Abstract：
Insect cells, especially Sf9 cells, are commonly used in biomanufacturing due to their advantages in high expression levels and post-translational modification. However, the development of stable expression cell lines via random integration tended to be unstable. Site-specific integration (SSI) is an alternative strategy. In this study, a φC31 -mediated cassette exchange system in Sf9 cells was established for SSI. The tagging cassette with the reporter gene egfp was randomly inserted into the cell genome. Potential platform cell lines were obtained by fluorescence-activated cell sorting (FACS) and single-cell cloning. Platform cell lines were selected by assessing the fluorescence expression, stability, and growth kinetics of cell lines. The selected platform cell lines were co-transfected with the φC31-containing plasmid and the targeting cassette. Green-fluorescence-negative clones were screened by hygromycin resistance and FACS. The resulting cell clones exhibited the expression properties of the platform cell lines. The rapid development of cell lines for the production of influenza subunit vaccines by the cassette exchange system demonstrated that the system constituted a versatile and reusable platform for the production of various recombinant proteins. Overall, the φC31-mediated cassette exchange system in Sf9 cells has the potential to facilitate and accelerate biologics development.
摘要：
昆虫细胞,尤其是Sf9细胞,由于它们在高表达水平和翻译后修饰方面的优势，通常用于生物制造。然而,通过随机整合的稳定表达细胞系的发展趋于不稳定。特定站点集成(SSI)是一种替代策略。在这项研究中,建立了Sf9细胞中φC31介导的盒式交换系统，用于SSI。将具有报告基因egfp的标记盒随机插入细胞基因组中。通过荧光激活细胞分选(FACS)和单细胞克隆获得潜在的平台细胞系。通过评估荧光表达选择平台细胞系，稳定性,和细胞系的生长动力学。选择的平台细胞系与含φC31的质粒和靶向盒共转染。通过潮霉素抗性和FACS筛选绿色荧光阴性克隆。得到的细胞克隆表现出平台细胞系的表达特性。通过盒交换系统用于生产流感亚单位疫苗的细胞系的快速发展表明,该系统构成了用于生产各种重组蛋白的通用且可重复使用的平台。总的来说,Sf9细胞中的φC31介导的盒交换系统具有促进和加速生物制品开发的潜力。本文受版权保护。保留所有权利。
