[Construction of recombinant adenovirus containing BMP-7 gene and its expression in proximal tubule epithelial cells].
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).



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.



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.



The bioactive recombinant adenovirus AdBMP-7 has been successfully constructed, which may be effective in inhibition of chronic renal fibrosis.
Yang
Su-wen
SW
Clinical Laboratory, Sir Run Run Shaw Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China. ysw6851@hotmail.com
Wang
Wei
W
Xu
Zai-chun
ZC
Zhu
Yun-yun
YY
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D055419:Bone Morphogenetic Protein 7 / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D055419:Bone Morphogenetic Protein 7; D011994:Recombinant Proteins