Construction of pcDNA3.1(+)/A2E eukaryotic expression vector and its expression on K562 cell.
- Author:
Si-Xi LIU
1
;
Jian-Pei FANG
;
Hong-Gui XU
;
Guo-Hua CHEN
;
Shao-Liang HUANG
Author Information
1. Department of Pediatrics, Shenzhen Children Hospital, Shenzhen, 518026, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
DNA, Complementary;
genetics;
Eukaryotic Cells;
metabolism;
Flow Cytometry;
Gene Expression;
Genetic Vectors;
genetics;
HLA Antigens;
biosynthesis;
genetics;
HLA-A2 Antigen;
biosynthesis;
genetics;
Histocompatibility Antigens Class I;
biosynthesis;
genetics;
Humans;
K562 Cells;
Polymerase Chain Reaction;
RNA, Messenger;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2005;13(3):464-467
- CountryChina
- Language:Chinese
-
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.
