Vennema H, Rijnbrand R, Heijnen L, Horzinek M C, Spaan W J
Department of Virology, Faculty of Veterinary Medicine, State University of Utrecht, The Netherlands.
Gene. 1991 Dec 15;108(2):201-9. doi: 10.1016/0378-1119(91)90435-e.
A recombinant vaccinia virus producing the bacteriophage T7 RNA polymerase was used to express foreign genes in eukaryotic cells. Translation efficiency in this expression system was enhanced significantly by employing the encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR) which confers cap-independent translation by directing internal initiation of translation. The enhancement was accomplished by fusing open reading frames (ORFs) to the N terminus of the EMCV polyprotein coding region, thus utilizing its highly efficient translation initiation site. Expression vectors were constructed to allow cloning in all three reading frames. As reporter genes, we used the lacZ gene and a number of genes encoding coronavirus structural proteins: among others the genes encoding glycoproteins with N-terminal signal sequences. The signal sequences of these glycoproteins are located internally in the primary translation product. We demonstrated that this did not interfere with translocation and glycosylation and yields biologically active proteins. The usefulness of sequences that direct internal initiation was extended by using EMCV UTRs to express two and three ORFs from polycistronic mRNAs.
一种产生噬菌体T7 RNA聚合酶的重组痘苗病毒被用于在真核细胞中表达外源基因。通过采用脑心肌炎病毒（EMCV）5'-非翻译区（UTR），该表达系统的翻译效率得到显著提高，该区域通过指导内部翻译起始赋予不依赖帽的翻译。这种提高是通过将开放阅读框（ORF）与EMCV多蛋白编码区的N端融合来实现的，从而利用其高效的翻译起始位点。构建表达载体以允许在所有三个阅读框中进行克隆。作为报告基因，我们使用了lacZ基因和一些编码冠状病毒结构蛋白的基因：其中包括编码具有N端信号序列的糖蛋白的基因。这些糖蛋白的信号序列位于初级翻译产物的内部。我们证明这不会干扰易位和糖基化，并产生具有生物活性的蛋白质。通过使用EMCV UTR从多顺反子mRNA表达两个和三个ORF，扩展了指导内部起始的序列的实用性。
