Name: a comparative approach to characterize the landscape of host-pathogen protein-protein interactions
Uploaded: 2013-07-18T12:00:00
Description: Watch this Scientific Journal Video about A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions at JoVE.com

这项工作是支持的，一部分资金从巴斯德研究所和国家法甲抗癌症（补助R05/75-129和RS07/75-75），协会倾LA RECHERCHE SUR LE癌症（赠款ARC A09 /补助1/5031和4867），和国家法新社​​德拉RECHERCHE（MIME 009 02 ANR09的ANR07冕026 01）。 MM收件人的MENRT奖学金。

1。酵母双杂交放映<ol><li>进行以下所需的解决方案： <ol><li>完成合成跌落式（SD）：与葡萄糖在1升水中溶解26.7克的最小SD基地。添加氨基酸混合物制备如下：将2克精氨酸盐酸盐，2克2克腺嘌呤硫酸盐，2克盐酸组氨酸异亮氨酸，2克，4克亮氨酸盐酸赖氨酸，将2克，蛋氨酸2克，苯丙氨酸3克G L 2丝氨酸，2 G L-苏氨酸， 色氨酸 3克，2克酪氨酸，1.2克尿嘧啶，缬氨?...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Java+Treeview--extensible+visualization+of+microarray+data.">Java Treeview--extensible visualization of microarray data.</a> Bioinformatics. 20, 3246-3248 (2004).</li><li>Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P. L., Ideker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoscape+2.8:+new+features+for+data+integration+and+network+visualization.">Cytoscape 2.8: new features for data integration and network visualization.</a> Bioinformatics. 27, 431-432 (2011).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. 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独立，酵母双杂交和哺乳动物的相互作用分析，，如GST拉下来，LUMIER或MAPPIT，已被证明是有效的工具来检测蛋白质 - 蛋白质相互作用，但假阳性和假阴性率高的限制与这些技术15的相互作用。此外，越来越多的证据正交的方法相结合，提高了可靠性，得到的交互数据集7。这里描述的的HT-GPCA技术的最新发展，不仅提高了检测的灵敏度二进制在哺乳动物细胞中蛋白质相互作用，但也?...

越来越多的数据收集产生的蛋白质 - 蛋白质相互作用图谱打开的角度来进一步了解病原体的感染。随着全球病原体感染的认识开始出现，它提供了访问连接时，病原体蛋白诱导人类蛋白质组1范围内的扰动。因此，它提供了一种方式来理解，病原体如何操纵宿主细胞的机械。特别是，一些病毒宿主相互作用网络的映射显示，病毒蛋白的优先目标主机蛋白高度蜂窝网络中的连接（集线器），或中央许多在一个网络中的路径（瓶颈蛋白）2-4。这些互动让病毒操纵重要的蜂窝流程，这是有助于复制并产生感染性子代。最近进行相关病毒与目标提取信息相对比较相互作用映射发病机制4。此外，研究宿主病原体相互作用景观一直延伸到许多病原体5。针对性的细胞因子识别提供洞察战略病原体感染细胞，并允许潜在的致病性标志物的检测。 酵母双杂交系统是目前最流行的方法来识别二进制交互作用，因为遗传筛查是一种高效，敏感的蛋白质 - 蛋白质相互作用的高通量映射工具。这里提出的方法，进一步提高个别Y2H放映通过评估多种病原体的蛋白质应变变种，从而提供比较概述病原宿主蛋白 - 蛋白相互作用。此外，酵母双杂交筛选的一个主要限制在于较高的假阴性率，因为它恢复约20％的总相互作用6。这意味着，只有一个子集的病理检测的相互作用一族变种可能逃脱检测与他人。因此，从所有双杂交放映个别合作伙伴，新兴的互动与研究菌株齐全的进一步挑战，提供比较相互作用数据库。因为它表明，结合不同的方法强烈地增加了鲁棒性的蛋白-蛋白相互作用的数据集7，此验证在哺乳动物细胞中进行了新开发的蛋白质片段互补法称为HT-GPCA 8。此细胞为基础的系统允许，Gaussia颤通过互补性分裂的蛋白质的相互作用的检测荧光素酶和已被设计为具有高通量的格式兼容。基于其发光技术及其背景噪音低，与其他基于荧光检测相比，HT-GPCA显示了惊人的高灵敏度。 总体而言，这种做法构成了一个高效工具来生成一个全面的映射宿主 - 病原体interplays的，它代表一个全球性的理解宿主细胞劫持的第一步。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast strains</td>
			<td>Clontech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minimal SD base</td>
			<td>US biological</td>
			<td>D9500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amino acids</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3-amino-1,2,4-Triazole (3-AT)</td>
			<td>Acros organics</td>
			<td>264571000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zymolase</td>
			<td>Seikagaku</td>
			<td>120491</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>31966</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BioWest</td>
			<td>S1810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffer Saline (PBS)</td>
			<td>Gibco-Life Technologies</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco-Life Technologies</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Renilla luciferase assay</td>
			<td>Promega</td>
			<td>E2820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White culture plate</td>
			<td>Greiner Bio-One</td>
			<td>655083</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-wellPCR plates</td>
			<td>4titude</td>
			<td>4t-i0730/C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator (30 &deg;C)</td>
			<td>Memmert</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator (37 &deg;C)</td>
			<td>Heraeus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Berthold</td>
			<td>Centro XS-LB 960</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a comparative approach to characterize the landscape of host-pathogen protein-protein interactions

聚集产生重大努力大型综合性蛋白质 - 蛋白质相互作用网络图。这是有助于理解病原体的宿主关系，基本上是由遗传放映酵母双杂交系统。由Gaussia基于荧光素酶片段互补法检测蛋白质相互作用最近有所改善，现在提供了发展的机会，中西医结合的比较interactomic的方法必须严格比较不同的病原体菌株变种对一组共同的细胞因子的蛋白质的互动型材。 本文特别着重于实用程序相结合的两个正交的方法生成的蛋白质-蛋白质相互作用的数据集：酵母双杂交（Y2H）和一个新的测定法，高通量Gaussia颤蛋白互补法（HT-GPCA）在哺乳动物细胞中进行。 NT“&gt;大型识别病原体蛋白细胞伙伴进行通过交配的酵母双杂交放映cDNA文库使用多个病原体应变变种。是进一步的交互合作伙伴上选择了高可信度的统计评分的一个子集验证整套的病原体变体的蛋白质，使用HT-GPCA成对的相互作用，在哺乳动物细胞中的两个互补的方法，此组合提高了鲁棒性的交互数据集，并允许严格的比较相互作用分析的性能，这样的比较interactomics构成一个可靠和强大战略破译任何病原主机interplays的的。

HT-GPCA的主要优势在于它的高灵敏度，假阳性和假阴性率HPV E2蛋白在图2（改编自参考文献13）所示的评估。要确定假阴性率，已知的相互作用进行了评估，从HPV16 E2 HT-GPCA（ 图2A）。满分18相互作用没有恢复（对应于22％的假阴性率）。假阳性相互作用测定为5.8％，使用12种HPV E2蛋白对细胞的蛋白质（ 图2B）一组随机的。此外，特异性强的方法是突出图2C</s...

Watch this Scientific Journal Video about A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions at JoVE.com

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

本文重点介绍高信心之间的相互作用数据库宿主与病原体的蛋白质结合使用两个正交的方法识别：酵母双杂交，然后通过高通量相互作用的检测在哺乳动物细胞中名为HT-GPCA。

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

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