Continuous high-titer HIV-1 vector production
Continuous high-titer HIV-1 vector production
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DOI：
10.1038/nbt815
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发表时间：
2003-05-01
影响因子：
46.9
通讯作者：
Collins, M
中科院分区：
文献类型：
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作者：
Ikeda, Y;Takeuchi, Y;Collins, M
目前，基于1型人类免疫缺陷病毒（HIV - 1）的载体是通过瞬时转染制成的，或者使用包装细胞系，在其中诱导HIV - 1 Gag和Pol蛋白的表达（1 - 3）。由稳定表达HIV - 1 Gag - Pol的细胞持续生产载体将能够快速且可重复地生产大量载体批次。然而，通过转染编码HIV - 1 Gag - Pol的质粒来制备稳定的HIV - 1包装细胞的尝试，导致细胞仅分泌低水平的p24抗原（20 - 80纳克/毫升）（4 - 6），这可能是因为HIV - 1蛋白酶的细胞毒性（7）。用HIV - 1感染细胞可导致稳定的病毒产生（8）；已经描述了可产生高达1000纳克/毫升分泌型p24抗原的细胞克隆（9）。在此我们报道，通过鼠白血病病毒（MLV）载体表达HIV - 1 Gag - Pol能够使HIV - 1 Gag组成型、长期、高水平（高达850纳克/毫升p24）表达。使用密码子优化的HIV - 1 GagPol（10）和γ逆转录病毒的包膜蛋白构建了稳定的包装细胞；这些生产细胞在培养中至少三个月内可产生高达10⁷个293T感染单位（i.u.）/毫升（20个293T i.u./细胞/天）。
Human immunodeficiency virus type 1 (HIV-1)-based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced(1-3). Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20-80 ng/ml)(4-6), possibly because of the cytotoxicity of HIV-1 protease(7). Infection of cells with HIV-1 can result in stable virus production(8); cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described(9). Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 GagPol(10) and envelope proteins of gammaretroviruses; these producer cells could make up to 10(7) 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.