title: Construction of recombinant adenovirus vector containing hBMP2 and hVEGF165 genes and its expression in rabbit Bone marrow mesenchymal stem cells.
abstract: To construct an adenovirus vector co-expressing human bone morphogenetic protein (hBMP2) and human vascular endothelial growth factor (hVEGF165) as well as green fluorescence protein (GFP) as a marker, with which the intracellular expression of the inserted genes could be identified in Bone marrow mesenchymal stem cells (BM-MSCs). BMP2 and VEGF165 genes were PCR amplified from a cDNA library and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. The virus solution (Ad-BMP2-VEGF165) was generated by co-transfecting HEK293 cells with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1, 3Cre. The virus solution was further purified and virus titer was determined accordingly. The expression of the target genes was subsequently detected and quantified in rabbit BM-MSCs by using real time PCR, ELISA and Western blotting. The recombinant adenovirus vector containing BMP2 and VEGF165 (Ad-BMP2-VEGF165) was successfully constructed, which was confirmed by Sanger sequencing, colony PCR, as well as visually detection of GFP, and the titer of the adenovirus was 1×10(10)PFU/mL, and the proteins level of BMP2 and VEGF165 secreted in the supernatant are significantly higher than the control. Recombinant adenovirus vector containing hBMP2 and hVEGF165 genes was successfully constructed. The transfection rate of BM-MSCs by the adenovirus was high (95% at 100 MOI) and the BMP2 and VEGF165 genes was highly expressed in the cells. The present study provides a method to efficiently express the target genes in BM-MSCs and an vector for further research of bone defect repair using dual genes of BMP2 and VEGF165.
authors.affiliation: Department of Orthopedics, Affiliated Hospital of Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Pathological Teaching and Research, Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Liu
authors.affiliation: Department of Orthopedics, Affiliated Hospital of Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China.
authors.forename: Qing-Wei
authors.initials: QW
authors.lastname: Li
authors.affiliation: Department of Orthopedics, Affiliated Hospital of Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China.
authors.forename: Guo-Wu
authors.initials: GW
authors.lastname: Chen
authors.affiliation: Department of Orthopedics, Affiliated Hospital of Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Orthopedics, Affiliated Hospital of Jining Medical University, Guhuai Road, Jining 272000, Shandong Province, China. Electronic address: blueskymaster@hotmail.com.
authors.forename: Chun-Yang
authors.initials: CY
authors.lastname: Meng
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A
keywords: Adenovirus vector; Bone defect; Bone morphogenetic protein; Vascular endothelial growth factor