先天性维生素K依赖性凝血因子缺乏症1细胞诊断体系的构建  

Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1

作　　者：高稳稳 刘红丽 苏改改 徐亚奇 王一伊 崔龙腾 黄蓉 杨海平[2] 高蒙 席守民[1] 沈国民[1] Gao Wenwen;Liu Hongli;Su Gaigai;Xu Yaqi;Wang Yiyi;Cui Longteng;Huang Rong;Yang Haiping;Gao Meng;Xi Shoumin;Shen Guomin(College of Medicine,Henan University of Science and Technology,Luoyang,Henan 471023,China;The First Affiliated Hospital of Henan University of Science and Technology,Luoyang,Henan 471023,China)

机构地区：[1]河南科技大学医学院,洛阳471023 [2]河南科技大学第一附属医院,洛阳471023

出　　处：《中华医学遗传学杂志》2020年第8期811-814,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金(81770140,31900412);河南省科技攻关项目(182102410079);河南省高校青年骨干教师(2017GGJS069)。

摘　　要：目的:建立可用于确诊先天性维生素K依赖性凝血因子缺乏症1(vitamin K-dependent coagulation factor deficiency 1,VKCFD1)疾病的细胞体系。方法:在稳定表达报告基因FIX-Gla-PC的HEK293细胞中,应用CRISPR/Cas9技术敲除GGCX基因;通过ELISA、DNA测序、Western印迹等方法鉴定GGCX基因敲除的单克隆细胞。用快速点变异的方法构建致病性GGCX变异体质粒;用ELISA方法检测GGCX变异对报告基因的影响。结果:首先用ELISA方法筛选到两株报告基因无活性的单克隆细胞;之后DNA测序结果和Western印迹验证GGCX基因发生了编辑和敲除;最后通过转染野生型GGCX基因,可以恢复报告基因的活性,提示成功筛选到两株GGCX基因敲除的单克隆细胞。在筛选到的细胞株中转染已知可导致VKCFD1的GGCX变异体,发现报告基因活性在致病性变异体中显著降低。结论:成功构建了检测GGCX活性的细胞体系,该体系可以用于诊断GGCX变异导致的VKCFD1。Objective To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1(VKCFD1).Methods In HEK293 cells stably expressing the reporter gene FⅨ-Gla-PC,theγ-glutamyl carboxylase(GGCX)gene was knocked out by using CRISPR/Cas9 technology.Enzyme-linked immunosorbent assay(ELISA),DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells.A quickchange point variant method was used to construct the GGCX variant.ELISA was used to assess the influence of GGCX variant on the activity of reporter gene.Results Two monoclonal cell lines with no reporter activity by ELISA was identified.Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting.The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene.Thereby two monoclonal cells with GGCX knockout were obtained.By comparing the wild-type and pathogenic GGCX variants,the reporter activity was decreased in the pathogenic variants significantly.Conclusion A cell-based system for the detection of GGCX activity was successfully developed,which can be used for the diagnosis of VKCFD1 caused by GGCX variants.

关 键 词：维生素K依赖性凝血因子缺乏症1 GGCX基因 酶联免疫吸附测定法 报告基因 

分 类 号：R596.1[医药卫生—内科学]