产气肠杆菌噬菌体生物学特性与基因组分析

发布时间：2018-05-31 02:41

  本文选题：噬菌体 + 产气肠杆菌　； 参考：《河南科技学院》2017年硕士论文

【摘要】：产气肠杆菌是一种重要的条件致病菌,可引起肺炎、菌血症、泌尿系统感染等,本研究以一株多重耐药产气肠杆菌为宿主,从污水中分离出一株噬菌体,并对该噬菌体的生物学特性和基因组进行分析,以期找到一种抗生素替代方案,为之后的噬菌体治疗做铺垫。主要内容与结果如下:1.产气肠杆菌噬菌体的分离从医院获得一株多重耐药产气肠杆菌3-SP,通过菌种鉴定,耐药性分析等,确定菌种信息,并将其保存于菌库。然后以该菌为宿主,以环境污水为样品,分离出一株烈性噬菌体,命名为phiEap-2,并制备好噬菌体原种悬浮液,保存于本实验室。2.产气肠杆菌噬菌体生物学特性研究在透射电镜下观察发现,phiEap-2为蝌蚪状,蛋白质衣壳的头部直径约55 nm,非收缩尾部长度约为117 nm,直径10 nm,可判断该噬菌体属于长尾噬菌体科。一步生长曲线的绘制表明,噬菌体phiEap-2的潜伏期为20min,爆发期约40min。稳定性研究表明,噬菌体phiEap-2对温度较为敏感,在高温(70-80℃)处理下会失去活性,而在人体体温(37℃)、室温(24℃)和低温(4℃)下,会保持较高的活性;噬菌体phiEap-2在pH3或pH=14时存活率急剧下降,总体来看,在碱性条件下该噬菌体的存活率比在酸性条件下的存活率高,这表明该噬菌体更加耐碱而不耐酸。宿主范围试验表明,该株噬菌体具有很强的专一性。3.产气肠杆菌噬菌体基因组分析经过提取与纯化,得到纯度较高的的噬菌体phiEap-2基因组,对其进行测序,并将其基因组全序列及注释信息上传至Genbank,序列号为KT287080。phiEap-2基因组长度为40491bp,GC含量为51.95%,属于线状双链DNA。对噬菌体的基因组进行预测之后,得到62个开放阅读框,其中23个开放阅读框的功能可以被注释,39个开放阅读框被注释为假定蛋白。分析表明,phiEap-2结构基因模块与噬菌体FSL SP-031和粘质沙雷氏菌噬菌体ETA有一定程度的序列保守性,虽然噬菌体之间存在着显著差异,但是结构基因模块是在这三株噬菌体(phiEap-2,FSL SP-031和ETA)基因组相同位置发现的,这表明phi Eap-2与沙门氏菌噬菌体FSL SP-031和粘质沙雷氏菌噬菌体ETA在进化上是相关的。
[Abstract]:Enterobacter aerogenes is an important conditional pathogen, which can cause pneumonia, bacteremia, urinary system infection, etc. In this study, a bacteriophage was isolated from sewage with a multidrug resistant Enterobacter aerogenes as host. The biological characteristics and genome of the bacteriophage were analyzed in order to find an alternative to antibiotics and pave the way for the later phage therapy. The main contents and results are as follows: 1. A multidrug resistant Enterobacter aerogenes 3-SP1 was isolated from the hospital. The information of the bacteria was determined by the identification of strains and the analysis of drug resistance, and stored in the library. Then a strong bacteriophage named phiEap-2 was isolated from environmental sewage and the suspension of bacteriophage was prepared and stored in our laboratory. The biological characteristics of Enterobacter aerogenes phage were observed under transmission electron microscope. It was found that phiEap-2 was tadpole shaped, the head diameter of protein capsid was about 55 nm, the length of non-contractile tail was about 117 nm, and the diameter was 10 nm. The one-step growth curve showed that the latent period of phage phiEap-2 was 20 min and the outbreak time was about 40 min. The stability study showed that the bacteriophage phiEap-2 was sensitive to temperature and could lose its activity under high temperature (70 ~ 80 鈩，

本文编号：1957996