The Construction,expression,purification And Bioassay Analysis Of RGD-nrhTNF Fusion Protein In E.coli

Tumor necrosis factor alpha, a cytokine mainly secreted by macrophages, is cytotoxic to various tumor cells and involved in immunological regulation. But when used in clinical, its side effects are usually intolerable. So it's usage is limited.In recent years, many works have been done to widen IMF's clinical use by (1)the construction of TNF mutants;(2)the construction of bi-function fusion proteins with other proteins;(3)the chemical modification ,such as PEG etc;(4)the combine administration with other cytokines .As we known ,integrin α v β3 play an important role in tumor angiogenesis. A peptide CRGDC, which can bind to activated integrin a v P 3 efficiently and specifically, is selected from phage display library. Then we synthesis the sequence encoding CRGDC residues and ligate it with 5' terminal of TNF gene. We hope that the product of this fusion gene can maintain the cytotoxic effect of TNF and furthermore homing TNF to newly developed tumor vascular.In our research, we constructed the fusion gene encoding CRGDC and TNFsuccessfully. The fusion gene was expressed in prokaryotic expression vector in E.coli efficiently and amounted to 30% of total protein content. Subsequently we established a cheap and easy procedure to purify RGD-nrhTNF from E.coli with normal pressure ion exchange chromatograph. RGD-nrhTNF was analysis by SDS-PAGE ,the purity is over 92% and the specific activity of purified RGD-nrhTNF reached 5.7 * 108,the protein recovery rate was 2.57%.At last, we establish animal tumor model in KM mice by inoculating SI80 cells. RGD-nrhTNF can inhibit tumor growth prominently and is superior to nrhTNF.