ABSTRACT
<p><b>OBJECTIVE</b>To construct a tetracycline-inducible eukaryotic expression vector of rat Smad7.</p><p><b>METHODS</b>The total RNA was extracted from normal rat kidney with Trizol agent. Rat Smad7 cDNA fragment was cloned by RT-PCR, and was inserted into the restriction site between Nhe I and Hind III of the inducible eukaryotic expression vector pBI-L by tetracycline. pBI-L-Smad7 was constructed by digestion and ligation, and detected by restriction endonuclease digestion and sequencing.</p><p><b>RESULTS</b>The recombinant eukaryotic expression vector pBI-L-Smad7 was constructed correctly as confirmed by restriction endonuclease digestion and sequencing. The fragment of pBI-L-Smad7 digested with restriction endonucleases and the sequence of inserted Smad7 cDNA were consistent with the results of theoretical analysis.</p><p><b>CONCLUSION</b>The tetracycline- inducible eukaryotic expression vector of rat Smad7, pBI-L-Smad7, is constructed successfully, which may facilitate further clinical study of Smad7 gene therapy for tissue and organ fibrosis.</p>
Subject(s)
Animals , Rats , Cloning, Molecular , DNA, Complementary , Genetics , Eukaryotic Cells , Metabolism , Gene Expression , Genetic Therapy , Genetic Vectors , Genetics , Rats, Sprague-Dawley , Smad7 Protein , Genetics , Tetracycline , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To construct a tetracycline-inducible eukaryotic expression vector containing human hepatocyte growth factor (HGF) cDNA.</p><p><b>METHODS</b>Human HGF cDNA fragment was obtained by PCR from pUC-SRalpha/HGF plasmid and inserted into the restriction site between Mlu I and Sal I of the tetracycline-inducible eukaryotic expression vector pBI-L. pBI-L-HGF was constructed by DNA recombination in vitro, and was identified by restriction endonucleases digestion and sequencing.</p><p><b>RESULTS</b>The fragment of pBI-L-HGF digested with restriction endonucleases well corresponded to expectation, and the sequence of inserted HGF cDNA was correct according to the GenBank.</p><p><b>CONCLUSION</b>The tetracycline-inducible eukaryotic expression vector of human HGF pBI-L-HGF has been constructed successfully, which allows further study of HGF gene therapy with much safety and easy manipulation.</p>