表达hβ2m-A24单链的RMA-S细胞系的构建及鉴定

Construction and identification of hβ2m-A24 single chain expressing cell line

目的构建人类白细胞抗原HLA-A24真核表达载体,并使其在小鼠细胞RMA-S获得稳定表达。方法采用PCR方法扩增出HLA-A24基因的重链,将其克隆到含轻链β2m的真核表达载体pcDNA3.1上,构建成表达完整HLA-24分子的真核表达载体。用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒,电转染RMA-S细胞,药物筛选稳定细胞系。蛋白印迹法和流式细胞鉴定HLA-A24分子在靶细胞表面的表达。结果成功构建了pcDNA3.1/A24真核表达载体,并使LA-A24分子在HLA-A24阴性的小鼠RMA-S细胞表面获得稳定表达。结论真核表达载体成功构建和稳定转染RMA-S细胞系的建立为进一步研究HLA-A24人群中多种疾病的细胞免疫、筛选和鉴定合适的T细胞抗原表位奠定了基础。

We aim to construct HLA-A24 eukaryotic expression vector and obtain stable expression on TAP-deficient RMA-S cells of HLA-A24 negative mouse.PCR technique was employed to amplify A24 gene,which was subsequently subcloned into vector pcDNA3.1（＋） for construct a eukaryotic expression namely pcDNA3.1（＋）/A24.Then,the recombinant plasmid was identified by restriction analysis and sequence analysis,and transferred into the target cells.The surface expression of linked chainβ2m-A24 was assessed by Western blot and flow cytometry.The results showed that HLA-A24 molecules were successfully expressed on RMA-S cells transfected with pcDNA3.1（＋）/A24.The construction of the eukaryotic expression vectors and the establishment of stable transfected RMA-S cell lines provide a solid foundation for further experimental studies on the function of HLA-A24genes.