Smith Daniel C., Marsden Catherine J., Lord J Michael, Roberts Lynne M.
Department of Biological Sciences, University of Warwick. Coventry, CV4 7AL. UK.
Biol Proced Online. 2003;5:13-19. doi: 10.1251/bpo42. Epub 2003 Feb 17.
Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass.
细胞毒素蓖麻毒素的去毒版本可将融合肽递送至靶细胞，从而导致主要组织相容性复合体（MHC）I类分子限制的抗原呈递[史密斯等人，《免疫学杂志》，2002年；169：99 - 107]。蓖麻毒素递送载体必须包含一个减毒的催化结构域以防止靶细胞死亡，并且融合肽表位必须保持完整以便递送至MHC I类分子并进行功能性装载。本文描述了融合蛋白在大肠杆菌中的表达及通过阳离子交换色谱法进行的纯化。在用于递送之前，必须通过N - 糖苷酶测定在体外以及通过细胞毒性测定在体内对载体的活性进行表征。必须使用质谱法通过将实际质量与预测质量进行比较来确认完整表位的存在。
