逆转录病毒载体介导人凝血因子Ⅷ在肝细胞中的表达  

Retroviral-mediated high efficient expression of human coagulation factor Ⅷ in vitro

作　　者：刘昶[1] 纪艳超[1] 潘尚哈[2] 

机构地区：[1]哈尔滨医科大学附属第四医院普外科,黑龙江哈尔滨150001 [2]哈尔滨医科大学附属第一医院中心实验室

出　　处：《胃肠病学和肝病学杂志》2010年第5期447-449,共3页Chinese Journal of Gastroenterology and Hepatology

基　　金：黑龙江省青年基金资助项目(QC04C17)

摘　　要：目的建立逆转录病毒载体介导的人凝血因子Ⅷ（FⅧ）鼠肝细胞BRL-3A。方法将一B区缺失（760~1 639 aa）的人FⅧcDNA（FⅧBD cDNA）克隆至逆转录病毒载体pLNCX2,构建重组表达载体pLNCX2-FⅧBD。感染BRL-3A细胞,分别采用一期法、ELISA法检测细胞培养上清中人FⅧ的凝血活性（FⅧ：C）和抗原含量（FⅧ：Ag）。结果 pLNCX2-FⅧBD在鼠肝细胞BRL-3A细胞中获得良好表达,在24 h内每毫升中106细胞表达FⅧ：C为1.12 U,FⅧ：Ag为350 ng。结论逆转录病毒载体能够介导人FⅧ在肝细胞中的表达,为血友病A的基因治疗奠定了基础。Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ.Methods The retroviral vector LNC-ⅧBD was generated by cloning a B-domain-deleted FⅧ cDNA（760aa～1639aa） into retroviral vector pLNCX2.The antigen and procoagulant activity of human FⅧ in the cell culture medium were measured by ELISA assay and one-stage method,respectively.Results Human FⅧ was expressed in BRL-3A cells.The procoagulant activity of secreted FⅧ was up to 1.12 U,and the FⅧ antigen was 350 ng by 106 cells/mL in 24 hours,respectively.Conclusion The constructed retroviral vector is able to generate high level expression of human FⅧ in BRL-3A cell line,and it may have the potential utility in the gene therapy for Hemophilia A.

关 键 词：血友病A 逆转录病毒 血液凝固因子Ⅷ 基因表达 

分 类 号：Q78[生物学—分子生物学]