Jinn Tzyy-Rong, Wu Chi-Ming, Tu Wu-Chun, Ko Jiunn-Liang, Tzen Jason T C
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.
Biosci Biotechnol Biochem. 2006 Nov;70(11):2627-34. doi: 10.1271/bbb.60232. Epub 2006 Nov 7.
The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.
在华人社会中，多种灵芝品种的子实体传统上一直被当作高档功能性食品补充剂食用。在松杉灵芝（松杉树芝）中发现的一种真菌免疫调节蛋白FIP-gts，被认为对癌症和自身免疫性疾病具有治疗作用。为了生产用于口服评估的活性FIP-gts，通过杆状病毒表达系统在Sf21昆虫细胞中表达了一种在其C末端融合有6His标签的重组FIP-gts（rFIP-gts）。通过一步镍亲和层析获得了高产率（约70%）和高纯度（约90%）的rFIP-gts。通过蛋白质免疫印迹和基质辅助激光解吸电离质谱分析验证了收获的rFIP-gts的正确性。当Sf21细胞以感染复数10感染72小时时，观察到rFIP-gts的最佳表达，产量高达47.2微克/3×10⁶个感染细胞。纯化的rFIP-gts的免疫调节活性通过检测从小鼠脾细胞释放的白细胞介素2来测定。与在大肠杆菌细胞中产生的rFIP-gts相比，在Sf21细胞中产生的rFIP-gts具有明显更高的特异性免疫调节活性。
