Cloning of Human NKp30 gene and construction of its eukaryotic expression vector

Objective To clone the gene of human NKp30 and construct its eukaryotic expression vector in order to provide a basis for further study of anti-tumor immune response of NK cells.Methods Peripheral blood mononuclear cells(PBMC)were extracted from a health volunteer′s peripheral venous blood.Total RNA was isolated from PBMC.A hNKp30 DNA fragment,about 606 bp was amplified from the total RNA by PCR and cloned to plasmid pMD18-T,and the cloned DNA fragment was sequenced.The recombinant plasmid pMD18-T-hNKp30 was digested with XhoI、EcoRI,then hNKp44 fragmentwas isolated and inserted to the corresponding restriction site on eukaryotic expression vector pIRES2-EGFP.The recombinant expression plasmid was transfected into COS-7 cells.Results The length of DNA fragment amplified by PCR was consistent with that of hNKp30 cDNA.DNA sequencing of pMD18-T-hNKp30 revealed that the cloned DNA sequence was identical to that of reported hNKp30 cDNA.After the recombinant expression plasmid was transfected into COS-7 cells,hNKp30 was instantaneous expressed and transfected in vitro.Conclusion The eukaryotic expression vector can be constructed successfully by reconstruction technology,which may provide basis for the study of biological activity.