Wang Yuancheng, Wang Feng, Wang Riyuan, Zhao Ping, Xia Qingyou
State key laboratory of silkworm genome biology, Southwest University, Chongqing, China.
College of biology and technology, Southwest University, Chongqing, China.
Sci Rep. 2015 Nov 5;5:16273. doi: 10.1038/srep16273.
Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.
家蚕的基础研究和应用研究已进入功能基因组学时代。在此，我们报道了一种基于2A自切割肽（2A）的多基因表达系统（MGES），该系统可调控转基因家蚕丝腺中多个基因靶点的同时表达和切割。首先，发现甘氨酸 - 丝氨酸 - 甘氨酸间隔区（GSG）可显著提高2A的切割效率。然后，分析了六种带有GSG的2A的切割效率。在我们测试的所有昆虫细胞系中，最短的猪捷申病毒1型2A（P2A - GSG）表现出最高的切割效率。接下来，P2A - GSG成功切割了与人类酸性成纤维细胞生长因子（20.2 kDa）融合基因相连的人工人血清白蛋白（66 kDa）以及与EGFP融合基因相连的家蚕卵黄原蛋白受体片段（196 kD），重要的是，卵黄原蛋白受体蛋白分泌到了细胞外。此外，P2A - GSG利用我们的丝胶蛋白1表达系统成功介导了DsRed和EGFP融合基因在家蚕丝腺中的同时表达和切割，并导致其分泌到转基因家蚕的茧中。我们预测，MGES将成为基因功能研究以及医学、化妆品和其他生物医学领域各种功能性丝绸材料创新研究的有效工具。
