Construction And Characteristics Of SEGFP-CD40L Fusion Gene

[Objective] To construct an eukaryotic expression vector encoding a fusion gene consisting of enhanced green fluorescent protein (EGFP) and human extracellular domain of CD40L (hecdCD40L), and investigate the effects of the expression product of the fusion gene on phenotypes and function of human dendritic cells (DCs).[Methods] hecdCD40L was cloned from human PBMCs by RT-PCR. The signal peptide of murine vascular endothelial cell growth factor receptor-2 (SigmKDR) and EGFP were fused with or without hecdCD40L, and then the fused fragments was inserted into the eukaryotic expression vector pcDNA3.1(-) myc-6×His to construct pcDNA3.1 SigmKDR-EGFP-myc-6×His (sEGFP) and pcDNA3.1 sEGFP-hecdCD40L-myc-6×His. After transfection of COS-7 cells with the vectors by lipofectamine 2000TM, the monoclonal COS-7 cells stably expressing sEGFP or sEGFP-hecdCD40L fusion proteins were obtained via G418-resistant screening and sorting of flow cytometer (FACS). The expression efficiencies of the fusion genes were analyzed by FACS and the fusion proteins in the cells and supernatants were detected by Western Blotting, respectively. Uptake rates, phenotypic changes, and secretion of interleukin-12 (IL-12) in DCs were analyzed by FCAS and ELISA after co-incubation of DCs with the sEGFP or sEGFP-hecdCD40L proteins depurated and concentrated by Ni-NTA Agarose and ultrafiltration.[Results] After the transfection of the pcDNA3.1 sEGFP-myc-6×His or pcDNA3.1 sEGFP-hecdCD40L-myc-6×His into COS-7 cells, the monoclonal COS-7 cells expressing the fusion proteins were obtained by G418-resistant screening and flow cytometer sorting, in which the cells expressed EGFP reached up to 95% by the analysis of FACS. Western blotting showed the expression of the fusion proteins in the cells and its supernatants. In addition, uptake rates of sEGFP or sEGFP-hecdCD40L by DCs were 28.6% and 56.9%, respectively, in 2 hrs after co-incubation of sEGFP or sEGFP-hecdCD40L with DCs. The increased expression of CD80, CD83, and HLA-DR and secretion of IL-12 on DCs were detected by FCAS and ELISA in 24 hrs after co-incubation with sEGFP-hecdCD40L protein. However, the sEGFP protein had no such an effect on DCs.[Conclusion] The fusion protein consisting of SigmKDR, EGFP, hecdCD40L can be expressed in the eukaryotic cells and secreted out of the cells. The secreting protein can be targeted to DCs, inducing maturation of DCs, up-regulating costimulatory and MHC-II molecules on DCs, and stimulating the IL-12 secretion of DCs.