Freese A, Zavazava N
Institute of Immunology, University of Kiel, Germany.
Transpl Int. 1996;9 Suppl 1:S352-5. doi: 10.1007/978-3-662-00818-8_87.
A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.
使用FMOC（9-芴甲氧羰基）技术，通过自动肽合成仪合成了一系列源自HLA - B7和HLA - A2分子α1和α2区域的肽。肽通过反相高压液相色谱（HPLC）进行分析和纯化。肽纯度>96%。在体内测试了B7肽对混合淋巴细胞培养物的影响。含有B7肽的培养物中的同种异体反应强烈增强。最有效的肽抑制细胞毒性T淋巴细胞（CTL）溶解。数据表明这些肽具有免疫原性，并且它们通过直接和间接途径都能被识别。此外，研究了肽识别的机制。我们将肽B7（HLA - B7中的62 - 70位氨基酸残基）和肽A2（HLA - B2中的62 - 70位氨基酸残基）与异硫氰酸荧光素（FITC）共价偶联。将B7特异性CTL与这些肽在37℃下孵育90分钟，然后通过流式细胞术测量细胞荧光。60%的CTL结合了B7肽，而作为阴性对照的A2肽仅10%的CTL结合。正在通过免疫沉淀来表征肽所结合的配体的分子大小。
