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目的 瞬时转染重组人凝血因子X(rhFX)到HEK293细胞，优化转染条件，体外构建高效表达rhFX的方法，检测rhFX活性。方法 pc DNA3.1-EGFP与F10基因构建真核表达载体pc DNA3.1-EGFP-FX并转染HEK293细胞，验证转染系统的有效性。pc DNA3.1与F10基因片段构建重组表达载体pc DNA3.1-FX，脂质体方法转染HEK293细胞，Western blot和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测细胞上清液中rhFX的表达。优化转染条件，ELISA测定rhFX的浓度;凝血酶原时间(PT)和发色底物法测定rhFX凝血生物活性。结果 在HEK293细胞上清液中成功表达rhFX。在细胞密度80%，质粒DNA与聚乙烯亚胺(PEI)的比例为1∶2，转染后第3天，rhFX表达量最高。ELISA检测3次上清液表达量最高为5.20 ng/mL。与对照组pc DNA3.1空载体转染收集的上清液相比，实验组rhFX的PT时间更短(P<0.000 1)，基于rhFX的发色底物法测得依度沙班的半数抑制浓度(IC50)为1.449 nmol/L，与文献报导的0.78 nmol/L处于同一数量级。结论 使用HEK293细胞成功表达出具有生物活性的rhFX蛋白，为进一步推动rhFX药物的研发奠定基础。
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 < 0. 0001) compared to control supernatant from pcDNA3. 1 empty vector-transfected cells. The half maximal inhibitory concentration(IC50) 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.
[1] Ichinose A,Osaki T,Souri M. Autoimmune coagulation factor X deficiency as a rare acquired hemorrhagic disorder:a literature review[J]. Thromb Haemost,2022,122(3):320-8. doi:10.1055/a-1496-8527.
[2] Peyvandi F,Auerswald G,Austin S K,et al. Diagnosis,therapeutic advances,and key recommendations for the management of factor X deficiency[J]. Blood Rev,2021,50:100833. doi:10.1016/j. blre. 2021. 100833.
[3] Escobar M A,Kavakli K. Plasma-derived human factor X concentrate for the treatment of patients with hereditary factor X deficiency[J]. Haemophilia,2024,30(1):59-67. doi:10. 1111/hae.14894.
[4] Santana J M,Brovman E Y. Prothrombin complex concentrates in cardiac surgery:where are we?[J]. Curr Opin Anaesthesiol,2022, 35(1):36-41. doi:10. 1097/ACO.0000000000001074.
[5] Payne J,Batsuli G,Leavitt A D,et al. A review of the pharmacokinetics,efficacy and safety of high-purity factor X for the prophylactic treatment of hereditary factor X deficiency[J]. Haemophilia,2022,28(4):523-31. doi:10. 1111/hae. 14570.
[6]李敏，吴日伟．凝血因子药物市场分析[J]．中国生物工程杂志，2017,37(5):133-9. doi:10. 13523/j. cb. 20170517.[6] Li M,Wu R W. The analysis of coagulation factors market[J].China Biotechnol,2017,37(5):133-9. doi:10. 13523/j. cb.20170517.
[7] Xue F,Zhao X,Sun J,et al. Pharmacokinetic,efficacy and safety evaluation of B-domain-deleted recombinant FVIII(SCT800)for prophylactic treatment in adolescent and adult patients with severe haemophilia A[J]. Haemophilia,2021,27(5):814-22.doi:10. 1111/hae. 14350.
[8] Shapiro A D,Chambost H,Ozelo M C,et al. Recombinant factor IX Fc for major surgery in hemophilia B:factor IX plasma activity levels and effective hemostasis[J]. Res Pract Thromb Haemost,2023,7(6):102169. doi:10. 1016/j. rpth. 2023. 102169.
[9]李敏．中国血液制品行业分析[J]．中国生物工程杂志，2016,36(9):119-25. doi:10. 13523/j. cb. 20160915.[9] Li M. The analysis of Chinese blood biologicals industry[J]. China Biotechnol,2016,36(9):119-25. doi:10. 13523/j. cb.20160915.
[10]朱跃跃，卞茂红，谢如锋．红细胞微粒的促凝血性质与小鼠急性肺损伤的关系[J]．安徽医科大学学报，2020,55(1):89-94. doi:10. 19405/j. cnki. issn1000-1492. 2020. 01. 019.[10] Zhu Y Y,Bian M H,Xie R F. Relationship between procoagulant properties of red blood cell-derived microparticles and acute lung injury in mice[J]. Acta Univ Med Anhui,2020,55(1):89-94. doi:10. 19405/j. cnki. issn1000-1492. 2020. 01. 019.
[11] Valentino L A,Kaczmarek R,Pierce G F,et al. Hemophilia gene therapy:first,do no harm[J]. J Thromb Haemost,2023,21(9):2354-61. doi:10. 1016/j. jtha. 2023. 06. 016.
[12] Camire R M. Blood coagulation factor X:molecular biology,inherited disease, and engineered therapeutics[J]. J Thromb Thrombolysis,2021,52(2):383-90. doi:10. 1007/s11239-021-02456-w.
[13]李心宇．抗O型口蹄疫病毒重组蛋白疫苗的研制及凝血因子X的构建和表达[D]．长春:吉林大学，2004. doi:10. 7666/d.Y966945.[13] Li X Y. The development of recombinant protein vaccine againsttype“O”of foot-and-mouth disease virus and theconstruction and expression of coagulation factor X. Changchun:Jilin University,2004. doi:10. 7666/d. Y966945.
[14] Feng Y,Ma J,Tang L V,et al. Characterization of a missense mutation in the catalytic domain and a splicing mutation of coagulation factor X compound heterozygous in a Chinese pedigree[J].Genes(Basel), 2021, 12(10):1521. doi:10. 3390/genes12101521.
[15] Siddiqui F,Hoppensteadt D,Jeske W,et al. Factor Xa inhibitory profile of apixaban,betrixaban,edoxaban,and rivaroxaban does not fully reflect their biologic spectrum[J]. Clin Appl Thromb Hemost, 2019, 25:1076029619847524. doi:10. 1177/1076029619847524.
基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.09.003
中图分类号:R927
引用信息:
[1]冯佳宁,邹森,赵泽林,等.重组人凝血因子X在HEK293细胞中的表达及活性检测[J].安徽医科大学学报,2025,60(09):1583-1590.DOI:10.19405/j.cnki.issn1000-1492.2025.09.003.
基金信息:
国家自然科学基金项目(编号:82373767、82301185)~~