Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (50): 9419-94.doi: 10.3969/j.issn.1673-8225.2011.50.027

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Construction and identification of recombinant adeno-associated virus vector with human bone morphogenetic protein 4/7 fusion gene

Yuan Shao-hui, Liu Wei, Wu Bin-qi, Han Xi-guang, Bi Zheng-gang   

  1. First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin  150001, Heilongjiang Province, China
  • Received:2011-06-18 Revised:2011-09-20 Online:2011-12-10 Published:2011-12-10
  • Contact: Bi Zheng-gang, First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China yuansh1970@sina.com
  • About author:Yuan Shao-hui☆, Ph.D., First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China yuansh1970@sina.com
  • Supported by:

    the Natural Science Foundation of Heilongjiang Province, No. D200876*; Science and Technology Research Project of Education Department of Heilongjiang Province, No. 11531149*

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Abstract:

BACKGROUND: Studies have shown that the activity of bone morphogenetic protein (BMP) heterodimer is 20 times higher than that of BMP homodimer, but related BMP 4/7 fusion gene-associated virus vector has been rarely reported.
OBJECTIVE: To construct the recombinant adeno-associated virus vector with human BMP 4/7 fusion gene (AAV-BMP-4/7) and to detect its expression.
METHODS: Human BMP 4, 7 mature peptide gene was amplified. Plasmids pGEM-human BMP 4, 7 were constructed, respectively. BMP 4/7 fusion gene was harvested using DNA ligation and then cloned to harvest pGEM-BMP-4/7 plasmid for gene sequencing. HEK293 cells were transfected with adeno-associated virus (AAV) particles to harvest viral vector AAV-BMP-4/7. BMP-4/7 fusion gene protein expression in E. coli was detected by SDS-PAGE. AAV-BMP-4/7 of different multiplicities of infection (MOI) was used to transfect bone marrow stromal cells (BMSCs) for 3 days. Total cellular RNA and total cellular protein were extracted. BMP-4/7 fusion gene expression in BMSCs was detected by RT-PCR and ELISA.
RESULTS AND CONCLUSION: The constructed recombinant pSNAV-BMP-4/7 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-BMP-4/7 was correct. Ttranscription bands of BMP-4/7 fusion gene in BMSCs were detected by RT-PCR. ELISA showed that BMP-4/7 protein in the AAV-transfected BMSCs was gradually increased with the increase in MOI (P < 0.01). These results showed that the AAV-BMP-4/7 was successfully constructed.

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