Title: Cloning of human NKp46 gene and its expression and purification in E.coli
Abstract: Objective:To clone the gene of human NKp46,express and purify the recombinant human NKp46 in E.coli.Methods:A hNKp46 DNA fragment,with a length of about 900?bp,was amplified from the total RNA of peripheral blood mononuclear cells by RT-PCR and cloned to plasmid pMD18-T,and then the cloned DNA fragment was sequenced.The recombinant plasmid pMD18-T-hNKp46 was digested with EcoRⅠ and NcoⅠ,andthen hNKp46 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pET30a(+).The recombinant plasmid pET30a(+)-hNKp46 was identified by enzymogram 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,and the expressed protein was purified by His·Bind Purification Kit.Results:The length of DNA fragment amplified by RT-PCR was consistent with that of hNKp46 cDNA.DNA sequencing of pMD18-T-hNKp46 revealed that the cloned DNA sequence was identical to that of reported hNKp46 cDNA.SDS-PAGE proved that expressed product,with a relative molecular weight of 38.5?kD,contained about 40% of total somatic protein.Western Blotting showed that the recombinant protein could specifically bind to anti-His·Tag antibody.The recombinant protein was obtained by purification with 95.5% final purity and 40% recovery rate.Conclusion:A recombinant bacterial strain for expressing hNKp46 is successfully constructed,and its recombinant protein is purified.
Publication Year: 2006
Publication Date: 2006-01-01
Language: en
Type: article
Access and Citation
AI Researcher Chatbot
Get quick answers to your questions about the article from our AI researcher chatbot