Division of Mucosal Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan; MG Pharma Inc., 7-7-25, Saito-Asagi, Ibaraki, Osaka, Japan.
Division of Mucosal Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan; International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, Japan.
J Biotechnol. 2014 Apr 10;175:45-52. doi: 10.1016/j.jbiotec.2014.02.005. Epub 2014 Feb 15.
Tumor necrosis factor alpha (TNF) plays a pivotal role in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although anti-TNF antibody therapy is now commonly used to treat patients suffering from these inflammatory conditions, the cost of treatment continues to be a concern. Here, we developed a rice transgenic system for the production of a llama variable domain of a heavy-chain antibody fragment (VHH) specific for mouse TNF in rice seeds (MucoRice-mTNF-VHH). MucoRice-mTNF-VHH was produced at high levels in the rice seeds when we used our most recent transgene-overexpression system with RNA interference technology that suppresses the production of major rice endogenous storage proteins while enhancing the expression of the transgene-derived protein. Production levels of mTNF-VHH in rice seeds reached an average of 1.45% (w/w). Further, approximately 91% of mTNF-VHH was released easily when the powder form of MucoRice-mTNF-VHH was mixed with PBS. mTNF-VHH purified by means of single-step gel filtration from rice PBS extract showed high neutralizing activity in an in vitro mTNF cytotoxicity assay using WEHI164 cells. In addition, purified mTNF-VHH suppressed progression of collagen-induced arthritis in mice. These results show that this rice-expression system is useful for the production of neutralizing VHH antibody specific for mTNF.
肿瘤坏死因子-α（TNF）在类风湿关节炎和克罗恩病等慢性炎症性疾病中发挥着关键作用。尽管抗 TNF 抗体疗法现在常用于治疗患有这些炎症性疾病的患者，但治疗费用仍然是一个问题。在这里，我们开发了一种在水稻种子中生产针对小鼠 TNF 的 llama 重链抗体片段（VHH）的水稻转基因系统（MucoRice-mTNF-VHH）。当我们使用我们最新的带有 RNA 干扰技术的转基因过表达系统时，MucoRice-mTNF-VHH 在水稻种子中高水平产生，该系统抑制主要的水稻内源储存蛋白的产生，同时增强转基因蛋白的表达。MucoRice-mTNF-VHH 在水稻种子中的产量平均达到 1.45%（w/w）。此外，当 MucoRice-mTNF-VHH 的粉末形式与 PBS 混合时，约 91%的 mTNF-VHH 容易释放。通过从水稻 PBS 提取物中单步凝胶过滤纯化的 mTNF-VHH 在使用 WEHI164 细胞的体外 mTNF 细胞毒性测定中显示出高中和活性。此外，纯化的 mTNF-VHH 抑制了胶原诱导性关节炎在小鼠中的进展。这些结果表明，该水稻表达系统可用于生产针对 mTNF 的中和 VHH 抗体。
