Name: rescue of recombinant newcastle disease virus from cdna
Uploaded: 2013-10-11T23:30:00
Description: Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

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。 <p class=...

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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?...

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, ...

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

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

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

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

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

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

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

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

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

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

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

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 ...