Name: screening and identification of small peptides targeting fibroblast growth factor receptor2 using a phage display peptide library
Uploaded: 2019-09-30T15:00:00
Description: Watch this Scientific Journal Video about Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library at JoVE.com

这项工作得到了广州市科技计划（第201620160403039号）的支持。

1. 试剂制备
<ol><li>LB（溶菌肉汤）介质：在H2O高压灭菌剂100 mL中溶解1克胰腺酮、0.5克酵母提取物和0.5克NaCl，并储存在4°C处。</li>
	<li>四环素库存：在1：1乙醇中制备20mg/mL：H2O.在-20°C储存，并在使用前涡流。</li>
	<li>IPTG/X-gal 溶液：将 0.5 g 的 IPTG（二丙基 β-D-1-硫丙酮）和 0.4 g X-gal（5-溴-4-氯-3-indolyl β-D-角质苷）混合在 10 mL 的 DMF（二甲基甲酰胺）中。溶液可在黑暗中在-20°C下储存...

<ol>
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M., Highsmith, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+display+technology:+clinical+applications+and+recent+innovations.">Phage display technology: clinical applications and recent innovations.</a> Clinical Biochemistry. 35, 425-445 (2002).</li><li>Pasqualini, R., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+targeting+In+vivo+using+phage+display+peptide+libraries.">Organ targeting In vivo using phage display peptide libraries.</a> Nature. 380 (6572), 364-366 (1996).</li><li>Liu, R., Li, X., Xiao, W., Lam, K. 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组合噬菌体库是一种强大而有效的工具，用于高通量筛选新型肽，这种肽可以结合目标分子并调节其功能13。目前，噬菌体显示肽库有着广泛的应用。例如，它们可用于选择与受体蛋白23结合的生物活性肽23、非蛋白靶点24、25、疾病特异性抗原模仿13、细胞特异性肽26、<sup cl...

纤维细胞生长因子受体（FGFRs）在细胞增殖、伤口愈合和体内血管生成中起着关键作用。在各种肿瘤2、3、4、5中观察到的FGFR信号异常激活包括基因扩增、基因突变、染色体畸变和过量配体分泌6.许多针对FGFRs的抑制剂在临床试验中显示出有希望的治疗效果，主要分为三类：（1）小分子激酶抑制剂，它与FGFR的细胞内域结合，（2）针对细胞外段和（3）FGF配体陷阱6。虽然几种小分子激酶抑制剂在体外和体内均具有良好的治疗效果，但大部分靶点特异性较差，并表现出高血压8等不良反应。大多数拮抗剂是单克隆抗体9、10和多肽11。肽具有比小分子的优势，由于其特异性和较低的副作用。与蛋白质药物12相比，它们还保留了细胞渗透性，不会在特定器官中积累。因此，靶向小肽是有效和前瞻性的治疗剂。
噬菌体显示技术是一个简单但强大的工具，用于识别小肽，它可以结合到给定的分子13，14，15。我们使用一个噬菌体显示肽库，它基于一个简单的M13噬菌体，在尾部显示超过109个不同的肽序列，以便与目标分子结合（见材料表）16。由于噬菌体对给定分子的亲和力高，未结合的噬菌体可以被冲走，并且只保留与所需短肽紧密结合的噬菌体。给定的分子靶点可以固定蛋白质17，18，碳水化合物，培养细胞，甚至无机材料19，20。一个令人兴奋的案例是，使用噬菌体显示技术21在体内选择器官特异性肽。噬菌体显示技术的优点包括高通量、易操作性、低成本和广泛的应用22。
在这项研究中，我们使用噬菌体显示库提供了筛选与固定蛋白（FGFR2）结合的小肽的详细方案。通过测量通过同质滴度测定仪（ITC）获得的肽对FGFR2的亲和力，以及通过细胞增殖测定的生物活性，也检查了该技术的疗效。该方法可以扩展用于筛选与碳水化合物、培养细胞甚至无机材料结合的小肽。

<table>
	<tbody>
		<tr>
			<td>0.22 &#956;m Filter</td>
			<td>Merck Millipore</td>
			<td>MPGP002A1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 cm2 Small dish</td>
			<td>Thermo</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>-96 gIII sequencing primer</td>
			<td></td>
			<td></td>
			<td>Synthesis from Sangon Biotech (Shanghai) Co., Ltd.</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Nest</td>
			<td>701001-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Beyotime</td>
			<td>ST004D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Tryptone</td>
			<td>Oxoid</td>
			<td>L0037</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c 3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-&#173;6587</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Biodragon</td>
			<td>BD-M10110</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK-8 kit</td>
			<td>DOJINDO</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>sh30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Newprobe</td>
			<td>PB10247</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576028</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 Protein</td>
			<td>Sino Biological Inc.</td>
			<td>10014-HNAE</td>
			<td>Purity &#62;95%</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Beyotime</td>
			<td>ST097</td>
			<td></td>
		</tr>
		<tr>
			<td>ITC200 system</td>
			<td>MicroCal Omega</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>NaI</td>
			<td>Bidepharm</td>
			<td>BD40879</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Guangzhou chemical reagent factory</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG&#8211;8000</td>
			<td>Sigma</td>
			<td>P2139-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ph.D.-7 phage display peptide library kit</td>
			<td>New England BioLabs</td>
			<td>E8100S</td>
			<td>Containing the Ph.D.-7 phage library, E. coli ER2738 host strain and M13KE control phage</td>
		</tr>
		<tr>
			<td>Recombinant FGFR2 extracellular domain proteins</td>
			<td>Sino Biological Inc.</td>
			<td>10824-H08H</td>
			<td>Purity &#62; 97%</td>
		</tr>
		<tr>
			<td>Small peptide</td>
			<td></td>
			<td></td>
			<td>Synthesis from GL Biochem Ltd. (Shanghai, China)</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>S-SHS-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>SLF-T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Beyotime</td>
			<td>ST825</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Beyotime</td>
			<td>ST912</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening and identification of small peptides targeting fibroblast growth factor receptor2 using a phage display peptide library

人类纤维细胞生长因子受体（FGFR）家族由四个成员组成，即FGFR1、FGFR2、FGFR3和FGFR4，它们参与各种生物活动，包括细胞增殖、存活、迁移和分化。由于突变或基因扩增事件，FGFR信号通路中的几个畸变在不同类型的癌症中被识别出来。因此，最近的研究侧重于开发涉及FGFRs治疗靶向的策略。 目前在临床前和临床开发的不同阶段，目前的FGFR抑制剂包括酪氨酸激酶的小分子抑制剂或单克隆抗体，管道中只有几种肽抑制剂。在这里，我们提供了一个协议，使用噬菌体显示技术来筛选小肽作为FGFR2的拮抗剂。简单地说，在涂有FGFR2的板中孵育出一个噬菌体显示肽库。随后，未结合的噬菌体被TBST（TBS = 0.1%[v/v]补间-20）洗掉，并结合噬菌体用0.2M甘氨酸-HCl缓冲液（pH 2.2）洗净。洗脱的噬菌体被进一步放大，并用作下一轮生物泛化的输入。经过三轮生物盘分，通过DNA测序确定了单个噬菌体克隆的肽序列。最后，对筛选的肽进行合成，分析亲和力和生物活性。

获得针对FGFR2的高亲和力小肽。
为了筛选针对FGFR2的噬菌体，本研究使用了一个Ph.D.-7库。工作流的原理图表示形式如图1所示。在此过程中，噬菌体输入（PFU）的数量保持不变，而FGFR2蛋白的涂层浓度逐渐降低。噬菌体命名结果表明，恢复的噬菌体数量逐渐增加，3轮后，与第一轮相比增加了65倍（表1）。
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Watch this Scientific Journal Video about Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library at JoVE.com

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

在此，我们提出了使用噬菌体显示肽库筛选与 FGFR2 结合的小肽的详细方案。我们进一步分析了所选肽在体外对FGFR2的亲和力及其抑制细胞增殖的能力。

使用噬菌体显示肽库筛选和识别针对纤维细胞生长因子受体2的小肽

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

与其他方法相比，在不知道水稀释结构和目标蛋白的形成的情况下，在定量和质量上更容易发现新的拮抗剂、激动剂和同性调节器。该协议效率高、易于处理、经济实惠且具有商业性。将这种高通量筛选方法与 ITC 分析相结合，可定量、定性地获得功能小肽。演示程序将是赵颖和王强，我们实验室的研究生。要开始此过程，请以每毫升 10 微克的浓度在涂层缓冲液中准备 FGFR2 溶液。将溶液的一毫升加入 35 平方厘米的盘子中，反复旋转，直到表面完全湿润。在四摄氏度下孵育过夜，摇晃。第二天，倒掉涂层溶液，加入准备好的阻解溶液。在四摄氏度下孵育至少一小时，然后丢弃阻塞溶液，用TBST快速洗盘子六次，同时确保盘子不会干涸。在一毫升的TBST中重建原始噬菌板库，并将其添加到涂层盘中。在室温下轻轻摇动摇床板两小时。在此之后，丢弃上经剂，用TBST洗10次。将一毫升0.2摩尔甘油HCL加入盘中，在室温下轻轻摇动10分钟，使装订的噬菌蛋白斑。将洗入消毒的微离心管中，并在 pH 9.1 下用 100 微升一摩尔 Tris-HCL 将其中和。首先，在使用前至少一小时，在37摄氏度下预热准备好的LB和IPTG X-Gal板。使用带滤芯的移液器尖端，在 LB 中准备 100 微升十倍稀释的 Eluent。在准备好的大肠杆菌培养达到中原阶段后，将培养的200微升分液放入消毒的微离出管中，每次洗出一个洗出的稀释液。要启动感染，在含有大肠杆菌培养的管子中加入每次稀释的50微升。涡流快速孵化，在室温下孵育五分钟。然后使用涂层棒将 LB 和 IPTG X-GAL 板涂覆为 90 微升的混合物。五分钟后，将盘子倒置，在37摄氏度下孵育。当斑块出现时，计算斑块数数。首先，在250毫升的埃伦迈尔烧瓶中稀释20毫升LB的隔夜培养。加入未增塑的伊卢，在37摄氏度下孵育，剧烈摇晃4-1~2小时。接下来，在12，000倍G和4摄氏度的离心，10分钟。将上一液转移到新鲜管中。使用相同条件再次离心并丢弃颗粒。将上半液的上部80%转移到新鲜管中，并加入20%聚乙烯二醇和2.5摩尔氯化钠的1/6体积。让噬菌菌在摄氏四度下过夜。第二天，将沉淀的噬菌（金）在12，000倍和4摄氏度的温度下离心15分钟。使用相同的条件再次丢弃上一代和离心机。使用移液器去除残留的上流液。将颗粒重新在一毫升 TBS 中，将上一液转移到微离心管中。在12，000倍G和4摄氏度的离心机5分钟。将上流液转移到新鲜的微离心管中，再加入1/6体积的20%聚乙二醇和2.5摩尔氯化钠，再次沉淀。在冰上孵育15至60分钟。接下来，在12，000倍的G和4摄氏度的离心机10分钟。使用相同的条件再次丢弃上一代和离心机。使用移液器去除残留的上流液。将颗粒重新在 200 微升 TBS 和离心机中再多一分钟，以去除杂质。然后将上流液转移到新鲜的微离心管中，以获得放大的吸水器。在这项研究中，噬菌蛋白的数量保持不变，而FGFR2蛋白的涂层浓度逐渐减少。噬菌体滴度结果表明，恢复的噬菌体数量逐渐增加，三轮后，与第一轮相比增加了65倍。然后进行 ITC 实验，以测量小肽与 FGFR2 的亲和力。结果表明，SP1肽对FGFR2具有很高的亲和力。此数据证明了筛选协议的效率。为了研究SP1肽的生物活性，使用CCK-8试剂盒进行成纤维细胞增殖测定。BALB/C3T3与SP1肽在不同浓度下孵育。结果表明，SP1肽抑制细胞的生长。这里必须避免野生型噬菌体的污染。确保使用防气溶胶移液器尖端。而通过获得噬菌的扩增而生长。此过程还可用于筛选与碳水化合物、培养细胞和组织结合的小肽。这些肽可以进一步放大诊断和靶向

Research

JoVE Journal

Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Generation and Recovery of ÃƒÅ½Ã‚Â²-cell Spheroids From Step-growth PEG-peptide Hydrogels

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...