Title: Construction of prokaryotic expression vector of human neuronal pentraxin 2 gene,and expression and purification of its recombinant protein
Abstract: Objective To construct the prokaryotic expression vector of human neuronal pentraxin 2(NPTX2) gene,induce the expression of the recombinant fusion protein in E.coli BL21,and purify the recombinant fusion protein.Methods The pReceiver-M15 plasmid containing full-length cDNA of NPTX2 was digested by restrictive enzyme EcoR I and Not I,and then cloned into the plasmid pGEX-4T-1 which contained the glutathione-s-transferase(GST).After restriction enzyme digestion identification,polymerase chain reaction(PCR) amplification and sequencing,the pGEX-4T-NPTX2 plasmid containing the correct target DNA fragments was obtained and transformed into E.coli BL21(DE3).By restriction enzyme digestion and sequencing,the positive transformed clones were identified.The expression of GST-NPTX2 fusion protein was induced with IPTG,and the products were analyzed by SDS-PAGE and Western blotting.The proteins were purified through affinity chromatography.Results The results of digestion and sequencing indicated that the recombinant prokaryotic vector pGEX-4T-NPTX2 was successfully constructed.After transformation with pGEX-4T-NPTX2 and induction with IPTG,the recombinant target protein of about 70kD was obtained by SDS-PAGE analysis,which was consistent with our anticipation.Western blotting suggested that the new band was purified recombinant GST-NPTX2 protein and possessed good immunogenicity.Conclusions The prokaryotic expression vector of human NPTX2 gene has been successfully constructed.The GST-NPTX2 fusion protein could be expressed in prokaryotic expression system of E.coli.Purified recombinant proteins are obtained through affinity chromatography,which is helpful for further study.
Publication Year: 2009
Publication Date: 2009-01-01
Language: en
Type: article
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