博士。 CJP Grimmelikhuijzen，从丹麦哥本哈根大学（丹麦）和迈克尔威廉姆森赞赏提供aequorin质粒。我们的合作者，罗纳德J博士Nachman ARS，USDA（美国德克萨斯州），是公认的肽合成和提供NOVOstar酶标仪。

1。建立稳定的细胞系<ol><li>克隆GPCR的利益，并把它插入到一个表达载体，结合各地的起始密码子在哺乳动物系统的最优核糖体结合“（科扎克，1986）5&#39;科扎克一致序列（GCCA / GCCATGG）。在这里，我们使用的蚊子激肽受体 （Holmes等，2003; Pietrantonio等，2005）。打勾激肽受体的质粒pcDNA3.1/Bm-KR，质粒pcDNA3.1/Aedae-KR。的pcDNA3.1（ - ）向量编码氨苄青霉素抗性选择在哺乳动物细胞中的细菌和新霉素（G418）电阻的选择。 </li><li>成长的CHO - K1空单元格（不含质粒）（ATCC，弗吉尼亚州马纳萨斯，美国）或其他所需的细胞株中的T - 25烧瓶（BD猎鹰）在37 ° C（在5％ 二氧化碳培养箱Holmes 等 ，2003）。除另有指明外，所有进一步孵化，在这些情况下应该做的。保持生长介质中的空细胞（10％胎牛血清的F12K培养基）1X抗生素Antimycotic（Invitrogen公司，CA）。 </li><li>要拆分单元格，温暖了所有的解决方案，以37 ° C删除旧的T - 25烧瓶中，用5 mL PBS冲洗，然后取出PBS。 trypsinize细胞，加2毫升PBS胰蛋白酶- EDTA（34毫升PBS，2毫升7.5％的碳酸氢钠，4毫升10X胰蛋白酶EDTA）2分钟。加入3毫升培养基和抽吸介质向上和向下混合细胞。传输介质与细胞成一个锥形管和离心2分钟〜200-300克（1000转）。弃上清，在5毫升培养基重新悬浮细胞。稀释悬浮细胞，以1:5或1:10的新鲜培养液的浓度，并转移在一个新的T - 25烧瓶5毫升。 </li><li>后细胞茁壮成长（2-3天左右），种子成的T - 25的组织培养瓶中的CHO - K1细胞和不含抗生素的增长它们在生长培养基过夜，直到他们在30％左右汇合（约18小时）。可通过倒置荧光显微镜下观察细胞的汇合程度。 </li><li>对每一个样品的准备下面的混合物： <ol><li>结合1-2微克DNA（例如：使用265μg/μlpcDNA3.1/Aedae-KR4μl）100μLOPTI - MEM我降低血清培养基（Invitrogen公司）。 </li><li>混入100μLF12K无血清培养6μL脂质体试剂（InvitrogenTM），比例为1:1的脂质体的DNA。在室温下孵育30-45分钟。 </li></ol>轻轻从1.5.1混合的解决方案，在下拉明智的时尚（总量= 200μL）1.5.2。让我们在室温下静置10-15分钟。 </li><li>从细胞中移除旧的生长介质，并用5毫升F12K无血清培养的细胞，然后取出F12K无血清培养。在15毫升管，轻轻混匀到1.8毫升新鲜F12K培养基转染混合物在下拉明智的时尚。然后，从步用PBS 1.5洗涤细胞，并添加新的细胞转染的解决方案。孵育18小时。 </li><li>更改介质F12K不含抗生素中加10％胎牛血清和孵育过夜。分割成两个T - 25烧瓶F12K培养基，加不加抗生素，另外18小时10％胎牛血清（分裂的细胞，请参见步骤1.3）的细胞。 </li><li>更换介质的选择性培养基（F12K培养基加上10％胎牛血清，800μg/mlGENETICIN Invitrogen公司）。使用3-4周的选择性培养基对培养细胞。随着时间的推移，这将选择有稳定纳入到他们的基因组DNA质粒的细胞。继续保持细胞维持液（F12K培养基加上10％的胎儿与GENETICIN400μg/ml牛血清）。定期冻结冻结媒体（20％二甲基亚砜的选择性培养基）以1:1的比例，以防止意外污染的情况下失去的细胞株。 </li><li>选择克隆的细胞株：作为第一步，trypsinize和离心机在步骤1.3中的细胞维持液1.8步。重新悬浮细胞，在维持液5毫升，0.5毫升的细胞暂停，并添加4.5毫升新鲜的维持液作10倍稀释。转移到96孔板，12口井的10倍稀释，每孔加入100μL选择单细胞。 </li><li>继续使这些细胞的10倍连续稀释100μL的每一个细胞（最终稀释度通常将在10 -11 10 -19;总人数的10倍连续稀释 〜19）的最后一个理论悬挂。紧随每个稀释度，转移到96孔板，12口井100μL的稀释。经过18小时的潜伏期，观察下一个倒光或荧光显微镜和标记井，似乎只包含一个单细胞，或包含，显然是从一个单细胞分成两个细胞的96孔板。保持每三天观察每天和改变介质。 </li><li>当井是80％confluENT（约一周），与200μLPBS冲洗标记的水井和trypsinize与100μLPBS胰蛋白酶- EDTA溶液。转移到一个6孔板，用1 ml维持液标记每个细胞。这些细胞成长为3天，然后转移到个人的T25烧瓶细胞。测试这些细胞使用钙激动剂肽（请参阅第2节）生物发光检测。 </li><li>选择1细胞中的钙的生物发光板检测的最高响应1.11步骤线，并执行第二次单细胞选择以下步骤1.9-1.11。定期冻结的细胞株。 </li><li>从步骤1.12所述的二次选择，选择2-3个细胞系中的钙的生物发光板检测的最高响应，并保持他们的文化，为进一步钙的生物发光板检测。跟踪通过号码。不时，从早期的通道冻结的细胞系，这样你可以随时回去给他们，如果有更多的通道细胞系一贯停止执行。 </li></ol> 2。钙的生物发光板检测<ol><li>结扎成记者感兴趣的基因的表达载体。在这里，我们使用aequorin质粒mtAEQ/pcDNA1（从博士。CJP Grimmelikhuijzen和丹麦哥本哈根大学的迈克尔威廉姆森，礼物）。变换在大肠杆菌中的质粒大肠杆菌细胞MC1061/PS（Invitrogen公司），净化他们使用QIAprep自旋小量制备试剂盒（QIAGEN公司） 。在最后一步洗脱无EDTA的，没有水用Tris缓冲质粒。 </li><li>种植步骤1.13细胞株，表达所需的受体在维持液。当细胞90％汇合，trypsinize，离心机，然后再暂停在步骤1.3维持液5毫升的细胞。显微镜下细胞计数（亮线Hemacytometer）稀释细胞（约维持液的10倍），计数细胞数。调整细胞数约为2 × 10 5细胞/毫升（平均20个细胞，在9个方格中的一个显示在Hemacytometer）。种子2毫升稀释细胞在媒体到六孔板的每个孔。孵育24小时（细胞应达到约60％汇合后孵化）。 </li><li>更改OPTI - MEM培养基中6井板的媒体。每口井，混合96μLOPTI - MEM 4μLFuGENE 6转染试剂（罗氏生物化学）在离心管中，并让在室温下坐了5分钟。添加到每管1微克aequorin / pcDNA 1质粒DNA，然后轻轻摇动1分钟的样品，在室温孵育15-20分钟。在滴加方式添加每个混合物，同时轻轻摇晃孔板每孔。 6小时孵育板和改变F12K无抗生素的培养基中含有10％胎牛血清的介质。 </li><li>在6孔板中的细胞24小时后孵化，trypsinize，离心和重新悬浮细胞作为第1.3步。计数的细胞数40万细胞/毫升步骤2.1，并转移到每孔96孔白色薄底部微孔板（中光学3610）100μL（共40000 cells/100μL）。孵育24小时，直到大约80％的细胞汇合。这是生物发光检测细胞的最佳浓度。 </li><li>钙的DMEM媒体在黑暗中含有5微米腔肠（Invitrogen公司）（Invitrogen）的准备（腔肠90μl/well是光敏感）。从2.4板，删除旧的媒体，并添加到每孔90μl。孵育3小时在黑暗中板在37 ° C和5％的CO 2，之后，在板的细胞准备进行测试。 </li></ol> 3。仪器操作和数据分析<ol><li>每个生物发光酶标仪是不同的。我们执行我们的实验使用NOVOstar（BMG Labtechnologies）bioluminensence模式酶标仪。如果您使用不同的仪器，你必须适应的协议。 </li><li>在使用前清除酶标仪泵（或总理泵）。板固定板之前关闭在房间里的光线。一旦该板块的持有人已关闭，关闭的灯。 </li><li>肽（在1.5 mL Eppendorf管）溶解在无钙DMEM媒体。设置“吸深度”和“定位”，使用前肽的解决方案。挑战10μL不同的肽浓度（FFFSWG - NH2，白，K1 - 3，或其他所需的肽）（10倍）的细胞，并立即开始录制的光发射。我们已建立记录每口井，每2秒的总时间为50秒的光发射（465 nm）的仪器。 </li><li>确保包括阳性对照，如积极的模拟（模拟FFFSWGa已被使用，塔内加- 巴盖什沃尔等，2009）和向量只转染的细胞，如阴性对照。阴性对照将在数据分析的必要设置基线的阈值（见代表性的结果）。一个不相关的，非活性肽也可以增加一条，作为阴性对照。 </li><li>运行完成后，洗净的仪器泵（或总理泵），然后把你的下一个肽样品。保存您的数据，并再次冲洗泵。 </li><li>数据处理：光发射的数据传输到Microsoft Excel数据表，从每口井。 </li><li>粘贴的数据从Excel Prism软件GraphPad软件公司（美国加利福尼亚州圣迭戈，）4.0。各肽浓度是X轴和生物发光单位是Y轴的。要正常化的数据，绘制日志响应曲线。选择一个非线性回归曲线拟合的分析（S形的剂量 - 反应方程可变斜率），以获取每个肽的浓度反应曲线。该方案地块最终的价值和让欧共体50。 </li><li>每次实验应重复进行数据分析的3倍。 </li></ol> 4。代表性的成果： 蚊子埃及伊蚊激肽受体的CHO - K1细胞中表达时，表现为multiligand受体和功能三个endogenours白激肽，激肽1-3 Aedae为1 nm的低浓度，测试单独​​使用钙的生物发光板检测。图1显示了效力的排名顺序获得Aedae - K - 3&gt; Aedae - K - 2&gt; Aedae - K - 1，根据各自的 EC 50值Aedae - K - 3，16.04 NM; Aedae - K - 2，26.6 nm和Aedae - K - 1，48.85纳米，有统计学显着性差异（P &lt;0.05）（Pietrantonio 等 ，2005）。 我们也采用了这种检测，以确定哪些肽残留量激肽的肽受体相互作用的关键。昆虫肽肽C端五肽，又称为核心的生物活性，所需的最小序列。表1，激肽的肽核心类似物合成的核心肽五肽FFSWGa丙氨酸更换系列和钙的生物发光板检测（塔内加-巴盖什沃尔等，2006）测试。我们发现，1苯丙氨酸和色氨酸的氨基酸，两种受体的昆虫激肽的活性至关重要。 该试剂盒还可以用来测试加强生物稳定性设计的肽。表2显示含有氨基异丁酸（AIB）蜱重组激肽受体和图2测试设计的肽类似物，显示上打勾肽钙CHO - K1细胞表达的受体的6个α-氨基异丁酸类似物的活性比较生物发光板法（塔内加-巴盖什沃尔等 ，2009）。六肽模拟FFFSWGa添加为受体活性的阳性对照。模拟FF [AIB] WGA导致比这六肽控制模拟较为活跃。两个aminoisobutyric酸的替代品，[AIB] DT [AIB] WGA，模拟测试（表2和图2）的双取代类似物的最有力的。 这个实验已可应用于见Nachman和Pietantonio（2010），Nachman 等更多的例子。 （2009年），塔内加-巴盖什沃尔等 。 （2008A），塔内加-巴盖什沃尔等 。 （2008B）。 <img src="/files/ftp_upload/2732/2732fig1.jpg" alt="图1"/> 图1。估计白激肽CHO - K1钙的生物发光板检测E10细胞的有效浓度（EC 50）。浓度反应曲线的Y轴是从每个观察到的最大反应的百分比表示的生物发光单位肽。数据点代表平均在3次独立实验获得六个复制。酒吧代表标准错误。 （一）估计Aedae - K1 EC 50 = 48纳米。 （二）估计Aedae - K2 EC 50 = 26纳米。 （三）估计Aedae - K3 EC 50 = 16纳米。欧共体50 Aedae - K3 &lt;欧共体50 Aedae - K2 &lt;欧共体50 Aedae - K1，P &lt;0.05。 GraphPad棱镜4.0软件统计分析和图形。 <img src="/files/ftp_upload/2732/2732fig2.jpg" alt="图2"/> 图2。蜱激肽受体表达CHO - K1细胞钙的生物发光板检测的6个α-氨基异丁酸类似物的活性比较。Y轴代表观察到的生物发光的浓度百分比表示每个模拟％ ，最大的生物发光单位与观察每个模拟测试的所有浓度之间的最大响应。 GraphPad棱镜4.0软件进行统计分析和图形。 <table border="1"><tbody><tr><td></td><td colspan="2">勾选受体细胞系</td><td colspan="2">蚊子受体细胞系</td></TR&gt; <tr><td>肽</td><td>欧共体50（NM） </td><td > 1毫米的最大的生物发光反应</td><td>欧共体50（NM） </td><td> 1毫米的最大的生物发光反应</td></tr><tr><td> AFSWGa </td><td>我</td><td>我</td><td>我</td><td>我</td></tr><tr><td> FASWGa </td><td> 586 </td><td> 5600 </td><td> ND </td><td> 400 </td></tr><tr><td> FFAWGa </td><td> 64 </td><td> 12800 </td><td> 621 </td><td> 3050 </td></tr><tr><td> FFSAGa </td><td>我</td><td>我</td><td>我</td><td>我</td></tr><tr><td> FFSWAa </td><td> 417 </td><td> 10600 </td><td> 2800 </td><td> 1830 </td></tr><tr><td> FFSWGa </td><td> 590 </td><td> 10800 </td><td> ND </td><td> 525 </td></tr><tr><td> FSWGa </td><td>我</td><td>我</td><td>我</td><td>我</td></tr><tr><td> FFSWa </td><td>我</td><td>我</td><td>我</td><td>我</td></tr><tr><td> FFSWG - OH </td><td>我</td><td>我</td><td>我</td><td>我</td></tr><tr><td> FFFSWGa </td><td> 259 </td><td> 13000 </td><td> 562 </td><td>万</td></tr><tr><td> FF [AIB] WGA </td><td> 29 </td><td> 12700 </td><td> 445 </td><td> 9300 </td></tr></tbody></table> 表1。估计的效力（EC 50）和最大的生物发光反应，蜱及蚊虫受体转染细胞系的所有测试肽*. *欧共体50估计诱导半最大反应所需的浓度。我：无效，如果生物发光反应是少于300个单位（矢量只转染的细胞水平）。答：肽FFSWGa各自的残留物已被丙氨酸取代的位置。 ND：模拟测试，但要么不十分活跃或不活跃，在较低molarities，从而不能确定的EC50。 <table border="1"><tbody><tr><td> K - AIB - 1 </td><td> [AIB] DT [AIB] WGA </td></tr><tr><td> K - AIB - 2 </td><td> [αMEF] DT [AIB] WGA </td></tr><tr><td> K - AIB - 3 </td><td> AC - R [AIB] DT [AIB] WGA </td></tr><tr><td> K - AIB - 4 </td><td> AC - R [β3F] DT [AIB] WGA </td></tr><tr><td> K - AIB - 5 </td><td> [AIB] RFF [AIB] WGA </td></tr><tr><td> K - AIB - 6 </td><td> [AIB - AIB - AIB - AIB] RFF [AIB] WGA </td></tr></tbody></table> 表2。肽类似物（K）的含有氨基异丁酸（AIB）测试上打勾重组激肽受体。 AC：乙酰;α我：α甲基-苯丙氨酸;β3F：β3-苯丙氨酸;一个：酰胺。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+insect+kinin+analogs+with+cis-peptide+bond,+type+VI-turn+motifs+identifies+optimal+stereochemistry+for+interaction+with+a+recombinant+arthropod+kinin+receptor+from+the+southern+cattle+tick+Boophilus+microplus.">Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.</a> Peptides. 29, 295-301 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Williams, H., Reyes-Rangel, G., Juaristi, E., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+selective+and+non-selective,+biostable+beta-amino+acid+agonists+of+recombinant+insect+kinin+receptors+from+the+southern+cattle+tick+Boophilus+microplus+and+mosquito+Aedes+aegypti.">Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti.</a> Peptides. 29, 302-309 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structure-activity+analysis+of+insect+kinin+core+analogs+on+recombinant+kinin+receptors+drom+southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+and+mosquito+Aedes+aegypti+(Diptera:+Culicidae).">Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors drom southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae).</a> Archives of Insect Biochemistry and Physiology. 62, 128-140 (2006).</li><li>Pietrantonio, P. V., Jagge, C., Taneja-Bageshwar, S., Nachman, R. J., Barhoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+a+multiligand+receptor+for+the+three+Aedes+kinins.">The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.</a> Insect Molecular Biology. 14, 55-67 (2005).</li><li>Holmes, S. P., Barhoumi, R., Nachman, R. J., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+a+G+protein-coupled+receptor+from+the+Southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+identifies+it+as+the+first+arthropod+myokinin+receptor.">Functional analysis of a G protein-coupled receptor from the Southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.</a> Insect Molecular Biology. 12, 27-38 (2003).</li><li>Staubli, F., Jorgensen, T. J. D., Cazzamali, G., Williamson, M., Lenz, C., Sondergaard, L., Roepstorff, P., Grimmelikhuijzen, C. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+the+insect+adipokinetic+hormone+receptors.">Molecular identification of the insect adipokinetic hormone receptors.</a> Proceedings of the National Academy of Sciences USA. 99, 3446-3451 (2002).</li><li>Stables, J., Green, A., Marshall, F., Fraser, N., Knight, E., Sautel, M., Milligan, G., Lee, M., Rees, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioluminescent+assay+for+agonist+activity+at+potentially+any+G-protein+coupled+receptor.">A bioluminescent assay for agonist activity at potentially any G-protein coupled receptor.</a> Analytical Biochemistry. 252, 115-126 (1997).</li></ol>

我们能够执行的蛛形纲（蜱，螨虫和蜘蛛），蜱激肽受体的发现的神经肽受体的功能特性，使用该协议。这种方法有三个主要的应用。第一，这项技术可应用于配基活性受体通过测量deorphanization。二，法可以解决配体 - 受体结构 - 活性关系（SAR）。三，方法可用于药物发现。此外，该协议可用于研究活动在几乎所有的GPCR受体激动剂或拮抗剂。我们开始筛选小型图书馆，以适应本议定书。我们利用该细胞系不表达无处不在的G 蛋白摹16。我们并不需要它，因为节肢动物激肽受体的信号通过Gq蛋白和细胞内钙离子级联和他们保存在哺乳动物细胞中的信号属性，如下所示。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> ABCD1234 </td><td></td></tr><tr><td> CHO - K1细胞</td><td> ATCC </td><td> CCL - 61 </td><td>美国弗吉尼亚州马纳萨斯</td></tr><tr><td> F12K培养基</td><td> Invitrogen公司</td><td> 21127 </td><td></td></tr><tr><td>胎牛血清</td><td> Sigma - Aldrich公司</td><td> F0643 </td><td></td></tr><tr><td>胰蛋白酶EDTA（10X） </td><td> Invitrogen公司</td><td> 15400 </td><td></td></tr><tr><td>抗生素的Antimycotic </td><td> Invitrogen公司</td><td> 15240 </td><td></td></tr><tr><td> OPTI - MEM我降低血清培养基</td><td> Invitrogen公司</td><td> 31985 </td><td></td></tr><tr><td>脂质体试剂</td><td> Invitrogen公司</td><td> 18292-011 </td><td></td></tr><tr><td> GENETICIN </td><td> Invitrogen公司</td><td> 10131035 </td><td></td></tr><tr><td> MC1061/P3 Ultracomp </td><td> Invitrogen公司</td><td> C663 - 03 </td><td></td></tr><tr><td> QIAprep自旋小量制备试剂盒</td><td> Qiagen公司的公司</td><td> 19064 </td><td></td></tr><tr><td> FuGENE 6转染试剂</td><td>罗氏公司</td><td> 11 814 443 001 </td><td></td></tr><tr><td> 96孔白色薄底部microtitere板</td><td>配角</td><td> 3610 </td><td></td></tr><tr><td>无钙的DMEM媒体</td><td> Invitrogen公司</td><td> 21068 </td><td></td></tr><tr><td>腔肠</td><td> Invitrogen公司</td><td> C - 2944 </td><td></td></tr><tr><td>光亮线Hemacytometer </td><td> Hausser科学</td><td></td><td>霍舍姆，PA </td></tr><tr><td> NOVOstar </td><td> BMG公司Labtechnologies </td><td></td><td></td></tr><tr><td> Prism软件4.0 </td><td> GraphPad软件公司</td><td></td><td>美国加利福尼亚州圣迭戈， </td></tr><tr><td> T - 25和T - 75培养瓶</td><td>屋宇署猎鹰</td><td> 353014和353135 </td><td></td></tr></tbody></table>

a calcium bioluminescence assay for functional analysis of mosquito (aedes aegypti) and tick (rhipicephalus microplus) g protein-coupled receptors

节肢动物激素受体调节许多重要的生理和行为的过程，因为他们的新农药的潜在目标。其中大部分属于G蛋白偶联受体（GPCRs）超家族。我们一直专注于描绘从蜱和蚊子节肢动物激肽受体。节肢动物激肽与myotropic，利尿，和神经递质功能的多功能神经肽。在这里，为昆虫激肽的结构 - 活性关系系统的分析方法上两个异肽受体表达系统的描述。我们myotropic，利尿，和/或虱子和蚊子的消化过程可能破坏生物稳定性的激肽类似物的发展提供了重要的相关信息。 从南部的牛蜱， 小牛蜱microplus（Canestrini），和蚊子埃及伊蚊 （林奈），激肽受体稳定表达的哺乳动物细胞系CHO - K1。完成了这些受体的功能分析使用的钙的生物发光板检测措施细胞内的生物发光后肽的应用程序，这些重组细胞的细胞质中钙的含量来确定。这个方法需要的aequorin蛋白的优势，从发光水母中分离出一种photoprotein。我们aequorin质粒（mtAEQ/pcDNA1）瞬时转染稳定表达激肽受体的细胞株。这些细胞，然后用辅助因子腔肠，复合物与细胞内aequorin。这个键断裂中的钙存在，发光指示的钙离子浓度的发光水平。由于激肽受体通过细胞内钙离子的释放的信号，信号强度相关肽的药效。 此协议是一个合成几个先前所描述的的协议进行了修改，它提出通过功能板块分析（Staubly等人，2002年和马厩等 ，1997年在哺乳动物细胞系的稳定，对GPCRs的表达一步一步的指示。 ）。使用这种方法，我们能够建立稳定的细胞系表达的蚊子和蜱激肽受体，比较三个蚊子激肽的药效，确定配体 - 受体相互作用的关键氨基酸位置，并执行半通量筛选肽库。由于昆虫激肽容易快速内源性肽酶降解，它们在使用中受到严重限制为控制虫害或内分泌学研究的工具。因此，我们还测试了含氨基异丁酸（AIB），以提高其效力和生物稳定性的激肽类似物。这种肽抗模拟在生物稳定昆虫激肽类似物发展的一个重要的带头作用，并可能在神经肽为基础的节肢动物控制策略的发展援助。

Watch this Scientific Journal Video about A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors at JoVE.com

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

该协议提供克隆细胞系的选择和钙生物发光检测，以分析其同源GPCRs的合成节肢动物神经肽的构效关系的说明。此法可用于受体deorphanization和构效关系研究合成的模拟设计和肽/药物导致发现。

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

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Research

JoVE Journal

Immunology and Infection

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

15.5K 次观看.  Texas A&M University (TAMU). 该协议提供克隆细胞系的选择和钙生物发光检测，以分析其同源GPCRs的合成节肢动物神经肽的构效关系的说明。此法可用于受体deorphanization和构效关系研究合成的模拟设计和肽/药物导致发现。

视频: A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

科研

免疫与感染

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

饲养<em&gt;肩突硬蜱，</em&gt;黑腿蜱：饲养对小鼠未成熟阶段

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

在伊蚊中传播微孢子寄生虫Edhazardia aedis

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

在 伊 蚊中喂养和量化动物衍生的血液和人工膳食

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...