Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid.
Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid.
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DOI：
10.1186/s12985-015-0321-5
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发表时间：
2015-06-22
期刊：
影响因子：
4.8
通讯作者：
Curtiss R 3rd
中科院分区：
文献类型：
--
作者：
Zhang X;Curtiss R 3rd
目前的流感疫苗对季节性流感是有效的，但不能及时生产用于突然的大流行，也不能以具有成本效益的方式对大量鸟类进行免疫。我们提出了一种由细菌载体和质粒货物组成的新型流感疫苗。在免疫的受试者中，细菌载体侵入并将其货物释放到宿主细胞中，其中质粒表达病毒RNA和蛋白质以重建减毒流感病毒。在这里，我们的目的是构建一个小鼠PolI驱动的质粒，用于高效生产流感病毒。构建质粒以表达所有流感病毒RNA和蛋白质。在转染后第3天，该多合一质粒在幼仓鼠肾（BHK-21）细胞中产生105-106 50%组织培养感染剂量（TCID 50）/mL的甲型流感病毒，并且还在Madin-Darby犬肾（MDCK）和中国仓鼠卵巢（CHO）细胞中重建流感病毒。通过从一体化质粒中删除HA和NA盒来构建6单位质粒。用6单位质粒和编码HA或NA基因的另外两个质粒共转染BHK-21细胞，导致流感病毒滴度与通过1质粒方法产生的滴度相似。开发了一种多合一质粒和一种3质粒鼠PolI驱动的反向遗传学系统，并在BHK-21细胞中有效地重建了流感病毒。多合一质粒可以作为一种工具，以确定抑制病毒从大尺寸质粒产生的因素。此外，我们推荐了一种简单而稳健的“1 + 2”方法来产生流感疫苗种子病毒。
The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus. A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 105–106 50 % tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin–Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method. An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust “1 + 2” approach to generate influenza vaccine seed virus.
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影响因子：
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