Construction of replication-deficient recombinant adenovirus vector with hTFPI-2 gene by AdMax system and expression in U937 monocytes in vitro.
- Author:
Junjie PAN
1
;
Haiming SHI
;
Xinping LUO
;
Duan MA
;
Wang LIANG
;
Jin ZHANG
;
Jun ZHU
;
Jian LI
Author Information
1. Department of Cardiovascular Medicine, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
metabolism;
Defective Viruses;
genetics;
metabolism;
Genetic Vectors;
genetics;
Glycoproteins;
biosynthesis;
genetics;
Humans;
Monocytes;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
Transfection;
U937 Cells
- From:
Journal of Biomedical Engineering
2011;28(2):326-331
- CountryChina
- Language:Chinese
-
Abstract:
We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2 (hTFPI-2) gene by AdMax system in HEK293 cells. Firstly, we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI. After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector, we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2. It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing. Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxdeltaE1,3Cre by liposome lipofectamine2000, and finished the package of recombinant adenovirus Ad-hTFPI-2. The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2. Furthermore, we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification. The titre was 0.931 x 10(12) pfu/ml. Finally, we infected U937 monocytes by purified Ad-hTFPI-2, and determined the infection efficiency and the TFPI-2's level and activity. The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%. After infected by Ad-hTFPI-2, the TFPI-2's level in supernatant increased about 7 fold. Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin. The recombinant adenovirus with the hTFPI-2 gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in antiatherosclerosis.
