Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of Factor Ⅸ gene and expression in HEK-293 cells
10.3760/cma.j.issn.0253-2727.2016.11.009
- VernacularTitle: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
- Author:
Jianfang CHEN
1
;
Yaofang ZHANG
;
Jianmin KANG
;
Xiuyu QIN
;
Meifang WANG
;
Gang WANG
;
Linhua YANG
Author Information
1. 山西医科大学第二医院血液科
- Keywords:
Hemophilia B;
Factor Ⅸ;
Eukaryotic cells;
Plasmids
- From:
Chinese Journal of Hematology
2016;37(11):971-975
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct pIRES2-ZsGreen1/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-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 ZsGreenl protein by confocal laser microscope.The relative expression levels of F Ⅸ mRNA,protein and F Ⅸ activity (F Ⅸ ∶ 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 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,pIRES2-ZsGreen1/F Ⅸ,was successfully constructed,which provided experiment basement for the follow study on the location,function and molecular pathology of hemophilia B.
