凝血因子Ⅸ野生型pIRES2-ZsGreen1真核表达载体的构建及其在HEK-293细胞的表达

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.