稳定表达HLA-A*1101蛋白的K562细胞株的建立

摘要

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 G4I8 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 * AJ101 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 upto 88. 5% , which was obviously higher than pEGFP-N3* K562 cells (0. 698% ) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A * 1101 * K562 cells has been established and a subline of K562 cell expressing HLA-A* 1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.%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基因和蛋白的表述情况.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.