HLA-E真核表达载体的构建及其在K562细胞中的表达与鉴定

本研究旨在亚克隆HLA-E真核表达载体，并使其在HLA-I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／ E cDNA，与真核表达载体pcDNA3．1( )重组，构建成HLA—E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HLA-E特异的单克隆抗体K0126-3进行FACS检测，以观察HLA—E分子在靶细胞表面的表达情况。结果显示，HLA-E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HLA-E分子在HLA-I类阴性的K562细胞表面表达，为进一步研究HLA-E作用的分子机制以及探索HLA-E与NK受体之间的相互作用和HLA-E体外表达对NK细胞功能的影响奠定了基础。

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

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.