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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。
Objective To construct human leukocyte antigen HLA-A24 eukaryotic expression vector and to obtain stable expression of RMA-S in mouse cells. Methods The heavy chain of HLA-A24 gene was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1 containing light chain β2m to construct an eukaryotic expression vector expressing the complete HLA-24 gene. Restriction endonuclease digestion analysis and DNA sequence analysis identified the recombinant plasmid, electrotransfected into RMA-S cells, drug screening of stable cell lines. Western blot and flow cytometry to identify the expression of HLA-A24 on the surface of target cells. Results The pcDNA3.1 / A24 eukaryotic expression vector was successfully constructed and the LA-A24 molecule was stably expressed on the surface of RMA-S cells of HLA-A24-negative mice. Conclusion The successful construction of eukaryotic expression vector and the establishment of stably transfected RMA-S cell line lays the foundation for further study on cellular immunity of many diseases in HLA-A24 population, screening and identification of suitable T cell epitopes.