普通变形杆菌噬菌体的分离鉴定和生物学特性的研究

发布时间：2018-03-17 20:19

  本文选题：普通变形杆菌　切入点：噬菌体　出处：《吉林大学》2011年硕士论文　论文类型：学位论文

【摘要】：变形杆菌在自然界中分布较广泛是人体肠道的正常菌群。只有离开了正常寄居的肠道而进入人体其他部位时才引起感染。在医院内,长期接受广谱抗生素、激素、免疫抑制剂、抗肿瘤药等治疗的机体抵抗力下降的病人易感染,先进的检查治疗方法在某种程度上也为变形杆菌入侵人体提供了途径,使变形杆菌在医院内感染中占有重要地位。 噬菌体治疗已表现出许多突出的优越性,尤其是针对使用抗生素治疗动物细菌感染时易产生细菌耐药性及残留等缺点。经过大量的动物模型试验及人类临床治疗的实践,对于噬菌体治疗中存在的缺点和不足,人们已经开始在噬菌体的选择、宿主谱、作用效力及其作用的动力学原理等方面进行探索性的改造,从而获得较理想的治疗效果,维护人类及动物的健康。 实验中我们采用双层琼脂噬斑法分离、提纯变形杆菌噬菌体,根据《分子克隆指南手册》第二版λ噬菌体核酸提取方法提取核酸并进行酶切。测定该噬菌体的最佳感染复数,绘制一步生长曲线。 分析实验结果得出如下结论:本实验利用10株临床分离的变形杆菌为宿主菌,从居民污水中分离得到4株毒性噬菌体,分别命名为噬菌体Pr-v 01、噬菌体Pr-v 02、噬菌体Pr-v 03、噬菌体Pr-v 04,其形成噬斑为圆形、透明、边界清晰。其中噬菌体Pr-v 04滴度最高为2.06×1012PFU/ml,形成直径0.8～2.3mm的溶菌空斑。交叉实验显示噬菌体Pr-v 04裂解能力强、滴度高、较宽的裂解谱,故筛选为本实验的研究对象。电镜显示,噬菌体Pr-v 04蝌蚪形,头部呈多面体立体对称,直径约为40～50nm,有一短尾。基因组DNA经EcoRⅠ、HindⅢ和NedⅠ酶切后、证实为双链DNA,其基因组大小约为26kb,且有多个EcoRⅠ的酶切位点。噬菌体Pr-v 04感染宿主的一步生长曲线显示潜伏期为15min作用,爆发时间为18min左右,裂解量170左右。噬菌体Pr-v 04感染其宿主普通变形杆菌的最佳感染复数为0.1。在4℃条件下,噬斑可保存10个月左右,而噬菌体Pr-v 04与其宿主菌的混悬液可保存至少6个月。
[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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