Construction Of Eukaryotic Expression System For Endostatin And Its Experimental Anti-angiogenic And Tumor Inhibitory Effect

Objective To achieve expression of recombinant human endostatin(rhES) in a eukaryotic expressin system,the yeast strain Pichia pastoris,and to observe the inhibitory effect of rhES(Endostar) on the growth of corneal neovascularization and nude mouse breast cancer cell line MCF-7,and to lay foundation for futher clinical search.Methods Endostatin gene was cloned from the human liver by RT-PCR and recombined into the yeast vector pPIC9 after DNA was subsequenced to prove its correctness.The linearized recombinant plasmid was transformed into chromosome of Pichia pastoris GS115 strain by electroporation.The yeast strains with high soluble rhES expression were fermented in flasks and selected.The animal models were constructed and used to characterize activity of rhES(Endostar).Animal experiment(NO.1):Alkanli burn was used to induce neovascularization in 20 New Zealand white rabbits,which were divided into 2 groups at random,10 in each.0.05ml rhES(Endostar) and normal saline were respectively injected into subconjunctival tissues of rabbits in experiment group and control group,one time every other day.The area of CNV was taken pictures regularly.The microvessel quantity was counted by microscope with immunohistochemical CD34 assay.Animal experiment(NO.2):Human breast cancer model was established by hypodermic implantation of histologically intact tumor tissue into 30 nude mice,which were divided into 2 groups at random,15 in each.rhES (Endostar) and PBS were respectively injected into subcutaneous stratum of nude mice in experiment group and control group.Endostar was administered sc with dose of 10.0 0mg/kg one time every other day for seven weeks.Eight weeks after implantation,tumor size,inhibition rate,intratumoral microvessel density and apoptotic index were evaluated respectively after the mice were sacrificed.Result The endostatin cDNA was successfully cloned and eukaryotic expressin system carring rhES cDNA was constructed.The two yeast strains with soluble rhES expression were selected,and SDS-PAGE analysis showed that the expressed product was the larget protein of 20KD.Animal experiment NO.1 proved that the area of CNV between the experiment group and control group during 4,7,14,19th days were (4.06±0.52;5.67±0.61;17.71±1.65;12.09±1.32)mm~2,(4.80±0.63;8.48±1.03; 23.91±1.82;16.75±1.79) mm~2 respectively,and MVD were 3.60±0.94,5.87±1.44 respectively.Animal experiment NO.2 proved that the volums of tumors were 18 374.6±471.2.mm~3,43 604.7±495.1mm~3,MVD were 14.08±3.48,25.25±4.73,AI(%) were11.54±2.85,4.25±2.18 respetively between the experiment and control groups and the inhibitory rate was 57.86%.The diffence between the experiment group and control group was significant(P<0.01).Conclusions The successfully constructing of eukaryotic expressin system carring rhES gene makes it possible to express ES protein higher,which provides a theory foundation for durg manufacture to produce rhES in quentity;The rhES has the ability to restrain the growth of corneal neovascularization and breast cancer MCF-7 cells in nude mice,which is showing a wide perspective for future clinical applications.