[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.
Liu
Si-Xi
SX
Department of Pediatrics, Shenzhen Children Hospital, Shenzhen, 518026, China.
Fang
Jian-Pei
JP
Xu
Hong-Gui
HG
Chen
Guo-Hua
GH
Huang
Shao-Liang
SL
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006680:HLA Antigens / Q000096:biosynthesis / Q000235:genetics*; D015789:HLA-A2 Antigen / Q000096:biosynthesis / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D000097625:HLA-E Antigens
D018076:DNA, Complementary; D006680:HLA Antigens; D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000097625:HLA-E Antigens