Construction, Expression And Identification Of The Fusion Protein Consisting Of Soluble TNFR (P55) And IgGFc

Objective: To construct high efficiency prokaryotic expression vector which expresses the fusion protein consisting of soluble TNFR (P55) and IgGFc. Studying the immunological and biological activity of TNFR:IgGFc fusion protein in vitro, it will lay a good basis for in vivo observations. Methods: Complete extra-cellular domain of TNFR gene was amplified from human heart cDNA library by PCR, and then cloned into pGEM-T/IgGFc vector. With this template, no signal extra-cellular domain of TNFR and IgGFc fusion gene was amplified with high efficiency prokaryotic codons in the upstream primer, then subcloned into pUC19 vector. After sequencing, TNFR:IgGFc fusion gene was cloned into the prokaryotic expression vector pRsetA. After being transformed into E.coli BL21 (DE3), TNFR:IgGFc fusion gene was induced to express by IPTG .The fusion protein was characterized by ELISA and Western blot. The biological activity of the fusion protein in vitro was analysed by L929 cells. The fusion protein was also purified by FPLC MonoQ. Results: SDS-PAGE analysis showed a high expression rate of the products which constituted about 25 % of the total bacterial proteins. The fusion protein is in inclusion body forms. After renaturation, the fusion protein has the binding activity to TNF, and the biological activity of neutralizing TNF in vitro. Recombinant TNFR:IgGFc was purified to 70% purity by FPLC MonoQ. Conclusion: We have got the TNFR:IgGFc fusion protein from E.coli, which has the good immunological and biological activity. The purified protein would promote the further study on the mechanism of neutralizing TNF in vivo.