HLA-E真核表达载体的构建及其在K562细胞中的表达与鉴定  

Construction of pcDNA3.1(+)/A2E Eukaryotic Expression Vector and Its Expression on K562 cell

作　　者：刘四喜[1] 方建培[2] 徐宏贵[2] 陈国华[3] 黄绍良[2] 

机构地区：[1]深圳市儿童医院儿内科,深圳518026 [2]中山大学附属第二医院儿科 [3]惠州市中心医院儿科,惠州516001

出　　处：《中国实验血液学杂志》2005年第3期464-467,共4页Journal of Experimental Hematology

基　　金：广东省自然科学基金资助项目;编号31708

摘　　要：本研究旨在亚克隆HLAE真核表达载体,并使其在HLAI类阴性的靶细胞K562细胞上获得稳定表达。首先采用PCR方法从多顺反子表达载体(pG/A2E)扩增出目的片段A2/EcDNA,与真核表达载体pcDNA3.1(+)重组,构建成HLAE真核表达载体pcDNA3.1(+)/A2E,然后采用脂质体转染的方法将重组质粒转入K562细胞,最后经G418筛选及有限稀释,利用抗HLAE特异的单克隆抗体K01263进行FACS检测,以观察HLAE分子在靶细胞表面的表达情况。结果显示,HLAE分子在经pcDNA3.1(+)/A2E转染的靶细胞表面获得表达(27.76%),而经空载体pcDNA3.1(+)转染的靶细胞则未获得表达。结论:成功构建了pcDNA3.1(+)/A2E真核表达载体,并使HLAE分子在HLAI类阴性的K562细胞表面表达,为进一步研究HLAE作用的分子机制以及探索HLAE与NK受体之间的相互作用和HLAE体外表达对NK细胞功能的影响奠定了基础。To construct pcDNA3.1(+)/A2E eukaryotic expression vector and obtain a stable expression on HLA-I negative human K562 cell, PCR technique was employed to amplify A2E cDNA from the multi-cistron expression vector pG/A2E carrying HLA-E and HLA-A2 cDNA through internal ribozyme entry site (IRES), the cDNA was subcloned into vector pcDNA3.1(+), thus a eukaryotic expression was constructed and named pcDNA3.1(+)/A2E; then, the recombinant plasmid was transferred into the target cells, followed by screening with G418 and limiting dilution; finally, flow cytometry was adopted to detect HLA-E expression on the target cells. The results showed that HLA-E molecules were successfully expressed on K562 cells transfected with pcDNA3.1(+)/A2E (27.76%) and the expression of HLA-E molecules was not detected on K562 cells transfected with pcDNA3.1(+). It is concluded that the pcDNA 3.1(+)/A2E eukaryotic expression vector was successfully constructed and the HLA-E molecules were expressed on K562 cells. The data presented here would be expected to lay a good basis for the research of the molecular mechanism of HLA-E function and the interaction between HLA-E and the receptor on NK cells, as well as the influence of the expression of HLA-E in vitro on NK cells.

关 键 词：HLA-E 真核表达载体 K562细胞 先导肽 

分 类 号：R733.7[医药卫生—肿瘤] Q782[医药卫生—临床医学]