作者要感谢过去和现在的成员中博士的实验室。彼得·帕莱塞和阿道弗·加西亚 - 萨斯特雷对NDV的发展反向遗传学技术和技术援助。研究在AG-S实验室新城疫病毒则有部分资金由NIAD授予R01AI088770并通过卓越的国土安全科学与技术中心部门为新发现及动物动物疾病（CEEZAD，奖号2010-ST-061-AG001）。研究LM-S的实验室是由美国国立卫生研究院资助RO1 AI077719，R21NS075611-01，R03AI099681-01A1，卓越流感研究和监测（HHSN266200700008C）的NIAID的中心和罗切斯特中心大学的生物防御免疫模型（HHSN272201000055C）资助。

1。哺乳动物细胞的制备（图4A，第1天） 转染在6孔板中的前一天分割的HEp-2或A549细胞。细胞的密度应该达到80-90％汇合的第二天。通常，一个汇合100mm培养皿可分割成8个孔（大约1×10 6个细胞每孔）。对于每一个病毒被救出，2-4不同井应包括在内，以及作为对照的pCAGGS-GFP和pCITE-GFP 18，分别为2个额外的井，目的是监测转染和MVA-T7感染效率。 2。哺乳动物细胞的重组修饰的安卡拉牛痘病毒表达噬菌体T7 RNA聚合酶（MVA-T7）的感染（图4A，第2天） <ol><li>热身PBS 1x/BA/PS和媒体在37°C </li><li>洗涤细胞两次，用1毫升的PBS 1x/BA/PS的。 </li><li>感染培养的哺乳动物细胞用MVA-T7病毒在感染复数（MOI的）1 PFU /细胞以200μl的PBS / BA / PS为1小时，在室温下的最终体积。 </li><li>在病毒感染的潜伏期，如3的介绍准备转染混合物。 </li></ol> 3。哺乳动物细胞的转染（图4A，第2天） <ol><li>制备脂质体的:混合1-2微克LPF2000用250微升的OptiMEM每抢救和孵化5分钟，在室温下。 </li><li> DNA的制备:做一个质粒鸡尾酒每个救援50微升的OptiMEM的。添加0.4 pTM1-NP微克，0.2微克pTM1-P和0.2微克的pTM1-L每管。加1微克NDV的全长cDNA克隆被救出到每个管。还要准备两个控制转染，一个是2微克的pCAGGS-GFP的（检查转染效率），另一个是2微克pCITE - 绿色荧光蛋白（检查T7聚合酶由MVA-T7驱动的表达）。 </li><li>后LPF2000-OptiMEM中溶液已预温育5分钟（步骤3.1）中，添加250微升的溶液与DNA管（步骤3.2）后，于室温下20-30分钟。 </li><li>孵化与MVA-T7之后，吸清除病毒接种，并替换为DNA的LPF2000转染混合物（步骤3.3）。 </li><li>加1的DMEM 10％胎牛血清/ PS中的溶液，以每培养皿中。 </li><li>孵育感染/转染的细胞为6-8小时（或过夜），在37℃，5％CO 2，然后用1.5ml新鲜的DMEM 10％胎牛血清/ PS替换转染介质。 </li><li>在24小时后转染，对照孔可以在荧光显微镜下观察，以评估转染效率（将pCAGGS-GFP）和T7聚合酶活性（pCITE-GFP）（图5A）。继续感染/转染的细胞，如下所述禽流成纤维细胞共培养。 </li></ol> 4。哺乳动物细胞的禽细胞的共培养物（图4A，第3天） 通常情况下，鸡的汇合100mm组织培养皿（封闭式基金）Oř鸭（的DEF）胚胎成纤​​维细胞每两转染井使用。一定要做好准备，提前，足够100mm组织培养占所有救援尝试禽类细胞的菜肴。对于病毒的有效救援，DMEM 10％胎牛血清/ PS培养基补充尿囊液和30mM MgCl 2的5％。在这一点上，24小时圆周率，哺乳动物细胞可以开始显示由于MVA-T7感染，asevident 图5A中的细胞病变效应（CPE）。 <ol><li>热身PBS 1x和媒体至37°C。 </li><li>洗哺乳动物细胞，2倍，用1毫升的PBS 1X的。 </li><li>胰蛋白酶消化的哺乳动物细胞用0.2ml EDTA-胰蛋白酶直到它们分离。重悬细胞于1毫升的DMEM 10％FBS / PS的，5％的尿囊液，30毫米氯化镁2，并转移到100毫米组织培养皿。加3毫升同媒体。 </li><li>洗禽细胞，两次，用4ml PBS中的1x。 </li><li>胰蛋白酶消化禽细胞用1ml EDTA-胰蛋白酶孵育1-2分钟，在37℃，直到细胞分离。重悬日E细胞加入8毫升DMEM 10％FBS / PS，5％尿囊液，30毫摩尔MgCl 2和加入4毫升胰蛋白酶处理细胞，以在哺乳动物细胞中的共培养100mm组织培养皿为8的总体积毫升（4毫升哺乳动物，4毫升禽细胞）。 </li><li>用手轻轻摇动，以便有一个均匀分布，然后将它们放置在培养箱中37℃下3-4天共培养细胞在100mm培养皿。的标准的哺乳动物和鸟类细胞的共培养后3天或4天感染鸡蛋的多少取决于细胞病变效应（细胞变圆，死亡，从表面脱离， 等等 ）是在MVA-T7感染的病毒观察到的。 </li></ol> 5。鸡鸡胚感染（图4A，日6-7） <ol><li>后3-4天的哺乳动物和鸟类细胞的共培养，去掉1毫升的组织培养上清液，加至Eppendorf管中。 </li><li>离心组织培养上清液在12000转的长凳上1分钟顶离心以去除细胞碎片。 </li><li>使用光烛框，查看空气囊和尿囊腔之间的接口蜡烛鸡蛋。就在界面边界，避免血管本地化的标志。喷70％乙醇在鸡蛋建立无菌条件。轻轻地使在蛋壳的孔上所标记的点和接种500微升上清液用胰岛素（1毫升）的注射器，垂直地和直接地瞄准针头插入尿囊腔（图4B）。 </li><li>封住缺口与熔化的蜡或石蜡蛋壳。 </li><li>在37℃下孵育卵2-3天</li></ol> 6。从被感染的鸡鸡胚尿囊液（图4A，天8-10）收获<ol><li>为了杀死鸡胚和凝结的血液孵化卵感染2-4小时或过夜，4°C。 </li><li>喷雾卵用70％乙醇（以保持无菌条件下）。 </li><li>仔细挖掘鸡蛋的顶端部分，在空气腔，用勺子。一旦蛋壳破裂，取出碎片钳，充分暴露尿囊膜。 </li><li>通过切除尿囊膜与镊子和剪刀，避免损坏血管和蛋黄露出尿囊腔。 </li><li>轻轻地压下胚胎用锅铲，用10毫升吸管收集向上流动的尿囊液（每鸡蛋8-12毫升）。避免损坏蛋黄膜。尿囊液转移到置于冰上的15ml离心管（使用每个鸡蛋1管）。 </li><li>通过在1,500 rpm离心5分钟澄清的尿囊液。将上清转移到一个新的管，而不会干扰颗粒。 </li><li>店内样品在4℃可保存1周，直到检查他们为NDV的存在下，血凝试验。 </li></ol> 7。血凝（HA）含量病毒在allant存在从感染的鸡蛋乌苏流体可以肉眼通过其hemagglutinate火鸡红细胞（RBC）的能力来确定。在NDV的情况下，每毫升约10 6空斑形成单位（PFU），必须给在HA测定一个积极的信号。 HA测定在V形底96孔板中进行。阴性（PBS 1X，未感染的尿囊液）和阳性（从任何新城疫病毒尿囊液）对照样品应始终包含在任何HA试验，以验证它。要执行HA测定: <ol><li>移液管加入50μl每孔PBS中的1x在V型底96孔板中。 </li><li>移液管加入50μl尿囊液样品成在板的第一列的孔中。通过该板的其它部分进行2倍系列稀释液并丢弃最后，额外50从油井中的最后一列微升。 </li><li>免除50微升0.5％火鸡红细胞在PBS 1X每孔。轻轻拍打摇动板。 </li><li>将培养板在4℃（或冰）F或30-45分钟或直到一个明确的粒料形成在阴性对照孔。 </li></ol>

<ol>
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P., 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=Recovery+of+infectious+human+parainfluenza+virus+type+3+from+cDNA.">Recovery of infectious human parainfluenza virus type 3 from cDNA.</a> Virology. 235, 323-332 (1997).</li><li>Zhao, H., Peeters, B. 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+Newcastle+disease+virus+as+a+viral+vector:+effect+of+genomic+location+of+foreign+gene+on+gene+expression+and+virus+replication.">Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication.</a> J. Gen. Virol. 84, 781-788 (2003).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+least+six+nucleotides+preceding+the+AUG+initiator+codon+enhance+translation+in+mammalian+cells.">At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells.</a> J. Mol. Biol. 196, 947-950 (1987).</li><li>Calain, P., Roux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rule+of+six,+a+basic+feature+for+efficient+replication+of+Sendai+virus+defective+interfering+RNA.">The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.</a> J. Virol. 67, 4822-4830 (1993).</li><li>Wyatt, L. S., Moss, B., Rozenblatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-deficient+vaccinia+virus+encoding+bacteriophage+T7+RNA+polymerase+for+transient+gene+expression+in+mammalian+cells.">Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells.</a> Virology. 210, 202-205 (1995).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+review.+New+mammalian+expression+vectors.">Product review. New mammalian expression vectors.</a> Nature. 348, 91-92 (1990).</li><li>Conzelmann, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+genetics+of+mononegavirales.">Reverse genetics of mononegavirales.</a> Curr. Top. Microbiol. Immunol. 283, 1-41 (2004).</li></ol>

阿道夫加西亚 - 萨斯特雷是对那些由医学伊坎学校在西奈山资重组新城疫病毒专利的发明者。

有几个因素要考虑，以达到良好的效果，同时抢救新城疫病毒。首先，所使用的全长cDNA构建体需要被设计为允许的新的转基因/修改功能并入NDV基因组中。这意味着，如上所述，是（i）适当的基因端（GE），基因间（IG）和基因启动（GS）的序列，如果需要添加;（ii）概无公认的通用电气或GS序列到国外基因，以及（iii）完整的重组体基因组中遵循"规则6的"。 至于抢救过程中，...

新城疫病毒（NDV），属于Avulavirus 1属的禽副粘病毒，是一个经济有关，因而被广泛研究和监视系统的人畜共患剂，这会严重影响家禽养殖在世界各地。虽然不是人类的病原体，新城疫病毒也被彻底的研究超越了兽医领域既作为一种模式和副粘病毒由于其非常有趣，自然溶瘤性6。研究NDV大大受益于反向遗传学技术的发展为单链，非节段负链RNA病毒，首先由Conzelmann和coleagues 2描述了狂犬病毒。多种遗传修饰NDVs，携带外源基因或它们的野生型基因组的修饰已被广泛自从研究。这些重组病毒工作已到关键不同毒力因子的特征不仅新城疫病毒，还包括其他相关呼玛Ñ ​​病原体，如流感病毒7 -或紧急尼帕病毒8。此外，许多不同的研究已经探索了利用这些技术来提高NDV 6,9,10的先天抗肿瘤活性，主要是通过增强病毒的免疫刺激特性。对重组NDVs研究其他相关领域一直候选疫苗对其他病毒疾病如流感5,11,12的产生，艾滋病毒13，麻疹14，SARS 15，或所造成的呼吸sincytial病毒（RSV）16。其中由NDV所提供的各种值得注意的优点是，缺乏在人群中预先存在的免疫力，该外源遗传插入，缺乏recombinatory活性和总体高度的安全配置文件结合上述天然免疫刺激性质17的稳定性。这也是值得注意的P中的潜在用途的重组双价疫苗oultry，保护对阵双方新城疫和高致病性禽流感病毒11,12。这可能是降低后者的野生蔓延到家养动物，从而也有助于防止可怕的禽流感的可能种间跳跃到人类的机会的好方法。最后，记者表达NDV已被用于的先天免疫反应的评价，以及干扰素拮抗剂由多种病毒18-27编码的识别。 重组的救援过程中，非节段负链RNA病毒基本上由上人为地迫使在一个生产单元中的病毒复制循环通过转染编码cDNA的感染性最小的分子机制，被称为核糖核蛋白或RNP（图2）。的组成的RNP的病毒聚合酶（P和L蛋白）的，核蛋白（NP）和病毒的全长基因组RNA。这种RNA +反基因是个所需的互补RNA-基因组，其中，还与病毒RNP的蛋白质的其余部分相关联的E世代模板，概括同一感染复数与天然病毒会释放对细胞的感染在细胞质中（图2A ）。从该步骤起，该病毒周期可以自然地进行，重组病毒颗粒，encapsidating修饰的基因组中，将产生（图2B）。值得注意的是，基因组cDNA，而不是反基因cDNA的转染大大削弱或完全废除救援效率2,28-30。即使在反基因组的cDNA转染，重组RNA的衣壳化的效率成在转染细胞中的RNP可能是非常低的。正因为如此，救援协议对NDV通常包括用于通过与许可的细胞和/或通过共培养它们从最初转染的细胞中释放出来的少数病毒颗粒的扩增不同的步骤感染鸡胚的。 在此之前的救援，该cDNA可以通过标准克隆程序，以便产生所需的修改操作。而不同的基因产物和病毒的调节序列的特定突变可以被直接地实现这种方式，许多已发表的工作涉及重组NDV已需要增加一个新的转录单位的进新城疫病毒的基因组。像副粘病毒科的其他成员的NDV基因组编码8种不同的蛋白质分为六个转录单位，这是根据它们的位置相对于3&#39;端以递减的梯度对于病毒生命周期1关键差异表达。正因为如此，在新的转录单位的基因组中的位置必须仔细选择，以达到病毒复制的基因和减损的表达之间的平衡。 P和M基因之间插入已使用最多，吨霍夫等网站也进行了测试13,31。 无论插入，克隆到NDV基因需要遵循一些规则来生成一个rescuable结构:（i）任何新的基因被纳入NDV基因组有受到控制适当的信号，为病毒RNA依赖的RNA的聚合酶。这些序列必须将新的开放阅读框（ORF）的上游被添加，以便所述聚合酶能够识别先前的基因（GE）和新转基因（GS）的开始结束时，由单核苷酸基因间序列（IG）隔开。另外一个有效的科扎克（K）的顺序，以改善真核生物核糖体的翻译，还建议为更好的外源蛋白表达32;新城疫病毒（ii）有效的复制，作为副粘病毒科家族的大部分成员，是依赖于基因组的长度是多重的6 33，因此，任何插入NDV必须遵循这种"规则六"。如果需要，REQuired额外的核苷酸可以在下游添加新的ORF;及（iii）转基因的顺序应该进行检查，以发现可能的GE和类似可能损害抢救效率，转基因表达和/或病毒的生存能力序列GS。如果存在，这些序列必须由沉默突变被删除。重组的全长cDNA下列上述规则的产生是为了有效地产生基因修饰的NDV如这里详细说明的第一个步骤。 系统中的所有DNA构建体是下的T7 RNA聚合酶启动子（ 图3）的控制。这细胞质聚合酶设置在反受共感染的重组修饰的安卡拉牛痘病毒（MVA-T7）34。 图3A显示了pNDV-B1的质粒，其编码全长反基因组cDNA的5。 图3B示出pTM1质粒编码的NP， P和L的ORFs。质粒pCITE-GFP的编码，UNDER T7启动子，绿色荧光蛋白（GFP），并且将pCAGGS GFP 18，其编码相同的ORF下的鸡β肌动蛋白启动子35，用作对照。在这个协议中，我们表明，从轻型NDV株Hitchner B1 5（ 图4）中的cDNA拯救重组NDV的过程。

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

新城疫病毒（NDV），原型的副粘病毒科家族1的Avulavirus属的成员，是一种非节段，负义，单链的，有包膜的RNA病毒（图1）具有潜在的应用，作为疫苗的载体和治疗人类疾病。深入探索这些应用程序的建立反向遗传学技术来营救质粒编码的完整基因组为2-5的cDNA的重组病毒后，只有成为可能。病毒cDNA可以通过标准克隆程序来改变病毒的基因型和/或以包括新的转录单位可以方便地修改在体外 。这种转基因病毒救援提供了一个有价值的工具，以了解影响感染的多个阶段，以及允许向量为抗原的表达和传递的发展和提高的因素接种疫苗和疗法。在这里，我们描述了一个协议，用于重组NDVs抢救。

NDV的救援是一个行之有效的程序，即有机会获得该病毒的完整基因实验室常规进行。然而，该方法的本征随机性质使得难以达到100％的救援效率。监测的早期步骤的过程中，特别是转染效率和感染MVA-T7，有助于识别可能存在的问题。 图5A显示标准的转染和转染/感染效率，这是足以让一个成功的NDV救援。该轮扩增中禽哺乳动物共培养和鸡胚后，获救病毒的存在是由在HA测定阳性孔进行?...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

新城疫病毒（NDV）已被广泛研究，在过去的几年里，以开发新的载体接种疫苗和治疗，等等。这些研究已经可能由于技术由cDNA拯救重组病毒，如那些我们在这里描述。

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, ...

rescue-of-recombinant-newcastle-disease-virus-from-cdna

Research

JoVE Journal

Immunology and Infection

19.2K 次观看.  Icahn School of Medicine at Mount Sinai. 新城疫病毒（NDV）已被广泛研究，在过去的几年里，以开发新的载体接种疫苗和治疗，等等。这些研究已经可能由于技术由cDNA拯救重组病毒，如那些我们在这里描述。 

视频: Rescue of Recombinant Newcastle Disease Virus from cDNA

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

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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 ...

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

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

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 ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...