Objective    To construct an eukaryotic expression vector encoding a fusion gene consisting of enhanced green fluorescent protein (EGFP) and h

uman extracellular domain of CD40L (hecdCD40L)

and to 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 peripheral blood mononuclear cells (PBMC) by RT-PCR. The signal peptide of murine vascular endothelial cell growth factor receptor-2 (Sig

mKDR

) and EGFP were fused with or without hecdCD40L

and then the fused fragments was inserted into the eukaryotic expression vector pcDNA3.1(-) to construct pcDNA3.1 Sig

mKDR

EGFP (sEGFP) and pcDNA3.1 sEGFP-hecdCD40L. After transfection of COS-7 cells with the vectors by lipofectamine 2000

TM

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 or pcDNA3.1 sEGFP-hecdCD40L 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 to sEGFP or sEGFP-hecdCD40L by DCs were 28.6% and 56.9%

respectively

in 2 h 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 h after co-incubation with sEGFP-hecdCD40L protein. However

the sEGFP prot

ein had no such an effect on DCs.      Conclusion    The fusion protein consisting of Sig

mKDR

EGFP

hecdCD40L is expressed in the eukaryotic cells and secreted out of the cells. The secreting protein can be targeted to DCs

and then induce maturation of DCs

up-regulate costimulatory and MHC-Ⅱ molecules on DCs

and stimulate the IL-12 secretion of DCs.