Construction of recombinant adenoviruses carrying human BMPª²7 and its expression in rabbit bone marrow stem cells

    YU Bing, FAN Qinª²Yu, MA Baoª²An, ZHOU Yong, ZHANG Mingª²Hua, LONG Hua, YAN Lu

    1PLA Institute of Orthopedics Oncology, Tangdu Hospital, Fourth Military Medical University, Xi¡¯an 710038 , China, 2Center of Rehabilitation, Xijing Hospital, Fourth Military Medical University, Xi¡¯an 710033, China

    ¡¾Abstract¡¿ AIM: To construct the replicationª²deficient recombinant adenoviruses AdBMPª²7 and to investigate BMPª²7 expression of AdBMPª²7 in rabbit bone marrow stem cells (BMSc). METHODS: Human BMPª²7 cDNA was amplified by RTª²PCR method from human fetal kidney and cloned into shuttle vector to generate pAdTrackª²CMVª²BMPª²7 which was linearized by PmeI and then transformed into AdEasierª²1 competent cells bearing adenoviral backbone plasmid to obtain recombinant adenoviral plasmid pAdBMPª²7. The recombinants were selected and further confirmed by restriction endonuclease analysis. The pAdBMPª²7 was then linearized with pacI enzyme and transfected into 293 cells to get packed adenoviruses which were then transduced into rabbit BMSc. The mRNA expression of BMPª²7 in rabbit BMSc was detected using RTª²PCR method and the expression of green fluorescence protein was observed under fluorescent microscope. RESULTS: A 1296 bp cDNA was amplified by RTª²PCR from human fetal kidney and its sequence analysis was documented as expected. The recombinant plasmid pAdBMPª²7 was established by homologous recombination and confirmed by restriction endonuclease digestion. GFP expression could be observed on the third day after the packing of the linearized pAdBMPª²7 in 293 cells and 3¡Á109 efu/L titer of Adª²BMPª²7 was obtained by CsCl gradient purification. Expressions of BMPª²7 were confirmed by RTª²PCR and observed with fluorescent microscope 72 hours after the rabbit BMSc were infected by the viruses. CONCLUSION: Human BMPª²7 gene is successfully cloned and its recombinant adenoviruses are constructed. Our data may offer a novel approach to local gene therapy of bone regeneration. ......