Shi Gengshou, Chen Hongxing, Wu Xiaojie, Zhou Yanrong, Liu Zhuguo, Zheng Tao, Huang Peitang
Cell Engineering Department, Beijing Institute of Biotechnology, No. 20 Dongdajie Street, 100071, Beijing, China.
Transgenic Res. 2009 Aug;18(4):573-82. doi: 10.1007/s11248-009-9248-1. Epub 2009 Feb 15.
The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP-hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins.
目前通过乳腺生物反应器技术生产药物受到外源蛋白表达水平低的限制。我们在此描述一种解决该问题的新方法。开发了一种连续三步的缺口修复策略，以将人乳铁蛋白（hLF）的基因组编码序列从起始密码子到终止密码子精确地替换小鼠乳清酸性蛋白（mWAP）基因座中的基因组编码序列。构建了一个50 kb的mWAP-hLF杂交基因座，并产生了相应的转基因小鼠。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质免疫印迹法证明了牛奶中重组人乳铁蛋白（rhLF）的极高水平表达。ELISA测定表明，五个转基因品系中的表达水平在16.7至29.8 g/l之间。重要的是，表达的rhLF保持了与天然hLF相同的抗菌活性。我们的策略很可能也可用于其他有价值药物蛋白的高效表达。
