Expression and optimization of recombinant human coagulation factor X in HEK293 cells
10.19405/j.cnki.issn1000-1492.2025.09.003
- VernacularTitle:重组人凝血因子 X 在 HEK293 细胞中的表达及活性检测
- Author:
Jianing Feng
1
;
Sen Zou
2
;
Zelin Zhao
1
;
Xiaoxiao Li
1
;
Zhifei Zhang
1
;
Zhaoyong Yang
2
Author Information
1. College of Pharmacy，North China University of Science and Technology，Tangshan 063000
2. Institute of Medicinal Biotechnology，Chinese Academy of Medical Sciences and Peking Union Medical College，Beijing 100050
- Publication Type:Journal Article
- Keywords:
recombinant human coagulation factor X;
instantaneous expression;
expression optimization;
HEK293 cells;
coagulation factor X deficiency;
prothrombin time
- From:
Acta Universitatis Medicinalis Anhui
2025;60(9):1583-1590
- CountryChina
- Language:Chinese
-
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.
- Full text:202603161102387147重组人凝血因子X在HEK293细胞中的表达及活性检测_冯佳宁.pdf
