Expression And Purification A Novel Human STGFβRⅡ And Interferon Gamma Fusion Protein And Researches On Antifibrosis Mechanism In Vitro

Hepatic fibrosis is caused by an imbalance between the synthesis and degradation of extracellular matrix (ECM), which leads to its excessive deposition. Studies indicated that activated hepatic stellate cell (HSC) is the primary ECM-producing cells in hepatic fibrogenesis;TGFβ1 is known to be the most potent pro-fibrogenic cytokine, which activates HSC and enhances ECM synthesis and depostition. Thus, the TGFβ/Smad signal transduction axis has become an important target for anti-fibrosis therapy. Both the extracellular region of the type Ⅱ transformin growth factor beta receptor (sTGFβR Ⅱ) and human interferon gamma (hIFN-γ) have been proved to be anti-fibrotic, the former competes with TGFβR Ⅱ for TGFβ1 and the latter inhibits Smad3, which indirectly inhibits TGFβ activity and HSC activation. sTGFpR Ⅱ and hIFN-γ act at different sites to block the activity of TGFβ, and the activation of HSC, so the combined application of them may produce a synergic effect and in turn reduce their required dose to lower the side effect.ObjectiveTo construct the eukaryotic plasmid that encoding sTGFβRⅡ and hIFN-γrecombinant gene, express the confusion protein in the DG44 cells. Select stable efficient sTGFpR II /hlFN Y fusion protein expression DG44 cell line and establish large-scale culture system, determine the protein's biological activity and research into anti-fibrosis mechanism in vitro.MethodThe eukaryotic expression plasmid PCDNA3.1 — sTGF P RII /hlFN-y was constructed by inserting the chimerical sTGFpRII/MFN-y gene. Select stable efficient sTGFpRH /hlFN-y fusion protein expression DG44 cell line after cotransfected with the constructed vector and PSV2-dhfr pasmid .Large-scale culture with roller bottle and purify the confusion protein. The cell culture supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot for the level of sTGFpRH and hlFN-y . And we also determined the biological activity and the antifibrotic effect of the fusion protein in vitro.HSC at one definite differentiation stage that responded most sensitively to TGF 0 1 was selected as the cell model for the following study. The confusion protein was incubated with TGF P 1 stimulated HSC , the a -SMAn Desmim and collogen expression were checked with the Cell immunofluorescence assay .The cytoplasmic and nuclear proteins were extracted , and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot Results.Result1. PCDNA3.1(+) —sTGF P RII /hlFN-y plasmid was verified by double digesting and DNA sequence analysis. The results were consistent with those expected and constructed the plasmid PCDNA3.1(+)-sTGF P RJI /hlFN-y successfully.2. After the PCDNA3.1(+) - sTGF P RII /hlFN-y chimerical plasmid transfected into DG44 cells ,we analysised the protein that expressed. ELISA resultsshowed that the mean expressive levels of hlFN-y and sTGFpR II these of the DG44 cell being transfected with the recombinant plasmid were were significantly high than cells transfected by PCDNA3.1(+) plasmid and DG44 only;The specific protein of sTGFpR II and hlFN-y was observed in same site in western blotting. After restricted diluted culturing , the level of hlFN-y can reach 6ug/105cells/ml with roller bottle culture.lt showes stable efficient cell line was established.The fusion protein and TGFpi was clotivated with the mink lung epithelial cells (MvlLu) , and could abrogate the growth -inhibitory effects of TGF- P 1 on MvlLu;The fusion protein clotivated with the lymphocyte, the NK cells activity was significant elevated compared with control.3. The fusion protein and TGFpl was clotivated with HSC and could significant reduce HSC expressing Col I -. CoinL a -SMA % Desmin that induced by TGFpi and could also significant restrain smad2/3 phosphorylation.Conclusion1. successfully constructed the eukaryotic expression plasmid PCDNA3.1(+) -sTGFPRII/MFN-y.2. obtained DG44 cell line that expressed sTGFpRII /hlFN-y fusion protein stably and efficiently.3. established large-scale roller bottle cuture system.4. The sTGFpRII /hlFN-y fusion protein holds the biological activity of sTGFpR II and hlFN-y correspondingly5. The sTGFpRII MFN-y fusion protein can inhibitor the HSC activation in vitro, and have the potential antifibrotic effect. The fusion protein exerts anti-fibrosis effect by restraining TGFpl/smad signaling.