[Expression and activity identification of a fusion protein for promoting adenovirus infection efficiency of dendritic cells].

OBJECTIVE

To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purity the CT40L fusion protein and verify its antigenicity.

METHODS

Gene sequences of Coxsackie and adenovirus receptor (CAR), bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a(+) to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 (DE3) and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA.

RESULTS

The recombinant expression vector was verified correct by double digestion with Nco I and EcoR I. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity.

CONCLUSION

The prokaryotic expression plasmid pET42a-Ct40L was successfully constructed and expressed in E. coli, and the purified fusion protein was proved to have a good antigenicity.