本文選題：普通變形桿菌　切入點(diǎn)：噬菌體　出處：《吉林大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)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(kāi)了正常寄居的腸道而進(jìn)入人體其他部位時(shí)才引起感染。在醫(yī)院內(nèi),長(zhǎng)期接受廣譜抗生素、激素、免疫抑制劑、抗腫瘤藥等治療的機(jī)體抵抗力下降的病人易感染,先進(jìn)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(nèi)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(duì)使用抗生素治療動(dòng)物細(xì)菌感染時(shí)易產(chǎn)生細(xì)菌耐藥性及殘留等缺點(diǎn)。經(jīng)過(guò)大量的動(dòng)物模型試驗(yàn)及人類(lèi)臨床治療的實(shí)踐,對(duì)于噬菌體治療中存在的缺點(diǎn)和不足,人們已經(jīng)開(kāi)始在噬菌體的選擇、宿主譜、作用效力及其作用的動(dòng)力學(xué)原理等方面進(jìn)行探索性的改造,從而獲得較理想的治療效果,維護(hù)人類(lèi)及動(dòng)物的健康。 實(shí)驗(yàn)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(cè)》第二版λ噬菌體核酸提取方法提取核酸并進(jìn)行酶切。測(cè)定該噬菌體的最佳感染復(fù)數(shù),繪制一步生長(zhǎng)曲線。 分析實(shí)驗(yàn)結(jié)果得出如下結(jié)論:本實(shí)驗(yàn)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(shí)驗(yàn)顯示噬菌體Pr-v 04裂解能力強(qiáng)、滴度高、較寬的裂解譜,故篩選為本實(shí)驗(yàn)的研究對(duì)象。電鏡顯示,噬菌體Pr-v 04蝌蚪形,頭部呈多面體立體對(duì)稱(chēng),直徑約為40～50nm,有一短尾�；蚪MDNA經(jīng)EcoRⅠ、HindⅢ和NedⅠ酶切后、證實(shí)為雙鏈DNA,其基因組大小約為26kb,且有多個(gè)EcoRⅠ的酶切位點(diǎn)。噬菌體Pr-v 04感染宿主的一步生長(zhǎng)曲線顯示潛伏期為15min作用,爆發(fā)時(shí)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(fù)數(shù)為0.1。在4℃條件下,噬斑可保存10個(gè)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(gè)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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