人fgl2凝血酶原酶基因的真核表达及其凝血功能研究  

Eukaryotic Expression of fgl2 Prothrombinase and Its Coagulation Activity

作　　者：李学军[1] 宋善俊[2] 魏文宁[2] 李永敢[1] 孙碧红[1] 

机构地区：[1]广西壮族自治区人民医院血液科,南宁530021 [2]华中科技大学同济医学院附属协和医院血液科,武汉430022

出　　处：《华中科技大学学报（医学版）》2011年第1期80-83,87,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：广西壮族自治区自然科学基金资助项目(No.桂科自0447025)

摘　　要：目的将人fgl2凝血酶原酶基因进行真核表达,了解表达蛋白的分布特点,初步研究其凝血活性。方法将pcDNA3-fgl2重组质粒转化大肠埃希菌,利用脂质体转染至HL-60细胞;RT-PCR法检测其fgl2凝血酶原酶mRNA的表达,间接免疫荧光法标记fgl2凝血酶原酶蛋白,应用激光共聚焦显微镜观察其分布,流式细胞仪检测fgl2凝血酶原酶蛋白在细胞膜的表达;并通过检测转染细胞的促凝活性(PCA)分析其凝血功能。结果 pcDNA3-fgl2真核表达质粒在HL-60细胞中进行了瞬时表达,转染细胞可见明显的fgl2凝血酶原酶mRNA条带。激光共聚焦显微镜观察到转染细胞胞质中可见明显的绿色荧光,流式细胞仪在转染细胞胞膜上未检测到明显的蛋白表达。转染pcDNA3-fgl2重组质粒细胞的PCA明显增高且依赖于凝血酶原。结论成功地将fgl2凝血酶原酶基因在HL-60细胞中进行了瞬时表达;fgl2凝血酶原酶主要在胞质中表达,细胞膜上无分布并可能分泌到细胞外;凝血功能分析显示fgl2凝血酶原酶具有直接激活凝血酶原的功能。Objective To investigate the eukaryotic expression of human fgl2 prothrombinase cDNA and the distribution characteristics of fgl2 protein,and to primarily study its coagulation activities.Methods The recombinant eukaryotic expression plasmid pcDNA3-fgl2 was transformed into E.coli DH5α and extracted on large scale,then transfected into HL-60 cell strain with liposomes Lipofectamine.The expression of fgl2 prothrombinase mRNA was detected by RT-PCR.fgl2 protein was marked with anti-fgl2 prothrombinase monoclonal antibody by indirect immunofluorescent methods.Confocal laser microscope and flow cytometer were used to analyze its expression characteristics.The coagulation activity of transfected cells was evaluated by its procoagulant activity（PCA）.Results The eukaryotic expression plasmid pcDNA3-fgl2 was transiently expressed in HL-60 cell strain.The mRNA levels of fgl2 prothrombinase and PCA of transfected cells were significantly higher than those of un-transfected cells.Bright green florescence in transfected cells was observed under confocal laser microscope in cytoplasm.fgl2 prothrombinase was not detected on cytomembrane of transfected cells by flow cytometer.PCA of tranfected cells were dependent on prothrombin.Conclusion The eukaryotic expression plasmid pcDNA3-fgl2 has been successfully transiently expressed in HL-60 strain.fgl2 prothrombinase might be expressed mainly in cytoplasm and then excrete out of cells.It could directly activate prothrombin.

关 键 词：凝血酶原酶 fgl2 真核表达 促凝活性 激光共聚焦显微镜 

分 类 号：R343.1[医药卫生—基础医学]