Prokaryotic expression and purification of recombinant human Importin α and preparation of its polyclonal antibody

Objective To express recombinant Importin α and prepare anti-Importin α polyclonal antibody.Methods The Importin α gene was obtained by RT-PCR from the total RNA of human hepatoma cell HepG2 and cloned into the vetor pGEX-6P-1.The recombinant plasmid pGEX-6P-1-Importin α was transferred into E.coli BL21 and the GST-Importin α fusion protein was expressed by inducing with IPTG.The fusion protein was purified by glutathione sepharose 4B affinity chromatography.Mice were immunized with recombinant Importin α,and the titer of the polyclonal antiserum was detected by ELISA.Results The length of the RT-PCR product of Importin α was consistent with the expected.The purified GST-Importin α fusion protein was 83kD demonstrated by SDS-PAGE,mass spectrometry and western blot.Antiserum was obtained by immunizing in mice and had a high titer determined by ELISA.Conclusion The recombinant Importin α is prokaryotically expressed and purified,and polyclonal antibody is obtained.Our study lays the foundation for further research of importing system and its application.