笔者想感谢所有成员的SSGCID财团。所有团队成员的努力，在翡翠生物的巨大成就SSGCID的目标成为可能。资助这项研究是根据联邦的合同号HHSN272200700057C从国立过敏和传染病研究所，国立健康和卫生与人类服务部。

在图1中，提出的协议的概述。 分子生物学 1。构造设计使用基因Composer软件的设计蛋白质结构和密码子设计合成的基因序列。基因作曲软件的使用已经提供详细3。 <ol><li>使用对齐查看器模块和构建蛋白质序列进行比较，并确定蛋白质结构设计模块。对准目标的氨基酸序列的两个主要和在蛋白质数据库（PDB）的同系物的三维结构元件，如果有的话（ 图2）。 </li><li>使用对应的信息，使通过选择保护的一级结构和同系物的三维结构的基础上的新的末端结构导向的结构设计。 </li><li>设计插入PCR（IPCR）和矢量PCR（VPCR）的扩增引物（终端的引物）。 </li><li>使用基因Composer的蛋白质-DNA算法，背构建的氨基酸序列翻译成密码子设计的核酸序列。 </li><li>使用正确的密码子使用表（CUT），以优化在大肠杆菌中的表达序列大肠杆菌 。 </li><li>实际上，克隆插入pET28载体修改，将N-端的6个组氨酸标签和Smt3/SUMO融合蛋白，易于纯化允许。 </li><li>将合成的基因顺序与DNA 2.0集成DNA技术的引物和秩序。 </li></ol> 2。聚合酶不完整的引物延伸（PIPE）的克隆<ol><li>准备引物和基因<ol><li>含有引物，1分钟，以1,000 rpm离心供应商提供的板。 </li><li>将引物浓度为100μM，并加入50μl的TE缓冲液中。 </li><li>稀释的引物，以10μM去离子（DI）水，在96孔V形底板。 </li><li>在1,300 rpm的转速下，持续1分钟，在1.5毫升管中离心供应商提供的基因。 </li><li&gt;使用TE缓冲液中，使每个管中的DNA浓度为50纳克/微升。 </li><li>在1.5ml管中，使稀释液的每种引物，至10毫微克/微升。 </li><li>在-20℃下在不使用时的存储区的引物和基因。 </li></ol></li><li>准备插入PCR（IPCR） <ol><li>解冻PFU主一小瓶冰混合，在室温下保持基因和引物。 </li><li>创建一个板的地图分配井的一组引物和构造。 </li><li> 13微升去离子水加入到一个96孔PCR板的各孔中。 </li><li>添加5μl的正向和反向引物的5微升在96 - 孔板的每一个反应板图，以确保彼此之间以及改变提示。 </li><li>每个全长基因中加入2μl适当根据板图。 </li><li>加入25微升的PFU预混，确保每口井每口井之间变更的提示。 </li><li>循环反应，使用下列的PCR反应条件： <ol class="lalpha"><li> 95℃2分钟</LI&gt; <li> 95℃30秒</li><li> 50℃45秒</li><li> 68℃3分钟</li><li> 4℃∞ </li></ol></li><li>重复步骤BD为25个周期。 </li><li>取10μl的每个iPCR的反应的一个新的96孔PCR板。 </li><li>将3微升的6X负载染料每个样品。 </li><li>分隔每个样品在1％TAE乙锭琼脂糖凝胶在110 V到100-500个基点的DNA阶梯旁，确认片段扩增。 </li><li>商店iPCR的产品在不使用时，在-20°C（避免冻融尽可能）。 </li></ol></li></ol> 3。准备矢量PCR（VPCR） <ol><li>启动过夜培养转化E.大肠杆菌载体质粒pET28。 <ol><li>两个5毫升管2-YT肉汤接种50微克/毫升卡那霉素。 </li><li>成长过夜培养物在37℃下摇床，以220rpm的转速。 </li></ol></li><li>自旋向下培养过夜生长后，在3,000 rpm下离心15分钟。 </li><li>使用Qiagen公司的QIA准备Spin Miniprep试剂盒提取pET28载体，按照制造商的说明，从细菌粒料。 </li><li>设置限制性内切酶消化提取pET28质粒。 <ol><li> 2.2微升10X的BamHI缓冲液和1μlBamHI和HindIII加入20μl的pET28载体。 </li><li>孵育反应1小时，在37°C。 </li></ol></li><li>单独的消化产物在凝胶上。 <ol><li>请参阅加强2.2.10。 </li><li>带从凝胶中切割的载体和纯化，使用QIAquick凝胶提取试剂盒，按照制造商的说明。 </li></ol></li><li>使用的NanoDrop，量化DNA浓度。 </li><li>稀切向量至10毫微克/微升。储存在-20℃下在不使用时。 </li><li>准备的VPCR底漆。 <ol><li> 1分钟在1300转离心IDT提供oligonucliotides的。 </li><li>把用DI水浓度为100μM。 </li><li>准备10μM稀释1.5毫升管的正向和反向引物。 </li><li>存储区的引物和引物的稀释液于-20℃下</li></ol></li><li>解冻PFU主冰和解冻的模板和引物在室温下混合。 </li><li>设置的VPCR反应在96孔PCR板： <ol><li>在第一行中的96 - 孔板结合VPCR正向和反向引物和60微升24微升消化的pET28模板（10毫微克/微升）。 </li><li>使用12头多道移液器，12μl引物和模板预混转移到其余的每个板。这会导致在板的各孔中的引物和模板的预混液12微升。 </li><li> 13微升去离子水加入到各孔中。 </li><li>添加25微升的PFU主混合到每口井。 </li></ol></li><li>循环通过PCR反应条件下，使用在步骤2.2.7。 </li><li>所有的VPCR到15毫升猎鹰管反应池。 </li><li>验证分离10微升池的PCR产物在凝胶上（pET28矢量消化的预期长度的扩增片段约6 KB）。 <ol><li>请参阅加强2.2.10。 </li></ol></li><li>准备合并板材。 <ol><li> VPCR的产品的等分试样3微升到在96孔V形底板的各孔中。 </li><li>存储板在-20°C，直到合并iPCR的产品。 </li></ol></li></ol> 4。合并iPCR和VPCR产品<ol><li>解冻的iPCR的产品和预先分装的VPCR 96孔板，在室温下合并。 </li><li>加入3微升每个iPCR的产品其各自以及合并板。 </li><li>转换板合并成十大化学感受细胞。 </li><li>每个合并反应成一个单一的50微升管供应商提供的化学感受细胞中加入2μl，并与制造商提供的协议继续进行。 </li><li>准备过夜培养每个构造转化平板。 </li><li>等分试样5 ml TB肉汤中（50微克/毫升卡那霉素）从25毫升无菌储层到每口井的深井块。 </li><li>使用免缝勒技术，挑一个孤立的殖民地从每个转换板和接种适当的井深井块。 </li><li>量块一个Airpore盖。 </li><li>摇块以220rpm的转速，于37℃培养过夜。 </li><li>沉淀细胞，经离心分离30分钟，每分钟4000转的块。 </li><li>倒掉上清液，轻拍干用纸巾块的顶部。 </li><li>小量制备使用Qiagen公司的96孔真空装置根据制造商的指示。 </li></ol> 5。准备甘油股票成功克隆的构建<ol><li>变换成功地克隆序列验证到BL21（DE3）化学感受态细胞中的DNA，根据制造商的指示。 </li><li>对于每一个构建体中，挑取单个从BL21（DE3）转化和接种到1ml的2-YT肉汤（50微克/毫升卡那霉素）中的单菌落。 </li><li>摇动培养3-4小时，在220 rpm下在37℃下</li><li>标签1.5毫升螺丝帽管具有独特的构造的识别号码，细胞株和日期。添加500μl的50％甘油和500微升细胞培养颠倒几次。立即干冰或在-80℃冷冻储存甘油股票。 </li></ol> 6。表达式测试<table border="1"><tbody><tr><td> 裂解缓冲 </td><td> 洗涤液 </td><td> 洗脱缓冲 </td></tr><tr><td>的50mM的NaH 2 PO 4，pH 8.0的 300 mM氯化钠 10 mM咪唑 1％吐温20的 2毫米氯化镁2 0.1微升/毫升的BENZONASE 1毫克/毫升溶菌酶</td><td>的50mM的NaH 2 PO 4，pH 8.0的 300 mM氯化钠 20 mM咪唑 0.05％吐温20的</td><td> 25毫摩尔Tris，pH值8.0 300 mM氯化钠 250 mM咪唑 0.05％吐温20的</td></tr></tbody></table> *的添加BENZONASE，溶菌酶，和蛋白酶抑制剂之前裂解。 <ol><li>从甘油库存数的样品到卡那霉素选择性琼脂孵育过夜，在37°C。 </li><li>在一个96孔圆底块，接种1.2毫升TB肉汤（含50毫克/毫升卡那霉素），补充了0.5％葡萄糖，用新鲜生长的大肠杆菌启动非诱导的预培养大肠杆菌分离。一夜之间增长在37℃，220rpm振荡</li><li>过夜培养后，开始诱导培养接种TB发酵液的1.2毫升（50毫克/毫升卡那霉素）补充Novagen公司隔夜Express系统1（根据制造商的协议）与预培养40微升。 </li><li>种植小规模诱导培养48小时，在20℃，220rpm振荡。 </li><li>收获细胞在4000转离心15分钟，倒出上清液，并储存于-20°C加工前至少1小时。 </li><li>在96孔的块，加入300μl裂解缓冲液中重悬细胞沉淀。 </lI&gt; <li>细胞裂解缓冲液中，在室温下孵育30分钟，然后剧烈振摇30分钟，在室温下的机械裂解。 </li><li>澄清的粗裂解液在4℃下以4000 rpm离心30分钟</li><li>使用一个多通道的移液管转移到96孔平底托盘（Qiagen公司）的澄清的裂解液200微升（可溶组分）。对于每个孔含有一个示例中，加入40微升的Ni-NTA亲和磁性珠（Qiagen公司）。 </li><li>轻轻搅拌1小时摇臂板在16°C。 </li><li>将磁板后板（QIAGEN），除去未结合的可溶部分。照顾没有吸取任何的Ni-NTA珠。 </li><li>拆下该板，后板，轻轻地悬浮在200μL洗涤缓冲液珠。移液器上下，持续30秒，然后将板后板。 </li><li>取出洗净缓冲区，并重复步骤6.12。 </li><li>删除板后板和洗脱的Ni-NTA势必TA通过用50μl洗脱缓冲液中5分钟洗涤rget蛋白。 </li><li>结果平底板磁性后板，并转移到一个新的96孔V形底板洗脱。 </li><li> 20微升的洗脱转移到一个新的96孔V形底板上，并和反应1微升ULP1蛋白酶。 </li><li>根据制造商的协议，分析洗脱，洗脱+ ULP1馏分毛细管电泳使用芯片实验室90。 </li><li>另外，所有零碎的表达测试，可以通过SDS-PAGE分析。 </li></ol> 7。大规模发酵<ol><li>使用无菌枪头从甘油股票获得一刮，接种100毫升结核病肉汤（50毫克/毫升卡那霉素）和成长过夜。摇在220 rpm和37°C </li><li>过夜培养后，展开接种结核病肉汤1升，的EMD自动感应解决方案（参见制造商的协议）（50毫克/毫升卡那霉素）在一个2 L莫名其妙的烧瓶用10毫升的预培养预培养（1:100稀释）。 </li><li>摇动扩大1升培养在37℃下摇床的温度改变到第20℃下0.6的光密度（OD 600）时，就达到了。 </li><li>过夜培养后，取有代表性的10毫升，从每个构造表达测试。 </li><li>收获细胞贴5000转离心15分钟，弃上清。 </li><li>冷冻细胞糊状物于-80℃。 </li></ol>蛋白质纯化缓冲区： <table border="1"><tbody><tr><td> 裂解缓冲液 </td><td> 缓冲液A（平衡） </td><td> 缓冲液B（洗脱） </td><td> 浆纱柱缓冲液 </td></tr><tr><td> 25毫摩尔Tris，pH值8.0 200 mM氯化钠 0.5％的甘油 0.02％CHAPS 10 mM咪唑 1毫米TCEP 50毫米精氨酸 5微升BENZONASE 百米克溶菌酶 3蛋白酶抑制剂片（不含EDTA） </td><td> 25毫摩尔Tris，pH值8.0 200 mM氯化钠 10 mM咪唑 1毫米TCEP 50毫米精氨酸 0.25％甘油</td><td> 25毫摩尔Tris，pH值8.0 200 mM氯化钠 200 mM咪唑 1毫米TCEP </td><td> 25毫摩尔Tris，pH值8.0 200 mM氯化钠 1％的甘油 1毫米TCEP </td></tr></tbody></table> *添加BENZONASE，溶菌酶，蛋白酶抑制剂药片立即松解术前每150毫升样品。 8。细胞裂解<ol><li> 2 L的裂解缓冲液中，不添加溶菌酶，蛋白酶抑制剂片剂或BENZONASE（每个采样将分别在150毫升的裂解缓冲液裂解）。 </li><li>粘贴在解冻和重悬细胞裂解液以1:5质量：体积比剧烈搅拌30分钟，在4°C。打破块松动双方烧杯中，用一个干净的锅铲。在这段时间内，制备Ni和DialySIS缓冲器</li><li>在冰，裂解细胞，使用一个Misonix的超声发生器（70％的电力，2秒/ 1秒脉冲​​3分钟），轻轻摇动容器，以防止过热。一个小的（200微升）原油裂解等分保存，供日后分析。 </li><li>澄清原油裂解18,000 XG离心35分钟，在4°C，收集上清液，并节省一笔不小（200微升）取供日后分析。商店颗粒在4℃下，直到它被证实蛋白质已被裂解成可溶部分。 </li></ol> 9。蛋白质厂商设置预运行<ol><li>与蛋白质制造商打开软件打开，初始化仪器。 </li><li>初始化完成后，将每个样品的单独一行，龙门1 5.0毫升GE医疗HisTrap FF镍螯合物柱（NI列）。 </li><li>通过每列运行的3-4倍柱体积（CV）的平衡缓冲液。 </li><li>素平衡和洗脱缓冲线。 </li><li> Equilibr吃了吸液通过一次列列。 </li></ol> 10。镍1（无）列<ol><li>每一列用20ml Milli-Q水洗涤，以除去存储缓冲器。运行5毫升缓冲液B，25毫升缓冲液A平衡。 </li><li>含溶解的蛋白质的一列，在2毫升/分钟的速率加载澄清的溶胞产物，然后按照所用缓冲液A洗涤一个15毫升的</li><li>与缓冲器中的分步梯度洗脱结合的蛋白A和B恭敬地由下面的比率：5毫升95:5 60:40，5毫升，10毫升0:100。收集各洗脱组分分开。 </li><li>分析：洗脱级分，得到粗提液，澄清的溶胞产物和流量通过用SDS-PAGE。普尔馏分含有蛋白质和使用的NanoDrop测量A 280来大致确定存在的蛋白质的量的。 </li></ol> 11。 ULP1裂解<ol><li>保持一个小等份（250微升）的的NI1列池随后的凝胶分析。把其余的的NI1池至10ml，加在1微升/ 5毫克总蛋白的泛素蛋白酶样蛋白酶1（ULP1）取出的SMT亲和标签。 </li><li> 2升4小时的缓冲液A透析妮池+ ULP1对10 kDa的分子量截止（截留分子量）的轰动板在4℃，在4°C。 </li><li>透析后，运行的SDS-PAGE NI1游泳池和NI1的游泳池+ ULP1确定如果ULP1裂解成功。 </li></ol> 12。 2镍（镍）列<ol><li>装入裂解蛋白相同的镍柱，并重复步骤9.3在1毫升/分钟的流量减少。裂断标记绑定到列，无标签的目标蛋白现在将流过。在一个新的容器收集流过。 </li><li>镍柱洗净，用3毫升缓冲液A后5毫升的缓冲液B洗脱他所有的标签和非特异性结合的蛋白质。收集每个部分分开。 </li><li>运行的SDS-PAGE镍流过，清洗和镍洗脱组分，验证ULP1裂解和公关的流量通过otein本。使用的NanoDrop测量280来大致确定蛋白质的存在。 </li></ol> 13。集中<ol><li>浓缩镍流通过（和Ni2洗脱蛋白是存在）至5ml的Amicon Ultra 10 kDa的截留分子量的离心管中。在4℃下在10分钟的时间间隔，在每分钟4000转的自旋混合用吸管每个自旋之间，防止蛋白质过度集中沿膜。 </li></ol> 14。尺寸排除色谱（SEC） <ol><li>设置在Sephacryl S-100 10/30 GL用200ml SEC缓冲液平衡，流速为0.5毫升/分钟的AKTApurifier系统（GE Healthcare公司）柱（GE Healthcare）。 </li><li>准备10毫升superloops上使用SEC柱根据制造商的指示。 </li><li>用5毫升注射器，装入样品到superloops，开始向SEC运行。 </li><li>在280 nm处的紫外吸光度跟踪监控，同时收集体积小FR动作。 </li><li>执行美国证券交易委员会通过SDS-PAGE分数。 </li><li>游泳池SEC分数强度最高的乐队。 </li><li>浓缩汇集SEC分数。请参阅步骤13.1。 </li><li>到100微升的样品，闪光冻结在液氮和存储在-80°C等分蛋白</li></ol>结晶 15。蛋白质结晶<ol><li>预先填充在96孔紧凑Jr的结晶板（绿宝石生物技术）每个库与80μl的结晶画面（绿宝石生物技术）给出。 </li><li>稀释蛋白质大小的缓冲区，以2-20毫克/毫升，储存在冰。 </li><li>免除0.4微升的蛋白质和0.4微升的结晶屏幕，并覆盖到每96孔清澈封箱胶带（曼乔）。 </li><li>板存放在16°C，而在未来的几个星期定期检查蛋白质结晶在解剖显微镜下。 </li></ol> 16。水晶收获&lt;/ P&gt; <ol><li>创建从母液中冷冻保护剂和乙二醇。切以及与目标蛋白质晶体的透明胶带覆盖。到一个空井，相应的结晶化条件下加入1.6微升，和0.4微升的1,2 - 亚乙基二醇得到的终浓度为20％的乙二醇和80％的结晶条件。注意：优化晶体衍射尝试不同的冷冻保护剂，如：甘油，油，低分子量的聚乙二醇，和/或在不同的冷冻保护剂的百分比。 </li><li>收获前盖上盖子用液氮杜瓦瓶充满ALS风格冰球降温。 </li><li>收获的晶体放置冷冻环的内径相匹配的尺寸的晶体上的磁性晶体棒（汉普顿研究），它直接从井溶液中舀。 </li><li>立即冷冻环蘸收获晶体的冷冻保护剂，然后淹没在ALS的风格冰球闪烁冻结的crystal。重复所需数量的晶体。 </li></ol> 17。筛选晶体/数据采集<ol><li>一旦收获是完全用冰球棒放置磁性冷冻ALS冰球冰球盖的。随着弯曲钳，翻转冰球倒挂。 </li><li>冰球转移到一个理学演员的杜瓦瓶，拧顽童推到冰球，冲关盖子把它留在杜瓦用大头针正视。 </li><li>屏幕使用JDirector软件，每个晶体根据以下参数：光束狭缝设置为0.5度，检测器设置到50毫米的距离，以70度的图像的步骤，和曝光的长度设置为30秒。 </li><li>运行你拍摄JDirector的决定什么是最好的晶体和策略是数据收集测试图像MOSFLM。 </li><li>收集完整的数据集的基础上从MOSFLM结果。 </li></ol> 18。数据处理/结构测定<ol><li>运行XDS / XSCALE 4的到处理的数据集。 </li><li>打开CCP4套件软件。 <ol><li>运行移相器5使用很高的同源性搜索模式，当计算出分子的替代解决方案。在这种情况下，我们使用的PDBID 3CW4为搜索模型6。 </li><li>运行REFMAC 7，完善的分子模型对数据集收集所观察到的反射。最终解决应根据最高外壳由以下参数：R因子&gt; 50％，I /西格玛&gt; 2和完整性，&gt; 90％。 </li></ol></li><li>构建3维电子密度模型与分子图形软件COOT的8。 </li><li>沉积在PDB结构之前验证9 MolProbity软件验证，质量，结构，适合沉积。 </li></ol>

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D. 66, 125-132 (2010).</li><li>McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phaser+crystallographic+software.">Phaser crystallographic software.</a> Appl. Cryst. 40, 658-674 (2007).</li><li>Kuzuhara, T., Kise, 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=Structural+basis+of+the+influenza+A+virus+RNA+polymerase+PB2+RNA-binding+domain+containing+the+pathogenicity-determinant+lysine+627+residue.">Structural basis of the influenza A virus RNA polymerase PB2 RNA-binding domain containing the pathogenicity-determinant lysine 627 residue.</a> J. Biol. Chem. 284, 6855-6860 (2009).</li><li>Murshudov, G. N., Skub&#224;k, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., Vagin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REFMAC5+for+the+refinement+of+macromolecular+crystal+structures.">REFMAC5 for the refinement of macromolecular crystal structures.</a> Acta Cryst. D. 67, 355-367 (2011).</li><li>Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+classical+density+modification.">Recent developments in classical density modification.</a> Acta Cryst. D. 66, 470-478 (2010).</li><li>Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., Richardson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MolProbity:+all-atom+structure+validation+for+macromolecular+crystallography.">MolProbity: all-atom structure validation for macromolecular crystallography.</a> Acta Cryst. D. 66, 12-21 (2010).</li><li>Mossessova, E., Lima, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ulp1-SUMO+crystal+structure+and+genetic+analysis+reveal+conserved+interactions+and+a+regulatory+element+essential+for+cell+growth+in+yeast.">Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.</a> Mol. Cell. 5, 865-876 (2000).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Cryst. D. 66, 486-501 (2010).</li><li>Raymond, A. C., Haffner, T. E., Ng, N., Lorimer, D., Staker, B. L., Stewart, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+design,+cloning+and+protein-expression+methods+for+high-value+targets+at+the+Seattle+Structural+Genomics+Center+for+Infectious+Disease.">Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease.</a> Acta Cryst. F. 67, 992-997 (2011).</li><li>Smith, E. R., Begley, D. W., Anderson, V., Raymond, A. C., Haffner, T. E., Robinson, J. I., Edwards, T. E., Duncan, N., Gerdts, C. J., Mixon, M. B., Nollert, P., Staker, B. L., Stewart, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protein+Maker:+an+automated+system+for+high-throughput+parallel+purification.">The Protein Maker: an automated system for high-throughput parallel purification.</a> Acta Cryst. F. 67, 1015-1021 (2011).</li></ol>

绿宝生物公司的员工

多目标并行处理的基于结构的药物设计在药物发现中起着重要的作用。 SSGCID致力于提供科学界与蛋白质三维结构从NIAID的类别为AC病原体。制作这种结构广泛地提供信息，最终将有助于加快基于结构的药物设计。 MTPP方法的第一个关键步骤是结构设计。每一个目标蛋白质的多重结构，增加成功的结构测定和增加周转的概率。这是不可避免的，一些蛋白质结构的管道阶段将失败。实施管道克隆方法支持MTPP方法允许在96孔板中没有劳动密集型的纯化步骤一代的许多构造。配对管克隆与蛋白表达的分析能力，在同一96孔板（卡尺实验室芯片90）进一步加快的总体流程。这些方法允许配对的结构，产生可溶性蛋白，确保大规模蛋白质生产和纯化的成功，可快速识别。 MTPP高通量的成功的一个重要方面是蛋白质制造商（美国专利第6818060号，翡翠生物）仪表。蛋白质的制造商是一个24通道平行液相色谱系统专门开发的高通量蛋白的生产和相关的结构基因组管道研究中的应用，以提高效率。使用先前描述的协议，用于蛋白设备单行FPLC系统相比，其优点是明显的。一个单个的人能净化并联多达48个目标，在8小时内。与此相反，一个人使用一个单一的线FPLC系统只能净化的和最多4个目标，在相同的时间内。高含量的纯度为每个目标实现与蛋白质制造商日益增长的蛋白质晶体结构分析在后来的成功是一个关键因素。 限制和故障排除求解三维结构，可通过X-射线晶体是一个多级着许多挑战，其中之一是不能获得大量的可溶性靶蛋白的努力。克服的溶解度问题，可以实现的一种策略是使用的另一种表达宿主为E。大肠杆菌细胞无法执行几个重要的真核翻译后修饰。在各酵母中的表达，昆虫和哺乳动物细胞系的能够执行这些翻译后修饰的往往是一个合适的替代品。有时靶蛋白表达，但完全不溶于标准的裂解条件下。蛋白质Maker可以替代细胞裂解条件下的快速检测是一种宝贵的资源Smith 等人所描述的。 2011 13。这一战略往往是必要的，以保持通过管道移动的目标。在任何结构基因组学研究的管道，标准化的协议可能不适合每一个目标，通过管道和目标，可能需要单独的优化。例如，我们选择使用20％的乙二醇为每个冷冻保护剂。替代冷冻保护剂或浓度的情况下，这种情况是不适合的，可能需要进行测试。 由于每个单独的靶蛋白的独特性质，速率限制和不可预知的步骤中确定的结构是结晶。 MTPP管道偏移通常成功率很低的蛋白质结晶优化从最初的稀疏矩阵屏幕。每个初始晶体市售稀疏矩阵屏幕击中进一步优化电子屏幕生成器（翡翠生物）。优化屏幕设计的ARound的条件的初始晶体命中的，改变浓度的缓冲液，盐，和添加剂。成功的优化屏幕产生适合衍射研究和结构测定的晶体。 提出由美国国家过敏和传染病研究所（NIAID）的结构基因组学计划提供资金绿宝生物和其他三个谁一起是SSGCID的（绿宝生物SeattleBiomed，华盛顿大学和西北太平洋国家实验室的西北太平洋机构） 。 NIAID的结构基因组计划的完成目标所需的国家的最先进的应用技术，每个成员的财团被选定为他们的专业知识。日期，SSGCID已交存461结构到PDB排名为世界第七大贡献者，并在2011年，最有生产力的。提供的协议和方法SSGCID的意图受益科学界和延续传染病的研究。

<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Vendor</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Primers</td>
 <td>IDT</td>
 <td></td>
 </tr>
 <tr>
 <td>Genes</td>
 <td>DNA 2.0</td>
 <td></td>
 </tr>
 <tr>
 <td>TE buffer</td>
 <td>Qiagen</td>
 <td>provided in kit</td>
 </tr>
 <tr>
 <td>96-well half skirt PCR plates</td>
 <td>VWR</td>
 <td>10011-248</td>
 </tr>
 <tr>
 <td>PFU Master Mix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>6X Orange Loading Dye</td>
 <td>Fermentas</td>
 <td>R0631</td>
 </tr>
 <tr>
 <td>10X TAE</td>
 <td>Teknova</td>
 <td>T0280</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Sigma-Aldrich</td>
 <td>A9414-10G</td>
 </tr>
 <tr>
 <td>pET28 vector</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>2-YT Broth</td>
 <td>VWR</td>
 <td>101446-848</td>
 </tr>
 <tr>
 <td>Kanamycin</td>
 <td>Teknova</td>
 <td>K2151 </td>
 </tr>
 <tr>
 <td>Restriction Enzymes</td>
 <td>Fermentas</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick Gel Extraction Kit </td>
 <td>Qiagen</td>
 <td>28704</td>
 </tr>
 <tr>
 <td>Top 10 chemically comp cells</td>
 <td>Invitrogen</td>
 <td>C4040-06</td>
 </tr>
 <tr>
 <td>Disposable Troughs (Sterile, 25 ml)</td>
 <td>VWR</td>
 <td>89094-662</td>
 </tr>
 <tr>
 <td>Airpore covers (Rayon films for bio cultures)</td>
 <td>VWR</td>
 <td>60941-086</td>
 </tr>
 <tr>
 <td>24-well blocks</td>
 <td>VWR</td>
 <td>13503-188</td>
 </tr>
 <tr>
 <td>QIAvac 96</td>
 <td>Qiagen</td>
 <td>19504</td>
 </tr>
 <tr>
 <td>BL21(DE3) cells chemcomp (phageR)</td>
 <td>NEB</td>
 <td>C2527H</td>
 </tr>
 <tr>
 <td>50% Glycerol</td>
 <td>VWR</td>
 <td>100217-622</td>
 </tr>
 <tr>
 <td>TB Media</td>
 <td>Teknova</td>
 <td>T7060</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td></td>
 </tr>
 <tr>
 <td>1 M Tris pH 8.0</td>
 <td>Mediatech</td>
 <td>46-031-CM</td>
 </tr>
 <tr>
 <td>5 M NaCl</td>
 <td>Teknova</td>
 <td>S0251</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Aldrich</td>
 <td>G7893-4L</td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>JT Baker</td>
 <td>4145-01</td>
 </tr>
 <tr>
 <td>Imidazole</td>
 <td>Sigma</td>
 <td>56749-1KG</td>
 </tr>
 <tr>
 <td>TCEP</td>
 <td>Amresco</td>
 <td>K831-10G</td>
 </tr>
 <tr>
 <td>L-arginine</td>
 <td>Amresco</td>
 <td>0877-500G</td>
 </tr>
 <tr>
 <td>Benzonase</td>
 <td>EMD</td>
 <td>70746-3</td>
 </tr>
 <tr>
 <td>Lysozyme</td>
 <td>USB</td>
 <td>1864525GM</td>
 </tr>
 <tr>
 <td>10 kDa MWCO dialysis tubing</td>
 <td>Thermo</td>
 <td>68100</td>
 </tr>
 <tr>
 <td>Amicon Ultra 10 ka MWCO concentrators</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>HisTrap FF columns</td>
 <td>GE</td>
 <td>17-5255-01</td>
 </tr>
 <tr>
 <td>HiTrap Chelating columns</td>
 <td>GE</td>
 <td>17/0408-01</td>
 </tr>
 <tr>
 <td>Compact Jr crystallization plates </td>
 <td>Emerald Bio</td>
 <td>EBS-XJR</td>
 </tr>
 <tr>
 <td>Crystalization screens</td>
 <td>Emerald Bio</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene Glycol 100%</td>
 <td>Emerald Bio</td>
 <td>EBS-250-EGLY</td>
 </tr>
 <tr>
 <td>Crystal Wand Magnetic Straight </td>
 <td>Hampton Research</td>
 <td>HR4-729</td>
 </tr>
 <tr>
 <td>Mounted CryoLoop 0.1-0.2 mm</td>
 <td>Hampton Research</td>
 <td>HR4-955</td>
 </tr>
 <tr>
 <td>ALS style puck</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Puck Wand</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Bent Tongs</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Puck Pusher</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

multi-target parallel processing approach for gene-to-structure determination of the influenza polymerase pb2 subunit

大流行暴发的高致病性流感病毒株能引起广泛的发病率和死亡率在全球人群。仅在美国，平均41,400人死亡，186万人住院，是由流感病毒感染引起的，每年1。点突变在聚合酶碱性蛋白亚基（PB2）已与适应的病毒感染在人类2。从这些研究结果揭示的生物学意义PB2作为一个致病因素，从而突出它的潜力作为一种抗病毒药物的目标。 提出由美国国家过敏和传染病研究所（NIAID）的结构基因组学计划提供资金绿宝生物和其他三个西北太平洋机构一起组成了西雅图的结构基因组学中心传染病（SSGCID）。致力于提供科学界三个SSGCID维E蛋白结构NIAID类别AC病原体的。这样的结构信息提供给科学界提供加速基于结构的药物设计。 基于结构的药物设计在药物发展中起着重要的作用。并行追求多个目标，目标的途径或整个蛋白家族的新先导化合物的发现成功的机会大大增加。翡翠生物已开发出一种高通量，多目标并行处理流水线（MTPP）基因到结构的决心，以支持该财团。在这里，我们描述了协议，用于确定从四个不同的A型流感毒株PB2亚基的结构。

的结果如下示出预期的结果，所描述的协议和PB2，所观察到的结果的情况下。 用Gene作曲者，，五个完整长度的目标的流感病毒聚合酶PB2亚单位的氨基酸序列的设计（ 图2）。 PB2序列翻译并受到许多工程步骤3，导致密码子优化的统一的序列在大肠杆菌中的表达成功地克隆到大肠杆菌从iPCR的产品（ 图3b），一共有34构造的变形pET28载体系统10，用6倍的N-末端-SMT使用PIPE克隆3，如在图3a所示的融合标签。克隆的工作流程的概要如图4所示。 克隆成功后，每个结构的微观尺度的蛋白表达在BL21（DE3） 大肠杆菌测试大肠杆菌细胞。细胞生长在补充Novagen公司隔夜快递1介质（根据制造商的协议）的TB培养基摇床中设定在220rpm的转速在20℃下48小时。生长后，收获细胞，并测试可溶性蛋白表达，用毛细管电泳用钳芯片实验室90。 14 34 PB2导致可溶性靶蛋白的结构和进入大规模发酵。每个构造大规模的培养物生长在Novagen公司的隔夜快1个中等大小的TB培养基中，根据制造商的协议。生长后，通过离心收获细胞，并储存于-80℃。大规模蛋白质的表达，每一种文化被证实通过SDS-PAGE分析（ 图5），然后再进行大规模纯化。 使用蛋白设备进行14 PB2构造的并行纯化。人澄清的裂解液升14个结构运行通过螯合镍列。确定哪些馏分含有目标蛋白通过SDS-PAGE后，相应的级分对每个样品的组分和各浓度由A 280读数确定。除去的6倍他SMT的标签进行过夜透析和第二镍柱ULP1此外。通过SDS-PAGE（ 图6）进行确认的SMT标签去除，每个样品用10 kDa的的Amicon超离心管浓缩。浓度的Amicon超离心管后，碾过每个样品一个上浆列，实现晶体纯度。第二个浓度进行结晶所必需的电平，以增加产品的蛋白质浓度。所有14个构造成功纯化，并订立结晶试验。 结晶开始解冻先前FROZEN蛋白。结晶在16°C在一个气候控制室内进行专门设计的板（翡翠生物）坐滴气相扩散（ 图7）。四个稀疏矩阵屏幕进行初步筛选; JCSG +条约，精灵全，CryoFull（翡翠生物），扩展纽曼战略。从相应的水库使用96以及紧凑型的JR结晶板（翡翠生物）的crystallant（或水库解决方案）用0.4微升0.4微升的蛋白质溶液，然后混合。 900的十四个纯化的样品产生的晶体，适合于衍射研究（ 图8）。 5结晶在CuKα射线的波长，使用在Rigaku超亮FR-E +旋转阳极X-射线发生器配备锇VARIMAX高频光学系统和一个土星944 +的CCD检测器9的结构（ 图9上的一个在内部衍射数据集收集）。每个数据集XDS / XSCALE 4的加工&lt;<html> / SUP&gt;缩放导致最后的解决。从的CCP4套房7与移相器 5进行尝试解决结构分子置换。细化REFMAC 7和手动重建与库特 11后，获得最终的模型。的结构进行了评估和几何纠正和健身与MolProbity 9 PB2亚基共四个结构（ 图10），并存入PDB 图11说明MTPP管道在每个阶段的整体结果。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/4225/4225fig1highres.jpg" src="/files/ftp_upload/4225/4225fig1.jpg" /> 图1。概述的SSGCID多目标并行处理翡翠生物的基因结构途径。 内容“的FO：保持together.within”=“总是”&gt; <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/4225/4225fig2highres.jpg" src="/files/ftp_upload/4225/4225fig2.jpg" /> 图2。对准Viewer和蛋白质构建基因Composer软件设计模块。氨基酸基地建设的目标是显示绿色（中间窗口）和黄金（底部窗口）所示结构制导截断替代结构，对齐多个流感病毒PB2序列相比，序列和二级结构元素从PDBID 3CW4的C-末端结构域。域的知识结构和二级结构元素使N-末端截短基因作曲设计模块内选择所需的氨基酸残基上按一下滑鼠右键， <a href="http://www.jove.com/files/ftp_upload/4225/4225fig2large.jpg" target="_blank">点击这里查看大图</a> 。 图3a。其中，所述的合成基因插入物（橙色）设计（橙红色）和反向（橙色蓝色线）的引物，以产生插入PCR材料被放大，示出管克隆的表达载体扩增的反向（红-黑线）和远期（蓝黑色线）引物来生成矢量PCR材料。终端序列iPCR的产品是互补的终端序列的VPCR产品（红色的iPCR补充红色的iPCR的VPCR和蓝色补充蓝色VPCR）。这允许的iPCR和VPCR产品进行退火，以形成主机化学感受态大肠杆菌 BL21（DE3）转化成时，被复制的质粒大肠杆菌细胞。 <img alt="图3b" src="/files/ftp_upload/4225/4225fig3b.jpg" /> 图3b。琼脂糖凝胶电泳分析的iPCR生产<html> iPCR的产品成功为代表的强大的乐队，而PB2亚基TS。的iPCR的失败可能被看作是微弱或油污带。 ，iPCR的产品质量一般可以与克隆成功。分子量标记物在千道尔顿。图转载自雷蒙德等人，2011年12。 <img alt="图4" src="/files/ftp_upload/4225/4225fig4.jpg" /> 图4。目标PB2蛋白基因工程步骤进行，用Gene作曲软件工程的核酸序列，为每个目标建立后，6-7的其他蛋白质的构建体设计的每一个。 34的结构，其中14个是可行的目标，产生可溶性蛋白在大肠杆菌导致多目标并行处理设计和克隆基因的初始步骤大肠杆菌 。 <html>再5“SRC =”/ files/ftp_upload/4225/4225fig5.jpg“/&gt; 图5。代表性的SDS-PAGE分析显示出强大的蛋白表达（预期的大小为25.76 kDa），溶于约50％（第4泳道）及约50％的6倍的His-SMT标签裂解洗脱蛋白（泳道7）大规模发酵。 <img alt="图6" src="/files/ftp_upload/4225/4225fig6.jpg" /> 图6。三个结构的聚合酶PB2亚基的SDS-PAGE结果，泳道1：分子量标记（标记为在左侧以kDa）;泳道2，6，10，汇集蛋白从镍1列中;泳道3，第7， 11，流量通过裂解缓冲液A从镍2蛋白泳道4，8，12，6倍他SMT贴片在缓冲区B从镍2去除。 d/4225/4225fig7.jpg“/&gt; 图7。坐滴法蒸汽扩散示意图。蛋白质结晶坐滴法属于类蒸汽扩散。此方法需要纯化的样品的蛋白质和沉淀含有相似的条件下在较高的浓度具有较大的储层平衡。由于水的蒸发，从蛋白质样品和传输到储存器，沉淀剂浓度增加蛋白质结晶的最佳水平。 <img alt="图8" src="/files/ftp_upload/4225/4225fig8.jpg" /> 图8。从流感病毒株的聚合酶PB2亚单位的蛋白质晶体。 <img alt="图9" src="/files/ftp_upload/4225/4225fig9.jpg" /> 图9所示。 X-射线衍射图像的聚合酶PB2亚单位从应变流感病毒。 <img alt="图10" fo:content-width="4in" fo:src="/files/ftp_upload/4225/4225fig10highres.jpg" src="/files/ftp_upload/4225/4225fig10.jpg" /> 图10。发带的图分子在晶体不对称单位中的4 PB2结构。色在彩虹图案与对应的PDB代码的二级结构（一）3K2V（A/Yokohama/2017/2003/H3N2）（二）3KHW（A /墨西哥/ InDRE4487/2009/H1N1）（）3KC6“（A/Vietnam/1203/2004/H5N1）（D）3L56（A/Vietnam/1203/2004/H5N1）。 <img alt="图11" fo:content-width="6in" fo:src="/files/ftp_upload/4225/4225fig11highres.jpg" src="/files/ftp_upload/4225/4225fig11.jpg" /> 图11。该结构的结果分析流感PB2所述的方法的目标。ê测定管道中所示的五个步骤：克隆，溶解度，纯化，结晶和结构测定。

Watch this Scientific Journal Video about Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit at JoVE.com

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

基于结构的药物设计在药物发展中起着重要的作用。并行追求多个目标，率先发现的成功的机会大大增加。下面的文章强调了西雅图结构基因组学中心传染病流感A亚基PB2基因到结构的确定采用了多目标的方法。

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

multi-target-parallel-processing-approach-for-gene-to-structure

Research

JoVE Journal

Immunology and Infection

13.0K 次观看.  Emerald Bio. 基于结构的药物设计在药物发展中起着重要的作用。并行追求多个目标，率先发现的成功的机会大大增加。下面的文章强调了西雅图结构基因组学中心传染病流感A亚基PB2基因到结构的确定采用了多目标的方法。

视频: Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...