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摘要：随着“超级细菌”的不断涌现，人类想要再回到“前抗生素时代”已经是无法企及的愿望了，于是我们再次将目光转移到噬菌体治疗 （phage therapy） 上。 肺炎克雷伯杆菌 （Klebsiella pneumonia）是常见的条件致病菌，也是医院内常见的耐药菌株，对于常见的抗生素都具有耐药性，已逐步成为人类健康的一大威胁。本项目通过从生活污水水样中以肺炎克雷伯杆菌 A2312-2 为宿主菌，分离纯化得到一株噬菌体 DL23，并进行其宿主谱、一步生长曲线及吸附率方面的生物学特性分析。之后，利用转座子插入技术筛选出噬菌体裂解该肺炎克雷伯杆菌的受体突变株 D1-2 和 W2-6，肺炎克雷伯杆菌并通过滴板法、噬菌体增殖以及吸附率实验对受体进行鉴定。结果表明，肺炎克雷伯杆菌噬菌体 DL23 的受体基因与肽聚糖合成酶（peptidoglycan synthase）相关，即该噬菌体 DL23 裂解该宿主菌 A2312-2的受体是细胞壁。该结果为噬菌体混合制剂治疗肺炎克雷伯杆菌引起的疾病奠定了基础。 25793
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毕业论文关键词：肺炎克雷伯杆菌；噬菌体；受体基因；噬菌体治疗
Isolation and Characterization of Bacteriophages against Klebsiella pneumonia and Identification of Receptor Abstract：With the emergence of "superbugs", the desire to go back to "the pre antibiotic era" may not become true, so we attached our attention to phage therapy. Klebsiella pneumoniae is a kind of common opportunistic pathogenic bacteria, and it is also a kind of common antibiotic resistant bacteria in hospital. It becomes a major threat to human health gradually. In this project, the isolation and purification of bacteriophage DL23 was completed by the treatment of sewage water samples in the use of the host bacteria Klebsiella pneumoniae A2312-2. In addition, the biological characteristics of the phage were also worked out, such as host spectrum, one-step growth curve and absorption rate. After using the transposon insertion method, we searched for the recipient mutant strains of Klebsiella pneumoniae, and then we identified the receptor by the means of drop plate method, phage proliferation and the absorption rate of phage. The results showed that the receptor gene of bacteriophage DL23 on Klebsiella pneumoniae was related to peptidoglycan synthase, so cell wall was the receptor of bacteriophage DL23. The results provided a scientific basis for the bacterial treatment of Klebsiella pneumoniae.
Keywords: Klebsiella pneumoniae; Bacteriophage; Receptor gene; Phage therapy
目 录
摘要 . 3
关键词 . 3
Abstract .. 3
Keywords 3
0 引言 . 4
1 材料与方法 . 4
1.1 材料 .. 4
1.1.1 菌株 ... 4
1.1.2 主要试剂 ... 4
1.1.3 污水样品 ... 5
1.2 实验方法 .. 5
1.2.1 噬菌体的分离与纯化 5
1.2.2 噬菌体的生物学特性 6
1.2.3 转座子库的建立 6
1.2.4 噬菌体受体的筛选 7
1.2.5 受体的鉴定 ... 8
2 结果 . 9
2.1 分离与纯化 .. 9
2.2 噬菌体的保存 .. 9
2.3 噬菌体的富集及滴度的测定 ... 9
2.4 噬菌体DL23 的生物学特性 . 10
2.4.1 噬菌体的宿主谱.. 10
2.4.2 一步生长曲线 . 11
2.5 受体基因的筛选及鉴定. 11
2.5.1 转座子库建库结果.. 11
2.5.2 受体突变株验证.. 11
2.5.3 随机引物PCR结果 12
2.5.4 测序结果及BLAST分析 ... 13
3 讨论 ... 13
致谢 ... 15
参考文献 ... 16
0 引言
肺炎克雷伯杆菌（Klebsiella pneumoniae）属革兰氏阴性杆菌，是肠杆菌科克雷伯氏菌