稳定表达HLA-A＊1101蛋白的K562细胞株的建立

Establishment of Stable Subline of K562 Cells Expressing Human Leucocyte Antigen A1101

本研究目的是建立稳定表达HLA-A*1101分子的K562细胞株,为研究慢性髓系白血病(CML)HLA-I限制性抗原特异T细胞的细胞毒作用提供靶细胞。利用RT-PCR从CML患者外周血单个细胞中扩增HLA-A*1101基因全长序列;利用重组PCR将2A肽连接子(D-V-E-X-N-P-G-P)基因连接到HLA-A*1101的3'端,并将其定向克隆入带有增强型绿色荧光蛋白基因的真核表达载体pEGFP-N3,构成HLA-A*1101-T2A-EGFP转录盒的重组表达载体;利用电穿孔技术将pEGFP-N3或重组质粒转染入K562细胞,利用荧光显微镜监测绿色荧光蛋白的表达,通过G418筛选出有效转染pEGFP-N3或HLA-A*1101-T2A-EGFP载体的K 562细胞株;利用RT-PCR和流式细胞术检测HLA-A*1101基因和蛋白的表达情况。结果表明,成功构建了以2A肽基因为连接子的HLA-A*1101-T2A-EGFP真核表达载体;重组质粒转染的K562细胞经G418筛选2个月后,获得2株表达绿色荧光蛋白的K562稳定细胞株HLA-A*1101+K562和pEGFP-N3+K562。RT-PCR检测证明,HLA-A*1101+K562细胞表达HLA-A*1101基因,而pEGFP-N3+K562细胞则不表达HLA-A*1101;流式细胞术分析显示,HLA-A*1101和GFP蛋白双阳性HLA-A*1101+K562细胞占88.5%,明显高于pEGFP-N3+K562细胞(0.698%)。结论:建立了一个简单有效地筛选HLA-A*1101+K562细胞株的方法,并成功地建立了膜稳定表达HLA-A*1101蛋白的K562细胞株,为进一步研究CML的特异性细胞免疫提供工具细胞。

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A＊1101 protein,which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte（CTL） effects against chronic myeloid leukemia（CML）.The HLA-A＊1101 protein encoding gene was amplified from peripheral blood mononuclear cell（PBMNC） of CML patient by RT-PCR;the 2A peptide linker（D-V-E-X-N-P-G-P）gene was linked to the 3′ terminal of the HLA-A＊1101 gene by recombinant PCR,then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene,and the eukaryotic recombinant expression vector containing HLA-A＊1101-T2A-EGFP transcription box was constructed;the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells.The expression of GFP was monitored by fluorescence microscopy,finally stably transfected sublines of K562 cells containing HLA-A＊1101 gene,and of K562 containing pEGFP-N3 vector were obtained by G418 selection;the transcriptional or translational expression of HLA-A＊1101 gene was detected with RT-PCR and flow cytometry respectively.The results indicated that the eukaryotic expression vector HLA-A＊1101-T2A-EGFP plasmid was successfully constructed;after G418 selection for 2 months,two sublines of K562 cells（HLA-A＊1101＋K562,pEGFP-N3＋K562） expressing GFP were constructed.The expression of HLA-A＊A1101 gene could be determined in HLA-A＊1101＋K562 cell line by RT-PCR,while the pEGFP-N3＋K562 cells could not express HLA-A＊A1101 gene.HLA-A＊1101 protein and GFP double positive HLA-A＊1101＋K562 cells were up