概要

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

Published: November 01, 2011
doi:

概要

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。

Abstract

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BunyaviridaePhlebovirus,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。

Protocol

1。 2质粒DNA的重组的MP – 12编码NSS突变(S)的回收传播幼仓鼠肾(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…

Discussion

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34 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?…

開示

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Name of the reagent Company Catalogue number Comments (optional)
Minimum Essential Medium (MEM)-alpha Invitrogen 32561037  
Dulbecco’s modified minimum essential medium Invitrogen 11965092  
Modified Eagle Medium (MEM 2x) Invitrogen 11935046  
Penicillin-Streptomycin Invitrogen 15140122  
Hygromycin B Cellgro 30-240-CR  
Tryptose phosphate broth MP biomedicals 1682149  
Noble agar VWR 101170-362  
TransIT-LT1 Mirus MIR2300  
Opti-MEM Invitrogen 31985070  
Aerosol tight lid Eppendorf C-2223-25  
0.33% neutral red solution Sigma Aldrich N2889-100ML  
C57/WT MEF cells InvivoGen mef-c57wt  
Blasticidin S InvivoGen Ant-bl-1  
Zeocin InvivoGen ant-zn-1  
QUANTI-Blue InvivoGen rep-qb1  
BHK/T7-9 cells15 Gifu university, Japan    
Vero E6 cells ATCC CRL-1586  

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記事を引用
Kalveram, B., Lihoradova, O., Indran, S. V., Ikegami, T. Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy. J. Vis. Exp. (57), e3400, doi:10.3791/3400 (2011).

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