Name: using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy
Uploaded: 2011-11-01T12:00:00
Description: Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

这项工作是由赠款5号U54通过西方优秀的区域中心（WRCE）AI057156 - 07，R01从国家过敏和传染病研究所AI08764301 - A1，西利疫苗开发中心，在大学内部资金德克萨斯州医学科。

1。 2质粒DNA的重组的MP - 12编码NSS突变（S）的回收<ol><li>传播幼仓鼠肾（BHK）/ T7 - 15 9细胞，稳定表达T7 RNA聚合酶，到6厘米的菜最起码的基本介质中（MEM）-α（Invitrogen公司，CAT＃32561037），含10％胎牛血清（ FBS ），青霉素，链霉素（青霉素100 U /毫升，链霉素100微克/毫升）（Invitrogen公司，CAT＃15140122），和600微克/ ml的潮霉素B（Cellgro，猫＃30 - 240 - CR）。 *病毒回收效率在6厘...

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为RVFV反向遗传学系统已经开发了几个小组利用T7启动子2,4,5或鼠标3人4 POL -我子。在这个手稿中，我们描述了一个协议， 使用15个稳定表达T7 RNA聚合酶BHK/T7-9细胞产生重组RVFV MP - 12菌株。不同的病毒回收效率取决于BHK/T7-9细胞的条件，质粒量，转染细胞和数量等。我们总是放大的P0病毒在Vero E6细胞，获得高滴度实验的病毒储存。 I型干扰素主管的细胞，如人肺二倍体?...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>最低限度的基本培养基（MEM -α） </td><td> Invitrogen公司</td><td> 32561037 </td><td></td></tr><tr><td>贝科的修改最低限度的基本培养基</td><td> Invitrogen公司</td><td> 11965092 </td><td></td></tr><tr><td>改良Eagle培养基（MEM 2X） </td><td> Invitrogen公司</td><td> 11935046 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140122 </td><td></td></tr><tr><td>潮霉素B </td><td> Cellgro </td><td> 30 - 240 - CR </td><td></td></tr><tr><td> Tryptose磷酸盐肉汤</td><td> MP生物医学</td><td> 1682149 </td><td></td></tr><tr><td>高贵的琼脂</td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td>中转- LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> OPTI - MEM </td><td> Invitrogen公司</td><td> 31985070 </td><td></td></tr><tr><td>气溶胶紧盖子</td><td> Eppendorf公司</td><td> C - 2223 - 25 </td><td></td></tr><tr><td> 0.33％的中性红溶液</td><td>西格玛爱秩序</td><td> N2889 - 100ML </td><td></td></tr><tr><td> C57/WT MEF细胞</td><td> InvivoGen </td><td> MEF - c57wt </td><td></td></tr><tr><td>稻瘟素小号</td><td> InvivoGen </td><td> ANT - BL - 1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> ANT - ZN - 1 </td><td></td></tr><tr><td>定量蓝</td><td> InvivoGen </td><td> REP - QB1 </td><td></td></tr><tr><td> BHK/T7-9细胞15 </td><td>日本岐阜大学， </td><td></td><td></td></tr><tr><td> Vero E6细胞</td><td> ATCC </td><td> CRL - 1586 </td><td></td></tr></table>

using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy

裂谷热病毒（RVFV），从而导致出血热，神经系统疾病，或在人类中的盲目性，以及高利率流产和反刍动物的胎儿畸形1，已被列为作为HHS /美国农业部重叠选择代理和风险组3病原体。它属于在家庭Bunyaviridae属Phlebovirus，是这个家庭的最致命的成员之一。自2006年以来已开发的RVFV MP - 12疫苗株2,3以及野生型RVFV 4-6株，包括ZH548和ZH501，几个反向遗传学系统。 MP - 12株（这是一个危险群2病原体和非选择代理）是由几个突变，其M -和L段高度减毒，但仍带有剧毒分部的S - 3，其编码的RNA功能的毒力因素，NSS。 rMP12 C13type（C13type）进行帧删除NSS的ORF 69％缺乏所有已知的NSS功能，而它复制为efficient作为MP - 12在VeroE6细胞缺乏I型干扰素。 NSS包括干扰素（IFN）-βmRNA的7,8诱导的转录主机关闭，并促进在翻译后水平降解双链RNA依赖的蛋白激酶（PKR）9,10 IFN -β转录干扰素调节因子3（IRF - 3），NF - kB和激活蛋白-1（AP - 1），IFN -β结合IFN-alpha/beta受体（IFNAR）上调刺激IFN -α的转录虽然IRF - 3，NF - kB和激活的基因或其他干扰素刺激基因（ISGs）11，诱导宿主的抗病毒活性，而主机包括NSS IFN -β基因的转录抑制阻止病毒复制的基因upregulations这些ISGs蛋白1（AP - 1），可以通过RVFV7激活。 。因此，NSS是一个很好的的目标，以进一步削弱MP - 12，并取消IFN -β的抑制功能，以提高主机的先天免疫反应。这里，我们描述了产生重组MP - 12的编码突变NSS的协议，并提供一种检查方法，找出缺乏的功能，抑制IFN -βmRNA的合成NSS突变体的一个例子。除了 ​​其在先天免疫系统的重要作用，I型干扰素是重要的树突状细胞诱导适应性免疫反应 12-14的成熟。因此，NSS突变体诱导I型干扰素的进一步减弱，但在同一时间在刺激宿主的免疫反应更有效率比野生型MP - 12，这使得它们接种疫苗的方法理想人选。

Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

裂谷热病毒的MP - 12疫苗株反向遗传学系统是一个有用的工具，用于创建附加的MP - 12突变体具有更高的衰减性和免疫原性。我们描述了协议的产生和表征NSS突变株。

利用反向遗传学操作的裂谷热病毒MP - 12菌株，以提高疫苗的安全性和有效性的NSS基因

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

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Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

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 ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...