Construction of Human CD154 Gene Eukaryotic Green Fluorescent Protein Expression Vector pEGFP-N3-CD154

Objective To construct recombined eukaryotic green fluorescent protein expression vector pEGFP-N3-CD154.Methods Total RNA was extracted from peripheral blood mononuclear cells(PBMCs) which were activated with PHA in vitro.The amplification of CD154 cDNA open reading frame sequence was conducted using PT-PCR.The fragment of CD154 cDNA was cloned into pGEM T Easy vector,and then cloned directly into pEGFP-N3.The recombinant plasmid pEGFP-N3-CD154 was identified by restriction enzyme,PCR,and DNA sequence analyzing technique.Results CD154 gene sequence in the recombined vector was(identical) with the GenBank(NM-000074).Conclusion The recombined eukaryotic green fluorescent protein expression vector pEGFP-N3-CD154 can be obtained.The results provide basis for the further studies of the biological significance of human CD154 gene transfected cell in tumor immunotherapy.