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The use of bi-cistronic transfer vectors for the baculovirus expressionsystem

机译：双顺反子转移载体在杆状病毒表达系统中的应用

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摘要

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a Very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.
机译：在这次交流中，我们描述了杆状病毒表达系统（BVES）的双顺反子转移载体的构建，该载体比现有载体更具优势。新载体提供了分离重组病毒的简单方法。更具体地说，目的基因和报道基因荧光素酶（LUC）分别构成同一转录本的第一和第二顺反子。因此，在这种双顺反子病毒的感染过程中测得的LUC活性可以在线估算重组蛋白的水平，这对于大规模生产重组蛋白非常有用。为了实现第二个顺反子的表达，使用了脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）元件。但是，这种在哺乳动物系统中非常有效的元件并不能促进第二顺反子在源自不同昆虫物种的各种昆虫细胞系中的有效内部翻译。缺乏有效的内部翻译不是由于杆状病毒的繁殖，因为在无病毒的表达系统中也观察到了相同的现象。看来，有效的EMCV IRES活性必不可少的组分在昆虫细胞中缺失或存在限量，或者不相容。尽管如此，LUC放置在IRES元件的下游，或紧邻第一个顺反子的下游，其表达水平使其能够实现其设计的生物技术应用。

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