The aim of the present study was to construct a fast-acting， eukaryotic expression vector in eukaryotic cells based on transmembrane-tumor necrosis factor?α (TM?TNF?α) structure. Two types of recombinant eukaryotic expression vectors were constructed， pcDNA3.1-TM-enterokinase-TNF?α and pcDNA3.1?TM?Factor?Xa?TNF?α， according to the TNF?α transmembrane segments. Following the generation of these vectors， mouse embryonic 3T3 fibroblasts were transfected and reverse transcription?polymerase chain reaction and western blotting analyses were used to analyze mTNF?α mRNA and protein expression levels， respectively， in total cellular protein extracts and extracellular fluid. The biological activity of TNF-α in the extracellular fluid was then measured using an MTT assay. The vectors were successfully constructed， and mRNA and fusion proteins were detected in the 3T3 cells. Among the fusion proteins， the one observed in pcDNA3.1-TM-FactorXa-TNF-α-transfected 3T3 cells remained as a transmembrane protein. In addition， treatment of L929 cells with TNF?α derived extracellular fluid samples from pcDNA3.1?TM?FactorXa?TNF?α?transfected 3T3 cells was associated with a dose?dependent reduction in in cell?specific activity. The results indicate that proteins expressed using pcDNA3.1?TM?FactorXa?TNF?α vectors form transmembrane proteins. In addition， the results indicate that， only when coupled with FactorXa activity， the extracellular region of TM?TNF?α forms s?TNF?α， and the controlled expression of the fusion protein is initiated.