中文摘要：

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FⅨmRNA，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。

英文摘要：

Objective To construct pIRES2-ZsGreenl/FⅨ 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-ZsGreenl vector using the Infusion enzyme. The positive clones of eukaryotic expression vector of pIRES2- ZsGreenl/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 ZsGreenl 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, plRES2- ZsGreenl/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 F Ⅸ protein distributed in the surrounding of nucleus. F Ⅸ ： 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, plRES2- ZsGreenl/FⅨ, was successfully constructed, which provided experiment basement for the follow study on the location, function and molecular pathology of hemophilia B.