Objective: To construct pIRES2-ZsGreen1/F Ⅸ expression vector, using the pcDNA/FⅨ plasmid containing FⅨ cDNA as template, and express in HEK-293 cells. Methods: The total ORF of F Ⅸ gene was amplified from pcDNA/F Ⅸ plasmid, then the amplified fragment was clonded into the pIRES2-ZsGreen1 vector using the Infusion enzyme. The positive clones of eukaryotic expression vector of pIRES2-ZsGreen1/F Ⅸ were screened and expanded after transfection, then were constructed and confirmed by PCR and sequencing. Transient expression experiments were performed using HEK-293 cells transfected with the expression vectors and observed the expression of ZsGreen1 protein by confocal laser microscope. The relative expression levels of FⅨ mRNA, protein and FⅨ activity (FⅨ∶C) were detected by real time PCR (RT-PCR), immunofluorescence microscopy, One-Stage method, respectively. Results: The expression vector, pIRES2-ZsGreen1/F Ⅸ, was successfully constructed and expressed in HEK-293 cells. RT-PCR detected the expression of F Ⅸ mRNA in HEK-293 cells and the immunofluorescence microscopy showed FⅨ protein distributed in the surrounding of nucleus. FⅨ∶C of cell lysates and cell culture fluid transfected with the expression vectors were (92.03 ± 0.29)% and (86.89 ± 8.78)%, respectively; while both F Ⅸ∶C of cell lysates and cell culture fluid transfected with or without the expression vectors were 0. Conclusion: The experimental results showed the expression vector, pIRES2-ZsGreen1/FⅨ, was successfully constructed , which provided experiment basement for the follow study on the location, function and molecular pathology of hemophilia B.
目的: 以含有凝血因子Ⅸ（FⅨ）cDNA的pcDNA/FⅨ质粒为模板构建真核表达载体pIRES2-ZsGreen1/FⅨ并检测其在HEK-293细胞中的表达。
方法: 以pcDNA/FⅨ质粒为模板，扩增出目的基因FⅨ的开放阅读框（ORF）区，使用Infusion酶对线性pIRES2-ZsGreen1双酶切产物及FⅨORF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/FⅨ转染HEK-293细胞后，分别采用实时定量PCR、细胞免疫荧光法、一期法检测野生型FⅨ基因mRNA表达水平、蛋白的表达量及细胞裂解液、细胞培养液的FⅨ活性。
结果: 成功构建pIRES2-ZsGreen1/FⅨ并转染HEK-293细胞，实时定量PCR证实HEK-293细胞表达FⅨ mRNA，激光共聚焦显微镜下观察到FⅨ蛋白在细胞质中合成，野生型质粒pIRES2-ZsGreen1/FⅨ转染HEK-293细胞裂解液和细胞培养液的FⅨ活性分别为（92.03±0.29）%、（86.89±8.78）%，无转染的HEK-293细胞裂解液和培养液中FⅨ活性均为0。
结论: 成功构建FⅨ野生型pIRES2-ZsGreen1真核表达载体。