蜂毒磷脂酶A2(bvPLA2)是一种小,15kDa酶,通过界面结合水解许多磷脂。突变的bvPLA2H34Q(bvPLA2m),其中组氨酸34被谷氨酰胺取代,没有催化活性。该蛋白质已被证明是合适的膜锚,并已被建议作为合适的肿瘤抗原载体,用于开发新的基于树突状细胞的疫苗。要确认此功能,在这项研究中,融合蛋白PNY,由融合到bvPLA2mC末端的NY-ESO-1(NY(S))组成,是工程。bvPLA2m增强了NY(s)与人单核细胞衍生的树突状细胞(DC)膜的结合,一旦被细胞占据,与载体融合的抗原指向MHCI和MHCII肽加载区室。BvPLA2m被证明增加了NY(s)衍生的交叉呈现,限制性HLA-A*02肽,NY-ESO-1157-165(NY157-165),在T1细胞表面。负载有融合蛋白的DC以比负载有NY(s)的DC更高的效率诱导NY(s)特异性CD8+T细胞的交叉引发。这些NY(s)特异性CD8+T细胞系的65%也可以用肽脉冲的DC激活,NY157-165.在这些CD8+T细胞系中,两个能够识别人类黑色素瘤细胞系,SK-MEL-37,在HLA-A*02的背景下。仅诱导少量的bvPLA2mCD8+T细胞系,表明蛋白质的低免疫原性。结论bvPLA2m可作为膜结合载体促进MHCⅡ类肽提呈和MHCⅠ类肽交叉提呈。这样的系统可以,因此,进行细胞疫苗制备试验。
Bee venom phospholipase A2 (bvPLA2) is a small, 15kDa enzyme which hydrolyses many phospholipids through interfacial binding. The mutated bvPLA2H34Q (bvPLA2m), in which histidine-34 is replaced by glutamine, is not catalytically active. This protein has been shown to be a suitable membrane anchor and has been suggested as a suitable tumor-antigen vector for the development of novel dendritic cell-based vaccines. To confirm this feature, in this study the fusion protein PNY, composed of NY-ESO-1(NY(s)) fused to the C-terminus of bvPLA2m, was engineered. bvPLA2m enhanced the binding of NY(s) to the membrane of human monocyte-derived dendritic cells (DCs) and, once taken up by the cells, the antigen fused to the vector was directed to both MHC I and MHC II peptide-loading compartments. bvPLA2m was shown to increase the cross-presentation of the NY(s)-derived, restricted HLA-A*02 peptide, NY-ESO-1157-165(NY157-165), at the T1 cell surface. DCs loaded with the fusion protein induced cross-priming of NY(s)-specific CD8 + T-cells with greater efficiency than DCs loaded with NY(s). Sixty-five percent of these NY(s)-specific CD8+ T-cell lines could also be activated with the DCs pulsed with the peptide, NY157-165. Of these CD8+ T-cell lines, two were able to recognize the human melanoma cell line, SK-MEL-37, in a context of HLA-A*02. Only a small number of bvPLA2m CD8+ T-cell lines were induced, indicating the low immunogenicity of the protein. It was concluded that bvPLA2m can be used as a membrane-binding vector to promote MHC class II peptide presentation and MHC class I peptide cross-presentation. Such a system can, therefore, be tested for the preparation of cell-based vaccines.