Optimized production and coagulation activity of rhFVII in HEK293 cells for bleeding disorders

IF 1.8 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2026-03-01 Epub Date: 2025-12-03 DOI:10.1016/j.pep.2025.106867

Sen Zou , Xiaoxiao Li , Jiajun Liu , Shuai Fan , Zhaoyong Yang

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引用次数: 0

Abstract

Hemophilia, a genetic disorder characterized by impaired blood clotting, necessitates innovative treatments. This study optimized the expression of recombinant human coagulation factor VII (rhFVII) in HEK293 cells to enhance its therapeutic efficacy. The human FVII gene was cloned into the pcDNA3.1 vector, and transient transfection was performed with an optimized DNA-to-PEI ratio (1:2) at 70 % cell density. Stable FVII-expressing cell lines were selected with G418, confirmed via RT-PCR, and adapted to suspension culture. rhFVII expression was analyzed using SDS-PAGE, Western blot, and ELISA, while its coagulation activity was evaluated by prothrombin time tests. Transient transfection yielded rhFVII concentrations of 10.22 μg/L and clotting activity of 232.46 %. Stable monoclonal HEK293 cells in suspension produced rhFVII at 11.22 mg/L with maximum coagulation activity of 563.17 % and high stability and safety. The findings underscore the successful optimization of rhFVII expression in HEK293 cells, paving the way for advancements in biopharmaceutical production and improved treatment options for bleeding disorders. Future research should focus on in vivo validation and further refinement of culture conditions to maximize protein yield and stability.

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出血性疾病HEK293细胞rhFVII生成及凝血活性优化

血友病是一种以凝血功能受损为特征的遗传性疾病，需要创新的治疗方法。本研究通过优化重组人凝血因子VII （rhFVII）在HEK293细胞中的表达，提高其治疗效果。将人FVII基因克隆到pcDNA3.1载体中，在70%细胞密度下，以优化后的dna - pei比（1:2）瞬时转染。用G418筛选稳定表达fvii的细胞系，通过RT-PCR确认，并适应悬浮培养。采用SDS-PAGE、Western blot和ELISA检测rhFVII的表达，采用凝血酶原时间检测rhFVII的凝血活性。瞬时转染的rhFVII浓度为10.22 pg/L，凝血活性为232.46%。稳定的单克隆HEK293悬浮细胞在11.22 mg/L的浓度下产生rhFVII，最大凝血活性为563.17%，稳定性和安全性高。这些发现强调了rhFVII在HEK293细胞中表达的成功优化，为生物制药的进步铺平了道路，并改善了出血性疾病的治疗选择。未来的研究应集中在体内验证和进一步完善培养条件，以最大限度地提高蛋白质产量和稳定性。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.