Expression And Identification A Novel Human STGFβRⅡ And Interferon Gamma Fusion Protein

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; TGFpl is known to be the most potent pro-fibrogenic cytokine, which activates HSC and enhances ECM synthesis and depostition. Thus, the TGFP/HSC axis has become an important target for anti-fibrosis therapy. Both the extracellular region of the type II transformin growth factor beta receptor (sTGFpR II) and human interferon gamma (hlFN-y) have been proved to be anti-fibrotic, the former competes with TGFPRII for TGFpl and the latter inhibits the interaction between TGFPR complex and Smad3, which indirectly inhibits TGFp activity and HSC activation. sTGFpR II and hlFN-y act at different sites to block the activity of TGFP, 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 sTGFpRII and hlFN-y recombinant gene, express the confusion protein in the CHO S cells. Select stable efficient sTGFpRII /hlFN Y fusion protein expression CHO S cell line and determine the protein's biological activity and anti-fibrosis in vitro.To compare sTGF R II /hlFN-y fusion protein with sTGFpR II protein on the anti-fibrosis, the eukaryotic plasmid that encoding sTGF R II gene was constructed and expressed in the CHO cells, the sTGFpR II protein's biological activity was determined.MethodThe eukaryotic expression plasmid pSecTag2B - sTGF 3 RII /hlFN- v was constructed by inserting the chimerical sTGFpR II /hlFN V gene by two sub-clone. Select stable efficient sTGFpR II /hlFN Y fusion protein expression CHO S cell line after transfected with the constructed vector. The cell culture supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot for the level of sTGFpR II and hlFN y . And we also determined the biological activity and the antifibrotic effect of the fusion protein in vitro.sTGFpR II gene was cloned into pSecTag2B and the pSecTag2B-sTGFpR II eukaryotic expression vector was constructed which was transfected into the CHO cells to produce the desired protein. The sTGFpR II protein was confirmed by Western blotting analysis and it's biological function was determined.Result1. pSecTag2B - 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 pSecTag2B -sTGF P RII /hlFN- Y successfully.2. After the pSecTag2B-sTGF P RII /hlFN- Y chimerical plasmid transfected into CHO S cells ,we analysised the protein that expressed. ELISA results showed that the mean expressive levels of hlFN Y and sTGFpR II these of the CHO S cell being transfected with the recombinant plasmid were 1.97 卤 0.29 and1.77 卤 0.11, which were significantly high than cells transfected by pSecTag2B plasmid( 015 + 0^ 0.23 + 0.05 respectively)and CHO cells only; The specific protein of sTGFpR II and hlFN Y was observed in same site in western blotting;much of a -helix and P -pucker was observed in the protein's secondary structure that simulated by computer.3. After restricted diluted culturing, the level of hlFN Y maintain in 20ng/106cells/ml and can reach 30ng/106cells/ml, which indicated we obtained CHO S cell line that expressed fusion protein stably and efficiently4. The biological function of the fusion protein :the protein and TGF l wereclotivated with the mink lung epithelial cells (Mvl Lu), and could abrogate the growth -inhibitory effects of TGF- 3 1 on MviLu; the fusion protein was clotivated with the lymphocyte, the NK cells activity was significant elevated compared with control(p<0.05) .All of those indicated that the protein what we obtained owns the biological activation of sTGFpR II and hlFN- Y corresponding.5. The anti-fibr...