The Antithrombus And Antitumor Effects Of A Protein With Fibrinolytic Activity From Eupolyphaga Sinesis Walker And Its Gene Cloning And Expression

Eupolyphaga sinensis Walker, a Chinese traditional medicine, which is used to activate bloodflow and remove blood stasis as recorded in Chinese Pharmacopoeia （2000 edition）. It also exhibitsthe effects of melting vein thrombosis, restraining blood platelet aggregation and anticoagulationthrough the modem pharmacological researches. But the effective component and the underlyingmechanisms are not very clear. Here we purified a protein with fibrinolytic activity fromEupolyphaga sinesis Walker （EFP） and detected the antithrombotic, anticoagulatic andfibrinolytic effects respectively. We also analysed the activities of EFP on anti-angiogenesis andanticancer. Moreover, we cloned EFP gene and expressed it both in E. coli and in Yeast PichiaPastoris. The results are summarized as follows.The antithrombotic experiments in vivo indicated that EFP could inhibit the thrombus forming onmice tail induced by carrageenin, delay thrombin time in mice obviously, enhance tissueplasminogen activator （t-PA） activity and inhibit plasminogen activator inhibitor activitysignificantly. The results suggest that EFP could suppress thrombosis.The proliferation ability of human microvascular endothelial cell （MVEC）/n vitro was examinedby MTr assay. The result showed that EFP could inhibit MVEC proliferation efficiently, in adose-dependent manner. The inhibitory rate reached 45.93％under the experimentalconcentration （0.02-200μg/ml）. EFP could induce the apoptosis of MVEC as indicated by AnnexinV-FITC/PI fluorescence staining and single cell gel electrophoresis （SCGE） assay. The flowcytometry analysis showed that EFP could block the cell cycle at S and G₂/M phases, inhibit celldivision and proliferation. Embryo chorioallantoic membrane （CAM） test showed that EFP couldinhibit angiogenesis effectively in a dose-dependent manner.The anti-tumor experiments in vivo indicated that EFP could inhibit the tumor growth in S180and H22-bearing mice significantly, and the inhibition rate could reach 48.54％and 42.16％respectively. At the same time, we also examined MDA and SOD level in the mice’s liver, and theresults showed that EFP could enhance the antioxidation ability in H22-bearing mice. By measuringthe level of anti-H22 antibody in serum and the spleen index in H22-bearing mice, the resultsshowed that EFP could enhance the immunity in those mice. By analyzing the homogeneity and the conserved sequence of fibrinolytic enzyme amino acidsequences of several animals, the primers wcrc designed. A fragment of ElF cDNA sequence wasamplified by PCR. Because the 5’ primer is the sequence of N terminal, the fragment contains awhole 5’ terminal. Wc obtained the 3’ terminal of the cDNA sequence by using 3’RACE. Then thewhole cDNA sequence of ElF was acquired.With the gcnc of EFP cloned, we constructed a recombinant vector pET28a-EFP, and transformedit into E. coli Rossctta. Transformants were induced by IPTG. The target protein was examined bySDS-PAGE and Western blot analysis. Nearly 10％of the total bacterial protein was recombinantprotein expressed in inclusion body form but without activity.Then we rccombined EFP gcnc with Yeast Pichia Pastoris vector pPICZαA, and transformed itinto Yeast Pichia Pastoris, the transformants were screened and cultured by methanol. Expressionproducts wcrc analyzed by SDS-PAGE and Western blot, and the results showed that the targetprotein has bccn expressed and with fibrinolytic activity by a fibrin plate assay.All the results showed that EFP exhibits effects of anti-thrombus, anti-tumor andanti-angiogcncsis. The EFP gcnc has bccn cloned successfully, and EFP has bccn expressed byYeast Pichia Pastoris expression system.