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Article(id=1308841067395510517, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-1434, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1764604800000, receivedDateStr=2025-12-02, revisedDate=1768665600000, revisedDateStr=2026-01-18, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981627558, onlineDateStr=2026-09-21, pubDate=1778515200000, pubDateStr=2026-05-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981627558, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981627558, creator=13701087609, updateTime=1789981627558, updator=13701087609, issue=Issue{id=1308840985296204417, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='5', pageStart='1435', pageEnd='1880', issueExtLink='null', onlineDate='null', pubDate='1778515200000', pubDateStr='2026-05-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981607984, creator='13701087609', updateTime=1789984207975, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851890515968861, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851890515968862, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1712, endPage=1718, ext={EN=ArticleExt(id=1308841067613614326, articleId=1308841067395510517, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Stable expression and functional identification of recombinant human coagulation factor Ⅸ in HEK293 cells, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

This study aimed to construct a stable cell line of human embryonic kidney epithelial cells 293 (HEK293) expressing recombinant human coagulation factor Ⅸ (rhFⅨ) and to identify the procoagulant biological activity of the expressed product. The recombinant plasmid pcDNA3.1-FⅨ was transfected into HEK293 cells, and a monoclonal stable transfection cell line was obtained through geneticin (G418) pressure screening. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine whether the FⅨ gene was transcribed and expressed in the monoclonal stable transfection cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to identify the expression of rhFⅨ; the concentration of rhFⅨ was determined by bicinchoninic acid assay (BCA), and the procoagulant activity of rhFⅨ was determined by activated partial thromboplastin time (APTT). The results showed that after G418 (200 µg·mL-1) pressure screening, 5 monoclonal stable cell lines could be normally transcribed, and the protein expression could be achieved through the transcription-translation process; the protein in the cell supernatant was purified and showed a single band of rhFⅨ; the expression level of rhFⅨ reached (1.58 ± 0.10) mg·L-1, and the maximum procoagulant activity was (66.8 ± 0.5)%. The established HEK293 monoclonal stable transfection cell line can efficiently and stably express rhFⅨ and has good procoagulant activity, providing a cell source for promoting the pharmacological evaluation and preclinical research of rhFⅨ.

, authors=Qian WANG1, Sen ZOU2, Jia-nian ZHANG1, Zi-tao ZHANG1, Jia-jun LIU1, Li-hua ZHU1, *, Zhao-yong YANG2, *, authorsList=Qian WANG, Sen ZOU, Jia-nian ZHANG, Zi-tao ZHANG, Jia-jun LIU, Li-hua ZHU, Zhao-yong YANG, authorCompany=null, correspAuthors=Li-hua ZHU, Zhao-yong YANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1308841071132635395, articleId=1308841067395510517, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=重组人凝血因子Ⅸ在HEK293细胞中的稳定表达与功能鉴定, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本研究为构建表达重组人凝血因子Ⅸ (recombinant human coagulation factor Ⅸ, rhFⅨ) 的人源胚胎肾上皮细胞293 (human embryonic kidney 293 cells, HEK293) 稳定细胞株, 并鉴定表达产物的促凝血生物学活性, 将重组质粒pcDNA3.1-FⅨ转染HEK293细胞, 经遗传霉素(geneticin, G418) 压力筛选获得单克隆稳定转染细胞株。逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR) 鉴定F9基因在单克隆稳定转染细胞株中是否发生转录表达, 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 和Western blot鉴定rhFⅨ的表达; 二辛可宁酸法(bicinchoninic acid assay, BCA) 测定rhFⅨ的浓度, 活化部分凝血活酶时间(activated partial thromboplastin time, APTT) 测定rhFⅨ的促凝血活性。结果表明, G418 (200 µg·mL-1) 加压筛选后获得5株单克隆稳定细胞株能够正常转录, 且可通过转录翻译过程实现蛋白表达; 细胞上清液蛋白纯化后呈现rhFⅨ单一条带; rhFⅨ表达量最高达到(1.58 ± 0.10) mg·L-1, 凝血活性最高为(66.8 ± 0.5)%。所建立的HEK293单克隆稳定转染细胞株能够高效、稳定地表达rhFⅨ, 且具有良好的促凝活性, 为推进rhFⅨ的成药性评价与临床前研究提供了细胞来源。

, authors=王倩1, 邹森2, 张甲年1, 张子涛1, 刘佳骏1, 朱丽华1, *, 杨兆勇2, *, authorsList=王倩, 邹森, 张甲年, 张子涛, 刘佳骏, 朱丽华, 杨兆勇, authorCompany=null, correspAuthors=朱丽华, 杨兆勇, authorNote=

#

共同第一作者.

作者贡献:

王倩、邹森负责实验设计、实验开展、数据收集及文章撰写; 张甲年、张子涛、刘佳骏参与前期文献调研; 朱丽华、杨兆勇负责论文指导与技术支持, 统筹研究全过程。

, correspAuthorsNote=
朱丽华, Tel: 13932554697, E-mail:
杨兆勇, Tel: 18513385316, E-mail:
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A: The blank control group (pcDNA3.1-FⅨ) shows black and white color; B: Fluorescence color development in the blank control group; C: Black and white color development in the transient transfection group (pcDNA3.1-EGFP-FⅨ); D: Fluorescence coloration in the transient transfection group. Scale bar: 50 μm. FⅨ: Coagulation factor Ⅸ, figureFileSmall=HCwKJjxOeO0Y3RzNuWYZ+A==, figureFileBig=Nyn4FYy+AkAcJAyh9Om1QQ==, tableContent=null), ArticleFig(id=1308841076123857209, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841067395510517, language=EN, label=null, caption=null, figureFileSmall=RfTC8a1hayViE3nG1QlGgA==, figureFileBig=cktNKK6ZzCzgA1fP6RGOYA==, tableContent=null), ArticleFig(id=1308841076190966074, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841067395510517, language=CN, label=Fig.2, caption=Screening and identification of monoclonal stably transfected cell lines. 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A: Growth curves of normal HEK293 cells and monoclonal stably transfected cell lines; B: Cell viability of normal HEK293 cells and monoclonal stable transfected cell lines. ns: P > 0.05, figureFileSmall=e31/WpWVQAWP31esCl7wvg==, figureFileBig=cXrnDAh06t0MusCmwuArVg==, tableContent=null), ArticleFig(id=1308841076451012925, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841067395510517, language=EN, label=null, caption=null, figureFileSmall=ZFFp4G4btdp0vZuSRlY0/w==, figureFileBig=xcywzSjDlbYug4HNobNDuQ==, tableContent=null), ArticleFig(id=1308841076513927486, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841067395510517, language=CN, label=Fig.4, caption=Expression of the monoclonal stably transfected cell line rhFⅨ. 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重组人凝血因子Ⅸ在HEK293细胞中的稳定表达与功能鉴定
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王倩 1 , 邹森 2 , 张甲年 1 , 张子涛 1 , 刘佳骏 1 , 朱丽华 1, * , 杨兆勇 2, *
药学学报 | 研究论文 2026,61(5): 1712-1718
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药学学报 |研究论文 2026 , 61 (5) : 1712 -1718
重组人凝血因子Ⅸ在HEK293细胞中的稳定表达与功能鉴定
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王倩1, 邹森2, 张甲年1, 张子涛1, 刘佳骏1, 朱丽华1, * , 杨兆勇2, *
作者信息
  • 1华北理工大学, 河北 唐山 063000
  • 2中国医学科学院医药生物技术研究所, 北京 100050
通讯作者:
朱丽华, Tel: 13932554697, E-mail:
杨兆勇, Tel: 18513385316, E-mail:
作者简介:

#

共同第一作者.

作者贡献:

王倩、邹森负责实验设计、实验开展、数据收集及文章撰写; 张甲年、张子涛、刘佳骏参与前期文献调研; 朱丽华、杨兆勇负责论文指导与技术支持, 统筹研究全过程。

Stable expression and functional identification of recombinant human coagulation factor Ⅸ in HEK293 cells
Qian WANG1, Sen ZOU2, Jia-nian ZHANG1, Zi-tao ZHANG1, Jia-jun LIU1, Li-hua ZHU1, * , Zhao-yong YANG2, *
Affiliations
  • 1North China University of Science and Technology, Tangshan 063000, China
  • 2Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China
出版时间: 2026-05-12 doi: 10.16438/j.0513-4870.2025-1434
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摘要
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本研究为构建表达重组人凝血因子Ⅸ (recombinant human coagulation factor Ⅸ, rhFⅨ) 的人源胚胎肾上皮细胞293 (human embryonic kidney 293 cells, HEK293) 稳定细胞株, 并鉴定表达产物的促凝血生物学活性, 将重组质粒pcDNA3.1-FⅨ转染HEK293细胞, 经遗传霉素(geneticin, G418) 压力筛选获得单克隆稳定转染细胞株。逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR) 鉴定F9基因在单克隆稳定转染细胞株中是否发生转录表达, 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 和Western blot鉴定rhFⅨ的表达; 二辛可宁酸法(bicinchoninic acid assay, BCA) 测定rhFⅨ的浓度, 活化部分凝血活酶时间(activated partial thromboplastin time, APTT) 测定rhFⅨ的促凝血活性。结果表明, G418 (200 µg·mL-1) 加压筛选后获得5株单克隆稳定细胞株能够正常转录, 且可通过转录翻译过程实现蛋白表达; 细胞上清液蛋白纯化后呈现rhFⅨ单一条带; rhFⅨ表达量最高达到(1.58 ± 0.10) mg·L-1, 凝血活性最高为(66.8 ± 0.5)%。所建立的HEK293单克隆稳定转染细胞株能够高效、稳定地表达rhFⅨ, 且具有良好的促凝活性, 为推进rhFⅨ的成药性评价与临床前研究提供了细胞来源。

血友病B  /  重组人凝血因子Ⅸ  /  HEK293单克隆细胞株  /  稳定表达  /  凝血活性

This study aimed to construct a stable cell line of human embryonic kidney epithelial cells 293 (HEK293) expressing recombinant human coagulation factor Ⅸ (rhFⅨ) and to identify the procoagulant biological activity of the expressed product. The recombinant plasmid pcDNA3.1-FⅨ was transfected into HEK293 cells, and a monoclonal stable transfection cell line was obtained through geneticin (G418) pressure screening. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine whether the FⅨ gene was transcribed and expressed in the monoclonal stable transfection cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to identify the expression of rhFⅨ; the concentration of rhFⅨ was determined by bicinchoninic acid assay (BCA), and the procoagulant activity of rhFⅨ was determined by activated partial thromboplastin time (APTT). The results showed that after G418 (200 µg·mL-1) pressure screening, 5 monoclonal stable cell lines could be normally transcribed, and the protein expression could be achieved through the transcription-translation process; the protein in the cell supernatant was purified and showed a single band of rhFⅨ; the expression level of rhFⅨ reached (1.58 ± 0.10) mg·L-1, and the maximum procoagulant activity was (66.8 ± 0.5)%. The established HEK293 monoclonal stable transfection cell line can efficiently and stably express rhFⅨ and has good procoagulant activity, providing a cell source for promoting the pharmacological evaluation and preclinical research of rhFⅨ.

hemophilia B  /  recombinant human coagulation factor Ⅸ  /  HEK293 monoclonal cell line  /  stable expression  /  coagulation activity
王倩, 邹森, 张甲年, 张子涛, 刘佳骏, 朱丽华, 杨兆勇. 重组人凝血因子Ⅸ在HEK293细胞中的稳定表达与功能鉴定. 药学学报, 2026 , 61 (5) : 1712 -1718 . DOI: 10.16438/j.0513-4870.2025-1434
Qian WANG, Sen ZOU, Jia-nian ZHANG, Zi-tao ZHANG, Jia-jun LIU, Li-hua ZHU, Zhao-yong YANG. Stable expression and functional identification of recombinant human coagulation factor Ⅸ in HEK293 cells[J]. Acta Pharmaceutica Sinica, 2026 , 61 (5) : 1712 -1718 . DOI: 10.16438/j.0513-4870.2025-1434
正文
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血友病B, 即凝血因子Ⅸ (coagulation factor Ⅸ, FⅨ) 缺乏症, 是一种X连锁隐性疾病[1], 也是最常见的出血性疾病之一; 其临床特征是关节、肌肉和内脏自发性或长期出血。血友病B通常与关节反复出血引起的致残关节病有关[2], 有潜在的生命危险。该类患者根据患病程度分为三类, 即症状严重(FⅨ活性< 1%), 症状中等(FⅨ活性在1%~5%之间) 和症状轻度(FⅨ活性在5%~40%之间[3])。血友病B的临床治疗主要依靠新鲜血浆中的FⅨ, 但这些制剂中FⅨ的浓度较低, 无法满足临床需求[4]。
FⅨ是一种在肝脏合成的依赖维生素K的糖蛋白[5], 成熟过程需要多个复杂的翻译后修饰, 包括12个谷氨酸残基的γ-羧化、糖基化、残基的羟基化和磷酸化[6,7]。血浆中的成熟FⅨ蛋白是一种由415个氨基酸组成的单链蛋白质, 分子质量约为57 kDa。F9由多个结构域组成: N-末端GLA结构域、短疏水序列、两个EGF结构域、连接区、激活肽和C-末端丝氨酸蛋白酶结构域。钙离子对于GLA和EGF结构域的正确折叠是必不可少的, 其具有良好的钙结合位点[8,9]; 此外, 11个二硫键稳定了F9分子的整体结构[10]。
HEK293细胞来源于人类, 具有与人类合成自身蛋白类似的翻译后修饰程序, 是一种被授权用于生产治疗性重组蛋白的细胞系[11]。基于高转染效率及良好的瞬时表达特性, HEK293细胞系被普遍应用于治疗性蛋白的快速筛选与表达工艺开发[12], 加之能够适应无血清悬浮培养[13], 该细胞已成为大规模生产人重组蛋白的关键宿主。本研究选择HEK293细胞作为rhFⅨ的表达宿主, 聚焦于pcDNA3.1-FⅨ重组载体的构建, 筛选稳定表达rhFⅨ的HEK293单克隆细胞株, 对其表达水平及促凝血功能进行系统性评价, 为推进该蛋白的后续药物开发奠定基础。
材料与方法
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质粒及细胞  pcDNA3.1-FⅨ质粒和pcDNA3.1-EGFP-FⅨ质粒均由中国医学科学院医药生物技术研究所提供。人胚肾细胞系HEK293细胞购自美国典型培养物保藏中心(American Type Culture Collection, ATCC)。
主要试剂  Eagle必需培养基(Eagle's minimum essential medium, EMEM, 批号: 80203251) 购自美国ATCC; 0.25%胰蛋白酶(批号: 3066349)、胎牛血清(批号: N2785574P) 均购自美国Gibco公司; 聚乙烯亚胺(polyethyleneimine, PEI, 批号: 543794) 转染试剂购自美国MCE公司; PVDF膜(批号: FFP32)、G418 (批号: A315250901)、BCA蛋白浓度测定试剂盒(增强型) (批号: A224250708) 均购自上海碧云天生物技术有限公司; 小鼠抗6×His Tag单克隆抗体(批号: L123AA2022)、辣根过氧化物酶标记山羊抗小鼠IgG (批号: K603AA035) 均购自上海生工生命科学有限公司; One Step SuperRT-PCR Mix Kit (批号: 14231222001)、总RNA提取试剂盒(Solarbio, 批号: 240006003) 均购自北京索莱宝科技有限公司; 无内毒素质粒小提中量快速提取试剂盒(批号: B0821B) 购自天根生化科技(北京) 有限公司; 考马斯亮蓝快速染液(批号: 039C1196) 购自上海雅酶生物医药科技有限公司; TBST速溶颗粒(批号: 7209030551) 购自北京兰博利德商贸有限公司; APTT试剂盒(批号: 325032) 购自武汉中太生物技术有限公司; 标准血浆(批号: NP703-2)、乏Ⅸ因子血浆(批号: DPF09-12) 均购自上海船夫生物技术有限公司; DNA测序、基因和引物合成均由北京六合华大基因科技有限公司完成。
HEK293细胞培养及pcDNA3.1-EGFP-FⅨ转染  HEK293细胞在37 ℃、5% CO2的培养箱中贴壁培养。细胞融合度达到约90%时, 进行传代: 0.25%胰酶消化3 min, 完全培养基(含10%胎牛血清的EMEM基础培养基) 终止反应, 1 200 r·min-1离心3 min收集细胞。重悬离心后的细胞沉淀, 调整每毫升细胞为106个后接种至新培养瓶。细胞生长至对数生长期进行转染: 转染前2 h, 将培养基更换为无血清的EMEM基础培养基; 取10 μg经0.22 μm滤膜除菌的pcDNA3.1-EGFP-FⅨ质粒, 与1 mL EMEM培养基轻柔混匀; 另取25 μL PEI, 与1 mL EMEM培养基振荡混匀。将上述两种溶液等体积混合, 室温孵育30 min以形成DNA-PEI复合物。将2 mL复合物逐滴加入细胞培养瓶, 置于37 ℃、5% CO2条件下继续培养。以转染pcDNA3.1-FⅨ质粒的细胞为阴性对照, 转染流程同实验组, 转染后72 h观察并记录细胞荧光强度。
pcDNA3.1-FⅨ稳定转染及单克隆细胞株筛选  EMEM完全培养基重悬HEK293细胞, 以每毫升104个细胞将HEK293细胞接种于24孔板。24 h后, 更换含不同G418浓度梯度(0、100、200、300、400、500、600、700和800 µg·mL-1) 的完全培养基, 每个梯度重复3个复孔; 将14天左右能够杀死所有HEK293细胞的最小G418浓度作为最适宜的筛选浓度。
pcDNA3.1-FⅨ转染HEK293细胞48 h后, 更换为含有G418 (筛选浓度为200 µg·mL-1) 的EMEM完全培养基, 继续培养14天获得抗性菌落, 期间2~3天更换新鲜培养基。抗性细胞株经0.25%胰酶消化后进行细胞计数, 调整细胞浓度为每毫升960个并定容至96 mL, 以每孔100 μL的体系接种96孔板。将孔板置于37 ℃、5% CO2培养箱中过夜培养; 24 h后于倒置显微镜下观察, 标记仅含单个细胞的孔。后续每3天更换含G418的新鲜EMEM完全培养基, 待孔内单细胞增殖形成克隆且细胞密度达孔底80%时, 依次将细胞传代至24孔板、6孔板、25 cm2培养瓶及75 cm2培养瓶, 后续培养、传代及冻存操作同常规HEK293细胞。筛选5株促凝血活性较好的单克隆稳定转染细胞株, 进行扩大培养与连续传代。
rhFⅨ单克隆稳定转染细胞株的鉴定及活力检测  提取单克隆稳定转染细胞株总RNA为模板, 按照RT-PCR试剂盒说明书扩增F9基因。单克隆稳转株活力检测: 选取上述5株单克隆稳定转染细胞株经传代后进行增殖分析: 培养的第1~6日内每日计数, 并检测细胞活率, 以培养时间和细胞数量/细胞活率为变量绘制生长曲线。
rhFⅨ蛋白纯化、SDS-PAGE和Western blot检测  His Tag Ni-NTA树脂亲和层析法纯化rhFⅨ。首先以低浓度咪唑(50 mmol·L-1 NaH2PO4、300 mmol·L-1 NaCl、10 mmol·L-1咪唑、10%甘油, pH 8.0) 缓冲液平衡树脂, 将细胞上清与含蛋白酶抑制剂的同类缓冲液共孵育2 h后上柱, 依次采用中、高浓度咪唑(50 mmol·L-1 NaH2PO4、300 mmol·L-1 NaCl、20 mmol·L-1咪唑、10%甘油, pH 8.0; 50 mmol·L-1 NaH2PO4、300 mmol·L-1 NaCl、250 mmol·L-1咪唑、10%甘油, pH 8.0) 缓冲液进行洗涤和洗脱, 获得纯化蛋白。将纯化rhFⅨ样本与5× loading buffer按体积比4∶1混合, 煮沸6 min。SDS-PAGE电泳: 参数设置恒压130 V, 时间50 min, 使用考马斯亮蓝染液染色并凝胶分析。Western blot检测: 转膜时参数设置恒流350 mA, 90 min快速转膜; 摇床上5%脱脂奶粉室温封闭2 h; 一抗4 ℃孵育过夜, 二抗室温孵育1 h, 结束后立即显影。
rhFⅨ浓度检测  BCA试剂盒测定蛋白浓度: 依试剂盒说明绘制标准曲线。分别收集连续传代10次细胞、冻存复苏细胞的培养上清液各50 mL, 经浓缩纯化至200 μL; 取rhFⅨ样品20 μL加入酶标孔, 添加200 μL工作液, 37 ℃孵育30 min, 于A562 nm波长读取吸光度值, 代入标准曲线计算蛋白浓度。重复独立制备3次单克隆稳转细胞株的rhFⅨ样本检测浓度。以转染pcDNA3.1空质粒的HEK293细胞上清为阴性对照。
rhFⅨ凝血生物活性检测  rhFⅨ凝血生物活性的检测方法参照2020年版《中华人民共和国药典》[14]进行。标准人血浆配制系列浓度标准品, 以凝血活性百分比的对数值为X轴, 相应凝固时间的对数值为Y轴, 绘制标准曲线。分别取25 μL上述纯化后的rhFⅨ样品及空白对照样本, 各与25 μL乏FⅨ血浆混匀; 加入50 μL APTT试剂后, 于半自动凝血分析仪37 ℃孵育3 min, 加入50 μL预热CaCl2试剂, 记录体系凝固时间。将凝血时间对数值代入标准曲线方程, 计算rhFⅨ相对标准血浆的百分比活性。以pcDNA3.1空载体质粒转染细胞的上清液作为阴性对照。
统计学分析  图表绘制和统计处理均由GraphPad Prism 9.5.0完成。呈正态分布的数据表示为(均值±标准差)。多组均值的差异性通过单因素方差分析(one-way ANOVA) 进行判断, 并运用Dunnett's法对实验组与对照组做进一步比较。P < 0.05表示差异有统计学意义。
结果
收起
1　pcDNA3.1-EGFP-FⅨ质粒瞬时转染HEK293细胞
为验证真核蛋白表达系统的有效性, 本研究将pcDNA3.1-EGFP-FⅨ质粒转染至HEK293细胞, 转染72 h后进行荧光观察, 测得转染效率为(41.7 ± 0.5)%。实验结果表明: 转染pcDNA3.1-FⅨ质粒的阴性对照组(图1A、B) 细胞无荧光信号检出, 转染组(图1C、D) 细胞可见显著绿色荧光, 表明EGFP-FⅨ融合蛋白成功表达。
2　pcDNA3.1-FⅨ单克隆细胞株的筛选及鉴定
在本实验中, G418筛选单克隆的最适宜浓度为200 µg·mL-1; 加压筛选获得20株单克隆稳定转染细胞株。依据上清中rhFⅨ蛋白的凝血时间(图2A), 挑选5株生长状态好、凝血活性高的单克隆稳定转染细胞株(FⅨ-17、FⅨ-28、FⅨ-35、FⅨ-57、FⅨ-62) 继续扩大培养。提取单克隆稳定转染细胞株总RNA, RT-PCR产物经1%琼脂糖凝胶, 目的片段位于1 200 bp附近(图2B), 提示F9基因在HEK293单克隆稳定株中能够正常转录, 且可通过转录翻译过程实现蛋白表达。
3　rhFⅨ单克隆稳定转染细胞株的活力检测
为评估单克隆稳定转染细胞株的活力, 分别对传代后的5株单克隆稳定转染细胞株的生长速度和细胞活力进行检测。结果显示, 稳定转染株的增殖速率(图3A) 及其活率(图3B) 与正常HEK293细胞相比无显著差异(P > 0.05)。
4　rhFⅨ蛋白纯化、SDS-PAGE和Western blot检测
收集5株rhFⅨ单克隆稳定转染细胞株培养上清并经镍柱纯化, SDS-PAGE (图4A) 和Western blot (图4B) 显示, 纯化后Elution中rhFⅨ均为特异性条带, 分子质量约为57 kDa (与预期相符), 表明rhFⅨ蛋白成功表达。
5　单克隆稳定转染细胞株表达rhFⅨ的浓度检测
分别收集5株单克隆稳定转染细胞株连续传代10次及冻存复苏后的培养基上清, 进行3次独立重复实验测定rhFⅨ的表达浓度。依据BCA标准曲线(R2 = 0.995 2) (图5A) 计算可得, 5株连续传代后的单克隆稳定转染细胞株产生的rhFⅨ浓度分别为(1.15 ± 0.05)、(1.47 ± 0.04)、(1.58 ± 0.10)、(0.95 ± 0.03) 和(1.34 ± 0.04) mg·L-1, 5株单克隆稳定转染细胞株在续传代与冻存复苏后, 其rhFⅨ的表达量未呈现显著性差异(P > 0.05) (图5B)。
6　单克隆稳转细胞株表达rhFⅨ的促凝血活性测定
一期凝固法检测rhFⅨ的促凝血生物学活性。基于标准血浆的活性标准曲线(图6A) 测定5株单克隆稳定转染细胞株经连续传代10次细胞及冻存复苏后的促凝血活性。结果显示, 对照组细胞上清促凝血时间大于120 s (图6B); 5株单克隆稳定转染细胞株分泌的rhFⅨ较对照组促凝血时间显著缩短; 其中, FⅨ-35促凝血时间最短(77.8 ± 0.1) s, 对应的促凝血活性达到(66.8 ± 0.5)% (图6C)。此外, 连续传代与冻存复苏后, 各细胞株表达蛋白的促凝血活性未出现统计学差异, 表明5株单克隆稳定转染细胞株可稳定分泌具有高效促凝血活性的rhFⅨ。
讨论
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血友病A和B是由凝血因子Ⅷ (coagulation factor Ⅷ, FⅧ) 或FⅨ缺乏活性引起的X连锁遗传性出血性疾病[15]。迄今为止, 血友病的治疗主要采用血浆来源或重组FⅧ/FⅨ浓缩物的替代疗法, 包括半衰期延长的替代疗法[16]。血源性FⅨ的应用面临多重固有局限。首要风险在于生物安全性, 难以完全规避已知病原体残留或未知新发病原体污染的理论可能。另一显著局限在于生产层面, 其供应高度依赖有限的人血浆资源; 此外, 复杂的纯化与病毒灭活工艺使得生产成本居高不下, 而终产品中残留的异源血浆蛋白还可能引入潜在的免疫原性风险。
目前已有4家企业的rhFⅨ上市[17], 包括辉瑞公司的Bene hFⅨ、百健艾迪公司的Alprolix、百特公司的Rixubis及诺和诺德公司的Rebinyn, 其中Bene hFⅨ、Rixubis和Rebinyn均在中国仓鼠卵巢(Chinese hamster ovary, CHO) 细胞中表达制备。治疗性重组蛋白药物领域在近年实现了高速发展, 其主流生产技术以哺乳动物细胞表达体系为主[18]。CHO细胞可能产生非人源糖型(如α-1,3-半乳糖苷酶), 该糖型易被人体免疫系统识别, 增加重组蛋白的免疫原性风险, 进而影响蛋白体内安全性与疗效稳定性; HEK293细胞具有高密度培养易实现、转染效率优异、可高效表达并分泌重组蛋白, 且能完成与人类高度相似的翻译后修饰等优势, 因此被广泛应用于复杂重组蛋白的制备[11]。因此, 开发HEK293源性的rhFⅨ具有重要的意义。国内报道rhFⅨ的表达水平普遍不高: 研究者[19]在小鼠脂肪干细胞中测得rhFⅨ的表达量为(21.33 ± 3.93) ng·(106个细胞/24 h)-1; 另有学者在小鼠脂肪干细胞中所获rhFⅨ的稳定表达量的最高值为(81.62 ± 8.82) μg·L-1[20], 均未能突破毫克级瓶颈。与之相比, 本研究采用HEK293细胞平台, 将rhFⅨ的表达量提升至(1.58 ± 0.10) mg·L-1, 较先前研究显著提高。
本研究基于HEK293系统, 通过EGFP-FⅨ融合蛋白确认其真核表达效能。所选pcDNA3.1表达载体含有neoR抗性筛选标签, pcDNA3.1-FⅨ成功转染HEK293细胞后具有抗遗传霉素的抗性。稳定转染的细胞在含有G418的培养基中正常存活, 基因没有整合至细胞者则在筛选过程中死亡[21]。通过有限稀释法成功分离获得20株单克隆稳定转染细胞株, 并系统评估其rhFⅨ凝血功能活性及表达水平。各单克隆稳定转染细胞株间rhFⅨ表达水平与凝血活性呈非完全一致性相关, 可能与不同克隆稳定转染细胞株的翻译后修饰功能异质性有关。本实验采用经典有限稀释法进行阳性克隆筛选[22], 在维持操作简便性的同时确保单克隆来源可靠。
活化部分凝血活酶时间实验是评估rhFⅨ的凝血生物学活性的经典方法, 其原理基于在枸橼酸钠抗凝的血浆中, 先后加入部分凝血活酶(提供磷脂表面) 和活化剂(如白陶土, 激活内源性凝血因子), 经孵育后, 再加入钙离子启动被刻意中断的凝血过程。通过精确测定从加入钙离子到纤维蛋白凝块形成所需的时间, 来评估FⅨ的功能状态。本研究通过测定活化部分凝血活酶时间验证了rhFⅨ的功能活性及其治疗应用潜力。
本研究构建的单克隆稳定转染HEK293细胞株分泌的rhFⅨ可显著缩短凝血时间, 其凝血活性最高达(66.8 ± 0.5)%, 相较于国内文献报道的8.5%活性水平[19]提升近8倍, 充分证实单克隆稳定转染细胞体系可有效增强rhFⅨ的凝血功能活性。然而, 该活性水平仍略低于已上市rhFⅨ产品, 这一差异可能与HEK293工程细胞的蛋白翻译后修饰效率、纯化过程中目标蛋白的构象稳定性以及体外活性检测体系的反应条件相关; 通过优化细胞培养工艺以提升蛋白表达质量、改进纯化流程减少活性损失、建立更贴近体内生理状态的活性评估模型等策略, 可进一步提高rhFⅨ的活性水平与产品应用潜力。
综上, 本研究成功建立了基于HEK293细胞的真核稳定转染平台, 通过系统性筛选获得了兼具高水平表达和高效凝血活性的单克隆细胞株, 为rhFⅨ的大规模制备奠定了关键技术基础。后续研究工作将重点开展贴壁HEK293细胞的悬浮适应性驯化, 并进一步优化表达条件, 旨在构建可规模化放大、适用于高密度培养的rhFⅨ生产体系。本研究的最终目标是获得符合药品生产质量管理规范要求的悬浮单克隆细胞株, 为重组凝血因子类药物的工业化生产提供标准化、质量可控的细胞工厂平台。

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doi: 10.16438/j.0513-4870.2025-1434
  • 接收时间：2025-12-02
  • 首发时间：2026-09-21
  • 出版时间：2026-05-12
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  • 收稿日期：2025-12-02
  • 修回日期：2026-01-18
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    1华北理工大学, 河北 唐山 063000
    2中国医学科学院医药生物技术研究所, 北京 100050

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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