Name: identification of nucleolar factors during hiv-1 replication through rev immunoprecipitation and mass spectrometry
Uploaded: 2019-06-26T12:30:00
Description: Watch this Scientific Journal Video about Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry at JoVE.com

作者感谢芭芭拉·费尔伯博士和乔治·帕夫拉基斯博士为国家卫生研究院(NIH)艾滋病研究和参考试剂项目、国家过敏和传染病研究所提供的HLfB粘附性培养疾病(NIAID),NIH。作者还承认NIH、格兰特AI042552和AI029329提供的财务来源。

1. 细胞培养
<ol><li>在Dulbeco的改性Eagle介质(DMEM)中保持HLfB,辅以10%胎儿牛血清(FBS)、2mM L-谷氨酰胺和1mM丙酸钠,在组织培养处理的100毫米板中。在加湿的培养箱中保持细胞培养在37°C,提供5%CO2。通过汇入细胞密度为1 x 106细胞/mL。</li>
	<li>丢弃细胞培养基。用 10 mL 的 1x 磷酸盐缓冲盐水 (PBS) 轻轻冲洗细胞。在不破坏细胞层的情况下取出并丢弃 1x PBS。</li>
	<li>向细胞中加入2 mL的1x胰蛋白酶...

<ol>
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对在HIV-1存在的情况下比较Rev-NoLS突变和WT Rev的质谱分析,以了解病毒复制周期中涉及的核因子。这将确定病毒感染所需的核苷酸成分。核卵石B23对Rev-NoLS具有很高的亲和力,在Rev3的核粒定位和Rev绑定HIV mRNA 22的核细胞质迁移中具有功能。B23与Rev-NoLS突变的亲和力,其中含有单个或多个精氨酸替代物,在病毒产生过程中通过Rev-3'flag的免疫沉淀进行评估(?...

核细胞被假定为病毒复制所需的各种细胞宿主和病毒因子的相互作用场。核细胞是一个复杂的结构,分为三个不同的隔间:纤维室,密集的纤维室和粒状隔间。HIV-1 Rev 蛋白专门在粒状隔间内进行本地化;但是,此本地化模式的原因未知。在 NoLS 序列中存在单点突变(Rev 突变 4、5 和 6)的情况下,Rev 保持核苷酸模式,并且以前曾证明可以挽救 HIV-1HXB2复制,但与 WT Rev 1 相比,效率降低.所有单点突变都无法维持HIV-1NL4-3感染周期。在NoLS序列中存在多个单点突变(Rev-NoLS突变2和9)时,已观察到Rev分散在整个细胞核和细胞质,并且未能挽救HIV-1HXB2复制1。此蛋白质组学研究的目标是破译与 Rev 介导的 HIV-1 感染途径有关的核细胞因子和非核细胞因子。Rev 免疫沉淀条件通过与核蛋白 B23 磷蛋白的相互作用进行优化,该蛋白先前已被证明在存在核突变时与 Rev 失去交互作用。
过去,对细胞因子进行了广泛的研究;然而,这是在没有病毒发病机制的情况下完成的。在这项研究中,通过HIV-1复制过程中的Rev相互作用,一种蛋白质是核磷蛋白B23,也称为核对霉素(NPM)、核糖核酸或NO38(两栖动物2、3)、4.B23表示为三种同种体(NPM1、NPM2和NPM3)-核素/核质核伴生家族的所有成员5、6。NPM1分子伴菜功能在核小体的适当组装,在染色质高阶结构7,8参与的蛋白质/核酸复合物的形成,以及防止聚集和通过N端核心域(残留物1-120)错误折叠目标蛋白9。NPM1功能通过核细胞核和细胞质10、11、内转录间隔序列中前核糖体RNA的处理,扩展到核糖体成因12,13,并逮捕核蛋白聚合期间核细胞组装14,15。NPM1与抑制凋亡16和肿瘤抑制剂ARF 17、18和p5319的致稳定有关,揭示了其作为致癌因子和肿瘤抑制器的双重作用。NPM1参与基因组稳定性、中位复制和转录的细胞活动。NPM1在细胞周期相间的核糖核酸中,在线虫期间沿染色体边缘,在线虫发生结束时在核细胞体(PNB)中发现。NPM2和NPM3的研究不如NPM1,在恶性肿瘤20期间,NPM1的表达水平发生了改变。
NPM1被记录在各种核/核细胞质穿梭中,通过内部NES和NLS9,21,并以前曾报告推动HIV-1 Tat和Rev蛋白的核进口。在B23结合域-β-乳酸酶融合蛋白的存在下,Tat在细胞质内错位,失去转活活性;这表明Tat对B232有很强的亲和力。另一项研究在缺乏含有RRE的mRNA的情况下建立了Rev/B23稳定复合物。在RRE mRNA的存在下,Rev与B23分离,优选与HIV RRE结合,导致B232的位移。在亚核水平上,Tat转能和B23HIV mRNA的转用交换过程在哪里发生,目前不得而知。两种蛋白质都假定通过B23相互作用同时进入核素。预期其他宿主细胞蛋白参与HIV核蛋白通路。本蛋白质组学调查中描述的方法将有助于阐明在HIV-1发病机制过程中涉及的核细胞因子与宿主细胞因子的相互作用。
蛋白质组学研究是通过表达Rev NoLS单点突变(M4、M5和M6)和HIV-1HXB2生产的多种精氨酸替代(M2和M9)开始的。在此模型中,一个HeLa细胞系可稳稳地表达Rev缺陷HIV-1HXB2(HLfB)转染WT Rev和Rev核糖核细胞突变,这些突变在3'末端含有标志标记。WT Rev 的存在将允许病毒复制发生在 HLfB 培养,与不挽救 Rev 缺乏 (M2 和 M9) 的 Rev-NoLS 突变相比,或允许病毒复制发生,但效率不如 WT Rev (M4、M5 和 M6)1。细胞莱沙在病毒增殖后48小时后在Rev表达的情况下被收集,并在免疫沉淀后使用针对Rev/B23相互作用优化的莱沙缓冲液进行免疫沉淀。介绍了使用不同盐浓度的莱沙缓冲液优化,并在银色或库马西染色SDS-PAGE凝胶中比较和分析HIV-1 Rev的蛋白质洗脱方法。第一种蛋白质组学方法涉及通过串联质谱直接分析来自表达的WT Rev、M2、M6和M9的洗脱样品。第二种方法将WT Rev、M4、M5和M6的改性与第一种方法进行比较。与WT Rev相比,对Rev-NoLS突变的肽亲和力进行了分析,并显示了蛋白质识别概率。这些方法揭示了在HIV-1复制期间参与HIV-1 mRNA传输和与Rev拼接的潜在因素(核核和非核苷酸)。总体而言,所述细胞溶解、IP和洗脱条件适用于感兴趣的病毒蛋白,用于了解激活和调节感染途径的宿主细胞因子。这也适用于研究各种疾病模型的持久性所需的细胞宿主因素。在此蛋白质组学模型中,HIV-1 Rev IP 针对 B23 相互作用进行了优化,以阐明与核细胞质穿梭活动和 HIV-1 mRNA 结合有关的核苷酸因子。此外,可以开发与HLfB细胞系类似的细胞系,以稳稳地表达缺乏关键蛋白质的传染病模型,以研究感兴趣的感染途径。

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			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A955-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Acrylamide:Bisacrylamide</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ammonium bicarbonate</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A643-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ammonium persulfate</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">7727-54-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">ANTI-Flag M2 affinity gel</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag M2 mouse monoclonal IgG</td>
			<td>Sigma-Aldrich</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BioMax MS film</td>
			<td style="width:321px;">Carestream</td>
			<td style="width:119px;">8294985</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Bio-Rad Protein Assay Dye Reagent Concentrate, 450 mL</td>
			<td style="width:321px;">Bio-Rad</td>
			<td style="width:119px;">5000006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">B23 mouse monoclonal IgG</td>
			<td style="width:321px;">Santa Cruz Biotechnologies</td>
			<td style="width:119px;">sc-47725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Bromophenol blue</td>
			<td style="width:321px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Carnation non-fat powdered milk</td>
			<td style="width:321px;">Nestle</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Cell scraper</td>
			<td style="width:321px;">ThermoFisher Scientific</td>
			<td style="width:119px;">179693PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">C18IonKey nanoTile column</td>
			<td style="width:321px;">Waters</td>
			<td>186003763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 100-mm TC-treated culture dishes</td>
			<td>Fisher Scientific</td>
			<td>08-772-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Thermo Scientific</td>
			<td>J1539714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">1 x DPBS</td>
			<td style="width:321px;">Corning</td>
			<td style="width:119px;">21-030-CVRS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">ECL Estern blotting substrate</td>
			<td style="width:321px;">Pierce</td>
			<td style="width:119px;">32106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Ethanol, 200 proof</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A409-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">FBS</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Formic Acid</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">A117-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">GelCode blue stain reagent</td>
			<td style="width:321px;">ThermoFisher</td>
			<td style="width:119px;">24590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Glycerol</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">goat-anti-mouse IgG-HRP</td>
			<td style="width:321px;">Santa Cruz Biotechnologies</td>
			<td style="width:119px;">sc-2005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Iodoacetamide</td>
			<td style="width:321px;">ACROS Organics</td>
			<td style="width:119px;">122270050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">KimWipe delicate task wiper</td>
			<td style="width:321px;">Kimberly Clark Professional</td>
			<td style="width:119px;">34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">L-glutamine</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Methanol</td>
			<td style="width:321px;">Fisher Chemical</td>
			<td style="width:119px;">67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">NanoAcuity UPLC</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Pierce Silver Stain Kit</td>
			<td style="width:321px;">Thermo Scientific</td>
			<td style="width:119px;">24600df</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">15-mL Polypropylene conical tube</td>
			<td style="width:321px;">Falcon</td>
			<td style="width:119px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Prestained Protein Ladder, 10 to 180 kDa</td>
			<td style="width:321px;">Thermo Scientific</td>
			<td style="width:119px;">26616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Protease inhibitor cocktail</td>
			<td style="width:321px;">Roche</td>
			<td style="width:119px;">4693132001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Purified BSA</td>
			<td style="width:321px;">New England Biolabs</td>
			<td style="width:119px;">B9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">PVDF&#160; Western blotting membrane</td>
			<td style="width:321px;">Roche</td>
			<td style="width:119px;">3010040001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Sodium Pyruvate</td>
			<td style="width:321px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">10 x TBS</td>
			<td style="width:321px;">Fisher Bioreagents</td>
			<td style="width:119px;">BP2471500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">TEMED</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610880edu</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Triton X-100 detergent solution</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1610407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizaic source</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">trypsin-EDTA</td>
			<td style="width:321px;">Corning</td>
			<td style="width:119px;">25-051-CIS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Tween 20</td>
			<td style="width:321px;">BioRad</td>
			<td style="width:119px;">1706531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Synapt G2 mass spectrometer</td>
			<td style="width:321px;">Waters</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Whatman filter paper</td>
			<td style="width:321px;">Tisch Scientific</td>
			<td>10427813</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of nucleolar factors during hiv-1 replication through rev immunoprecipitation and mass spectrometry

HIV-1感染周期需要病毒蛋白与宿主因子相互作用,以促进病毒复制、包装和释放。感染周期进一步要求形成具有HIV-1 RNA的病毒/宿主蛋白复合物,以调节拼接和使核细胞质迁移。HIV-1 Rev蛋白通过具有非电子cis作用靶点-Rev响应元件(RRE)的多面化实现HIV-1 mRNA的核出口。富精素图案(ARM)的COOH-总站内存在核定位信号(NoLS),允许在核素中积累Rev/RRE复合物。除了调解与mRNA无关的核出口和拼接外,还推测核因子通过各种其他功能支持HIV-1的传染周期。在存在HIV-1复制的质谱中,我们描述了一种野生型(WT)Rev的免疫沉淀方法,与Rev核苷酸突变(删除和单点Rev-NoLS突变)相比。与核细胞质迁移相关的核核素因子(核细胞因子B23和核素C23)以及细胞拼接因子,在Rev-NoLS突变存在时失去与Rev的相互作用。各种其他核因子,如snoRNA C/D盒58,被识别失去与Rev突变的相互作用,但他们在HIV-1复制周期的功能仍然未知。这里介绍的结果表明,这种方法用于识别维持HIV-1感染周期的病毒/宿主核素因子。该方法中使用的概念适用于需要描述研究不足的通路的其他病毒和疾病模型。

与WT Rev.WT Rev-3'标志和pcDNA-标志相比,Rev-NoLS单点和多点精氨酸突变与各种亚细胞定位模式相对应,在与细胞宿主因子相互作用的能力方面进行了检查在HLfB文化中表达向量。蛋白质复合物从总细胞赖沙处理,并沾染银染色试剂。Rev-NoLS-3'标志在三种不同的液化缓冲条件下可检测到(约18 kDa),其中含有图1中不同浓度的NaCl(137 mM、200 mM和300 mM)。B23在含低盐浓度(137 mM,通道2⁄4)的莱?...

Watch this Scientific Journal Video about Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry at JoVE.com

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

在这里,我们描述了在质谱中存在HIV-1复制的Rev免疫沉淀。所述方法可用于识别艾滋病毒-1感染周期中涉及的核素因子,并适用于其他疾病模型,用于对研究不足的途径进行表征。

通过转版免疫沉淀和质谱测定,在HIV-1复制过程中核子因子的识别

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

我们的协议为识别和鉴定维持HIV-1感染周期的病毒、核和非核宿主因子提供了替代方法。这种方法使用的概念适用于需要对未研究途径进行表征的其他病毒和疾病模型。介绍了用于修改其他蛋白质模型的故障排除技术。展示这个程序的将是希望之城贝克曼研究所分子和细胞生物学部的科学家李海堂和Pritsana Chomchan，以及希望之城蛋白质组学核心和分子免疫学系成员的罗杰·摩尔。首先，在三个100毫米培养板中稳定地表达HLfB细胞系培养，以每毫升密度的2倍到10至6个细胞。当细胞达到适当的浓度时，在15毫升管中混合20微克含有Rev核定位信号的质粒，与15毫升管中1.76毫升TE 79/10的TE 79/10突变，然后加入240微升的两摩尔氯化钙，进行彻底的混合。接下来，将管内的东西滴入一个新的15毫升管，含有两毫升的2x HBS，然后混合在涡流机上。在室温下30分钟后，旋涡降水。将悬浮液滴到三个 100 毫米的 HLfB 培养板中每个板上添加一毫升，同时轻轻旋转介质。在细胞培养箱中孵育6小时后，用10毫升新鲜培养培养物取代转染混合物，将细胞返回孵化器42小时。转染48小时后，将每个盘子放在生物危害二级组织培养罩内的冰床上。取出细胞培养基，用10毫升预冷PBS轻轻冲洗细胞，而不会破坏细胞层。冲洗细胞后丢弃 PBS。接下来，每盘用一毫升的解液缓冲液，辅以蛋白酶抑制剂鸡尾酒来治疗细胞。倾斜板将细胞聚集到池中，使用单元格刮刀手动破坏图层。使用1000微升微管，将每个板的落酸放入一个15毫升的预冷管中，在冰上孵育15分钟，每5分钟进行一次涡旋。在孵育结束时，将酸盐分成三个1.5毫升的微离心管，通过离心收集酸盐。将上清液转移到新的15毫升管中，获得病毒蛋白酸盐浓度，如手稿中所述。对于Rev核定位信号三质标志的共生沉淀，每次洗涤用500微升新鲜解合缓冲液，补充蛋白酶抑制剂鸡尾酒，三次冲洗25微升M2亲和凝胶珠。在冷室或冰上使用试剂执行这些步骤。最后一次冲洗后，在五毫升的解解缓冲液的最后体积中，将每毫升病毒蛋白解酸的1毫克浓度加入冲洗珠。在四摄氏度下旋转，孵育反应三小时。在孵育结束时，通过离心将病毒蛋白分解结成珠子颗粒。收集上清液，测量免疫后沉淀的细胞蛋白浓度。有关样品存储说明，请参阅手稿。将750微升的解液缓冲液加入病毒蛋白解结珠颗粒中，将珠子转移到1.5毫升微离心管中，并在4摄氏度的旋转器上清洗珠子5分钟。如刚刚演示，通过离心收集珠子，在旋转器上再洗两次。最后一次洗涤后，使用捏的、长的凝胶加载尖端，从珠子-共性沉淀物中去除任何解液缓冲液的痕迹。将珠子重新在 55 微升 2 倍加载缓冲器中。然后，在95摄氏度下将样品煮沸10分钟。将 25 微升的 Eluate 加载到一个西侧印迹凝胶和一个库马西染色 SDS-PAGE 凝胶上。完成SDS-PAGE凝胶电泳后，在25毫升超纯水中冲洗凝胶三次，15分钟。上次洗涤后，在凝胶上加入100毫升的库马西染色试剂。有关完整的染色协议，请参阅手稿。为了消化凝胶带，首先使用干净的剃须刀刀片将凝胶带从凝胶的整个通道（代表每个突变）切成大约五毫米的立方体。将每个带子放在一个干净的 0.5 毫升微离心管中。在室温下用100毫摩尔碳酸氢铵覆盖带子两次，每次洗涤15分钟。然后，在真空离心机中干燥凝胶片五分钟。为了减少蛋白质，在100毫摩尔碳酸氢铵中用10毫摩尔二甲酰胺覆盖干凝胶片，在56摄氏度下孵育一小时，然后在水中用100毫摩尔多乙酰胺进行烷基化，在黑暗中室温下在水中燃烧一小时。接下来，收缩和重新垫凝胶片两次与乙酮，其次是100毫升碳酸氢铵，两者都在室温下轻轻摇动15分钟。第二次再垫后，在真空离心机中干燥凝胶片五分钟。在100毫摩尔碳酸氢铵中，每微升50毫微克的凝胶片中，用50毫微克。五分钟后，用100毫摩尔碳酸氢铵覆盖凝胶片，让它们完全重新井，添加额外的100毫摩尔碳酸氢铵，使肿胀的凝胶片完全覆盖。然后，在37摄氏度下孵育凝胶片，第二天早上停止反应，将1/10的样品体积与10%的甲酸在水中产生反应，然后从每个管中收集上流液。在含有不同浓度的氯化钠的三种不同的解解缓冲条件下，可检测出三极核定位信号三极标志。B23在含有低盐浓度的解液缓冲液中也可检测，在含有中等盐浓度的解液缓冲液中几乎检测不到，在含有高盐浓度的解液缓冲液中检测不到。B23 检测在含有低氯化钠浓度的解液缓冲液中，具有野生型 Rev 三极旗，与 Rev 核定位信号 M1 三极旗失去亲和力。B23 与野生型 Rev 三极旗的亲和力也随着盐浓度的升高而降低，并且 PCDNA 阴性对照在两种解解缓冲条件下免疫沉淀后不产生非特异性免疫检测。在2倍样品加载缓冲液中煮沸后，可检测出复核定位信号三等信号，而B23在37摄氏度和95摄氏度的样品缓冲液孵化条件下可检测。免疫沉淀和银染色后，如证明，野生型 Rev 和 Rev 核定位信号 M2、M6 和 M9 可检测为 18 千吨。在37千代顿标记物上也观察到与B23蛋白对应的波段。使用 Coomassie 试剂进行染色进一步表明，在 18 千度（18 千吨）存在且不存在突变的情况下，Rev 核定位信号三极标志的可检测性。在潜在结合因素的故障排除和筛选过程中，对与疾病模型相关的参考使用尽可能多的正向和负控制。所有删除和单点突变可以通过与野生型 Rev 进行多样化来检查显性阴性活性，并用于逮捕 HIV-1 复制。

Research

JoVE Journal

Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...