OVA66抗原肽与HLA-A2分子结合性和结合稳定性的研究

The Binding Ability and Binding Stability between OVA66 Peptides and HLAA * 0201 Molecule

目的:建立利用T2细胞体外筛选肿瘤相关抗原OVA66中HLA-A2限制性抗原肽表位的实验方法。方法:应用计算机预测获得OVA66抗原中多个不同分值的九肽分子,在不同浓度(0.2μg/ml,2μg/ml,5μg/ml和20μg/ml)下与T2细胞共同孵育4h,并分别在其后的0,2,4h测定T2细胞表面HLA-A2分子的平均表达强度,抗原肽与HLA-A2的亲和力以20％MFI_(max)时的抗原肽作用浓度表示;抗原肽与HLA-A2分子结合稳定性以MFI(4h)/MFI(0 h)的百分比表示。结果:经计算机预测所获得的分值不同的4种候选OVA66抗原肽表位与T2细胞表面HLA-A＊0201的结合能力和结合稳定性显示出差别,并且与预测的分值不尽符合,其中L235和L238与HLA-A＊0201的结合力优于L236和L237。结论:利用T2细胞可以在体外初步筛选获得与HLA-A＊0201具有高亲和力的抗原肽。

Objective: To establish the methods for screening the HLA-A2-restricted peptides on T2-dependent cell model. Methods: The binding ability and binding stability of peptides to HLA-A2 molecule was determined by measuring peptide-induced expression of HLA-A2 molecules on TAP-deficient cell line-T2 cells. Briefly, n cells were incubated with OVA66 candidate peptides at different concentration (0. 2μg/ml, 2μg/ml, 5μg/ml, 20μg/ml) and different time. After incubation, expression of HLA-A2 molecules on T2 cells was detected by flow cytometry with murine mAb BB7. 2 against human HLA-A2 molecule. The binding ability of peptides was calculated with peptide concentration of 20% MFI_(max), while the binding stability of peptides was calculated with percentage of MFI (4h)/MFI (0h). Results: Four OVA66 candidate peptides were assayed with this method. Compared with reference peptide, two peptides (L235 and L238) displayed strong binding ability and good stability, while other two peptides (L236 and L237) exhibited low affinity to HLA-A2 molecules on T2 cells. Conclusion: With T2-dependent cell model, we could test the binding ability and binding stability of peptides before inducing peptide-specific T cell lines in vitro.