Name: expression and purification of virus-like particles for vaccination
Uploaded: 2016-06-02T15:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

我们要感谢罗斯实验室谁帮助优化效率最高和产量协议的前成员。

流感H3N2 VLP的产生1.真核表达系统<ol><li>亚克隆病毒结构的糖蛋白血凝素（HA），神经氨酸酶（NA），和人免疫缺陷病毒（HIV）的Gag的p55到真核表达载体中，如先前所描述pTR600 7 </li><li>扩增的DNA在化学感受大肠杆菌（ 例如 ，DH5α）和使用质粒纯化试剂盒按照制造商的说明分离转染级的质粒。的DNA的量依赖于产率和用户的需要。 </li><li>保持哺乳动物细胞系，293T，在含有10％胎牛血清（...

<ol>
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J., 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=Balanced+Hemagglutinin+and+Neuraminidase+Activities+Are+Critical+for+Efficient+Replication+of+Influenza+A+Virus.">Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus.</a> J Virol. 74, 6015-6020 (2000).</li><li>Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 463, 191-222 (2009).</li><li>Pijlman, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enveloped+virus-like+particles+as+vaccines+against+pathogenic+arboviruses.">Enveloped virus-like particles as vaccines against pathogenic arboviruses.</a> Biotechnol J. 10, 659-670 (2015).</li><li>Park, J. K., 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=Protective+efficacy+of+crude+virus-like+particle+vaccine+against+HPAI+H5N1+in+chickens+and+its+application+on+DIVA+strategy.">Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy.</a> Influenza Other Respir Viruses. 7, 340-348 (2013).</li><li>Gangadharan, D., Smith, J., Weyant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+Recommendations+for+Work+with+Influenza+Viruses+Containing+a+Hemagglutinin+from+the+A/goose/Guangdong/1/96+Lineage.+MMWR.+Recommendations+and+reports+:+Morbidity+and+mortality+weekly+report.">Biosafety Recommendations for Work with Influenza Viruses Containing a Hemagglutinin from the A/goose/Guangdong/1/96 Lineage. MMWR. 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我们使用以上描述的技术成功表达并纯化的SVP和各种病原体多种结构蛋白组成的VLP。在一般情况下，我们使用哺乳动物表达系统，以产生我们的VLP。然而，在我们的手中，哺乳动物细胞衍生CHIK VLP收益率较低。使用重组杆状病毒昆虫细胞系统时，CHIK VLP产量为更加强劲。在一般情况下，杆状病毒 - 昆虫细胞系统产生的同时控制下更高量的重组蛋白，这可能是由于，并且还由于高水平的外源基因的表...

病毒样颗粒（VLP）是人类疫苗接种核准技术。事实上，一些比较现代感许可的疫苗，包括人乳头瘤病毒（HPV）和肝炎Β的（HepΒ）疫苗采用这种方法。的VLP从能够自组装的结构蛋白形成。组装的粒子模仿病毒形态，但不能感染或复制，因为它们缺乏病毒基因组。的VLP可表达并从许多原核和真核系统的纯化。对文献的审查表明，不同的表达系统按以下标准使用：细菌- 28％，酵母- 20％，植物- 9％，昆虫- 28％，和哺乳动物- 15％1。值得注意的是，基于L1衣壳蛋白HPV疫苗是在酵母中（Gardasil的），或在昆虫细胞系统（的Cervarix）2制备。 HepΒ疫苗，Recombivax和的Engerix-Β，也产生了酵母和组成HepΒ表面抗原3的4。 我们使用哺乳动物和昆虫细胞表达系统来产生需要用于组装多个结构蛋白的共表达的VLP。我们的工作主要集中在设计，生产和净化基于VLP的疫苗对人类病原体：流感病毒，呼吸道合胞病毒（RSV），登革热病毒（DENV）和基孔肯雅病毒（CHIKV）。我们的方法是足够的灵活性，以允许从多个表达质粒的多种结构蛋白的共表达，或单个表达质粒（ 图1）。先前，我们制备并纯化的H5N1的VLP从编码人胚胎肾293T细胞5,6-流感血凝素（HA），神经氨酸酶（NA），和基质（M1）的质粒的共表达装配。用于在哺乳动物细胞中表达的基因进行密码子优化，并克隆到pTR600含有巨细胞病毒立即早期启动子加上内含子的用于启动创见一个真核表达载体真核刀片ription和转录7终止牛生长激素聚腺苷酸化信号。用于产生人类季节性流感亚型H3N2的VLP的本研究中使用的HA，NA（ 图1A）和另一种基质蛋白，HIV的Gag的p55，三个质粒共表达了类似的方法，并已被证明产生大小颗粒流感8种相似的病毒样颗粒。虽然流感疫苗主要诱导抗HA抗体，增加流感病毒神经氨酸酶介导唾液酸酶的活性，使VLP转染细胞的9，也提出了另外的免疫原性目标崭露头角。以产生的RSV的VLP，我们还选择的HIV Gag的的无关核心来设计呈现完全RSV表面糖蛋白，以进一步展示使用HIV Gag的VLP形成灵活性，如先前描述和表征通过电子显微镜6,10原型疫苗。其他先前已经表明，病毒样颗粒呈现呼吸道合胞病毒糖蛋白可以利用新城疫病毒（NDV）11和流感基质12多种病毒组件进行组装。全长表面糖蛋白的序列在本研究中利用保留天然构象可能是必要的功能性受体结合和抗体识别测定通过酶联免疫吸附试验（ELISA）。 我们使用单一质粒表达系统来生成颗粒的例子是DENV和CHIKV。在登革病毒的情况下，我们可以生产亚病毒颗粒（的SVP）从含有的prM / E的结构基因的表达盒13中的单个质粒中的293T细胞无衣壳。术语SVP用于表示在病毒结构蛋白的组装缺少核心或衣壳蛋白。 CHIK的VLP也可使用含有CHIKV结构基因盒，编码衣壳和包膜蛋白的单个质粒产生的，或在我NSECT细胞通过用重组杆状病毒编码相同的结构基因盒为昆虫细胞表达（ 图1B - C）的优化感染。 表达办法上面讨论的最终结果是VLP的释放到可随后通过超速离心通过20％甘油缓冲纯化细胞培养液中。在这份报告中，我们提出的方法来表达和哺乳动物和昆虫细胞系统净化这些病毒样颗粒。

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Plasmid Maxi Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">12163</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM</td>
			<td style="width:167px;">Cellgro</td>
			<td>10-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Lipofectamine</td>
			<td style="width:167px;">Life Technologies</td>
			<td>L3000075</td>
			<td>Lipofectamine 2000, Lipofectamine 3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opti-MEM I</td>
			<td style="width:167px;">Life Technologies</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Bac-to-Bac Baculovirus Expression System</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:119px;">10359-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cimarec I 6 multipoint stirrer plate</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyclear ultracentrifuge tubes</td>
			<td style="width:167px;">Seton</td>
			<td style="width:119px;">7025</td>
			<td>Confirm appropriate tubes for ultracentrifuge and bucket size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">23235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate citrate buffer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P4922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">o-phenylenediamine dihydrochloride&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P8287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.22 &#956;m vacuum filter top (500 mL)</td>
			<td style="width:167px;">Nalgene</td>
			<td style="width:119px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">H2SO4</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">339741&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sf-900 II SFM</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10902-096</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression and purification of virus-like particles for vaccination

病毒样颗粒（VLPs）和亚病毒颗粒（的SVP）是另一种方法，以病毒疫苗的设计，在使用活病原体提供了更高的生物安全性和稳定性的优点。非感染和自组装，的VLP被用来呈现结构蛋白作为免疫原，绕过活病原体或用于抗原递送的重组病毒载体的需要。在这篇文章中，我们证明VLP设计和开发的不同阶段的临床前动物试验未来的应用。该过程包括以下阶段：抗原的选择，在选择的细胞系的VLP / SVP的纯化抗原的表达，并量化为抗原剂量。我们演示如何使用哺乳动物和昆虫细胞系，为我们的抗原表达和展示方法产量的差异。提出的方法可以应用于各种病原体，并且可以通过替换与免疫原性str中的抗原来实现感兴趣的用户的微生物的uctural蛋白质。病毒样颗粒和的SVP协助抗原特性和最佳候选疫苗的选择。

VLP产量分别由病毒抗原结构的设计变量。在这个协议中，我们已经证明了在上清液生产SVP或VLP的纯化通过超速离心使用昆虫和哺乳动物细胞的。 DENV的prM / E的结构基因的表达盒的四种亚型被用来构建DENV的SVP（界定为1-4）的版本于表1，并在0.6毫升卷证明1.1-2.6毫克总蛋白的范围内。对于需要三个基因构建病毒样颗粒，我们发现1优化的DNA转染比：1：2的HA：NA：加...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

这里，我们提出用于合成使用任一杆状病毒或哺乳动物表达系统，并超速离心纯化病毒样颗粒的协议。这种高度可定制的方式被用来识别在一个安全的，灵活的方式的病毒抗原作为疫苗的目标。

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Purification of Transcripts and Metabolites from Drosophila Heads

Purification of Transcripts and Metabolites from Drosophila Heads

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Metabolic Rate in Drosophila using Respirometry

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...