Construction of eukaryotic expression plasmid of Smad-7 and study on its function as TGF-β1 inhibitor

Abstract

Objective To construct eukaryctie expression vectors stably expressing Smad-7 and study its function as TGF-β1 inhibi- tor. Methods Smad-7 genes were obtained by PCR from fetal liver cDNA library and inserted in to eukaryotic expression plasmid pcD- NA3. 1/myc-his-B (-). The constructed plasmids containing right sequence of target genes were transfected into human mesangial cells by lipofectamine. The ceils were selected by G418, the expression of Smad-7 was detected by Western-blotting. Cell proliferation was detected by MTT method, and cell cycle was detected by Flow Cytometer to study its function as TGF-β1 inhibitor. Results We successfully con- struct Smad-7 expression plesmid. Smad-7 can inhibit the effects of TGF-[M induced GI cell cycle block and apoptosis. Conclusion The eukaryotic expression vector of Smad-7 has been successfully constructed. Over-expression of exogenous Smad-7 can inhibit the effects of TGF-β1 induced GI cell cycle block and apoptosis. Key words: DNA-binding proteins; Kidney; Transforming growth factor beta