title: [Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of Factor Ⅸ gene and expression in HEK-293 cells].
abstract: 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.
authors.affiliation: Department of Hematology, the Second Hospital of Shanxi Medical University, Taiyuan 030001, China.
authors.forename: J F
authors.initials: JF
authors.lastname: Chen
authors.forename: Y F
authors.initials: YF
authors.lastname: Zhang
authors.forename: J M
authors.initials: JM
authors.lastname: Kang
authors.forename: X Y
authors.initials: XY
authors.lastname: Qin
authors.forename: M F
authors.initials: MF
authors.lastname: Wang
authors.forename: G
authors.initials: G
authors.lastname: Wang
authors.forename: L H
authors.initials: LH
authors.lastname: Yang
mesh_terms: D056890:Eukaryota; D005164:Factor IX / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids; D012333:RNA, Messenger; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005164:Factor IX