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Published on: July 31, 2012
[Construction of recombinant eukaryotic expression vector of NKG2D gene and its expression in COS-7 cells]
Ying-xia Liu1, Guo-ling Hu, Min Liu
1Institute of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.
Objective:
To construct recombinant eukaryotic expression vector of NKG2D, and to examine its expression in COS-7 cells.
Methods:
Human NKG2D cDNA was obtained from peripheral blood mononuclear cells using RT-PCR, and then the target gene was cloned into pMD18-T vector. A eukaryotic expression plasmid containing target gene was constructed by DNA recombinant technique, and was confirmed by double restriction enzymes digestion and DNA sequence analysis. The recombinant plasmid with encapsuled lipofectamine was transfected into COS-7 cells, and the transfected COS- The 7 cells containing expressive target gene were confirmed by RT-PCR and flow cytometry.
Results:
The sequence of NKG2D cDNA obtained from the recombinant eukaryotic expression vector was identical with that published on GeneBank. The NKG2D gene was expressed successfully in COS-7 cells.
Conclusion:
The recombinant expression plasmid containing NKG2D gene is constructed successfully. After being transfected to COS-7 cells, the NKG2D protein is expressed by the engineering COS-7 cells line, which lays a foundation for further studying biological activity of NKG2D.
