Construction, Expression And Purification Of RGD-FasL And Analysis Its Function

The malignant tumor seriously threaten the health of mankind.The commonly used cancer therapy methods include surgery,radiotherapy and chemotherapy.However, the side effect of radiotherapy and chemotherapy is serious,because normal cells were simultaneously killed.Carcinoma of the liver is the common malignant tumor that does harm to the human beings'health,liver cancer is the second position lethal factor in all tumors,which is next only to lung cancer in city or cancer of the stomach in rural.Therefore the research of treating liver cancer is urgent.Now surgical operation, chemotherapy,radiotherapy and biotherapy are four kinds of main ways to treat it,In recent years,with progress of the pathogenesis and treatment research of liver cancer,it has being known that the unusual mechanism of cell apoptosis occurring in liver cancer cells,which would be of importance to develop new treatment method.Recently,we have been trying to induce cancer cell apoptosis for Anti-cancer therapy.Tumor targeting therapy is a novel method which selectively kills tumor cells by means of specific tumor targeting vehicles,without harming normal cells,and is a central challenge for improving cancer therapies.FasL is cytotoxic to various tumor cells and involved in immunological regulation. But when used in clinical,its side effects are usually intolerable.So its usage is limited.In recent years,many works have been done to widen FasL's clinical use by①the construction of FasL mutants;②the construction of bi-function fusion Proteins with other Proteins;Tumor targeting therapy is a novel method which selectively kills tumor cells by means of specific tumor targeting vehicles,without harming normal cells,and is a central challenge for improving cancer therapies.Integrins are a family of cell surface receptors withαandβsubunits.Integrinαvβ3 expressed on proliferating but not on quiescent endothelial cells.So integrinαvβ3 can highly expressed on endothelial cells of tumor blood vessels or tumor cells.It is a new targe for cancer therapy.The RGD(arginine-glycine-aspartic acid)sequence is known to serve a recognition moiety between the ligands and integrins.Present data obtained from phagdisplay techniques found that ACDCRGDCFCG(RGD-4C) can bind selectively toαvβ3 integrin.So RGD-4C is a promising homing peptide for tumor targeting therapy.In this work,we fused FasL with the RGD peptide,a ligand ofαvβ3 integrins,by recombinant DNA technology.We constructed E.coli expression system of the fusion protein RGD-FasL.Then we studied its dual activity in vitro,inducing tumor cells apoptosis(H22 and H9101)and the property of tumor targeting.We amplified the fusion gene RGD- FasL by PCR,then cloned the gene RGD- FasL into the express vectors pGEX-5x-1.The recombined vectors were identified through restriction endonuclease analysis and gene sequencing.A single positive E.coli BL21DE(3) of pGEX-SX-1/RGD-FasL clone was incubated in shaken flasks at 30℃in LB medium for 12 h,inducing by 0.5mmol/L IPTG..Several conditions,such as IPTG concentration and inducing time,temperature of induction were optimized. The proteins were expressed mainly as inclusion bodies with the yield of more than 30%of total bacterial proteins.The fusion protein was purified by GST affinity column(according to the manufacturer's protocols of Gen Script Corporation) and identified by SDS-page electrophoresis.On SDS-PAGE,the mobility of the purified protein was found to correspond to a molecular weight of 62 kDa as expected and no degradation was observed.After purification,the purity of the proteins were all more than 95%.MTT demonstrated that the fusion protein RGD- FasL could induce tumor cells(H22 and H9101)apoptosis and suppress the growth of tumor cells in vitro.The expression of Fas and CD61 on the surface of hepatoma cells were assayed by FCM,And we study on adhesive properties of RGD-FasL in vitro.To quantify apoptosis,we utilized the annexin V-FITC staining assay,which reports the loss of phosphatidylserine asymmetry of plasma membrane at the early stage of apoptosis. TUNEL methods were used to detect and quantify myocardial apoptosis in all experimental cell lines,based on labeling of DNA strand breaks.In the H22(mouse liver cancer)-bearing mouse models,the tumor growth inhibitions of RGD- FasL were 62.5%,which is better than group treated with FasL(growth inhibitions 51.3%) In histological studies,the selective localization of the fusion proteins was observed,and tumor cells around the occluded vessels appeared damaged.No thrombosis in blood vessels and cell damage,or other side effects were observed in normal tissues.We can conclude that the fusion protein RGD- FasL could induce tumor cells apoptosis and result in growth suppression in vitro,without harming normal cells,and have the characteristic of tumor targeting.This study shed light on the possible usage of RGD-FasL in further investigation of hepatocellular carcinoma,which provides a voluble way for the design of new gene therapy.