HUANG Xianghui 1 , SHI Zhibin 1 , WANG Kunzheng 1 , DANG Xiaoqian 1 , YANG Pei 1 , YU Pengbo 2
  • 1Department of Orthopedics, the Second Affiliated Hospital of School of Medicine, Xi’an Jiaotong University, Xi’an Shaanxi, 710004, P.R.China;;
  • 2Laboratory of Virology, Shaanxi Provincial Center for Disease Control and Prevention.;
Adeno-associated virus; VEGF; BMP; Internal ribosome entry site
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To construct the recombinant adeno-associated virus (rAAV) vector co-expressinghVEGF165 and hBMP-7 depending on internal ribosome entry site (IRES) sequence, to measure the virus titer and to ver ify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid pIRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were ampl ified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein (GFP) labeled rAAVIRES
GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packaging
was monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells.
The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7
was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAVhVEGF165- IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of 5.5 × 1011vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement

Citation： HUANG Xianghui,SHI Zhibin,WANG Kunzheng,DANG Xiaoqian,YANG Pei,YU Pengbo. CONSTRUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR CO-EXPRESSING hVEGF165 ANDhBMP-7 GENES. Chinese Journal of Reparative and Reconstructive Surgery, 2008, 22(7): 807-813. doi: Copy