ABSTRACT
<p><b>OBJECTIVE</b>To express a bioactive fusion protein comprising rat soluble transforming growth factor beta type II receptor and interferon gamma(rsTGFbetaR II-IFN gamma) in mammalian cells.</p><p><b>METHODS</b>mRNA was extracted from rat liver and the sTGFbetaR II-IFN gamma genes amplified by RT-PCR, then the two gene segments were cloned into the same pSecTag2A expression vector, and pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid was obtained, which was later transfected into CHO cells using liposomes. The expression of pSecTag2A/rsTGFbetaR II-IFN gamma in the supernatant was detected by ELISA and Western blotting. The bioactivities of the fusion protein were tested by sTGF betaR II-IFN gamma and IFN gamma bioassays.</p><p><b>RESULTS</b>pSecTag2A/rsTGFbetaR II-IFN gamma transfectants expressed rsTGF betaR II-IFN gamma fusion protein. The purified fusion protein exhibited anti-viral activity and antagonized the proliferation-inhibitive effect of TGFbeta1 on CCL-64 cells. It inhibits the HSC activation in vitro.</p><p><b>CONCLUSION</b>The pSecTag2A/rsTGFbetaR II-IFN gamma recombinant plasmid constructed in this study can express bioactive rsTGFbetaR II-IFN gamma fusion protein.</p>
Subject(s)
Animals , Cricetinae , Rats , Amino Acid Sequence , Base Sequence , CHO Cells , Cloning, Molecular , Eukaryotic Cells , Metabolism , Genetic Vectors , Hepatocytes , Metabolism , Interferon-gamma , Genetics , Molecular Sequence Data , Plasmids , Genetics , Protein Serine-Threonine Kinases , Receptors, Transforming Growth Factor beta , Genetics , Recombinant Fusion Proteins , Genetics , Recombinant ProteinsABSTRACT
<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector encoding the gene of extracellular region of type II transforming growth factor beta receptor (sTGFbetaR II), to express the protein in CHO cell line and to determine its biological activity.</p><p><b>METHODS</b>The extracellular region (amino acids 1-159) of the human TGFbetaR II cDNA was amplified by PCR from a TGFbetaR II chimeric plasmid,and the eukaryotic expression plasmid pCDNA3.1/myc-his(-)B-sTGFbetaR II(pCDNA-sTGFbetaR II) was constructed by inserting the sTGFbetaR II cDNA into the EcoR I/Hind III-digested pCDNA. The DNA sequence was confirmed by double digestion and the pCDNA-sTGFbetaR II plasmid was transfected into the CHO cell line. The sTGFbetaR II protein was confirmed by Western blotting analysis and its biological function was determined.</p><p><b>RESULTS</b>The specific protein was observed in western blotting, and the protein abrogated the growth-inhibitory effects of TGF-beta1 on mink lung epithelial cells (Mv1Lu).</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid pCDNA-sTGFbetaR II has been successfully constructed and the sTGFsTGFbetaR II protein with biological activity obtained.</p>