噬菌體藥理學及其實驗方法（簡體書）

噬菌體是細菌特異性病毒，也是潛在的活體抗菌藥物，必將在防治細菌感染和污染中發揮重要作用，亟待開展藥理學研究和規範。
本書以噬菌體藥理學為主線，串聯噬菌體分離、生物學性質、噬菌體-宿主相互作用、噬菌體評價方法、生物信息學、噬菌體應用的技術方法。本書將對提高動物保護、植物保護、畜牧業、農業和食品行業人員的噬菌體研發和應用能力，實施減抗降抗，控制超級細菌等發揮積極作用。
本書適用於從事細菌生物技術研究的技術人員。

在細菌抗藥性日愈嚴重的當下，噬菌體治療是對抗細菌感染更有前景的治療方法。在我國這樣一個人口眾多的農業大國，細菌抗藥性的問題尤其尖銳，細菌抗藥性使得一個個曾經有效的抗生素失效，對人類的健康生存提出了巨大的挑戰。噬菌體治療從原理上與抗生素不同，克服了細菌抗藥性，對其進行深入的研究與應用，將使人們掌握更有效的對抗細菌方法，因而意義深遠。

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

緒論：噬菌體群體治療藥理學方法和框架1

第1部分基礎噬菌體學23
1水和土壤中噬菌體的富集25
2環境樣品中噬菌體的分離方法31
3溶原誘導法分離噬菌體39
4氯化鐵絮凝回收海水中的噬菌體46
5陰離子交換色譜法純化噬菌體52
6噬菌體計數——雙層平板法60
7噬菌體計數——直接塗板法66
8噬菌體的計數——微量滴定板法69
9噬菌體的電鏡觀察72
10噬菌體宿主範圍和裂解率的測定82
11噬菌體對細胞吸附率的測定88
12一步生長曲線測定92
13利用純化受體測定噬菌體的失活動力學97
14噬菌體突變株的構建100
15裂解性噬菌體的普遍性轉導110
16噬菌體的高通量篩選119
17家蠶幼蟲模型在噬菌體治療實驗中的應用123
18大蠟螟幼蟲替代動物作為感染模型分析病原菌致病力的應用130
19果蠅感染模型和評價噬菌體抗菌效果的應用137
20浮萍和紫苜蓿作為細菌感染模型系統的應用143
21雞胚致死試驗評估噬菌體治療效果的應用149
22噬菌體-生物膜相互作用的評價技術154
23使用噬菌體FISH法檢測單細胞水平的噬菌體感染161
24酶譜分析噬菌體裂解蛋白的肽聚糖水解活性177
25即食食品中噬菌體效果的定量測定183

第2部分噬菌體生物信息學189
26噬菌體的分類與命名191
27病毒組研究方法194
28常見細菌的噬菌體201
29噬菌體研究的網絡資源219
30噬菌體表征的必要步驟：生物學、分類學和基因組分析224
31使用RAST對噬菌體基因組注釋238
32噬菌體基因組數據的可視化分析：比較基因組學和作圖244
33噬菌體裂解物的制備和DNA純化260
34脈衝場凝膠電泳（PFGE）測定噬菌體基因組大小266
35噬菌體基因組末端的高通量測序分析271
36通過分析有尾噬菌體基因組的末端確定DNA包裝策略290
37來自單一噬菌斑的雙鏈DNA噬菌體的基因組測序304
38利用感染裂解性噬菌體的細胞構建cDNA文庫以使用RNA-Seq進行轉錄組分析308
39熒光定量PCR測定宿主菌和噬菌體mRNA的表達315
40噬菌體的純化以及噬菌體結構蛋白的SDS-PAGE分析324
41噬菌體蛋白質組學：質譜的應用332

第3部分噬菌體應用341
42噬菌體治療343
43使用Strep-tagⅡ純化分析噬菌體-宿主的蛋白質-蛋白質相互作用349
44用於分枝桿菌藥物敏感性試驗的熒光桿菌噬菌體363
45工程噬菌體生物傳感器370
46將噬菌體基因組導入大腸埃希菌的電穿孔法379
47枯草芽孢桿菌噬菌體SPO1的定點突變384
48電穿孔DNA噬菌體重組技術（BRED）在裂解性噬菌體基因操作中的應用391
49用親和層析法分離競爭性噬菌體修飾展示的T4噬菌體401
50用於檢測和生物防控的噬菌體及其蛋白質的固定化407
51銅綠假單胞菌噬菌體中編碼生長抑制性ORFan的篩選420
52噬菌體展示技術432