Name: construction of synthetic phage displayed fab library with tailored diversity
Uploaded: 2018-05-01T14:30:00
Description: Watch this Scientific Journal Video about Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity at JoVE.com

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。

注: 过滤无菌提示必须在处理噬菌体时使用, 以避免污染的吸管枪和周围地区。在处理细菌和噬菌体实验时, 必须使用无菌区或罩。噬菌体实验区必须使用2% 的月桂酸钠 (SDS), 其次是70% 乙醇, 以避免噬菌体污染。对于在本协议中进行串行稀释, 每个稀释都应使用新的提示。
1.大肠杆菌SS320 电子主管细胞制剂
<ol><li>预热的 LB/新年琼脂盘 (准备和储存在4°c 小于1周) 在37°c 孵?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+antibody+molecule.">Anatomy of the antibody molecule.</a> Mol Immunol. 31 (3), 169-217 (1994).</li><li>Virnekas, B., 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=Trinucleotide+phosphoramidites:+ideal+reagents+for+the+synthesis+of+mixed+oligonucleotides+for+random+mutagenesis.">Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.</a> Nucleic Acids Res. 22 (25), 5600-5607 (1994).</li><li>Fellouse, F. A., Sidhu, S. 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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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 ...

Research

JoVE Journal

Immunology and Infection

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

“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

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

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

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

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

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

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

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

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

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

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

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

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 ...