| Objective To purificate the recombinant fusion protein of Agkihpin a arginine esterase from snake Venom,research on the biological activity and influence factors of enzyme activity of the protein,and to investigate its effect on the growth of liver cells and hepatoma cell.Methods 1.The recombinant gene His6-Agkihpin in BL21(DE3)would express the protein by induction of IPTG,and would be detected by SDS-PAGE and Western blot.2.(1).Purify the recombinant fusion protein His6-Agkihpin with Ni2+ affinity chromatography gel,and then identificate the purity of protein by SDS-PAGE.(2).Determine the arginine esterase activity of the purified recombinant fusion protein His6-Agkihpin with TAME as the reaction substrate;(3).Determine the changes of enzyme activity through the effects of different temperature,pH,metalion,protease inhibitor on recombinant fusion protein His6-Agkihpin;(4).With viper venom,saline,natural purification Agkihpin as control,respectively with lecithin plate and agarose-fibrin plate methods to detect the phospholipase A2 activity and fibrinolytic activity of recombinant fusion protein His6-Agkihpin;(5).0.4%fibrinogen solution mixed with recombinant fusion protein His6-Agkihpin under the condition of 3 7 ? bain-marie,then identificated the influence to fibrinogen by SDS-PAGE.3.Culture liver cells L-02 and hepatoma cells BEL-7404,then affected with different concentrations of fusion protein His6-Agkihpin by 72h,the cellularvitality was detected by CCK8,the impacts on proliferation and migration of cell were detected by cell count.Results 1.After induced by IPTG,SDS-PAGE appears obvious protein band at?30 KDa,Western Blot also identificated recombinant fusion protein His6-Agkihpin bands in the corresponding position,and results showed the recombinant fusion protein His6-Agkihpin was inclusion body protein.2.Purified recombinant fusion protein His6-Agkihpin identified by SDS-PAGE,at?30KDa appeared the protein bands and basically reached the electrophoretic homogeneity;The TAME method to measure the arginine esterase activity was 8.723 U/mg.3.(1).The recombinant fusion protein His6-Agkihpin reflected the highest activity at temperature of 37—and pH 8.0.(2).Several different metal ions were used in experiment could enhance on the arginine esterase activity of recombinant fusion protein His6-Agkihpin,calcium ions in which the strongest effect.(3).EDTA has inhibition effect on the enzyme activity,while the other inhibitors had little effect.4.Using lecithin plate,it was found that there are transparent circle from crude venom,natural and recombinant fusion Agkihpin around the hole which resulted the activity of phospholipase A2.5.Using agarose-fibrin plate,it was found that there are transparent circle from crude venom,natural and recombinant fusion protein His6-Agkihpin around the hole which resulted in the fibrinolytic activity.6.SDS-PAGE to identify the fibrinogenolytic activity of recombinant fusion protein His6-Agkihpin,and the hydrolysis starts from 0.5h till 8h the a band had been completely Hydrolyzed.7.Compared with the control group,the cellular vitality of BEL-7404 reduced obviously(P<0.01)with the increaseing of concentration of His6-Agkihpin(P<0.05,r=0808),and the inhibiting rate ranged from 11.69%to 48.52%,after the treatment of 0.207?4.13 ?g/ml His6-Agkihpin;the proliferation of BEL-7404 was inhibited obviously in a concentration-depended manner(P<0.05),and the inhibiting rate ranged from 20.47%?76.76%.56.36%?79.38%and 11.79%?45.44%respectively,after treated with 1.033?4.13?g/ml His6-Agkihpin respectively for 24h,48h and 72h;the concentration of 3.23 ?g/ml and 4.13?g/ml His6-Agkihpin can cause casualty of BEL-7404 in a rate of 19.73%and 50.22%;the migration of BEL-7404 reduced obviously with the increasing of concentration of His6-Agkihpin(p<0.05,r=0.759),and the inhibiting rate ranged from 8.7±0.52%to 52.58±7.8%after 24h,while 11.49±0.89%to 92.43±7.46%after 48h.8.The recombinant fusion protein His6-Agkihpin promoted the cellular vitality,proliferation and migration of L-02 cells to a certain degree at concentration from 0.207 to 4.13 ?g/ml.Conclusions 1.We purified electrophoretically the pure recombinant fusion protein--His6-Agkihpin successfully.2.The emzymatic activity of His6-Agkihpin affected by reactaion temperature and pH.His6-Agkihpin reflected the highest arginine esterase activity at 37?,pH 8.0.3.His6-Agkihpin was a kind of metal protein,its vitality was promoted by metal ions,espicailly by Ca2+;and EDTA inhibited the enzyme activity obviously.4.His6-Agkihpin had the activities of phospholipase A2,ibrinolysin and thrombin-like enzyme.5.The recombinant fusion protein His6-Agkihpin at the concentration from 0.207 to 4.13?g/ml could inhibit the cellular vitality,proliferation and migration of hepatoma cells BEL-7404 significantly,while promote that of L-02 cells to a certain degree. |
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