Construction of mammalian cell expression vector of human CD154 gene from active peripheral blood mononuclear cell and analysis of its sequence

ObjectiveTo obtain mammalian cell expression vector of human CD154 gene MethodsA 820 bp cDNA fragment was amplified by RT PCR method from total RNA of human peripheral blood mononuclear cell(PBMC) activated with 10 ng/mL PMA and 1 μg/mL PHA for 8 hours The fragment was cloned into pcDNA3 1(+) plasmids The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes Bam H Ⅰ and Eco R Ⅰ and sequenced by Sangers dideory mediated chain termination ResultsThis cDNA fragment included 820 bp entire coding region and a part of the 3 non coding region The recombinant mammalian cell expression vector of pcDNA3 1(+)/hCD154 was constructed, the sequence of the insert was identical to the published sequence encoding human CD154 antigen ConclusionThe recombinant mammalian cell expression vector of pcDNA3 1(+)/hCD154 was successfully constructed