这项工作是由NIH / NIGMS / MBRS（格兰特58268），美国国立卫生研究院/（格兰特G12-RR03034）NCRR / RCMI的的资金补助GRA.VAC08.W，乔治亚研究联盟，NIH / NIAID / NRSA的的补助F31AI091484，埃默里CFAR授予P30支持A1050409。进行这项调查是在设施建造与研究设施改善格兰特＃C06 RR18386支持NIH / NCRR。 Jurkat细胞，20组HIV-1 Nef的肽，以及来自美国国立卫生研究院艾滋病研究和参考试剂计划，（在Rockville，MD）获得兔抗HIV-1 Nef的抗血清。

I.使用短肽在生物分析一.1。测绘使用肽的生物功能图案<ol><li>治疗细胞与NEF扫描的肽<ol><li>促使扫描感兴趣区域的一组肽。对于我们的实验中，20聚体肽，带有10个氨基酸重叠，跨越整个HIV-1 Nef蛋白（205个氨基酸），得到，艾滋病试剂从程序。单独测定的肽，如下所示: </li><li>加入10毫微克/毫升的细胞培养液中的肽。 </li><li>添加10ml的培养基中每板Jurkat细胞培养。 </li><li>培养24小时，在37°C。 </li></ol></li><li>终端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测细胞凋亡<ol><li>用1X PBS洗涤细胞，并在1X PBS，pH为7.4，用4％多聚甲醛在室温下固定30分钟。 </li><li>用1X PBS洗涤，并用0.1％的通透的Triton X-100和10分钟，在室温下。 </li><li>用1X PBS冲洗细胞两次。 </li><li>上的计算机控制的显微镜系统中，通过荧光染色的细胞计数。 </li></ol></li></ol> 1.2。竞争性抑制肽分析<ol><li>转肽使用的战车蛋白交货试剂盒<ol><li>每转染反应混合物，稀释的1μgwtNef-GFP质粒DNA和100​​ng的来自从SMR域，无论是野生型肽或肽含有丙氨酸的单个氨基酸替代的的SMR动机中，至终体积为100μl 1X PBS。 </li><li> 6微升的1/10稀释的战车试剂稀释用无菌水至终体积为100μl，并结合肽稀释的DNA混合。 </li><li>孵育30分钟，在室温下，以允许的车辆/ DNA-肽复合物的形成。 </li><li>颗粒3×10 5 Jurkat细胞在600×g离心5分钟，通过离心分离。 </li><li>用1X PBS洗涤细胞两次，战车/ DNA-肽复合物和悬浮。 </li><li>的悬浮液中加入400μl的无血清RPMI 1640培养液，在培养板中的细胞在37℃下孵育2小时。 </li><li>加入1毫升新鲜的含10％FBS的RPMI 1640培养基中，以每块板，孵育细胞48小时，在37℃下</li><li>收获细胞和荧光显微镜确定转染效率。 </li></ol></li><li> NEF-GFP荧光酶标仪测定分泌肽抑制<ol><li>从收获的细胞，收集无细胞的条件培养基，并转移100μl到个别的96孔黑色微量滴定板的孔中。 </li><li>在媒体上GFP荧光激发波长为485 nm，发射波长为515 nm的相对荧光单位一个的TECAN GENEios荧光计测量。 </li><li>设置在控制水平的GFP荧光（介质从细胞中共同转染与Nef的GFP和一个加扰的肽）在100％。 </li><li>与此相比，在共转染各种SMR肽确定可在碱GFP分泌的更改介质从细胞水平的GFP荧光。 </li></ol></li></ol> .3。隔离使用肽作为诱饵的动机具有约束力的合作伙伴<ol><li> SMR肽的免疫共沉淀<ol><li> Jurkat细胞生长的RPMI 1640培养基中含10％FBS在37℃下的T-75组织培养瓶，和沉淀细胞通过离心分离20分钟，在4℃下以1,000×g的确定转染效率，通过将板在荧光显微镜下，捕获图像，随后的总的和标记的细胞进行计数，并确定标记的细胞的百分比。 </li><li>在1毫升的1X PBS重悬细胞沉淀，并将其转移至一个1.5毫升的Eppendorf管中。微型离心机在1,000×g离心1分钟。悬浮颗粒在1毫升的α-FLAG裂解缓冲液轻柔吹打。裂解缓冲液:将50毫米的Tris HCl pH值7.4，150毫米钠叶绿素oride [氯化钠]，1mM的EDTA，1％的Triton X-100，用1mM苯甲基磺酰氟PMSF和10微升/毫升的蛋白酶抑制剂鸡尾酒用于哺乳动物细胞和组织提取物[图; Sigma公司。使用前，请立即。 </li><li>差异的离心悬浮液在2000×g离心5分钟，并以13,000×g离心10分钟沉淀细胞碎片和DNA，分别。收集上清液（以下简称为"裂解物"）。 </li><li>添加的细胞裂解液的蛋白和1毫克的SMRwt或SMRmut肽的抗-FLAG M2亲和凝胶（以下称为"树脂"，Sigma社制）的20μl的1微克。请注意，这里使用两个SMRwt SMRmut有一个标志标签嵌入肽序列。把混合物在合作的IP缓冲液（α-FLAG的裂解缓冲液减去PMSF和PIC）为1毫升，至终体积。孵育混合物在4℃过夜，最终高端旋转。作为阴性对照，孵育在没有任一肽与树脂的溶胞产物。 </li><li>佩尔吨树脂的微量离心5000 - 8000×g离心30秒。允许树脂解决2分钟前处理样品，然后吸出上清液。窄枪头，或微量注射器，取出上清液小心，不转让任何树脂。窄枪头可以使用镊子捏住开口的塑料枪头，直到局部封闭。四次洗涤树脂，用500μl洗涤缓冲液（的50mM Tris HCl pH值7.4，150 mM氯化钠）。 3X FLAG肽（Sigma公司），在50微升的洗涤缓冲液中15微克洗脱结合的细胞蛋白，并孵育30分钟，在室温下摇臂/摇床。 </li><li>浓缩洗脱液，用丙酮沉淀，然后在Laemmli样品缓冲液（Bio-Rad公司）煮沸。在4-20％的Tris-HCl标准预制凝胶（Bio-Rad公司），分离出的蛋白质通过SDS-PAGE。染色凝胶用考马斯亮蓝R-250（Bio-Rad公司）。注:为质谱鉴定实验（ <stron克&gt;图4A），每一个条件（IP与SMRwt，SMRmut，或无肽）一式四份进行，从所有四个重复的洗脱液合并的丙酮沉淀之前为4毫克总蛋白输入/共条件）。 </li></ol></li><li> NEF-GFP蛋白的免疫共沉淀<ol><li>混合50微升的Dynabeads G蛋白磁珠（Invitrogen公司）用2微克的鼠单克隆抗GFP在1毫升的PBS洗涤，用0.02％吐温20（PBS-T）。在1.5毫升的Eppendorf管端部上下旋转孵育混合物在4℃下1小时</li><li>分隔的有孔玻璃珠从缓冲管1.5毫升商Magnasphere技术磁性分离架（Promega公司，麦迪逊，威斯康星州），使磁性下拉随后除去上清液。洗的抗GFP涂层的有孔玻璃珠，用1ml的PBS-T使用轻柔吹打。 </li><li> NEF-GFP转染Jurkat细胞裂解1毫升1X RIPA +缓冲轻柔吹打和孵化15米在冰上。的RIPA缓冲液10X的RIPA裂解缓冲液，pH值7.4 [Millipore公司，旅游Bedford，MA] 1:10稀释于无菌水，0.02％叠氮化钠[Sigma公司]，和0.1％十二烷基硫酸钠[SDS]，用1mM PMSF和10微升/毫升PIC立即加入后方可使用。 </li><li>颗粒细胞碎片在2000×g离心10分钟，通过微量离心。收集上清液（以下简称为"裂解物"）。 </li><li>加入1毫克的细胞裂解液的蛋白的抗GFP包被的磁珠，使终体积为1毫升1X RIPA +缓冲，并与端部上下旋转孵育混合物在4℃下1小时注:1毫克的裂解液蛋白质对于肽竞争研究，可能会预先用2微克的SMRwt或SMRmut肽孵育的1×RIPA +缓冲液，之前被加入到的抗GFP涂层的有孔玻璃珠在1毫升。 </li><li>通过再悬浮在1毫升的洗涤缓冲液（洗珠的50mM Tris HCl pH值7.4，150 mM氯化钠，0.1％吐温20）。端部上下旋转5分钟，在4℃下孵育分离的珠FROM洗净，用磁分离架。分别含有250 mM和300 mM氯化钠，用洗涤缓冲液洗珠的第二和第三次。 </li><li>重悬在200μlPBS中的有孔玻璃珠，将混合物转移到一个干净的试管中，然后分离从洗涤的磁性底座上的有孔玻璃珠。熬的有孔玻璃珠在50μl的Laemmli样品缓冲液中5分钟，在95℃，使混合物在室温下冷却10分钟，然后将混合物放置在磁性支架分离，并收集上清液（"洗脱物"） 。 </li><li> 8-16％的Tris-HCl标准预制凝胶上，用SDS-PAGE分离蛋白洗脱液。 </li></ol></li><li>质谱法<ol><li>海关考马斯亮蓝染色凝胶蛋白条带的兴趣。 </li><li>在10mM的1,4 - 二硫苏糖醇（DTT）的减少样品在37℃下45分钟</li><li>样品在25℃下45分钟，在55 mM的碘乙酰胺烷基化</li><li>消化样品测序级尝试过夜PSIN（Promega公司）在37℃下</li><li>脱盐样品肽ZipTip微量层析柱C-18（Millipore公司）。混合样品肽与α-CHC矩阵（安捷伦科技公司，加利福尼亚州圣克拉拉），并发现它们到MTP目标框架III（布鲁克·道尔顿公司，比尔里卡，MA）。 </li><li>执行串联基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/TOF）飞行使用布鲁克·道尔顿的ultraflex III TOF / TOF质谱分析。 </li><li> NCBI的非冗余和SWISS-PROT数据库使用吉祥物的算法（ <a href="http://www.matrixscience.com" target="_blank">www.matrixscience.com</a> ）的MS / MS数据进行比较。 </li></ol></li><li>免疫印迹分析<ol><li>在4-20％或8-16％的Tris-HCl标准预制凝胶（Bio-Rad公司），通过SDS-PAGE分离出的蛋白样品。带电泳转移到硝酸纤维素膜上。 </li><li>洗膜在Tris缓冲盐水（TBS; Bio-Rad公司），持续5分钟。用5％脱脂牛奶座TTBS（TBS，0.1％吐温20），在室温下摇动1小时。 </li><li>处理膜的免疫印迹法，使用特定的初级抗体在5％脱脂乳中于4℃过夜振荡。洗20分钟，随后的二次HRP标记的IgG抗体（H + L）抗体在室温下1小时，在5％脱脂奶粉在TTBS。在TTBS洗涤30-60分钟。 </li><li>检测蛋白条带，免疫印迹的鲁米诺试剂（圣克鲁斯生物技术公司，圣克鲁斯，加利福尼亚州）。过滤器暴露感光胶片。注:在一些实验中，剥离剂，用于去除膜（皮尔斯，Rockford，伊利诺州），随后用不同的小学和中学的抗体的杂交。 </li><li>扫描的X光片到Adobe公司的Photoshop 5.0.2。使用ImageJ软件（美国国立卫生研究院，贝塞斯达，MD）进行密度测定分析。 </li></ol></li></ol>二。使用抗体抑制功能分析二.1。共转染的抗体指使用的战车蛋白交货试剂盒<ol><li>每转染反应混合物，稀释的wtNef-GFP质粒DNA的1微克，1微克任一反mortalin的抗体或抗α-微管蛋白抗体，用1X PBS，至终体积为100μl。注意:这是以前发现，共转染的抗α-微管蛋白抗体有没有可察觉的影响对exNef分泌，因此，它作为一个有效的在这些实验中的阴性对照。 </li><li> 6微升未稀释的战车试剂稀释用无菌水至终体积为100μl，并结合DNA抗体的稀释混合。 </li><li>孵育30分钟，在室温下，以允许的车辆/ DNA抗体复合物的形成。 </li><li>颗粒3×10 5 Jurkat细胞在600×g离心5分钟，通过离心分离。 </li><li>用1X PBS洗涤细胞两次，和悬浮战车/ DNA抗体复合物。 </li><li>加入400微升无血清的RPMI 1640培养基中，该悬浮液中，并孵育的c在培养板中的细胞l在37℃下搅拌2小时。 </li><li>加入1毫升新鲜的含10％FBS的RPMI 1640培养基中，以每块板，孵育细胞48小时，在37℃下</li></ol> .2。 NEF-GFP分泌荧光酶标仪测定抗体抑制<ol><li>从收获的细胞，收集无细胞的条件培养基，并转移100μl到个别的96孔黑色微量滴定板的孔中。 </li><li>在媒体上GFP荧光激发波长为485 nm，发射波长为515 nm的相对荧光单位一个的TECAN GENEios荧光计测量。 </li><li>设置在控制（介质从细胞共转染碱-GFP和抗α-微管蛋白抗体），在100％的GFP荧光的水平。 </li><li>与此相比，从共转染的细胞与抗mortalin抗体确定可在碱GFP分泌的更改在媒体GFP荧光的水平。 </li></ol>

<ol>
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Invest. 116 (6), 1514-1524 (2006).</li><li>Mueller, Y. M., Do, D. H., Altork, S. R., Artlett, C. M., Gracely, E. J., Katsetos, C. D., Legido, A., Villinger, F., Altman, J. D., Brown, C. R., Lewis, M. G., Katsikis, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+treatment+during+acute+simian+immunodeficiency+virus+(SIV)+infection+increases+viral+set+point+and+accelerates+disease+progression+despite+the+induction+of+stronger+SIV-specific+CD8++T+cell+responses.">IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses.</a> J. Immunol. 180 (1), 350-360 (2008).</li><li>Ali, S. A., Huang, M. B., Campbell, P. E., Roth, W. W., Campbell, T., Khan, M., Newman, G., Powell, F., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Characterization+of+HIV+Type+1+Nef-Induced+Vesicle+Secretion.">Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion.</a> AIDS Res. Hum. Retroviruses. 26 (2), 173-192 (2010).</li><li>Raymond, A. D., Campbell-Sims, T. C., Khan, M., Lang, M., Huang, M. B., Bond, V. C., Powell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+Type+1+Nef+Is+Released+from+Infected+Cells+in+CD45++Microvesicles+and+Is+Present+in+the+Plasma+of+HIV-Infected+Individuals.">HIV Type 1 Nef Is Released from Infected Cells in CD45+ Microvesicles and Is Present in the Plasma of HIV-Infected Individuals.</a> AIDS. 27 (2), 167-178 (2011).</li><li>James, C. O., Huang, M. -. B., Khan, M., Garcia-Barrio, M., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Nef+Protein+Targets+CD4++T+Cells+for+Apoptosis+by+Interacting+with+CXCR4+Surface+Receptors.">Extracellular Nef Protein Targets CD4+ T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors.</a> J. Virol. 78 (6), 3099-3109 (2004).</li><li>Huang, M. B., Jin, L. L., James, C. O., Khan, M., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Nef-CXCR4+Interactions+Important+for+Apoptosis+Induction.">Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction.</a> J. Virol. 78 (20), 11084-11096 (2004).</li><li>Heveker, N., Montes, M., Germeroth, L., Amara, A., Trautmann, A., Alizon, M., Schneider-Mergener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+the+signalling+and+antiviral+properties+of+SDF-1-derived+small+peptides.">Dissociation of the signalling and antiviral properties of SDF-1-derived small peptides.</a> Curr. Biol. 8 (7), 369-376 (1998).</li><li>Shelton, M. N., Huang, M. B., Ali, S. A., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMR-derived+peptide+disrupts+HIV-1+Nef's+interaction+with+mortalin+and+blocks+virus+and+Nef+exosome+release.">SMR-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release.</a> J. Virol. 86 (1), 406-419 (2012).</li><li>Ptak, R. G., Fu, W., Sanders-Beer, B. E., Dickerson, J. E., Pinney, J. W., Robertson, D. L., Rozanov, M. N., Katz, K. S., Maglott, D. R., Pruitt, K. D., Dieffenbach, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataloguing+the+HIV+type+1+human+protein+interaction+network.">Cataloguing the HIV type 1 human protein interaction network.</a> AIDS Res. Hum. Retroviruses. 24 (12), 1497-1502 (2008).</li><li>Shugars, D. C., Smith, M. S., Glueck, D. H., Nantermet, P. V., Seillier-Moiseiwitsch, F., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+human+immunodeficiency+virus+type+1+nef+gene+sequences+present+in+vivo.">Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.</a> J. Virol. 67 (8), 4639-4650 (1993).</li></ol>

作者宣称，他们有没有竞争经济利益。

理解Nef的能力去操纵的exosomal贩运途径将是有益的工程新型抑制剂的exNef分泌机制。应减少抑制exNef分泌的CD4 + T细胞耗竭和中央情报局驱动艾滋病毒/艾滋病的发病机制。为了实现这一目标，我们制定了一些新颖的试剂和方法，让我们来分析的的遗传学exNef分泌，并开始确定所涉及的细胞蛋白质。这种方法也导致了发展的第一个直接针对exNef分泌，使SMRwt的肽抑制剂。 我们工作的...

随着抗逆转录病毒疗法的问世，在西方世界的艾滋病疫情已经放缓，但不会削减，和艾滋病毒的蔓延仍然是一个主要的健康负担，在世界各地。除了轻微有效RV144泰国试用，艾滋病毒疫苗迄今未能保护免受感染。因此，成额外的，潜在的治疗靶点的研究仍是必要的。 随着CD4 + T细胞耗竭，持续性全身免疫激活是一个标志性的艾滋病毒感染。这种慢性免疫激活（CIA）导致增加细胞的新陈代谢，激活和分化的淋巴细胞亚群，细胞的疲惫和衰老，并杀害的T细胞和B细胞通过激活诱导的细胞死亡（AICD）1,2,3，它是公认为疾病进展4,5,6,7,8,9,10,11,12最强的预测之一。然而，机制中央情报局（CIA）和CD4在HIV感染的T细胞耗竭，仍然完全阐明。 从我们的实验室和其他证据，导致疾病进展到一个模型（ 图1），其中HIV蛋白Nef的（负调控因子）诱导其分泌外来HIV-1感染细胞13,14。在这些含Nef的的外来（exNef）诱导细胞凋亡中的细胞谱系数，包括未感染的CD4 + T细胞15,16。另外，在单核细胞/巨噬细胞exNef改变基因表达模式， 如细 ​​胞因子的表达，出现不定期的免疫激活诱导的状态。该机构的证据表明在中央情报局（CIA）和CD4 + T细胞耗竭exNef了重要作用。 理解Nef的能力去操纵的exosomal贩运途径将是有益的工程新型抑制剂的exNef分泌机制。应减少抑制exNef分泌的CD4 + T细胞耗竭中情局驱动艾滋病毒/艾滋病的发病机制。 为了收集证据，导致我们的模型为艾滋病毒/艾滋病疾病的进展和后续的数据，建到这个模型，我们开发了一些新颖的试剂和方法，使我们能够分析遗传学的exNef分泌，并开始确定细胞的蛋白质参与。在最初的工作中，我们发现，Nef蛋白诱导旁观者细胞凋亡，并释放到细胞外从奈夫转染和HIV感染的细胞15。肽SDF-1α（基质细胞衍生因子-1，与替代拼接阿尔法），来自先前的研究显示，保留绑定和信令活动分子全长17。我们推测的Nef肽可能保留一些细胞凋亡活性全蛋白，，这些肽必然会包含NEF凋亡域。要识别这些肽，因此奈夫凋亡域（S），我们获得了一组20例HIV-1 Nef的扫描肽从美国国立卫生研究院艾滋病研究和参考试剂计划。在这些20个氨基酸的肽，每一个重叠的10个氨基酸，它的邻居，被命名为他们的最后的氨基酸， 即 N20跨度Nef的氨基酸1-20，N30的跨度的Nef的氨基酸11-30，等16的数量。我们发现，将T细胞暴露以重叠的两个不同的10个氨基酸的结构域的特异性肽全长的Nef蛋白在这些细胞中诱导细胞凋亡的细胞外。随后的分析显示这些Nef的衍生细胞凋亡肽的物理交互的趋化因子受体CXCR4的T细胞的表面上，结合动力学，使这些肽的竞争性抑制CXCR4和其天然配体SDF-1α之间的结合的能力。最后，NEF源性凋亡肽与CXCR4的相互作用被发现在这些T细胞，导致细胞凋亡诱导应激反应。这方面的证据使我们能够QuIC的地图NEF千光年的功能域;一个过程，会花费更长的使用标准DNA诱变技术，如丙氨酸扫描诱变。它还表明，这些短Nef的衍生肽保留了​​细胞凋亡的生物学功能域的全长蛋白。 经鉴定外奈夫的作用， 即 T细胞凋亡，我们寻求更好的理解奈夫如何从细胞分泌。我们使用了一系列突变的Nef的构造，映射Nef蛋白高度保守的基序的N-末端区域的同时感染HIV Nef的，和:相当于猕猴SIV 的mac（猴免疫缺陷病毒）Nef的，这是至关重要的为分泌exNef 13。这些图案之一，分泌修改地区（SMR 66VGFPV70），是特别重要的，因为任何其五个氨基酸要么大大减少或取消exNef，分泌丙氨酸更换。当传递到细胞通过Active Motif公司的C结果发现，包含连接到一个FLAG肽序列（SMRwt）的SMR的肽hariot蛋白交货试剂从Nef的转染和HIV感染的细胞18抑制exNef分泌。根据我们以往的经验与肽上，我们决定使用这种肽，阐明分子和机制的Nef的SMR的作用在exNef分泌相关。 ，我们使用肽作为我们的"诱饵蛋白"SMRwt，共同免疫沉淀的细胞的结合配偶体的SMR来自未感染的T细胞的溶胞产物18。 FLAG肽序列提供了一个方便的把手，使用抗FLAG亲和树脂捕捉SMRwt的肽。我们以前发现，一个单一的缬氨酸，丙氨酸突变在SMRwt肽废除其抑制分泌exNef的足够确定了方便，具有高度特异性的控制的肽（SMRmut），我们用来排除联合免疫沉淀的蛋白质，不特定的SMR。使用有s的肽pecific领域的利益，而不是全长蛋白允许，我们绕过筛选几十种细胞因子结合到其他领域奈夫19。 一旦我们确定了的SMR结合合作伙伴，一个合乎逻辑的下一步是要表明细胞结合合作伙伴是非常重要的生物学功能18。的标准程序来完成，这是使用目标蛋白特异性的miRNA或siRNA，并随后测定效果的生物学功能拦截蛋白水平。我们进行的miRNA的拦截，从而抑制靶mRNA的翻译，降低生产该目标，在这种情况下，SMR-结合蛋白。这种减少的目标蛋白是一种间接的影响，并可能有一个延迟效应对目标蛋白的生物学功能发挥了作用。因此，我们还采用了不常见的抗体，以确定是否直接破坏的活性的抑制技术目标蛋白的生物学功能降低或消除了。在此过程中，对目标蛋白的抗体的转染进入细胞内使用战车试剂的，并直接与目标蛋白的任一螯合剂从它的网站的功能，或阻断其相关的结合结构域。抑制靶蛋白通过此程序直接破坏其功能，可以互补RNA拦截程序，进一步确认的目标蛋白的生物学功能的重要性。 虽然战车试剂在进入细胞提供的肽和蛋白质是有效的，这个过程是耗时的，并限制类型的实验， 例如，长期或反复暴露在体内动物研究中可以执行的。因此，我们增加了一个细胞穿透肽（CPP）序列的SMRwt肽（SMRwt CPP）18生成肽，可以采取细胞被动从培养基。这个版本是有效的，因为前者在抑制exNef分泌。 从这些发表的实验证据表明含有特定的功能图案，小肽拮抗的全长蛋白的功能通过竞争性抑制，并分离的蛋白质结合这些图案的能力。人们期望，这些技术在许多实验的协议应该是有用的。他们也应该是有效的工程新型肽抑制剂的许多细胞过程中的功能，可以进一步增强联动CPP序列。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>20mer peptide set with 10 amino acid overlap</td>
 <td>NIH AIDS Research and Reference Reagent Program</td>
 <td>4641</td>
 <td></td>
 </tr>
 <tr>
 <td>TUNEL Assay</td>
 <td>Roche</td>
 <td>11 684 809 910</td>
 <td></td>
 </tr>
 <tr>
 <td>Chariot Protein Delivery Reagent</td>
 <td>Active Motif</td>
 <td>30100</td>
 <td></td>
 </tr>
 <tr>
 <td>Tecan GENEios fluorimeter</td>
 <td> (Tecan Group, Switzerland)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>96-well black microtiter plate</td>
 <td>Corning</td>
 <td>3792</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-FLAG M2 Affinity Gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads Protein G magnetic beads </td>
 <td>Invitrogen</td>
 <td>100.03D</td>
 <td></td>
 </tr>
 <tr>
 <td>MagnaSphere Technology Magnetic Separation Stand (two position)</td>
 <td>Promega Corp., Madison, WI</td>
 <td>Z5332</td>
 <td></td>
 </tr>
 <tr>
 <td>C-18 ZipTip </td>
 <td>Millipore</td>
 <td>ZTC18S096</td>
 <td>C18 Resin (0.6 &mu;l or 0.2 &mu;l bed volumes). Oligonucleotides or small (&lt;50 kDa) proteins/ peptides in aqueous solution</td>
 </tr>
 <tr>
 <td>MALDI TOF/TOF</td>
 <td>Bruker Daltonics ultraflex III TOF/TOF</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

peptide-based identification of functional motifs and their binding partners

全长蛋白的基序发现，在我们的例子中的HIV-1 Nef的是来自于特定的短肽，不仅保留其生物学功能，但也可以竞争性抑制的全长蛋白的功能。 20 Nef的扫描肽，20个氨基酸的长度与每个重叠10个氨基酸，它的邻居，一组被用来确定负责其诱导细胞凋亡的Nef的图案。包含这些细胞凋亡的基序的肽的诱导细胞凋亡的水平相媲美Nef蛋白的全长。第二肽，来自分泌修改地区（SMR）的Nef保留奈夫的分泌的外来（exNef）细胞的蛋白质参与互动的能力。作为"诱饵"蛋白此SMRwt肽免疫共沉淀实验中分离的细胞蛋白特异性结合的Nef的SMR动机。蛋白转染和抗体抑制使用的物理破坏的相互作用的赌注测定碱之间mortalin，隔离SMR-结合蛋白之一的，而且效果与的基于荧光exNef分泌测定。该SMRwt肽的能力胜过全长Nef的细胞蛋白质结合的SMR动机，使第一抑制剂分泌exNef的。因此，利用这里描述的技术，利用图案全长蛋白中发现的来自于特定的短肽的独特性能，可加速蛋白质中的功能性基序的识别和致病性的功能的肽类抑制剂的发展。

使用肽的生物功能图案映射。两个区域被确定在NEF蛋白诱导细胞凋亡。观察细胞凋亡肽驱动（ 图2）开始用肽N50（AA30-50），峰值在N60（AA40-60）和7610（AA50-70），并减少在肽N100（AA80-100）的背景水平。的主要母题1（M1）峰值，围绕AA50-60，诱导80％全长Nef蛋白的细胞凋亡水平。第二个较小的跨越肽N180（aa160-180）和N190（aa170-190）观察凋亡峰。这一个小小的母题2（M2）的高峰期...

Watch this Scientific Journal Video about Peptide-based Identification of Functional Motifs and their Binding Partners at JoVE.com

基于肽的功能基序及其结合伙伴的鉴定|免疫学和感染|视频

Peptide-based Identification of Functional Motifs and their Binding Partners

解剖机制的分泌HIV-1 Nef的外来技术进行了阐述。来自NEF和蛋白转染的特殊短肽被利用来确定Nef的分泌修改区域的结构，功能和有约束力的合作伙伴。这些程序在许多机理研究具有普遍意义。

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

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12.4K 次观看.  Morehouse School of Medicine. 解剖机制的分泌HIV-1 Nef的外来技术进行了阐述。来自NEF和蛋白转染的特殊短肽被利用来确定Nef的分泌修改区域的结构，功能和有约束力的合作伙伴。这些程序在许多机理研究具有普遍意义。

视频: Peptide-based Identification of Functional Motifs and their Binding Partners

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

脊髓灰质炎病毒识别粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

科研

免疫与感染

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

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