采用核转染技术建立稳定表达可溶性人类白细胞抗原G_1的真核细胞系

ESTABLISHMENT OF EUKARYOTIC CELL LINE STABLY EXPRESSING SOLUBLE HUMAN LEUCOCYTE ANTIGEN G_1 BY NUCLEOFECTION

目的建立稳定表达可溶性人类白细胞抗原G1(solublehumanleucocyteantigenG1,sHLA-G1)的真核细胞系。方法采用核转染技术将质粒pcDNA3-sHLA-G1转入不表达HLA-类分子的LCL721.221细胞,经G418筛选获得稳定转染细胞,通过RT-PCR及Dot-ELISA在基因和蛋白水平鉴定目的基因的表达。结果采用核转染法LCL721.221细胞转染效率可以达到约14%。RT-PCR检测发现了sHLA-G1基因特异性条带;采用Dot-ELISA方法利用sHLA-G1特异性抗体MEM-G/9检测,存在sHLA-G1蛋白。结论通过核转染方法成功建立了稳定表达sHLA-G1的真核细胞系。

Objective To establish a eukaryotic cell line that can express soluble human leucocyte antigen G1 （sHLA-G1） stably. Methods The recombinant plasmid pcDNA3-sHLA-G1 is transfected by a novel nonviral, electroporation-based gene transfer method termed nucleofection into the host cell lymphoblastoid cell line （LCL） 721. 221 which does not express any HLA-classical I molecules. After selection by G418, the cell line stably expressing sHLA-G1 is identified by RT-PCR and Dot-ELISA with HLA-G1 specific monoclonal antibody MEM-G/9. Results The efficiency of transfection for LCL721. 221 is about 14% by nucleofection. The specific band for sHLA-G1 was found by RT-PCR assay from the transfections and the protein of sHLA-G1 in the supernatant of the transfections was detected by Dot-ELISA assay. Both confirmed that the eukaryotic cell line expressing sHLA-G1 has been established successfully at genic and proteinic levels. Conclusion In this study, the eukaryotic cell line expressing sHLA-G1 have been established successfully by nucleofection.