Construction of pcDNA3.1(+)/A2E eukaryotic expression vector and its expression on K562 cell / 中国实验血液学杂志
Journal of Experimental Hematology
; (6): 464-467, 2005.
Article
in Zh
| WPRIM
| ID: wpr-356536
Responsible library:
WPRO
ABSTRACT
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.
Full text:
1
Index:
WPRIM
Main subject:
Recombinant Fusion Proteins
/
RNA, Messenger
/
Transfection
/
Histocompatibility Antigens Class I
/
Gene Expression
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HLA-A2 Antigen
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Polymerase Chain Reaction
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Cloning, Molecular
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DNA, Complementary
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K562 Cells
Limits:
Humans
Language:
Zh
Journal:
Journal of Experimental Hematology
Year:
2005
Type:
Article