凝血因子Ⅷ野生型pIRES2-ZsGreen1真核表达载体的构建及鉴定  

Construction and identification of wild-type pIRES2-ZsGreen1 eukaryotic expression vector of factor Ⅷ

作　　者：陈剑芳[1] 杨林花[1] 张耀方[1] 王梅芳[1] 康建民[1] 秦秀玉[1] 王刚[1] 

机构地区：[1]山西医科大学第二医院血液科,太原030001

出　　处：《中国药物与临床》2016年第9期1247-1249,共3页Chinese Remedies & Clinics

基　　金：国家自然科学基金(81270587);山西省青年科技研究基金(2014021040-1)

摘　　要：目的本研究以含有凝血因子Ⅷ(FⅧ)cDNA的pCI/FⅧ质粒为模板构建真核表达载体pIRES2-ZsGreen1/FⅧ并进行鉴定,在HEK-293细胞中表达。方法以pCI/FⅧ质粒为模板,扩增出FⅧ的开放阅读框(ORF)区,使用Infusion酶对线性pIRES2-ZsGreen1双酶切产物及FⅧORF扩增产物进行连接,连接产物进行转化后筛选阳性克隆,对阳性克隆进行DNA测序及凝胶电泳鉴定。野生型pIRES2-ZsGreen1/FⅧ转染HEK-293细胞后,采用实时定量聚合酶链反应(PCR)检测野生型FⅧ基因mRNA表达水平。结果成功构建pIRES2-ZsGreen1/FⅧ并转染入HEK-293细胞中,实时定量PCR检测FⅧmRNA在HEK-293细胞中的表达,激光共聚焦显微镜观察转染情况。结论为实时观察FⅧ真核表达载体在HEK-293细胞中的表达及FⅧ基因突变导致血友病A的分子发病机制的研究奠定实验基础。Objective To construct, identify a eukaryotic expression vector pIRES2-ZsGreen1/FⅧ using factorⅧ（FⅧ） cDNA-containing plasmid p CI/FⅧ as a template, and to express FⅧ m RNA in HEK-293 cells. Methods Using pCI/FⅧ plasmid as a template, the open reading frame（ORF） of FⅧ was amplified, and clone into the enzyme double digestion products of linear pIRES2-ZsGreen1 by Infusion. The eukaryotic expression vector of pIRES2- ZsGreen1/FⅧ was constructed and confirmed by DNA sequencing and gel electrophoresis. HEK-293 cells were transfected with wild-type pIRES2-ZsGreen1/FⅧ. Quantitative real-time PCR was used to detect the wild-type FⅧ gene mRNA expression level in the cells. Results We successfully constructed pIRES2-ZsGreen1/FⅧ and transfected it into HEK-293 cells, as detected by quantitative real-time PCR and laser scanning confocal microscopy. Conclusion This study laid down a foundation for real-time observation of FⅧ m RNA expression in HEK-293 cells via a eukaryotic expression vector and the molecular pathogenesis of hemophilia A caused by F Ⅷ mutation.

关 键 词：血友病A 因子Ⅷ 真核表达载体 分子发病机制 

分 类 号：R927[医药卫生—药学]