摘要
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.
摘要译文
昆虫细胞生物反应器中使用杆状病毒表达载体的重组蛋白质生产的有效放大受阻于产量随病毒传代增加而降低，所谓的通过效应。这种现象的特征是缺陷型干扰杆状病毒（DI）的产生和随后的积累，干扰基因组完整病毒的复制。提出了一种新的杆状病毒表达载体，该载体装配有双顺反子表达盒，该表达盒允许重组基因（GFP，第一顺反子）和来自单个信使RNA（mRNA）的基本杆状病毒基因（GP64，第二顺反子）。GP64的翻译由来自Rhopalosiphum padi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，一个显着的选择压力是放在整个双顺反子mRNA上，并因此维持外源基因。双顺反子表达载体优于对照杆状病毒载体，因为在持续病毒传代时GFP表达保持在高得多的水平。该稳定的载体的多功能性通过其在许多细胞系（包括Sf21，Sf9和High Five细胞）中增殖的能力来证明。这种新型杆状病毒载体对于病毒传代数量高的昆虫细胞生物反应器中的大规模重组蛋白生产特别有价值。
Gorben P.Pijlman[a][1];Els C.Roode[a];XiaoxiangFan[a];Lisa O.Roberts[b];Graham J.Belsham[c];Just M.Vlak[a];Monique M.van Oers[a];. Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript[J]. Journal of Biotechnology, 2006,123(1): 13-21