3C protease is one of the most important proteases in enteroviruses, which is responsible for several cleavages of the viral polyprotein, leading to virus maturation. It has several harmful effects on host cells, such as the induction of caspase-mediated apoptosis and the cleavage of various proteins like P53, TATA-binding protein (TBP), histone H3, octamer-binding protein 1 (Oct-I) and DNA polymerase III. In this study, we aimed to construct a structure to express enterovirus 71 3C protease, consisting of a 3C protease sequence and two kinds of linkers including rigid and cleavable. As a result of cloning the structure into a pEGFP-N1 vector, two forms of constructions were expressed and verified by western blotting analysis including a 3C-Linker-Cleavage site (3C-L-CL) and a 3C-Linker (3C-L) without a cleavage site. In 3C-L-CL construction, 3C protease is released from fused GFP by cleave cleavage site. In 3C-L construction, 3C protease was expressed in fusion with GFP. In fact, one of the constructions produced two forms of 3C proteases, a free 3C and a 3C fused with GFP. This study shows that free 3C is preferred over the second form (3C with linker) because it is not connected to other peptides, and its protease activity is not affected. The designed structures could be used as useful tools for 3C protease expression and investigation of its interactions with cellular signaling pathways such as interferon pathway.

中文翻译：

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设计表达肠道病毒71 3C蛋白酶的自切割结构

3C蛋白酶是肠病毒中最重要的蛋白酶之一，它负责病毒多蛋白的多次切割，导致病毒成熟。它对宿主细胞具有多种有害作用，例如诱导半胱天冬酶介导的细胞凋亡以及各种蛋白质的裂解，例如P53，TATA结合蛋白（TBP），组蛋白H3，八聚体结合蛋白1（Oct-1）和DNA聚合酶III。在这项研究中，我们旨在构建一种表达肠道病毒71 3C蛋白酶的结构，该结构由3C蛋白酶序列和两种连接子组成，包括刚性和可裂解的。将结构克隆到pEGFP-N1载体中的结果是，表达的两种形式的构建体均已通过Western blot分析证实，包括3C-Linker-Cleavage位点（3C-L-CL）和3C-Linker（3C-L ），但没有卵裂位点。在3C-L-CL结构中 3C蛋白酶通过切割切割位点从融合的GFP释放。在3C-L构建中，3C蛋白酶与GFP融合表达。实际上，一种构建体产生了两种形式的3C蛋白酶：游离的3C和与GFP融合的3C。这项研究表明，游离3C优于第二种形式（带有连接子的3C），因为它不与其他肽连接，并且其蛋白酶活性不受影响。设计的结构可用作3C蛋白酶表达和研究其与细胞信号传导途径（如干扰素途径）相互作用的有用工具。这项研究表明，游离3C优于第二种形式（带有连接子的3C），因为它不与其他肽连接，并且其蛋白酶活性不受影响。设计的结构可用作3C蛋白酶表达和研究其与细胞信号传导途径（如干扰素途径）相互作用的有用工具。这项研究表明，游离3C优于第二种形式（带有连接子的3C），因为它不与其他肽连接，并且其蛋白酶活性不受影响。设计的结构可用作3C蛋白酶表达和研究其与细胞信号传导途径（如干扰素途径）相互作用的有用工具。