这项工作得到了 NIH AI099854 和 AI126742 的支持。

1. 诱发 PFV 在大肠杆菌中的表达式
<ol><li>
添加1μ l 的 PFV 在表达质粒 (10 ng/μ l) 到20μ l 的大肠杆菌BL21 (DE3) pLysS (20 μ l 分的商业可用细胞有 > 2 x 106 CFU/微克质粒) 在1.5 毫升管。用手指轻轻敲击或轻拂管子。在冰上孵育5分钟。
	<ol>
<li>热休克整整三十年代在42˚C 水浴, 然后立即返回到冰2分钟。</li>
		<li>添加80μ l 室温超优化汤与物抑制 (SOC) 媒体。孵育为1小时在37˚C 以225转每分钟 (rpm) 震动。</li>
		<li>25μ l 的混合物在100毫米 x 15 毫米培养皿中, 含有25毫升的 Luria 肉汤 (lb) 琼脂 (1 L 磅琼脂:10 克 bacto-胰, 10 克氯化钠, 5 克酵母提取物, 15 克琼脂) 和100微克/毫升氨苄西林 (100 毫克/毫升的库存溶液)。</li>
		<li>在第二个相同的板上将剩余的75μ l 转化后的细胞板。在37° c 的夜间, 用盖子将盘子孵育。</li>
	</ol>
</li>
	<li>
检索已转换的大肠杆菌的板.使用一个单一的殖民地接种3毫升磅 (1 升磅:10 克 bacto-胰, 10 克氯化钠, 5 克酵母提取物) 补充100微克/毫升氨苄西林在14毫升圆底聚丙烯培养管。孵育为〜8小时在37˚C 与 225 rpm 晃动。
	<ol>
<li>在34毫升锥形瓶中加入100微克/毫升氨苄西林和34微克/毫升氯霉素 (250 毫克/毫升的库存溶液), 添加3毫升的养殖50毫升磅。在37° c 的夜间孵育培养, 225 rpm 晃动。</li>
		<li>将53毫升的养殖转移到1升磅、100微克/毫升氨苄青霉素和34微克/毫升氯霉素在4升锥形瓶中。孵育在37° c 与225转每分钟摇晃。</li>
		<li>定期测量区域性的 600 nm (OD600) 的光学密度。最初, 检查每个 h 的区域性 OD600 。当区域性达到所需的 OD600时, 请检查每15分钟不满。将区域性扩展到0.9 和1.0 之间的 OD600 。将烧瓶转到冰浴和旋流中, 以降低养殖温度。</li>
	</ol>
</li>
	<li>
将1毫升的培养基转移到1.5 毫升的试管中, 然后进行变性 SDS-page 分析。
	<ol>
<li>将1.5 毫升管中的细胞在室温下离心1分钟, 在离心 1.4万 x g。丢弃上清液。重150μ l 磷酸缓冲盐 (PBS) 中的颗粒移。PBS 可以是有或没有 CaCl2和氯化镁2。</li>
		<li>添加150μ l 2x sds-页样品缓冲液 (150 毫米三盐酸, pH 6.8, 1.2% sds, 30% 甘油, 15% β-基 (βME), 0.0018% 溴蓝色), 并通过吸管或涡流彻底混合。在-20 ° c 下煮沸3分钟。</li>
	</ol>
</li>
	<li>添加到细菌培养: 最后浓度0.25 毫米异丙基β-d-thiogalactopyranoside (IPTG, 0.25 米的股票溶液) 和50μ m ZnCl2 (50 mM 股票解决方案)。IPTG 诱导表达的 PFV在基因从 T7 启动子和 ZnCl2是补充为正确折叠的一个氨基终端锌指域在 PFV. 孵育文化在25° c 以震动在 225 rpm 为 4 h. 立刻把烧瓶转到冰浴。保存1毫升的文化为 SDS-页分析遵循相同的议定书, 如以上1.3。</li>
	<li>
将细菌培养转化为适当大小的离心瓶。例如, 1 L 文化可以转移到四无菌一次性250毫升锥形底聚丙烯瓶。旋转的瓶子在一个冷藏桌面离心机在 3000 x g 20 分钟在4˚C。通过浇注丢弃清。
	<ol>
<li>重所有的颗粒从 1 L 细菌培养在总容量25毫升 (6.25 毫升每瓶) 冷 PBS (有或没有 CaCl2和氯化镁2) 由移。将细菌悬浮液与一个50毫升的锥形管结合并转移。旋转在一个冷冻离心机在 3000 x g 为20分钟在4° c。通过浇注或移丢弃上清液。</li>
	</ol>
</li>
	<li>重在10毫升悬浮缓冲 (50 毫米三盐酸, pH 7.5, 500 毫米氯化钠, 和500μ m phenylmethylsulfanoxide (PMSF) 蛋白酶抑制剂) 移的细菌颗粒。通过将试管放入带有液态氮气的杜瓦瓶中, 使其凝固, 足以淹没50毫升管。小心地将管子从液氮中取出, 贮存在-80 ° c。 注: 细菌颗粒可在-80 ° c 贮存数月。在-80 ° c 贮藏两年后, 我们没有发现活性蛋白的损失。</li>
	<li>
用8.3 厘米宽, 7.3 厘米高, 0.75 毫米厚的 SDS-页状凝胶, 1.5 毫升6% 聚丙烯酰胺堆叠层 (125 mm 三盐酸, pH 6.8, 6% 丙烯酰胺, 0.1% SDS, 过硫酸铵, 0.1% TEMED) 和 0.001% ml 3.5 分析预感应和后诱导样品聚丙烯酰胺解决层 (375 毫米三盐酸, pH 8.8, 10% 丙烯酰胺, 0.1% SDS, 过硫酸铵, 0.1% TEMED)0.001%。
	<ol>
<li>倒入堆叠凝胶后, 插入10井梳。当凝胶有聚合, 组装页设备, 并添加足够的 SDS 页运行缓冲 (25 毫米三, 192 毫米甘氨酸, 0.1% sds)。</li>
		<li>装载10μ l 每个样品和4μ l prestained 蛋白质标准。运行在16.5 伏/厘米, 直到染料前到达底部的凝胶, 约60分钟。</li>
		<li>拆开凝胶板, 将凝胶转移到塑料浴缸。添加马斯亮明亮的蓝色染色液 (0.1% 马斯亮亮蓝色 R-250, 40% 甲醇, 10% 乙酸) 慷慨地覆盖在室温下20分钟的凝胶和染色。</li>
		<li>通过浇注和更换 destain 溶液 (40% 甲醇, 10% 醋酸) 除去染色液, 直到带很容易看到。通过浇注和更换去离子水去除脱色溶液。PFV 在后诱导样品中应易于识别。PFV 与 hexahistidine 标签有一个分子量 47374 Da。</li>
		<li>如果 PFV 不可见, 则从一个变换板中的一个不同的菌落开始重复感应。</li>
	</ol>
</li>
</ol>2. 镍亲和层析
<ol><li>在冰上解冻一个细菌颗粒。 注意: 这将需要很大的时间 (约 2-3 小时)。当颗粒解冻到一个细胞浆, 添加一个额外的500μ m PMSF (100 毫米库存溶液)。</li>
	<li>将50毫升的细菌浆管放在冰上。几种的细菌在30% 振幅为三十年代 (1 s, 1 s 关闭) 每丸从 1 L 培养使用 sonicator 与0.5 英寸直径的生物喇叭与0.125 英寸直径锥形 mircotip, 频率20赫和最大功率 400 W。</li>
	<li>将细胞样品转移到冷 ultracentrifuge 管。使用聚碳酸酯瓶组件 (25 毫米直径, 89 毫米高度) 与 a 型60钛固定角转子兼容。如果达到相同的引力力, 可选用其它管和转子。旋转 12万 x g 为60分钟在4° c。应该有一个明显的颗粒。上清的颜色可能是黄色的。</li>
	<li>将上清液倒入或移到冷的50毫升锥形管上。注意音量;此卷应近似于在对齐冻结之前使用的悬浮缓冲区的体积。如果上清液粘稠, 可能会被细菌 DNA 污染, 从而堵塞色谱柱。在这种情况下, 重复离心, 以颗粒的细菌 DNA。</li>
	<li>
准备500毫升缓冲器 A (50 毫米三盐酸, ph 7.5, 500 毫米氯化钠, 500 μ m PMSF) 和500毫升缓冲 B (50 毫米三盐酸, ph 7.5, 500 毫米氯化钠, 500 μ m PMSF, 200 毫米咪唑)。 注意: 在本协议中, 将快速蛋白质液相色谱 (FPLC) 系统、所有缓冲器和样品保存在4° c 的冷室中。<ol>
<li>无菌过滤器缓冲器 A 和 B 与0.2 μ m 一次性过滤器单位。根据 FPLC 系统, 分别用缓冲器 a 和缓冲器 b 清洗泵 a 和泵 b。流90% 缓冲器 a 和10% 缓冲器 B 通过系统, 直到电导率和 UV 读数稳定。最大流速将取决于仪器;使用5毫升/分钟的流量, 最大压力限制为1.0 兆帕斯卡。</li>
	</ol>
</li>
	<li>准备一个110毫米长, 5 毫米直径 FPLC 柱与1.5 毫升镍充电树脂 (最大结合容量50毫克/毫升, 最大压力 1 MPa)。该列可在提纯前的前一天准备好, 贮存在4˚C。</li>
	<li>将列连接到 FPLC 仪器。平衡20毫升的90% 缓冲器 a 和10% 缓冲 B (20 mM 咪唑) 的流量为0.5 毫升/分钟, 最大压力极限为0.5 兆帕斯卡。该仪器应收集 280 nm (一个280) 的电导率和紫外线吸收的实时数据。电导率和 UV 读数应稳定。如果这些读数没有稳定, 继续通过该列的流动缓冲区, 直到它们是稳定的。</li>
	<li>
在0.15 毫升/分钟的流速下, 将蛋白质样本 (每粒10毫升) 加载到柱上, 最大压力极限为0.5 兆帕斯卡。在整个净化过程中, 流动速率和柱压力保持不变。收集在50毫升管的流量。
	<ol>
<li>用20毫升90% 缓冲器 a 和10% 缓冲液冲洗柱 b. 在50毫升的管子里收集洗涤。洗蛋白质与 20 mL 线性梯度10% 到100% 缓冲 B (20 毫米到200毫米咪唑)。</li>
		<li>最后, 用5毫升100% 缓冲 B (200 mM 咪唑) 清洗柱。在0.27 毫升分数中收集渐变和最终洗涤。
</li>
	</ol>
</li>
	<li>
结合15μ l 2x SDS-页样本缓冲区与15μ l 的流动, 洗涤, 并峰值一个280分数。将样品煮沸3分钟。
	<ol>
<li>准备两 10% SDS 页凝胶, 如前所述的步骤 1.6, 除了15井梳子。将每个样品的10μ l 载入凝胶和4μ l prestained 蛋白标准。如步骤1.6 所述, 运行、着色和分析凝胶。</li>
		<li>在视觉检查的基础上, 将似乎包含几乎纯 PFV 的分数组合在一起。通过移测量体积, 通常为 8-10 毫升。</li>
		<li>使用分光光度计测量组合分数的一个280 , 并计算蛋白质中 PFV 的总量;我们的典型产量是每升10毫克的诱导培养。PFV 的消光系数与 hexahistidine 标记为 59360 cm-1 M-1。</li>
	</ol>
</li>
	<li>要删除 hexahistidine 标签, 补充 PFV 在10毫米糖 (德勤, 1 米的股票解决方案) 和0.1 毫米 EDTA, pH 8.0 (0.5 米库存解决方案)。添加15微克的人鼻 (HRV) 3C 蛋白酶每毫克 PFV 在. 孵育在4˚C 过夜。从 47374 da 到 44394 da, 裂解减少了分子量的 PFV, 并可通过 10% SDS 页分析进行验证。</li>
</ol>3. 肝素亲和层析
<ol><li>
准备250毫升缓冲剂 C (50 毫米三盐酸, ph 7.5, 10 毫米的, 0.1 毫米 edta, 500 μ m PMSF) 和250毫升缓冲 D (50 毫米三 hcl, ph 7.5, 10 毫米的数字式, 0.1 毫米 edta, 500 μ m PMSF, 1 米氯化钠)。 注: 在本协议中, FPLC 系统、缓冲器和样品保存在4˚C 的冷室中。<ol>
<li>无菌过滤器缓冲 C 和 D 与0.2 μ m 一次性过滤器单位。根据 FPLC 系统, 分别清洗泵 a 和泵 B 与缓冲器 C 和缓冲器 D。流80% 缓冲器 C 和20% 缓冲器 D 通过系统, 直到电导率和 UV 读数稳定。最大流速将取决于仪器;我们经常使用的流量为5毫升/分钟, 最大压力限制为1.0 兆帕斯卡。</li>
	</ol>
</li>
	<li>
准备一个80毫米长, 5 毫米直径 FPLC 柱与1毫升肝素琼脂树脂 (最大结合能力2.0 毫克的牛抗凝血酶 III 每毫升树脂; 最大压力 1.4 MPa)。该柱可在提纯前的前一天准备好, 贮存在4° c。将列连接到 FPLC 仪器。
	<ol>
<li>平衡20毫升的80% 缓冲 C 和20% 缓冲 D (200 毫米氯化钠) 的流量为0.5 毫升/分钟, 最大压力限制为1兆帕斯卡。该仪器应收集 280 nm (一个280) 的电导率和紫外线吸收的实时数据。电导率和 UV 读数应稳定。如果这些读数没有稳定, 继续通过该列的流动缓冲区, 直到它们是稳定的。</li>
	</ol>
</li>
	<li>通过增加1.5 卷缓冲 C, 将 PFV 的氯化钠浓度降低到200毫米。例如, 在中添加15毫升的缓冲 C 到10毫升 PFV。我们的总容量是典型地 ~ 25 毫升。</li>
	<li>
将稀释后的 PFV 在样品中, 以0.5 毫升/分钟的流速, 以最大压力极限1.0 兆帕斯卡的速度载入肝素琼脂柱。在整个净化过程中, 流动速率和柱压力保持不变。收集在50毫升锥形管的流量。
	<ol>
<li>用20毫升80% 缓冲 C 和20% 缓冲 D (200 毫米氯化钠) 冲洗柱, 在50毫升锥形管中收集洗涤液。洗 30 mL 线性梯度20% 到100% 缓冲 D (200 毫米到 1 M 氯化钠) 的专栏。</li>
		<li>用5毫升100% 缓冲器清洗柱 d. 在0.37 毫升分数中收集梯度和最终洗涤。</li>
	</ol>
</li>
	<li>分析负载, 流经, 洗涤和分数 8% SDS 页, 如所述, 在2.9。PFV 在以下的 hexahistidine 标签的裂解有一个分子量的 44394 Da 和灭绝系数保持59360厘米-1 M-1没有 hexahistidine 标记。将所有分数存储在4˚C, 直到完成核酸检测。</li>
</ol>4. 核酸测定
<ol><li>
确定肝素琼脂分数, 似乎含有几乎纯 PFV. 结合3μ l 肝素分数和 50 ng 3 kb 超质粒 DNA 在反应缓冲 (10 毫米 HEPES, pH 7.5, 110 毫米氯化钠, 5 mm MgSO4, 4 μ m ZnCl2, 10 毫米的数码化), 最后的体积为15μ l。孵育在37˚C 为90分钟, 包括一个没有 PFV 的负控制。
	<ol>
<li>停止反应与1毫克/毫升蛋白酶 K (20 毫克/毫升的股票溶液) 和 0.5% SDS (10% 库存溶液)。孵育在37° c 为 1 h. 样品可贮存在-20 ° c 供以后分析。</li>
	</ol>
</li>
	<li>
准备一个125毫升凝胶1% 重量每体积 (瓦特/v) 琼脂糖在1x 泰式缓冲器 (40 毫米三醋酸盐, 1 毫米 EDTA) 与0.1 µg/毫升溴溴化物 (10 毫克/毫升库存解决方案)13。融化琼脂糖溶液, 倒入15厘米 x 10 cm 凝胶铸造托盘。插入一个15井梳子与5毫米宽, 0.75 毫米厚实的井。与1.5 毫米厚井相比, 薄井具有更好的带分辨率。允许凝胶在室温下凝固。
	<ol>
<li>用0.1 µg/毫升溴溴化液浸泡在1x 泰的凝胶中。加入3.5 μ l 6x 负载染料 (50% 甘油, 0.15% 橙 G 染料) 的核酸测定反应。将整个卷装入凝胶。在恒定电压下运行凝胶, 10 伏每 cm (100 V) 在环境温度为 1 h 或直到橙色 G 染料前面到达胶凝体的末端。</li>
	</ol>
</li>
	<li>
立即图像琼脂糖凝胶与荧光扫描仪设置检测溴溴化物。线性 dna 应该以 3 kb 的大小运行, 超的 dna 应该跑得更快 (〜 2 kb), 而松弛的圆 dna 应该运行得更慢 (〜 3.5 kb)。
	<ol>
<li>利用图像分析软件, 计算各车道超、线性、松弛圆质粒的像素体积。调用像素卷的总和为这三脱氧核糖核酸形成 "总脱氧核糖核酸" 价值并且使用它计算线性和宽松圈子的百分比。例如, 松弛圆的像素体积除以该车道中的总 DNA 像素值, 乘以这个数100来确定一个百分比。含有污染核酸的分数与负控制相比, 显示的线性和松弛圆的百分比更高。</li>
	</ol>
</li>
	<li>结合肝素琼脂分数, 不显示污染核酸活动。透析在10毫米 6-8 kDa 分子量截止 (分子) 透析管在4° c 过夜对1毫米的 50 L 的-HCl, pH 7.5, 200 毫米氯化钠, 50 μ m ZnCl 2, 10 毫米.</li>
	<li>从透析管中回收样品中的无核酸 PFV。测量一个280并计算最终的蛋白质浓度。液氮中的分和冻结。商店等分在-80 ° c。</li>
</ol>

<ol>
	<li>Lopez, M. A., Mackler, R. M., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Virol Methods. 235, 134-138 (2016).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nat Methods. 12 (10), 901-902 (2015).</li><li>Gunn, K. H., Marko, J. F., Mondragon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+single-molecule+experiment+reveals+multiple-attempt+dynamics+of+type+IA+topoisomerases.">An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.</a> Nat Struct Mol Biol. 24 (5), 484-490 (2017).</li><li>Valkov, E., 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=Functional+and+structural+characterization+of+the+integrase+from+the+prototype+foamy+virus.">Functional and structural characterization of the integrase from the prototype foamy virus.</a> Nucleic Acids Res. 37 (1), 243-255 (2009).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Li, M., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+viral+DNA+ends+channels+the+HIV-1+integration+reaction+to+concerted+integration.">Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.</a> J Biol Chem. 280 (32), 29334-29339 (2005).</li><li>Li, M., Mizuuchi, M., Burke, T. R., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+reaction+pathway+and+critical+intermediates.">Retroviral DNA integration: reaction pathway and critical intermediates.</a> EMBO J. 25 (6), 1295-1304 (2006).</li><li>Sinha, S., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+immunodeficiency+virus+type+1+integrase+exhibits+a+capacity+for+full-site+integration+in+vitro+that+is+comparable+to+that+of+purified+preintegration+complexes+from+virus-infected+cells.">Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.</a> J Virol. 79 (13), 8208-8216 (2005).</li><li>Goodarzi, G., Im, G. J., Brackmann, K., Grandgenett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+integration+of+retrovirus-like+DNA+by+human+immunodeficiency+virus+type+1+integrase.">Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.</a> J Virol. 69 (10), 6090-6097 (1995).</li><li>Vora, A. C., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+catalytic+properties+of+retrovirus+integrase-DNA+complexes+capable+of+efficiently+performing+concerted+integration.">Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.</a> J Virol. 69 (12), 7483-7488 (1995).</li><li>Jones, N. D., 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nat Commun. 7, 11409 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li></ol>

作者没有什么可透露的。

与 dna 相互作用的重组蛋白, 如 dna 修复蛋白, 单分子显微镜应用的氧清除剂, 或逆转录病毒 integrases, 应无污染细菌核酸2,3。这些污染物可能会混淆的解释结果在散装生物化学或单一分子化验。
我们发现, 细菌核酸经常与 PFV 一起净化。然而, PFV 在显示一种亲和力的肝素琼脂, 是容易区分的细菌核酸污染物1。制备无核酸 PFV 的关键是对肝素琼脂梯度洗脱后各馏分中核酸活性的仔细定量。梯度必须足够浅, 使 PFV 的分离从核酸污染物;陡峭的梯度与有效的分离不相容。此外, 质粒 DNA 基质应具有较低的松弛圈百分比, 以减少核酸的背景。如果质粒 dna 基质具有很高的松弛圈背景, 则应进行质粒 dna 的新纯化。核酸活性测定揭示了污染的核酸和应丢弃的分数的存在。
在某些情况下, 大小排除色谱 (SEC) 可能是去除污染核酸2的有效方法。在4中, 以前的组在纯化 PFV 时使用了 SEC。SEC 的负载量有限, 并倾向于降低最终的蛋白质浓度。亲和性和离子交换色谱的主要优点是能够比 SEC 加载更大的容量。
对核酸活性的测试可以适应不同的蛋白质, 与细菌核酸的联合纯化。在这里, 我们发现, PFV 和污染核酸可能被分离的肝素琼脂色谱。我们以前已经表明, PFV 在核酸具有类似的剖面与单 S 阳离子和单 Q 阴离子交换色谱, 使这些树脂不适合这种蛋白质。根据所需蛋白质与核酸的亲和特性, 本协议可用于亲和或离子交换树脂和梯度洗脱。有效去除蛋白质中的核酸污染的关键是树脂亲和性的差异。每个分数必须被检测为核酸活动。本议定书的色谱树脂不能直接应用于所有与细菌核酸共纯化的蛋白质。然而, 使用亲和剂或离子交换树脂将核酸从感兴趣的蛋白质中分离出来, 并对核酸活性的成分进行测定的总体概念可以广泛适用。

蛋白质与 DNA 相互作用的生物化学和单分子研究需要格外纯的重组蛋白。污染核酸的细菌可以掩盖这些化验结果。从大肠杆菌(大肠杆菌) 中分离出的重组蛋白氧清除 protocatechuate-3,4-dioxygenase (PCD) 和原型泡沫病毒 (PFV) 整合 (in) 的制备中发现了污染核酸1,2,3。
逆转录病毒集成检测依赖于超 DNA 到有缺口或线性产品的转换, 作为活动的4。在细胞感染期间, 将病毒 cDNA 的两端连接到宿主染色质5。每个端接反应称为链转移。重组活性的测定可以加入两个 dna 齐聚物, 模仿病毒 cDNA 结束到目标 dna 在一致的集成反应中4,5,6,7,8。二者择一地重组在5月加入仅一个脱氧核糖核酸末端在一个非生理相关的半站点综合反应9,10。当超质粒 dna 是整合的目标时, 协同集成产品是线性化 dna 和半站点集成产品的放松圈。这些反应产物通过其相对流动性在琼脂糖凝胶电泳1。如果重组中有一个污染的核酸, 将有假放松圈或可能线性化的质粒混淆了实验结果。病毒 DNA 寡聚物可能被荧光标记, 以最终确定集成产品, 而不是核酸产品。然而, 在很大程度上有利于超的 DNA 靶点;任何超质粒通过污染核酸而导致的松弛圆或线性 DNA 的损失都可能扭曲数据11的结果和解释。因此, 在制剂中清除逆转录病毒的细菌核酸是势在必行的。
PFV 对肝素琼脂有不同的亲和力, 与细菌核酸1比较。PFV 和核酸可由肝素琼脂的线性梯度洗脱分离。核酸在 280 nm 峰值或通过分析十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 的紫外线 (UV) 吸收后不易检测到。相反, 核酸是通过核酸活性测定检测的, 采用超质粒转化为松弛的圆或线性产品。肝素琼脂色谱法测定核酸活性。PFV 和核酸污染物对单 Q 阴离子树脂亲和性无影响。单 S 阳离子树脂亲和性有很小的差异。然而, 细菌核酸和 PFV 的单 S 分辨率不会允许有效分离蛋白质。最终, 肝素琼脂亲和纯化提供了最好的分离细菌核酸从 PFV 在和具有无限的优势的负荷量。
对污染核酸活性的检测可以适应其他蛋白质。兴趣的蛋白质可能有其他的亲和力特征比 PFV 在;有兴趣的蛋白质和核酸污染物的结合特征的区别必须是经验主义地确定的。这种识别核酸污染的方法可适用于其他树脂, 包括单 S 阳离子或单 Q 阴离子交换树脂。亲和性和离子交换树脂可以提供一种可靠的方法来分离核酸污染的重组蛋白, 而不限制色谱中蛋白质的体积。

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detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

逆转录病毒原型 (PFV) 中的整合蛋白是研究反转录酶的机制。与其他病毒相比, PFV 在更易于溶解, 更适于实验操作。此外, 它是敏感的临床相关的人体免疫缺陷病毒 (HIV-1) 的抑制剂, 表明 PFV 的催化机制类似于 HIV-1 在. 在催化病毒互补 DNA (cDNA) 共价加入靶DNA 在一个称为链转移的过程中。这股转移反应引入了缺口的目标 DNA。综合反应产品的分析可以被混淆由核酸的存在相似地尼克脱氧核糖核酸。在大肠杆菌(大肠杆菌) 中, 一种细菌核酸已被证明与重组 PFV 联合纯化.在这里, 我们描述了一种方法, 以隔离 PFV 从污染核酸的肝素亲和层析。通过超质粒和琼脂糖凝胶电泳, 可以很容易地筛选出分数核酸污染。PFV 在和污染的核酸显示替代亲和力的肝素琼脂允许核酸自由制备重组 PFV 适合散装生物化学或单分子分析一体化。

重组 PFV 中经常被细菌核酸1污染。生化积分测定取决于超质粒 DNA 转化为松弛圆和线性产品的定量。污染核酸的存在可能导致这些化验的虚假定量。在大肠杆菌(图 1) 中, 首先用镍亲和色谱 (图 2) 对 PFV 的表达式进行了 hexahistidine 标记。与几乎纯 PFV 的分数相结合, hexahistidine 标记由蛋白酶劈开。我们已经确定, PFV 和污染核酸有不同的亲和力为肝素琼脂色谱 (图 3)1。PFV 在肝素琼脂色谱后, 用超质粒对松弛圆和线性化质粒进行测定, 核酸活性的产物 (图 4)。此分析允许在没有核酸的情况下识别蛋白质组分 (图 5)。这些分数可以结合, 透析, 并冻结未来的实验。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56605/56605fig1.jpg"> 图 1:在大肠杆菌中 PFV 的感应。细胞从文化在之前 (车道 2) 和以后 (车道 3) 归纳被分析为存在 PFV 在, 47374 Da. 样品用 10% SDS 页隔开, 用马斯亮鲜艳的蓝色染色。1车道, 分子量标记 (kDa)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56605/56605fig2.jpg"> 图 2:镍亲和色谱分数.用镍亲和层析法分离可溶性细菌裂解。PFV 与一个 hexahistidine 标记是洗与线性梯度20毫米到200毫米咪唑。分数由 10% SDS 页, 染色与马斯亮灿烂的蓝色。PFV 在, 47374 Da, 是容易地明显的在样品装载了到专栏 (装载), 但是不明显的在流动通过或洗涤。洗早期在咪唑梯度中的分数通常显示较慢的移动污染物近 75 kDa, 以及一些更快的移动污染物在或低于 25 kDa。在较高浓度的咪唑, 没有明显的污染物和 PFV 似乎是相对纯净。在这个例子中, 分数 #33 通过 #84 被结合和进一步纯化。分子量标记 (kDa) 在每个胶凝体的左侧。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56605/56605fig3.jpg"> 图 3: 肝素琼脂色谱 PFV 在1 。hexahistidine 标记的镍亲和性和蛋白酶去除后, PFV (44394 Da) 由肝素琼脂。蛋白质是洗与线性梯度从200毫米到 1 M 氯化钠。即使分数 #12 到 #44 被评价为 8% SDS 页染色马斯亮蓝色。分子量标记 (kDa) 在每个胶凝体的左侧。这些分数被测试为核酸活动。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56605/56605fig4.jpg"> 图 4:肝素琼脂分数的核酸测定1 。每个肝素琼脂分数被添加到超质粒 DNA 中。分数编号与图 3中所示的数字相关联。在孵化后, 核酸反应去, 用1% 琼脂糖与溴溴化物分离。阴性控制包括质粒 DNA 没有蛋白质 (仅目标) 和早先纯化狂放的类型 PFV 在已知无核酸 (PFV 在重量)。一个积极的控制, 污染核酸活动是以前的净化催化不活泼 PFV 在这是已知的污染核酸 (PFV 在 D128N)。DNA 大小标记以 kb 显示。超质粒 (SC)、线性质粒 (L) 和松弛圆 (RC) 被表明。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56605/56605fig5.jpg"> 图 5: 核酸化验的定量1. 琼脂糖凝胶扫描和像素值是量化的超 (SC), 线性 (L), 和放松圈 (RC) 在每条车道。小数与图3和4中所示的数字相关联。用像素值和所示方程计算了线性和松弛圆的百分比。例如, 分数 #12 带的像素值为: 817636 超质粒、50467线性质粒和112052松弛圆。分数 #12 的总 DNA 像素值是这些值的总和, 980155。该百分率的线性 dna 是50467乘以100的百分比和除以总 dna 值等于5.1%。松弛圆的百分比是112052乘以 100, 除以总的 DNA 值等于11.4%。线性和松弛圆百分比的总和是16.5%。阴性对照质粒 dna 无蛋白 (no, 底图) 表明, 这种制备的质粒包括4.4% 的总 DNA 为线性或松弛的圈子。第二个负控制是以前的野生类型 PFV 的纯化 (重量), 显示8.6% 的总 DNA 为线性或宽松的圈子。这两个控制车道表明的背景水平的缺口或线性化质粒目前与基板单独和与 PFV in。阳性对照是先前纯化的催化非活性 PFV (D128N), 其中有23% 的总 DNA 为线性和松弛圆。dna 暴露在肝素琼脂分数 #12 通过 #22 导致 > 10% 的 dna 作为线性和宽松的圈子。在本例中, 分数 #24 #42 显示 < 10% 转换为线性和宽松的圆圈。这些分数是结合和透析。等分被冷冻并贮存在-80 ° c 以备将来使用。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

重组原型泡沫病毒整合蛋白经常被细菌核酸在纯化过程中污染。这种方法识别核酸污染, 并将其从酶的最终制备中除去。

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

detection-removal-nuclease-contamination-during-purification

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.0K 次观看.  The Ohio State University College of Medicine. 重组原型泡沫病毒整合蛋白经常被细菌核酸在纯化过程中污染。这种方法识别核酸污染, 并将其从酶的最终制备中除去。

视频: Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...