Tetracycline-inducible packaging cell line for production of flavivirus replicon particles
Tetracycline-inducible packaging cell line for production of flavivirus replicon particles
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DOI：
10.1128/jvi.78.1.531-538.2004
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发表时间：
2004-01-01
影响因子：
5.4
通讯作者：
Khromykh, AA
中科院分区：
文献类型：
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作者：
Harvey, TJ;Liu, WJ;Khromykh, AA
我们以前开发的复制子载体来自澳大利亚黄病毒Kunjin，具有独特的noncytopathic性质，并已被证明直接在体外和体内编码的异源基因的长期高水平表达，并诱导强而持久的免疫反应，在小鼠中编码的免疫原。为了促进这些载体以病毒样颗粒（VLP）形式的进一步应用，我们现在已经产生了稳定的BHK包装细胞系tetKUNCPrME，其携带在四环素诱导型启动子控制下的Kunjin结构基因盒。从培养基中去除四环素导致产生Kunjin结构蛋白，其能够将转染和自扩增的Kunjin复制子RNA包装到分泌的VLP中，滴度高达1.6 × 10（9）VLP/ml。此外，来自tetKUNCPrME细胞的分泌的KUN复制子VLP可以在RNA转染后连续收获长达10天，产生每IO 6个转染细胞超过IO 1 t3个VLP的总产率。VLP在Vero细胞上传代或脑内注射到2至4日龄乳鼠中说明完全不存在任何感染性昆津病毒。tetKUNCPrME细胞还能够包装分别来自近亲和远亲黄病毒、西尼罗病毒和2型登革热病毒的复制子RNA。通过显示随着KUN VLP剂量的增加，CD 8（+）-T细胞对编码的外源蛋白的应答增加，证明了高滴度KUN复制子VLP的效用。在离体γ干扰素酶联免疫斑点试验中，单剂量2.5 × 10（7）个携带人呼吸道合胞病毒M2基因的VLP诱导每10（6）个脾细胞中有1，400个CD 8 T细胞。因此，包装细胞系代表了一个显着的进步，在非致细胞病变的昆津病毒复制子为基础的基因表达系统的发展，并可广泛适用于黄病毒RNA包装和病毒组装的基础研究，以及从不同的黄病毒复制子为基础的基因表达系统的发展。
We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.