KIR2DL3新变异体与配基HLA-C 结合特性的研究

The KIR2DL3 locus, in combination with its HLA-C ligand, has been associated with license for human natural killer (NK) cells. Four new variants KIR2DL3*019, *020,*022,*023 and control alleles (KIR2DL2*003and KIR2DL3*001) were chosen as the objective for this project. The different KIR2DL3 alleles were cloned into the retrovirus vector and transiently transfected into NKL cells, then analyzed the cytotoxicity assay for NKL cell interaction with the expression HLA-C 721.221 cells. The stable expression of different KIR alleles were also established with insect expression system in order to obtain recombinant soluble KIR molecules. The different binding characteristics between KIR alleles and HLA-C molecules were determined by luminex technology and combined with the structure analysis for the amino acid polymorphism of KIR and HLA interaction, which can clarify the recognition mechanism of KIR and its cognate ligands. The research will elucidate the binding characteristics between KIR2DL3 and its cognate HLA-C ligands, which provide the basis for the regulation for NK cell.

KIR2DL3不同等位基因和HLA-C结合特性的不同，将导致NK细胞产生不同的效应。本项目拟以实验室前期发现的4个新的变异体KIR2DL3*019、*020、*022、*023为研究对象，利用KIR2DL2*003、KIR2DL3*001作为对照，将KIR2DL3分子不同等位基因cDNA通过体外克隆在NKL细胞进行表达，并与表达HLA-C分子的721.221细胞直接作用，分析其细胞毒作用。同时构建昆虫表达系统获取重组的可溶性KIR2DL3分子，利用luminex 技术分析KIR2DL3不同等位基因编码抗原与HLA-C分子的结合特性，通过空间构象分析研究氨基酸多态性对KIR2DL3和HLA-C结合的影响。项目将阐明KIR2DL3与其特异性配体识别及结合的特性，为研究KIR2DL3与HLA-C对NK细胞的调节作用提供基础。

杀伤细胞免疫球蛋白样受体（KIR）属于免疫球蛋白样超家族，主要表达于NK 细胞和T细胞表面，KIR 可与靶细胞表面的HLA-I 类分子结合，传导激活或者抑制信号，从而调节NK 细胞或者T 细胞活性，在造血干细胞移植及NK 细胞抗肿瘤效应中具有重要意义。本研究在获取浙江人群HLA-C1和HLA-C2的频率分布的基础上，成功构建了包含不同等位基因型的HLA-C1和HLA-C2的真核表达体系，同时选取不同等位基因型KIR2DL3*001、 KIR2DL3*019、KIR2DL3*023为研究对象，通过构建KIR2DL3不同等位基因的逆转录病毒系统和昆虫细胞表达体系，制备小量蛋白，同时应用流式细胞术，比较KIR不同等位基因型与HLA-C 分子的结合特性，为阐明KIR与其特异性配体识别及结合的具体机制和研究KIR与HLA-C对NK 细胞的调节作用提供理论依据，同时对进一步研究造血干细胞移植中供者的选择、KIR 对造血干细胞移植预后的影响以及阐明某些与KIR 相关联疾病的机制有重要意义。