| It is significant to reverse the progress of hepatic fibrosis which paly an important role in the physiopathologic process of chronic hepatic disease. In this paper, a eukaryotic expression vector containing truncated type II human transforming growth factor was established to set foundation for further research.Objective: To construct a eukaryotic expression vector pEGFP/ΔTβRII without the cytoplasmic domain of the cDNA of the human transforming growth factor-beta type II receptor (TβRII), to express the fusion protein EGFP/ΔTβRII in human embryonic hepatic cell line L02 and to determine the biological activity of its extracellular domain.Methods: The cDNA of type II truncated receptor(ΔTβRII) that lacks the cytoplasmic domain was amplified by PCR from the plasmid H2-3FF, and two restriction sites (EcoR I and BamHI ) were introduced into it at the same time. The PCR product was inserted into the vector pEGFP-N2 to construct the eukaryotic expression plasmid pEGFP/ΔTβRII, which was identified by double enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into L02 cells by cation lipoid Lipofectamine2000, and the GFP fluorescence was detected under inverted fluorescence microscope after the transfection. To obtain stable cells line, G418 was added in RPMI-1640 culture medium to screen the positive cells. The biological activity of its extracellular domain was examined by MTT assay and flow cytometry (FCM).Results:①Recombinant plasmid, pEGFP/ΔTβRII, digested with EcoRI and BamHI, and two bands at 619bp and 4.7kb could be observed respectively by agarose gel electrophoresis. These results were consistent with expection and the result of DNA sequencing, which demonstrated the gene ofΔTβRII was cloned to the vector pEGFP-N2.②The recombinant plasmid was transfected into the human embryonic hepatic cell line L02 by lipoid, and the specific fusion protein, EGFP, was observed by inverted fluorescence microscope 24h later. The positive clone was obtained by G418 screening.③The optical density (OD) of the test group was significantly higher than that of the control group, however, decreased the G1-phase of cell cycle and improved the cell proliferation index(PrI),which demonstrated the fusion protein would attenuate the inhibitory effect of TGFβ1 on L02 cells significantly.Conclusion: The eukaryotic expression plasmid pEGFP/ΔTβRII has been constructed and the fusion protein EGFP/ΔTβRII was successfully expressed in cell L02. The extracellular domain of the fusion protein can bind cytokine TGFβ1 , attenuate the inhibitory effect of TGFβ1 on L02 cells significantly and these works will help to set up foundation for further research. |
