噬菌体药理学及其实验方法

在细菌抗药性日愈严重的当下，噬菌体治疗是对抗细菌感染更有前景的治疗方法。在我国这样一个人口众多的农业大国，细菌抗药性的问题尤其尖锐，细菌抗药性使得一个个曾经有效的抗生素失效，对人类的健康生存提出了巨大的挑战。噬菌体治疗从原理上与抗生素不同，克服了细菌抗药性，对其进行深入的研究与应用，将使人们掌握更有效的对抗细菌方法，因而意义深远。

Preface for Chinese Version of Book
It is a great pleasure for myself，and my co-editors of Bacteriophages：methods and protocols，that so many of the chapters that we commissioned and edited have now been translated into Chinese within this volume.Myself，and professors Andrew Kropinski and Rob Lavigne were aware when we started this project in 2005，that if we did not ‘save’ many of the protocols and methodologies that were being used by practitioners of phage biology，that they would be lost to history. This is because the leaders in the field were generally about to retire，and their protocols were dispersed in many discrete papers or not even written down formally. Furthermore，at the time although a few young research scientists coming into the field of bacteriophage biology，they did not have their own independent positions and so were not actively using or collating such information.
I am happy to say that almost two decades later the situation has completely reversed. The need and awareness to use bacteriophages has increased and expanded，which is reflected in funding and research initiatives within universities and companies across the world. There are also many new and thriving research groups dedicated fully on phage.The core techniques involved in working with bacteriophages were developed over decades and it is clearly extremely useful to build on these rather than having to reinvent them. This book hopes to support this by bringing you well ‘homed’ and fine-tuned methods to investigate all aspects of phage biology.
In addition to saving the traditional protocols，we also tried to ensure we captured many new methods as they were developed. We include in this book many cutting-edge and novel approaches that have allowed the field of bacteriophage biology to be really revolutionised. Our ability to sequence the genomes，transcriptomes and proteins of these organisms，and carry out work to understand the biology at a mechanistic level is unparalleled.Furthermore，our ability to explore issues surrounding the production and formulation of phages is now possible.
Although bacteriophages were discovered over a century ago and initially developed in the 1920s-30s，in light of the technical advancements in molecular biology and genetics，many of which we present here，it is likely that the 2020s-2030s will be the true ‘age of phage’.Therefore，phages will be able to be developed in a safe way which is fully conversant with an understanding of their biology.
We are happy that the work we did in gathering this material has been so meticulously translated by our Chinese colleagues，particularly Professor Yuqing Liu and his extended team，and we are grateful for all the work they did to do this. Global problems need global solutions，and we are pleased that our Chinese colleagues will have full access to this material. We wish you all the best of wishes and joy in discovering these protocols and in understanding，taming and exploiting your bacteriophages to prevent and control disease.

Martha R.J.Clokie

噬菌体是细菌特异性病毒，也是潜在的活体抗菌药物，必将在防治细菌感染和污染中发挥重要作用，亟待开展药理学研究和规范。本书以噬菌体药理学为主线，串联噬菌体分离、生物学性质，噬菌体-宿主相互作用，噬菌体评价方法、生物信息学，噬菌体应用的技术方法，将提高动物保护、植物保护、畜牧业、农业和食品行业人员的噬菌体研发和应用能力，在实施减抗降抗，控制超级细菌的国家战略中发挥积极作用。

绪论：噬菌体群体治疗药理学方法和框架1

第1部分基础噬菌体学23
1水和土壤中噬菌体的富集25
2环境样品中噬菌体的分离方法31
3溶原诱导法分离噬菌体39
4氯化铁絮凝回收海水中的噬菌体46
5阴离子交换色谱法纯化噬菌体52
6噬菌体计数——双层平板法60
7噬菌体计数——直接涂板法66
8噬菌体的计数——微量滴定板法69
9噬菌体的电镜观察72
10噬菌体宿主范围和裂解率的测定82
11噬菌体对细胞吸附率的测定88
12一步生长曲线测定92
13利用纯化受体测定噬菌体的失活动力学97
14噬菌体突变株的构建100
15裂解性噬菌体的普遍性转导110
16噬菌体的高通量筛选119
17家蚕幼虫模型在噬菌体治疗实验中的应用123
18大蜡螟幼虫替代动物作为感染模型分析病原菌致病力的应用130
19果蝇感染模型和评价噬菌体抗菌效果的应用137
20浮萍和紫苜蓿作为细菌感染模型系统的应用143
21鸡胚致死试验评估噬菌体治疗效果的应用149
22噬菌体-生物膜相互作用的评价技术154
23使用噬菌体FISH法检测单细胞水平的噬菌体感染161
24酶谱分析噬菌体裂解蛋白的肽聚糖水解活性177
25即食食品中噬菌体效果的定量测定183

第2部分噬菌体生物信息学189
26噬菌体的分类与命名191
27病毒组研究方法194
28常见细菌的噬菌体201
29噬菌体研究的网络资源219
30噬菌体表征的必要步骤：生物学、分类学和基因组分析224
31使用RAST对噬菌体基因组注释238
32噬菌体基因组数据的可视化分析：比较基因组学和作图244
33噬菌体裂解物的制备和DNA纯化260
34脉冲场凝胶电泳（PFGE）测定噬菌体基因组大小266
35噬菌体基因组末端的高通量测序分析271
36通过分析有尾噬菌体基因组的末端确定DNA包装策略290
37来自单一噬菌斑的双链DNA噬菌体的基因组测序304
38利用感染裂解性噬菌体的细胞构建cDNA文库以使用RNA-Seq进行转录组分析308
39荧光定量PCR测定宿主菌和噬菌体mRNA的表达315
40噬菌体的纯化以及噬菌体结构蛋白的SDS-PAGE分析324
41噬菌体蛋白质组学：质谱的应用332

第3部分噬菌体应用341
42噬菌体治疗343
43使用Strep-tagⅡ纯化分析噬菌体-宿主的蛋白质-蛋白质相互作用349
44用于分枝杆菌药物敏感性试验的荧光杆菌噬菌体363
45工程噬菌体生物传感器370
46将噬菌体基因组导入大肠埃希菌的电穿孔法379
47枯草芽孢杆菌噬菌体SPO1的定点突变384
48电穿孔DNA噬菌体重组技术（BRED）在裂解性噬菌体基因操作中的应用391
49用亲和层析法分离竞争性噬菌体修饰展示的T4噬菌体401
50用于检测和生物防控的噬菌体及其蛋白质的固定化407
51铜绿假单胞菌噬菌体中编码生长抑制性ORFan的筛选420
52噬菌体展示技术432