Name: development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle
Uploaded: 2017-02-01T13:00:00
Description: Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

This work was supported in part by the Research Program on Hepatitis from Japan Agency for Medical Research and Development (AMED) and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

1.生产重组乙型肝炎病毒的编码蛋白质记者<ol><li> HepG2细胞的制备 <ol><li>制备细胞培养基（Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），100U / ml青霉素，100微克/毫升链霉素和100单位/毫升的非必需氨基酸）。 </li><li>板4×10 6个 HepG2细胞中在10ml培养基的转染前一天10厘米胶原包被的培养皿。孵育HepG2细胞于37℃在湿润的5％CO 2培养箱。 注意：当大约4×...

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HBV基因组有四个主要的开放阅读框（ORFs），包括核心，聚合酶，表面和X的ORF（ 图1）。这四个HBV ORF的转录紧紧四个启动子调控：含有增强II的前核心/核心启动子，含有增强我18启动S1，S2启动子和X子。 3.5 kb和3.4 kb的mRNA的分别翻译成前C区和核心蛋白。大包膜蛋白（L）的从最大的亚基因组mRNA的（2.5kb的）产生的，而中间（M）和小的表面蛋白（S）是从短转录（2.1kb的?...

乙型肝炎病毒（HBV）慢性感染是慢性肝病1的主要危险因素。虽然目前的治疗策略是基于抑制通过干扰素刺激基因功能2的HBV POL功能和/或激活在感染个体的免疫反应的I型干扰素的施用以及间接抑制HBV的增殖的核苷酸类似物，这些治疗不能消除HBV的DNA完全3。而且，对防聚合酶剂耐药HBVS的出现是关注4。合并的抗病毒剂直接针对人免疫缺陷病毒（HIV）或丙型肝炎病毒（HCV）的生命周期的不同步骤的管理被证明成功地抑制或根除病毒（ES）。类似这样的想法，即直接作用于H的不同阶段抗HBV剂的开发BV的生命周期是建立未来的乙肝治疗方法很重要。 在一般情况下，建立在目标病毒的体外培养系统中的简单的便于防病毒代理的发展。然而，有至少两个障碍的体外培养系统的发展，以筛选抗HBV剂。首先是缺乏一个方便的体外细胞培养HBV感染/扩散制度。不像其他的病毒，如HIV和HCV，其在建立的细胞系增殖，它是很难培养的HBV 体外因为实验的限制，包括一个窄的宿主范围的。使用具体的细胞培养系统，例如人肝癌细胞系HepaRG，这是易受HBV感染，5，6，7已经开发了克服了这些问题。此外，PXB的细胞，从尿激酶TY孤立PE纤溶酶原激活物的转基因/原发性人肝细胞（PHH）接种的SCID小鼠，被证明是容易受到HBV感染和复制8。然而，在HepaRG HBV复制的水平依赖于培养后的细胞的分化状态，这会导致HBV感染/复制水平的不一致和不可再现的结果。 PXB通常用于HBV感染实验，但是由它的可用性的限制。四环素诱导型HBV的表达细胞株，HepAD38，也被广泛用于研究HBV复制，但这种系统只允许转录后，而不是在HBV感染9的进入步骤评价。最近，牛磺胆酸钠cotransporting多肽的识别（NTCP）用作HBV的功能性受体已经允许可变的HBV培养系统10的开发。事实上，在非易感肝癌细胞如的Huh7 NTCP表达和肝癌使HBV感染10，因此，乙肝病毒易感细胞系的选择也不断扩大，解决许多实验的限制。第二个问题是缺少一种简单的测定系统来评估HBV感染和复制的。 HBV感染的评价通常是通过分析HBV DNA，RNA和蛋白质进行。但是，这些病毒标志物的定量是费时，常常是昂贵和不总是简单的。因此，一个简单的测定系统的发展，诸如使用报告基因，可能克服与HBV的测定系统相关的问题。 但是，因为可被包装成一个HBV衣壳基因组大小被限制-小于3.7 kb的11 -报告基因的大小应该尽可能地短。此外，分散在整个基因组中的多个顺式元件，其是病毒复制所必需的存在，限制了可用于在位置<html>报道基因到基因组中的插入可以。若干报告已经试图插入外源基因，包括HIV-1的Tat，绿色荧光蛋白，和红色荧光蛋白，成HBV基因组11，12，13。然而，这些重组HBVS不是用于筛选HBV感染/复制，或用于影响HBV感染/复制因子的高通量筛选是有用的。这主要是因为重组病毒的生产效率低而造成低效病毒产生报道基因表达的降低强度的。 为了克服这些问题，我们构建了记者乙肝病毒生产的高产率。这种病毒是用于监测HBV复制循环的早期阶段，从入口到转录高度敏感。为了实现这一目标，NanoLuc（NL）被选择作为标记基因，因为它是一个小的（171个氨基酸）工程改造发光报告类=“外部参照”&gt; 14。另外，NL是约150倍萤火虫或Renilla荧光素酶亮，和发光反应是ATP的独立的，这表明假命中率将是低的为高通量筛选。重组乙肝病毒的生产效率为约1/5的父的HBV，和类似报告以前的HBV的重组病毒的水平;然而，NL的亮度克服病毒生产率的问题，因此它可以被用于抗HBV剂的大量筛选。 使用原代肝细胞的抗HBV剂的筛查，HepaRG，HepAD38和NTCP转导的肝细胞可能是抗HBV剂通过常规方法（多个）的筛选是有用的。然而，在这里所描述的系统具有各种优点，例如简单的处理，灵敏度高，且成本低的筛选。这些优势是适用于高通量分析制定和确定新HBV药物达到治疗目的。

<table border="0" cellpadding="0" cellspacing="0" width="781">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Nano-Glo Luciferase Assay Regent</td>
			<td style="width:201px;">Promega</td>
			<td style="width:160px;">N1110</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin Mixed Solution</td>
			<td>Nacalai tesque</td>
			<td>09367-34&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:332px;">100 mm/collagen-coated dish</td>
			<td>Iwaki</td>
			<td>4020-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000001&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 6000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>81255</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol (PEG) 8000&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Nacalai tesque</td>
			<td>31319-45&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5 mol/l-EDTA Solution</td>
			<td>Nacalai tesque</td>
			<td>06894-14&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Nacalai tesque</td>
			<td>35434-21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HP, 0.45 &#956;m, polyethethersulfone, filter</td>
			<td>Merck Millipore</td>
			<td>SLHP033RS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagen coated 96-well plate</td>
			<td>Corning</td>
			<td>NO3585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Passive Lysis 5xBuffer</td>
			<td>Promega</td>
			<td>E1941</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GloMax 96 Microplate Luminometer</td>
			<td>Promega</td>
			<td>E6501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Nacalai tesque</td>
			<td>30403-55</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luminometer plate</td>
			<td>Greiner bio-one</td>
			<td>655075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HepG2-NTCP1-myc-clone22</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV delta epsilon</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC1.2HBV/NL</td>
			<td>-</td>
			<td>-</td>
			<td>Reference 15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tube</td>
			<td>Violamo</td>
			<td>1-3500-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Myc antibody</td>
			<td>Sigma-Aldrich</td>
			<td>C3956</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBIG</td>
			<td>Japan Blood Products Organization</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFN-&#946;</td>
			<td>Mochida Pharmaceutical</td>
			<td>14987224005413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a hepatitis b virus reporter system to monitor the early stages of the replication cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

图1显示了HBV基因组，转录的RNA，病毒蛋白和它们的编码区的基因组的示意图。在基因组中的报告基因和它的位置也被指示。 图2示出了包含报告基因的HBV报告质粒和辅助质粒。所述pUC1.2HBV / NL记者从pUC1.2HBV 16的前C mRNA的转录起始位点删除核苷酸位置223-811，然后插入报告基因构建。通过定点诱变PCR产生与包装信号序列的点突变的pU...

Watch this Scientific Journal Video about Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle at JoVE.com

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

这里，我们描述了新开发的乙型肝炎病毒（HBV）报告系统来监视HBV生命周期的早期阶段。 该体外系统的简化将在使用高通量策略抗HBV剂的筛选帮助。

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Protocol for Analyzing Hepatitis C Virus Replication

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

小鼠胎肝文化系统解剖靶基因功能的终端红细胞生成的早期和晚期

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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

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

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Assembly and Purification of Prototype Foamy Virus Intasomes

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

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 ...