Etto Tamara L, Stewart Lisbeth A, Nguyen Thi H O, Williamson Nicholas A, Purcell Anthony W, Schwarer Anthony P
Immunotherapy Research Group, Bone Marrow Transplant Unit, Alfred Hospital, Melbourne, Australia.
Protein Expr Purif. 2007 Jul;54(1):176-82. doi: 10.1016/j.pep.2007.02.011. Epub 2007 Feb 27.
The minor histocompatibility antigen HA-1H is a potential immunotherapeutic molecule. It can be used as a target for graft versus leukaemia reactions to eliminate residual HA-1H expressing leukaemic cells in patients following haemopoietic stem cell transplantation, whereby HA-1H primed donor cells can be transferred into a patient via adoptive immunotherapy. However, thus far only synthetic peptides corresponding to a HLA-A *0201 restricted HA-1H epitope have been used to generate HA-1H specific T cells. We are the first laboratory to clone, express and purify a region of HA-1H using an Escherichia coli expression system. The recombinant HA-1H protein was purified under denaturing conditions and the affinity purification tag removed using thrombin to remove non-specific amino acids. The 92 amino acid recombinant protein was characterised by mass spectrometry. Our rationale is that by using a recombinant HA-1H protein rather than peptide, HA-1H specific T cells may be generated from presentation of multiple HA-1H epitopes complexed in different HLA molecules. A multi-epitope approach may have wider applicability and maybe more effective at leukaemia control. The recombinant HA-1H protein may also be used as a research tool to identify novel CD4(+) helper T cell and CD8(+) cytotoxic T cell epitopes.
次要组织相容性抗原HA-1H是一种潜在的免疫治疗分子。它可用作移植物抗白血病反应的靶点，以清除造血干细胞移植后患者体内残留的表达HA-1H的白血病细胞，从而可通过过继性免疫疗法将经HA-1H致敏的供体细胞转移到患者体内。然而，迄今为止，仅使用与HLA-A*0201限制性HA-1H表位相对应的合成肽来产生HA-1H特异性T细胞。我们是首个使用大肠杆菌表达系统克隆、表达和纯化HA-1H区域的实验室。重组HA-1H蛋白在变性条件下纯化，使用凝血酶去除亲和纯化标签以去除非特异性氨基酸。通过质谱对92个氨基酸的重组蛋白进行了表征。我们的理论依据是，通过使用重组HA-1H蛋白而非肽，可能从在不同HLA分子中复合的多个HA-1H表位的呈递中产生HA-1H特异性T细胞。多表位方法可能具有更广泛的适用性，并且在控制白血病方面可能更有效。重组HA-1H蛋白还可用作研究工具，以鉴定新的CD4(+)辅助性T细胞和CD8(+)细胞毒性T细胞表位。
