Zhang X, O'Shea H, Entwisle C, Boursnell M, Efstathiou S, Inglis S
Cantab Pharmaceuticals Research Ltd, Cambridge, UK.
J Gen Virol. 1998 Jan;79 ( Pt 1):125-31. doi: 10.1099/0022-1317-79-1-125.
Due to their simplicity and flexibility of genomic construction, herpes simplex virus (HSV) amplicon-based vectors are attractive vehicles for gene delivery. However, a significant problem faced in the generation of amplicon stocks is the low amplicon to helper virus (A/H) ratio. In order to improve the proportion of amplicons generated, a selection system for amplicon production was developed in which the HSV thymidine kinase (TK) gene is inserted into an amplicon plasmid and an HSV mutant with both TK and glycoprotein H (gH) genes deleted is used as a helper virus. Using a protocol in which amplicon stocks are passaged 2-3 times in BHK cells of TK- and gH+ genotype in the presence of selection medium containing methotrexate, stock preparations with high A/H ratio (up to 5:1) and high amplicon titre (>1 x 10(9) infectious units/ml) were generated. In vitro characterization demonstrated that a high level of biologically functional products can be efficiently produced from these amplicon constructs.
由于其基因组构建的简单性和灵活性，基于单纯疱疹病毒（HSV）扩增子的载体是有吸引力的基因传递工具。然而，在产生扩增子储备时面临的一个重大问题是扩增子与辅助病毒（A/H）的比例较低。为了提高产生的扩增子比例，开发了一种用于扩增子生产的选择系统，其中将HSV胸苷激酶（TK）基因插入扩增子质粒，并使用同时缺失TK和糖蛋白H（gH）基因的HSV突变体作为辅助病毒。使用一种方案，即在含有甲氨蝶呤的选择培养基存在下，扩增子储备在TK和gH+基因型的BHK细胞中传代2-3次，产生了具有高A/H比例（高达5:1）和高扩增子滴度（>1×10⁹感染单位/毫升）的储备制剂。体外表征表明，从这些扩增子构建体中可以高效产生高水平的生物功能产物。
