[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.