Conditional knockout tools: Application of site-specific incorporation of unnatural amino acid via genetic code expansion in viral and parasite vaccine development.
Conditional knockout tools: Application of site-specific incorporation of unnatural amino acid via genetic code expansion in viral and parasite vaccine development.
复制标题
DOI：
10.1016/j.synbio.2017.02.001
复制
发表时间：
2017-03
影响因子：
4.8
通讯作者：
Jiang L
中科院分区：
文献类型：
--
作者：
Liu P;Jiang L
最近，Si 等人。报道了他们尝试生产无法复制的活流感病毒疫苗1，作为灭活和减毒病毒疫苗的替代品。目标是利用全病毒活疫苗的全部传染性来引发保护性免疫，同时尽可能保持无毒力。在本报告中，作者探索了非天然氨基酸方法 2, 3 的位点特异性掺入作为条件敲除工具的应用。在此方法中，使用一对 tRNA-tRNA 合成酶，使用其中一个终止密码子来编码感兴趣的非天然氨基酸。该 tRNA-tRNA 合成酶对与宿主细胞中所有其他 tRNA-tRNA 合成酶对正交，以确保目标非天然氨基酸的位点特异性掺入（图 1）。问题之一是用于编码非天然氨基酸的成熟前终止密码子也可能是某些宿主基因的天然终止密码子。因此，引入这种tRNA-tRNA合成酶对并补充相应的非天然氨基酸可能会损害宿主细胞本身的功能。为了选择合适的病毒包装细胞系，作者将 Methanosarcinabarkeri MS 热解酰 tRNA 合成酶/tRNACUA 对和含有绿色荧光蛋白基因的琥珀抑制密码子整合到人胚胎肾 (HEK) 293T 细胞基因组中。通过选择非天然氨基酸依赖性 GFP 产生，经过 200 代传代后，作者获得了能够以与亲代细胞一样的效率介导流感 A/WSN/33 (H1N1: WSN) 病毒包装的细胞系。然后，作者将提前终止密码子 (PTC) 引入到流感病毒基因的各个位置，包括 NP、PB1、HA、NA、NS、PA、PB2、M1 和 M2。大多数这些 PTC 病毒在含有 1 mM 非天然氨基酸的 Methanosarcinabarkeri MS 热解氨酰 tRNA 合成酶/tRNACUA 对的转基因细胞系（而不是传统细胞系）中表现出细胞病变效应。相对于野生型病毒，在某些位置，PTC取代对病毒包装效率和复制动力学影响很小。确实存在较低水平的逃逸频率（7×10-10 至 5.9×10-7），这可能归因于琥珀密码子向有义密码子的回复。
Recently, Si et al. reported their attempts on generating live, while replication-incompetent influenza virus vaccine1 as an alternative to inactivated and attenuated virus vaccine. The goal is to make use of the full infectivity of live whole-viron vaccine to elicit protective immunity while remaining as avirulent as possible. In this report, the authors explored the application of the site-specific incorporation of unnatural amino acid approach2, 3 as a conditional knockout tool. In this method, a pair of tRNA-tRNA synthetase using one of the stop codons to encode for an unnatural amino acid of interest was used. This tRNA-tRNA synthetase pair is orthogonal to all of the other tRNA-tRNA synthetase pairs in the host cell to ensure the site-specific incorporation of the unnatural amino acid of interest (Fig. 1). One of the concerns is that the pre-matural stop codon used for encoding the unnatural amino acid might also be the natural stop codon for some of the host genes. As a result, introducing such tRNA-tRNA synthetase pair and the supplementation of the corresponding unnatural amino acid might impair the functions of the host cell itself. To select a proper cell line for viral packaging, the author integrated the Methanosarcinabarkeri MS pyrolysyl tRNA synthetase/tRNACUA pair, and an amber suppressor codon containing green fluorescent protein gene into the Human embryonic kidney (HEK) 293T cell genome. After 200 generations of passage by selecting the unnatural amino acid dependent GFP production, the authors obtained a cell line that can mediate influenza A/WSN/33 (H1N1: WSN) viral packaging in an efficiency as good as in the parental cells.The authors then introduced the premature termination codon (PTC) to various positions of the influenza viral genes, including NP, PB1, HA, NA, NS, PA, PB2, M1, and M2. The majority of these PTC viruses show cytopathic effect in the presence of 1 mM of unnatural amino acid in the Methanosarcinabarkeri MS pyrolysyl tRNA synthetase/tRNACUA pair containing transgenic cell line, not the conventional cell line. In some of the positions, PTC substitution has little effect on virus packaging efficiency and replication kinetics relative to the wild type virus. There is indeed a low level of escape frequency (7× 10-10 to 5.9× 10-7), which might be attributed to reversion of the amber codon to the sense
登录
查看更多内容
影响因子：
4.8
作者：
Keitany, Gladys J.;Vignali, Marissa;Wang, Ruobing
通讯作者：
Wang, Ruobing
产生甲型流感病毒作为活的但无法复制的病毒疫苗。
影响因子：
56.9
作者：
Si, Longlong;Xu, Huan;Zhou, Demin
通讯作者：
Zhou, Demin
影响因子：
56.9
作者：
Chin, JW;Cropp, TA;Schultz, PG
通讯作者：
Schultz, PG
影响因子：
46.9
作者：
Dow, Lukas E.;Fisher, Jonathan;O'Rourke, Kevin P.;Muley, Ashlesha;Kastenhuber, Edward R.;Livshits, Geulah;Tschaharganeh, Darjus F.;Socci, Nicholas D.;Lowe, Scott W.
通讯作者：
Lowe, Scott W.