳 1012 PFU / ml, forming bacteriolytic plaque with diameter of 0.82.3mm. Cross experiment showed that phage Pr-v 04 had strong cleavage ability, high titer and wide cleavage spectrum, so it was selected as the research object of this experiment. Electron microscope showed that the bacteriophage Pr-v 04 had strong cleavage ability, high titer and wide cleavage spectrum. The head of phage Pr-v 04 was polyhedron stereosymmetric, with a short tail and a diameter of 400.50nm.Genomic DNA was digested by EcoR 鈪，

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