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Published on: May 6, 2015
[Construction of recombinant adeno-associated virus vector with human bone morphogenetic protein]
Zhiyong Dong1, Zhaomin Zheng, Guanming Kuang
1Department of Orthopaedics, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou Guangdong, 510080, PR China. dongzhiyong12@yahoo.com.cn
Objective:
To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene (AAV-hBMP-4).
Methods:
The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal I site into downstream. The hBMP-4 gene was amplified with the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vector to construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR I and Sal I enzyme, the fragments were collected and linked with T4 DNA ligase at 16 C over night, recombinant plasmid pSNAV-hBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. These cells were infected with HSV1-rc/deltaUL2 which has the function of packaging and copying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed.
Results:
The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 was correct. The virus titer was about 1.5 x 10(12) microg/ml. The purity of the virus was more than 95% using the SDS-PAGE method.
Conclusion:
With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
