Construction, Expression, Purification, Bioactivity And Distribution Of GFE-1-rmhTNF Fusion Protein

Tumor necrosis factor alpha(TNF- a ), a multifunctional cytokine mainly secreted by macrophages and activated T cell.is cytotoxic to various tumor cells in vivo and vitro.lt is potential as an antitumor,antiviral and immunomodulater.But the side effect restrict it's use in clinic.At present.increasing the local concentration of TNF- a and the constrution of TNF- a mutants take on a better application perspective. rmhTNF is a TNF- a mutation which have higher biological activity and lower toxicity.Organ and tissue-specific molecular heterogeneity of the vasculature is genera, the CGFECVRQCPERC (termed GFE-1) peptide can selectively bind to mouse lung vasculature after an intravenous injection, and was screened out from phage displaying library.The receptor of GFE-1 is MDP(membrane dipeptidase),a 55-kDa lung cell-surface protein that selectively binds to the GFE-1 peptide.lt is a cell-surface zinc metalloprotease involved in the metabolism of glutathione, Leukotriene D4, and certain # -lactam antibiotics.In6 Biotechnology Center FMMUthis study,we constuct a fusion gene encoding GFE-1 and rmhTNF.The resultant recombined DNA was verified by sequencing analysis.Then the recombined gene was cloned into expression vector PBV220 and transformed into E.coli DH5 a for protein expression.The quantity of GFE-1-rmhTNF protein is 30% of total protein content.To purify the soluble GFE-1-rmhTNF from E.coli,The supernatant of the bacterial lysate was subsection salt out.Then with normal pressure ion-exchange chromatograph.we obtained GFE-1-rmhTNF protein with high purification and confirmed it's bioactivity with L929 cell line in vitro.The specific activity of purified GFE-1-rmh.TNF reached 2.5xl07IU/mL,the protein recovery rate was 2.58%.To investigate the distribution of GFE-1-rmhTNF in vivo. We injected GFE-1-rmhTNF into mice and analysed the concentration of several organs.The result indicated that GFE-1-rmhTNF can concentrate in lung specifically.