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Abstract：
Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.
摘要：
杆状病毒表达载体成功地用于真核细胞中复杂(糖)蛋白的商业生产。大肠杆菌中的单拷贝杆状病毒感染性克隆(杆粒)的基因组工程在杆状病毒生物学研究中具有重要的价值。但杆粒作为表达载体尚未广泛应用。第一代杆粒用于大规模蛋白质生产的重要限制是目的基因(GOI)表达的快速丧失。不稳定性是由bacmid主链中的mini-F复制子引起的，这对于昆虫细胞中的杆状病毒复制是非必需的，并在attTn7转座位点之间携带相邻的GOI。在本文中,我们测试了这样的假设，即attTn7转基因插入位点远离mini-F复制子的位置可以防止GOI的缺失，从而导致更高和延长的重组蛋白表达水平。我们应用λ红色基因组工程与SacB反向选择相结合，生成了一系列具有重新定位的attTn7位点的杆粒，并通过比较不同GOI的相对表达水平来测试其性能。我们得出的结论是，与原始bacmid相比，odv-e56（pif-5）基因座的GOI表达导致更高的总体表达水平，并且在连续传代中更稳定。最后,我们在Sf9昆虫细胞悬浮的生物反应器放大过程中评估了这种改进的下一代bacmid,以产生包膜基孔肯雅病毒样颗粒作为模型疫苗.
