Construction of recombinant adenovirus containing BMP-7 gene and its expression in proximal tubule epithelial cells / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector containing bone morphogenic protein-7 (BMP-7) gene and to identify its biological activities in proximal tubule epithelial cells (PTECs).</p><p><b>METHODS</b>Forty-six fragments of BMP-7 gene were obtained by PCR method and then were ligated to the full-length gene. The full length sequence of BMP-7 was subcloned into pBluescript II(+) vectors, and confirmed by sequencing. Double digested with Not I and Hind III, BMP-7 gene was inserted into pShuttle-CMV. EcoR I pre-linearized pShuttle plasmid was transformed into competence bacterium BJ5183 to obtain the recombinant adenovirus-BMP-7 by efficient homologous recombination. Then the AdBMP-7 was obtained by packaging Pac I linearized in 293 cells. Adenoviral titer was determined by adenovirus fluorescent detection kit. The protein expression of BMP-7 in PTECs was respectively detected by ELISA and Western blot. RT-PCR method was used for analyzing the alpha-smooth muscle action (alpha-SMA) expression in PTECs, which was treated consecutively with TGF-beta and AdBMP-7.</p><p><b>RESULTS</b>The recombinant plasmid AdBMP-7 was successfully generated, which increased BMP-7 protein expression levels in PTECs, and down-regulated TGF-beta-induced alpha-SMA expression.</p><p><b>CONCLUSION</b>The bioactive recombinant adenovirus AdBMP-7 has been successfully constructed, which may be effective in inhibition of chronic renal fibrosis.</p>