Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells.
10.12200/j.issn.1003-0034.2021.07.017
- Author:
Qian-Hui LI
1
;
Chun-Yang MENG
1
;
Feng-Yu MA
1
;
Cong ZHANG
1
Author Information
1. Department of Spine Surgery, Zhongda Hospital of Southeast University Medical School, Nanjing 210009, Jiangsu, China.
- Publication Type:Journal Article
- Keywords:
Adenovirus vector;
Bone marrow mesenchymal stem cells;
Bone morphogenetic protein;
Vascular endotheliel growth factor
- MeSH:
Adenoviridae/genetics*;
Animals;
Bone Marrow Cells;
Genetic Vectors/genetics*;
HEK293 Cells;
Humans;
Mesenchymal Stem Cells;
Rabbits;
Transfection
- From:
China Journal of Orthopaedics and Traumatology
2021;34(7):674-679
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.
METHODS:BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.
RESULTS:The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×10
CONCLUSION:Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.
