Construction of recombinant HIF-1α and NIS lentiviral expression plasmid and its functional identification
10.3760/cma.j.issn.2095-2848.2014.02.012
- VernacularTitle:重组HIF-1α及NIS慢病毒表达载体的构建及功能鉴定
- Author:
Shuo SHI
;
Rui GUO
;
Lihua WANG
;
Min ZHANG
;
Miao ZHANG
;
Ying MIAO
;
Biao LI
- Publication Type:Journal Article
- Keywords:
Hypoxia-inducible factor 1,alpha subunit;
DNA,recombinant;
Plasmids;
Transfection;
Sodium/iodide symporter
- From:
Chinese Journal of Nuclear Medicine and Molecular Imaging
2014;34(2):130-135
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-1α with the myosin light chain-2v(MLC-2v) as a promoter and to investigate the specific expression and feasibility of NIS as a reporter gene in cardiomyocytes.Methods The target gene HIF-1α and NIS were subcloned into the lentivirus (Lv)-elongation factor (EF)1-HIF-1α-internal ribosome entry site (IRES)-NIS and Lv-MLC-HIF-1α-IRES-NIS lentivirus vectors.The recombinated vectors were transfected into Hela cells by lipofectamine 2000.The expression of HIF-1α and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot.The H9C2 cells were exposed to different multiplicities of infection (MOI; 5,10,20,40) with packaged virus particles.The infection efficiency was detected by Western blot.MOI 20 was used for H9C2,NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot.The function and features of NIS protein were evaluated by dynamic iodine uptake and NaClO4 iodine uptake inhibition tests in vitro.Two-sample t test was used to analyze the data.Results The two recombinant lentivirus vectors were constructed successfully.The HIF-1α protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells.The grey levels of NIS and HIF-1α proteins in the positive control were 69.8 and 71.9,respectively,which were 109.4 and 92.7 after being prompted by EF1,and 141.9 and 132.4 by MLC-2v.The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter.The optimal MOI for the Lv-MLC-HIF-1α-IRES-NIS virus to infect H9C2 cells was 20.With the MOI of 20,the grey levels of NIS protein promoted by EF1 were 23.4,29.8 and 28.6 for H9C2,NIH-3T3 and L6 cells infected with Lv-EF1-HIF-1α-IRES-NIS virus,respectively.The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells than the other two cell lines.The grey level of NIS protein was 157.9 in H9C2 cells,178.8 in L6 cells and 217.3 in NIH-3T3 cells.The NIS protein expressed in infected H9C2 cells showed high radioiodine uptake.The peak of iodine uptake was 4 287.2 counts · min-1 at 40 min which was 16.85 times of the control group (254.4 counts · min-1) (t=5.34,P＜ 0.01).The inhibition rate of iodine uptake was up to 85.5％ (3 666.4/4 287.2,t=21.3,P＜0.01) by NaClO4.Conclusions MLC-2v promoter allows specific expression of the external gene HIF-1α and NIS in myocardium.The cardiomyocytes transfected with NIS gene acquires the function of iodine uptake.Therefore,NIS may have a potential to be the reporter gene to monitor the external gene therapy in ischemic cardiomyopathy.
