Wu C-M, Wu T-Y, Kao S-S, Ko J-L, Jinn T-R
Biopesticide Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taiwan.
J Appl Microbiol. 2008 May;104(5):1354-62. doi: 10.1111/j.1365-2672.2007.03686.x. Epub 2008 Feb 4.
To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications.
A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes.
A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein.
The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.
开发一种高效便捷的表达系统，以提供用于口服给药、药物研究及应用的高纯度、活性稳定的重组真菌免疫调节蛋白fve（rFip-fve）。
构建了一种重组病毒，其包含嵌合基因，该基因编码与Fip-fve-6His序列融合的家蚕素信号肽序列。利用镍螯合亲和柱从感染的Sf21细胞的上清液中纯化rFip-fve，并通过蛋白质免疫印迹法和基质辅助激光解吸电离质谱（MALDI-MS）分析进行验证。结果表明，产生了糖基化的成熟rFip-fve并分泌到感染细胞的上清液中。通过测量从小鼠脾细胞释放的白细胞介素-2的量来评估rFip-fve的免疫调节活性。
在杆状病毒/昆虫细胞系统中表达和纯化具有活性的rFip-fve用于药物应用和基因研究的可靠方案，是解决与rFip-fve蛋白的生产和活性相关潜在问题的可行手段。
在昆虫细胞中表达的rFip-fve的加工和修饰方式比在细菌细胞中更类似于其天然对应物。因此，与在大肠杆菌中产生的rFip-fve相比，在Sf21细胞中产生的rFip-fve的潜在应用可以得到更有效的评估。
