Cloning of human NKp44 gene and its expression and purification in E. coli

OBJECTIVE:To clone the gene of human NKp44 and express the recombinant human NKp44 in E.coli in order to provide a basis for further study of antitumor immune response of NK cells.METHODS:Peripheral blood mononuclear cells(PBMC)were prepared from a health volunteer's peripheral venous blood,cultured and introduced by IL-2.Total RNA was isolated from PBMC.A hNKp44 DNA fragment,with a length of about 860 bp,was amplified from the total RNA by nested PCR and cloned to plasmid pMD18-T,and the cloned DNA fragment was sequenced.The recombinant plasmid pMD18-T-hNKp44 was digested with EcoRⅠ and NcoⅠ,then hNKp44 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pET30a(+).The recombinant plasmid pET30a(+)-hNKp44 was identified by restriction analysis and transformed to E.coli BL21(DE3),and then its expression was induced by IPTG.The expressed product was identified by SDS-PAGE and Western Blotting.The expressed protein was purified by His·Bind Purification Kit.RESULTS:The length of DNA fragment amplified by nested PCR was consistent with that of hNKp44 cDNA.DNA sequencing of pMD18-T-hNKp44 revealed that the cloned DNA sequence was identical to that of reported hNKp44 cDNA.SDS-PAGE proved that expressed product,with a relative molecular weight of 35.4×103,contained about 40% of total somatic protein.Western Blot showed that the recombinant protein could specifically bind to anti-His·Tag antibody.The recombinant protein was obtained by purification with 95% of final purity and 40% of recovery rate.CONCLUSION:A recombinant bacterial strain for expressing hNKp44 is successfully constructed.