High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.
High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.
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DOI：
10.1371/journal.pone.0018556
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Choi SY
中科院分区：
文献类型：
--
作者：
Kim JH;Lee SR;Li LH;Park HJ;Park JH;Lee KY;Kim MK;Shin BA;Choi SY
当需要在细胞中表达多于一种基因时，已使用双顺反子或多顺反子表达载体。在用于构建双顺反子或多顺反子载体的各种策略中，内部核糖体进入位点（IRES）已被广泛使用。然而，由于IRES前后基因的大小和表达水平的差异，需要一种新的策略来取代IRES。自切割2A肽由于其小的尺寸和2A肽的上下游基因之间的高切割效率而可能是替代IRES的良好候选物。尽管2A肽具有优点，但其使用并不广泛，因为（i）没有公开可获得的携带2A肽基因的克隆载体，以及（ii）迄今为止报道的各种2A肽之间的切割效率的全面比较尚未在不同的背景下进行。在这里，我们产生了四个表达质粒，每个含有不同的2A肽来自口蹄疫病毒，马鼻炎A病毒，Thosea asigna病毒和猪捷申病毒-1，分别，并评估其切割效率在三个常用的人类细胞系，斑马鱼胚胎和成年小鼠。Western blotting和共聚焦显微镜分析表明，在四个2A中，来自猪捷申病毒-1（P2 A）的2A在所有检查的情况下具有最高的切割效率。我们预计，我们产生的2A携带克隆载体和我们证明的P2 A肽的最高效率将有助于生物医学研究人员在需要双顺反子或多顺反子表达时轻松采用2A技术。
When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.