〔Abstract〕 Objective To perform transient transfection of recombinant human Factor X(rhFX) into HEK293 cells,to optimize transfection parameters,to develop a high-yield in vitro expression system for rhFX production,and to assess the biological activity of expressed rhFX. Methods The eukaryotic expression vector pcDNA3. 1-EGFP-FX was constructed by inserting the F10 gene into pcDNA3. 1-EGFP and subsequently transfected into HEK293cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3. 1-FX was generated through ligation of the F10 gene fragment with pcDNA3. 1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Transfection conditions were systematically optimized,and rhFX concentration was quantified by enzyme-linked immunosorbent assay(ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time(PT) assay and chromogenic substrate method. Results rhFX was successfully expressed in the supernatant of HEK293 cells. rhFX was successfully expressed in the supernatant of HEK293 cells. The highest expression level of rhFX was achieved on the third day after transfection when the cell density was 80% and the ratio of plasmid DNA to polyethyleneimine(PEI) transfection reagent was 1 ∶ 2.Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5. 20 ng/mL in the supernatant.The prothrombin time(PT) of rhFX-containing supernatant was significantly shorter(P 50) of etoxaban was determined to be 1. 449 nmol/L using the chromogenic substrate method based on rhFX,which was in the same order of magnitude as the reported 0. 78 nmol/L in the literature. Conclusion HEK293cells successfully express biologically active recombinant human Factor X(rhFX) protein,laying a foundation for advancing the development of rhFX-based therapeutics.