Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene
- Author:
SONG KE
1
;
RAO NIANJING
;
CHEN MEILING
;
CAO YINGGUANG
Author Information
1. 华中科技大学同济医学院附属同济医院
- Keywords:
human bone morphogenetic protein 7;
adeno-associated virus;
jaw bone;
gone therapy
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2008;28(1):17-21
- CountryChina
- Language:Chinese
-
Abstract:
To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se- quence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombi- nant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated,the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×1011 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.
