Wu Tzong-Yuan, Wu Chih-Yu, Chen Ying-Ju, Chen Chung-Yung, Wang Chung-Hsiung
Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung-Li, Taiwan.
FEBS Lett. 2007 Jun 26;581(16):3120-6. doi: 10.1016/j.febslet.2007.05.070. Epub 2007 Jun 4.
A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' untranslated region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.
使用双顺反子杆状病毒表达载体和基于荧光蛋白的检测方法来鉴定在杆状病毒感染的昆虫细胞中具有内部翻译活性的序列。我们证明，裸刺蛾病毒（PnV）的5'非翻译区（5'UTR；473个核苷酸）和禾谷缢管蚜病毒（RhPV）的5'UTR（579个核苷酸），而非蟋蟀麻痹病毒的内部核糖体进入位点（IRES）序列，在杆状病毒感染的Sf21细胞中具有内部翻译活性。此外，我们发现包含预测的PnV开放阅读框（ORF）的前22个密码子（总共539个核苷酸）可使内部翻译活性提高约18倍。这是关于杆状病毒表达系统（BEVS）在艾弗病毒5'序列中具有内部翻译活性的首次报道，可能有助于开发可用于BEVS以生产多种蛋白质复合物的多顺反子杆状病毒转移载体。
