Pijlman Gorben P, Roode Els C, Fan Xiaoxiang, Roberts Lisa O, Belsham Graham J, Vlak Just M, van Oers Monique M
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
J Biotechnol. 2006 May 3;123(1):13-21. doi: 10.1016/j.jbiotec.2005.10.022. Epub 2005 Dec 20.
The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.
使用杆状病毒表达载体在昆虫细胞生物反应器中高效扩大重组蛋白生产规模，受到病毒传代增加导致产量降低（即所谓的传代效应）的阻碍。这种现象的特征是缺陷干扰杆状病毒（DIs）的产生和随后的积累，这些病毒会干扰基因组完整病毒的复制。本文介绍了一种新型杆状病毒表达载体，其配备了双顺反子表达盒，可从单个信使RNA（mRNA）同时表达重组基因（绿色荧光蛋白，第一个顺反子）和一个必需的杆状病毒基因（GP64，第二个顺反子）。GP64的翻译由来自禾谷缢管蚜病毒（RhPV）的内部核糖体进入位点（IRES）元件介导，而天然GP64基因被删除。通过这种方式，对整个双顺反子mRNA施加了显性选择压力，从而对外源基因的维持施加了选择压力。双顺反子表达载体优于对照杆状病毒载体，因为在病毒持续传代时绿色荧光蛋白表达保持在更高水平。这种稳定化载体的通用性通过其在包括Sf21、Sf9和High Five细胞在内的多种细胞系中增殖的能力得到证明。这种新型杆状病毒载体对于在病毒传代数较高的昆虫细胞生物反应器中大规模生产重组蛋白特别有价值。
