作者赞赏 Sidhu 实验室的弗雷德里克 Fellouse 博士对安德鲁·库恩克尔方法的综合构建。作者赞赏 Alevtina Pavlenco 夫人和 Sidhu 实验室的其他成员, 为制备高效的电子能力的大肠杆菌细胞和高质量的杜 ssDNA 提供了宝贵的帮助。这项工作得到中国国家自然科学基金 (批准号: 81572698, 31771006) 的支持, 并由 ShanghaiTech 大学 (批准号: F-0301-13-005) 到抗体工程实验室。

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Making and Using Antibodies: A Practical Handbook. , 157-180 (2006).</li><li>Fantini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+antibody+library+diversity+through+next+generation+sequencing+and+technical+error+compensation.">Assessment of antibody library diversity through next generation sequencing and technical error compensation.</a> PLoS One. 12 (5), e0177574 (2017).</li><li>Glanville, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing+in+library+selection+projects:+what+insight+does+it+bring?.">Deep sequencing in library selection projects: what insight does it bring?.</a> Curr Opin Struct Biol. 33, 146-160 (2015).</li><li>Perelson, A. 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作者没有什么可透露的。

构建高多样性、噬菌体展示的工厂图书馆, 需要质量控制检查点来监测施工过程的各个阶段, 包括电控细胞的能力、ssDNA 模板的质量、效率dsDNA 合成、电穿孔后滴度、晶圆折叠、CDRs 氨基酸多样性的序列分析。
ssDNA 的高产和纯度是高诱变率的必要条件。在我们的经验, 噬菌体诱导在25°c 隔夜可以产生更多的杜 ssDNA 比从噬菌体诱导在37摄氏度过夜。这与以前的报表19<...

抗体具有广泛的应用范围, 从基础研究到疾病诊断和治疗。截至 2016年, 美国食品药品监督管理局 (USFDA) 批准了60多抗体, 用于临床治疗自身免疫性疾病、癌症和传染性疾病1,2。
在 1975年, 科勒和米尔斯坦报告了一种技术, 连续产生抗体的单一克隆特异性从细胞源称为 ' 杂交瘤 ', 这一技术后来成为医药和工业的基石 3 ,4。这种方法产生的抗体需要各种步骤, 包括抗原生产、小鼠免疫、b 淋巴细胞的提取、b 细胞与骨髓瘤细胞的融合, 形成不朽的杂交瘤细胞、克隆选择和治疗应用,人性化是需要避免人类抗鼠抗体 (哈马)4,5。然而, 对于这种技术, 抗原, 包括毒素, 病原体和高度保守的蛋白质是相对无效的触发体内免疫应答的单克隆生产5。
在 1978年, 和记黄埔et报告使用寡核苷酸直接诱变的残留在单链噬菌体病毒6。在 1985年, 史密斯报告说, 外国基因片段可以融合在框架内的基因编码噬菌体涂层蛋白 III, 从而可以显示在噬菌体表面, 而不损害其传染性7。这些开创性的工作奠定了基础, 随后构建噬菌体显示抗体库的免疫, 幼稚, 和合成形式的单链可变片段 (抗体) 和抗原结合片段 (工厂) 的格式, 以治疗单克隆开发8,9。从技术角度看, 基于噬菌体的抗体开发提供了一种互补的方法, 以杂交瘤为基础的单克隆细胞的发展, 可以帮助规避的局限性, 一些抗原可以构成和人性化的过程,杂交瘤衍生的抗体通常需要5。截至 2016年, 已在市场上批准了6种噬菌体显示衍生抗体, 其中 Humira 是治疗类风湿性关节炎最成功的抗体之一, 许多噬菌体显示抗体候选者目前处于不同的临床阶段。调查10。
对于免疫和幼稚的噬菌体抗体库, 在光和重链中互补决定区域 (CDRs) 的多样性来源于自然免疫汇 (即, 从 B 细胞)。与此相反, 合成噬菌体抗体库中 CDRs 的多样性是完全人工的。图书馆建设的综合方法对序列多样性的设计提供了精确的控制, 为抗体结构和功能的机械研究提供了机会11,12。此外, 在图书馆建设之前, 可以优化合成库框架, 以方便下游、大型工业发展11、12。
在 1985年, 安德鲁·库恩克尔报告了单链 DNA (ssDNA) 基于模板的突变方法, 将站点定向突变引入到 M13 噬菌体中, 有效地13。这种方法后来被广泛应用于噬菌体展示库的建设。化学合成的 DNA 寡核苷酸设计, 以引入多样性的 CDRs, 被纳入一个 phagemid 与抗体骨干模板。在这个过程中, phagemid 被表达为一个尿的 ssDNA (杜 ssDNA), 寡核苷酸被退火到 CDRs, 并扩展到合成双链 dna (dsDNA) 在存在 T7 dna 聚合酶和 T4 dna 连接酶。最后, 生成的 ds DNA 可以通过电穿孔引入到大肠杆菌中. 
为高多样性, 噬菌体展示图书馆建设, 应仔细准备的两组分混合物的电主管细胞和共价键闭环 dsDNA (CCC-dsDNA) 的高压电穿孔。Sidhu et修改了传统方法制备的电控细胞和 DNA, 大大提高了图书馆的多样性14。
在本协议中, 我们描述了一种构建的合成噬菌体显示的工厂图书馆的多样性 109-1010获得单电穿孔。图 1显示了库结构的概述, 其中包括: 1) 高效电控电池的制备;2) 提取杜 ssDNA;3) 基于寡核苷酸定向诱变的安德鲁·库恩克尔方法;4) 电穿孔及库尺寸计算;5) 蛋白质 A/l-基 ELISA 用于折叠和功能多样性评价;和 6) DNA 序列分析的多样性。所有试剂、菌株和设备都列在材料的表中。表 1显示了试剂设置。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M H3PO4</td>
			<td>Fisher</td>
			<td>AC29570</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M Tris, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15568-025</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM ATP</td>
			<td>Invitrogen</td>
			<td>18330-019</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM dithiothreitol</td>
			<td>Fisher</td>
			<td>BP172</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mM dNTP mix</td>
			<td>GE Healthcare</td>
			<td>28-4065-60</td>
			<td>solution containing 25 mM each of dATP, dCTP, dGTP and dTTP.</td>
		</tr>
		<tr>
			<td>3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
			<td>Kirkegaard &#38; Perry Laboratories Inc</td>
			<td>50-76-02</td>
			<td></td>
		</tr>
		<tr>
			<td>50X TAE</td>
			<td>Invitrogen</td>
			<td>24710030</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher</td>
			<td>BP160</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin, carb</td>
			<td>Sigma</td>
			<td>C1389</td>
			<td>100 mg/mL in water,&#160; 0.22 &#956;m filter-sterilize, work concentration: 100 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Chloramphenicol, cmp</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>100 mg/mL in ethanol, 0.22 &#956;m filter-sterilize, work concentration: 10 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9620G</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated agar</td>
			<td>VWR</td>
			<td>J637-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2 peroxidase substrate</td>
			<td>Kirkegaard &#38; Perry Laboratories Inc</td>
			<td>50-65-02</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>795488</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin, kan</td>
			<td>Fisher</td>
			<td>AC61129</td>
			<td>50 mg/mL in water,&#160; 0.22 &#956;m filter-sterilize, work concentration: 50 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>94046</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Alfa Aesar</td>
			<td>U19C015</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Fisher</td>
			<td>ND2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>SS256</td>
			<td>! CAUTION NaOH causes burns.</td>
		</tr>
		<tr>
			<td>NON-Fat Powdered Milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Fisher</td>
			<td>BP233</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A-HRP conjugate</td>
			<td>Invitrogen</td>
			<td>101123</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin M13 Kit</td>
			<td>Qiagen</td>
			<td>22704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Protein L</td>
			<td>Fisher</td>
			<td>77679</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0274S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline, tet</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td>50 mg/mL in water, 0. 22 &#956;m filter-sterilize, work concentration: 10 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>0123-07-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3750</td>
			<td>25 mg/mL in ethanol, work concentration: 0.25 &#956;g/mL.</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>VWR</td>
			<td>DF0127-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli CJ236</td>
			<td>New England Biolabs</td>
			<td>E4141</td>
			<td>Genotype: dut-&#160; ung-&#160; thi-1 relA1 spoT1 mcrA/pCJ105(F&#39; camr). Used for preparation of dU-ssDNA.</td>
		</tr>
		<tr>
			<td>E.coli SS320</td>
			<td>Lucigen</td>
			<td>60512</td>
			<td>Genotype: [F&#39;proAB+lacIq lacZ&#916;M15 Tn10 (tetr)] hsdR mcrB araD139 &#916;(araABC-leu)7679 &#916;lacX74 galUgalK rpsL thi. Optimized for high-efficiency electroporation and filamentous bacteriophage production.</td>
		</tr>
		<tr>
			<td>M13KO7</td>
			<td>New England Biolabs</td>
			<td>N0315S</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2-cm gap electroporation cuvette</td>
			<td>BTX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well 2mL Deep-well plates</td>
			<td>Fisher</td>
			<td>278743</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Maxisorp immunoplates</td>
			<td>Nunc</td>
			<td>151759</td>
			<td></td>
		</tr>
		<tr>
			<td>Baffled flasks</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5811000096</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECM-630 electroporator</td>
			<td>BTX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stir bars</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Fisher centrifuge</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High speed shaker</td>
			<td>TAITEK</td>
			<td>MBR-034P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td>QILINBEIER</td>
			<td>QB-9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid handler for 96 and 384 wells</td>
			<td>RAININ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mutil-channel pipette</td>
			<td>RAININ</td>
			<td>E4XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon concentrator</td>
			<td>Merck</td>
			<td>UFC803096</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of synthetic phage displayed fab library with tailored diversity

基础研究和医学中单克隆抗体 (抗体) 的需求逐年增加。自1975年第一次报告以来, 杂交瘤技术一直是单克隆抗体发展的主要方法。作为一种替代技术, 噬菌体显示方法为单克隆的发展越来越有吸引力, 因为 Humira, 第一种噬菌体抗体和最畅销的抗体之一, 被批准用于临床治疗类风湿关节炎在2002年。噬菌体展示作为一种非动物性的单克隆技术, 它绕过了传统的基于杂交瘤技术的抗体开发所需要的抗原免疫原性、人性化和动物维护。在本协议中, 我们描述了一种构建的合成噬菌体显示的工厂图书馆的多样性 109-1010获得单电穿孔。本协议包括: 1) 高效电控细胞制剂;2) 提取含尿单链 DNA (杜 ssDNA);3) 基于寡核苷酸定向诱变的安德鲁·库恩克尔方法;4) 电穿孔及库尺寸计算;5) 蛋白质 A/l-基酶联免疫吸附试验 (ELISA) 用于折叠和功能多样性评价;和 6) DNA 序列分析的多样性。

按照工厂库结构的流程图 (参见图 1), 我们准备了 M13KO7 帮助噬菌体预感染的大肠杆菌SS320 电子主管细胞。当使用 phagemid 库构造骨干 (图 4) 时, 这些电子主管单元的效率估计为 2 X 109 cfu/µg。
通过对大肠杆菌CJ236 和大肠杆菌SS320 细胞的效价比较, 尿合并效率...

Watch this Scientific Journal Video about Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity at JoVE.com

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

本议定书描述了一个详细的程序, 以构建一个噬菌体显示合成抗体库, 具有量身定做的多样性。合成抗体具有广泛的应用, 从基础研究到疾病诊断和治疗。

具有量身定做多样性的合成噬菌体展示库的构建

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

The overall goal of this procedure is to provide readers detailed hands-on experience in constructing a phage displayed synthetic human antibody library with tailored diversity. Synthetic phage displayed antibody libraries have great potential for replace traditionally hybridoma-based techniques for therapeutic monoclonal antibody development by bypassing the need for animal immunization and the subsequent antibody humanization. The advantage of synthetic antibody library is that tailored diversity is introduced into an antibody framework using insight into positional diversity that support high affinity interaction and desirable protein properties.Beyond the practical utility, synthetic antibody libraries can also be used to experimentally probe the role of specific amino acids in biomolecular interactions, thus supporting their own continuous improvement. This procedure will be demonstrated by graduate student named Zhenwei Zhong and Doctor Shin-chen Hou from our laboratory. To be

construction-synthetic-phage-displayed-fab-library-with-tailored

Research

JoVE Journal

Immunology and Infection

13.4K 次观看.  ShanghaiTech University. The overall goal of this procedure is to provide readers detailed hands-on experience in constructing a phage displayed synthetic human antibody library with tailored diversity. Synthetic phage displayed antibody libraries have great potential for replace traditionally hybridoma-based techniques for therapeutic monoclonal antibody development by bypassing the need for animal immunization and the subsequent antibody humanization. The advantage of synthetic antibody library is that tailored div...

视频: Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

重叠肽库在蛋白质 Qa-1 表位中的映射

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...