通过“自切割”2A肽系统共表达功能性人血红素加氧酶1、胞外5'-核苷酸酶和胞外核苷三磷酸二磷酸水解酶-1。
Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system.
作者信息
De Giorgi Marco, Cinti Alessandro, Pelikant-Malecka Iwona, Chisci Elisa, Lavitrano Marialuisa, Giovannoni Roberto, Smolenski Ryszard T
机构信息
Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy; Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland.
Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.
出版信息
Plasmid. 2015 May;79:22-9. doi: 10.1016/j.plasmid.2015.03.004. Epub 2015 Mar 14.
We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.
我们开发了一种基于F2A的多顺反子系统，以评估三种对异种移植重要的蛋白质共表达的功能效应：血红素加氧酶1（HO1）、胞外5'-核苷酸酶（E5NT）和胞外核苷三磷酸二磷酸水解酶-1（ENTPD1）。我们构建的三顺反子p2A质粒能够在HEK293T细胞中有效驱动HO1、E5NT和ENTPD1的同时表达。所有三种过表达的蛋白质都具有相关的酶活性，而添加弗林蛋白酶切割位点会干扰蛋白质表达和活性。我们得出结论，我们的三顺反子p2A构建体对于测试HO1、E5NT和ENTPD1对异种排斥机制的联合保护作用是有效且最佳的。
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