Construction of prokaryotic expression vector of human neuronal pentraxin 2 gene,and expression and purification of its recombinant protein

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.