Ikeda Yasuhiro, Takeuchi Yasuhiro, Martin Francisco, Cosset Francois-Loic, Mitrophanous Kyriacos, Collins Mary
Department of Immunology and Molecular Pathology, Windeyer Institute, University College London, 46 Cleveland St., London W1T 4JF, UK.
Nat Biotechnol. 2003 May;21(5):569-72. doi: 10.1038/nbt815. Epub 2003 Apr 7.
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. 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), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. 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 Gag-Pol 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.
1型人类免疫缺陷病毒（HIV-1）载体目前通过瞬时转染或使用诱导HIV-1 Gag和Pol蛋白表达的包装细胞系来制备。由稳定表达HIV-1 Gag-Pol的细胞持续生产载体将允许快速且可重复地产生大量载体批次。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，得到的细胞仅分泌低水平的p24抗原（20 - 80 ng/ml），这可能是由于HIV-1蛋白酶的细胞毒性所致。用HIV-1感染细胞可导致稳定的病毒产生；已描述了能产生高达1000 ng/ml分泌型p24抗原的细胞克隆。在此我们报告，鼠白血病病毒（MLV）载体表达HIV-1 Gag-Pol可实现HIV-1 Gag的组成性、长期、高水平（高达850 ng/ml p24）表达。使用密码子优化的HIV-1 Gag-Pol和γ逆转录病毒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10^7个293T感染单位（i.u.）/ml（20个293T i.u./细胞/天）。
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