Prokaryotic expression and antigenicity of nucleocapsid protein gene from human metapneumovirus in Beijing
Objective To express the nucleocapsid protein(N) gene of human metapneumovirus(hMPV) recently identified in Beijing in E.coli.Methods N gene was amplified from recombinant plasmid pUCm-N_1816 by PCR with primers and then sub-cloned into the pET30a(+) vector, a prokaryotic expression vector, after dual-enzyme digestion with BamHⅠ and EcoRⅠ. The pET30a-N_1816 was transformed into E.coli BL21(DE3) and expressed. Target protein was characterized by SDS-PAGE electrophoresis and Western blot.Results The recombinant plasmid pET30a-N_1816 has correct open reading frame confirmed by dual-enzyme digestion analysis and sequencing. The fusion protein 6·His-N was produced and with 20% expression level in terms of total bacterial protein after inducing by 1 mmol/L IPTG at 37℃ for 6 h. A unique protein band of approximate 45 kD was characterized by SDS-PAGE electrophoresis and purified by Co~2+ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the nucleocapsid protein of hMPV and to human sera. Conclusion The recombinant plasmid pET30a-N_1816 was successfully constructed. The N gene was highly expressed in prokaryotic system and the expressed N protein has specific antigenic activities. It can be used for the further studying of hMPV infections in Beijing.