Oettinger H F, al-Sabbagh A, Jingwu Z, LaSalle J M, Weiner H L, Hafler D A
Department of Medicine, Brigham & Women's Hospital, Boston, MA.
J Neuroimmunol. 1993 May;44(2):157-62. doi: 10.1016/0165-5728(93)90037-y.
Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP). The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE. The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP. Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration. Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue. Purified recombinant MBP produced in E. coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed. Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed.
我们利用大肠杆菌中的诱导型表达载体表达、纯化了重组人髓鞘碱性蛋白（r-MBP）并对其进行了生物学特性鉴定。重组蛋白在阳离子交换色谱中与纯化的人MBP具有相似的亲和力，通过SDS-PAGE判断，其以单一的18.5 kDa条带洗脱。ELISA结果显示，重组蛋白与一组针对人MBP至少三种不同表位的六种单克隆抗体反应，表明其构象与天然MBP相似。此外，重组MBP可触发识别MBP的人T细胞克隆的增殖，可在SJL小鼠中诱导实验性自身免疫性脑脊髓炎（EAE），并且在经口服给予诱导耐受后能够抑制EAE。最重要的是，通过一种新的纯化方法，重组蛋白制剂中未检测到MBP的蛋白水解衍生片段，这相对于从富含蛋白酶的中枢神经系统组织中纯化的MBP具有显著优势。在需要不含降解产物的高度纯化蛋白的情况下，在大肠杆菌中产生的纯化重组MBP将作为一种生物试剂发挥作用。本文还讨论了其在抗原加工、MHC与TCR之间相互作用以及抗原驱动的免疫耐受研究中的相关性。
