ABSTRACT
<p><b>OBJECTIVE</b>To obtain the cDNA sequence encoding fibrinolytic enzyme from Eupolyphaga sinensis and express it in prokaryotic and eukaryotic expression system.</p><p><b>METHOD</b>The primers were designed according to the cDNA of other animals'fibrinolytic enzyme. The cDNA sequence was cloned by RT-PCR and 3 RACE.</p><p><b>RESULT</b>Sequence analysis revealed that the length of the cDNA fragment was 672 bp and encoded a protein of 224 amino acid residues, the N end amino acid sequence residues was IVGG in accordance with other fibrinolytic enzyme. The cDNA sequence was expressed in E. coli, inactive protein was obtained. While expressed in Pichia pastoris, recombinant protein had fibrinolytic activity.</p><p><b>CONCLUSION</b>The cDNA sequence of fibrinolytic enzyme from E. sinensis Walker was cloned and expressed for the first time and it proved a good basis for further functional study of the enzyme.</p>
Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cockroaches , Chemistry , Genetics , DNA, Complementary , Chemistry , Genetics , Fibrinolysin , Chemistry , Genetics , Metabolism , Gene Expression , Insect Proteins , Chemistry , Genetics , Metabolism , Molecular Sequence Data , Sequence AlignmentABSTRACT
Aim To explore the inhibitory effects and immunoregulatory activity of the extract from Eupolyphaga sinensis Walker (EFP) on the growth of liver cancer H22 in mice.Methods The chemical composition of extract from Eupolyphaga sinensis Walker(EFP)was separated with water extraction methods.The tumor bearing mouse model was constructed by injecting tumor cells subcutaneously.The EFP was given to the mouse by oral and hypodermic injection to observe its antitumor activity and immunoregulatory activity in vivo.The antibody level of anti-H22 in serum was measured with ELISA methods.The weight,tumor weight,spleen index,liver index were noted,and series of enzymes,such as,MDA,SOD,GPT and GOT activity of mouse serum and liver were measured.Results The extracts from Eupolyphaga sinensis Walker markedly inhibited the proliferation of tumor H22.The EFP constituent purified might be the main antitumor active part of Eupolyphaga sinensis Walker as shown by antitumor test in vitro.After treatment with EFP,the general condition of the test mice was much better than that of control mice,with spleen index,liver index increasing more obviously,antibody level of anti-H22,and MDA,SOD,GPT and GOT activity increasing more,and tumor growing more slowly.Conclusion EFP may be the main antitumor active part of Eupolyphaga sinensis Walker. EFP also has antitumor effect in vivo.EFP can exert antitumor effect in vivo by enhancing antibody level of anti-H22 and the series enzymes to be related to immunity activity.
ABSTRACT
Aim The aim is to purify fibrinolytic enzyme from Scolopendra subspinipes mutilans L.Koch and to study the thrombolytic and anticoagulant effect.Methods Scolopendra subspinipes mutilans L.Koch fibrinolytic enzyme(SSFE) was purified by ammonium sulphate precipitation,DEAE-cellulose and SephadexG-75 column chromatography from Scolopendra subspinipes mutilans L.Kochby.And fibrinolytic activity was determined by fibrin plate.The anticoagulant effect was measured on mice with haemolytic test and hemorrhagic test.The thrombolytic effect was measured with rats In vitro and in vivo,and the activated partial thromboplastin time(APTT),plasma prothrombin time(PT),thrombin time(TT) were measured.Results SSFE was single component with fibrinolytic activity and without any hemolyzation and hemorrhagic activity.All doses of SSFE(2,5,10 mg?kg-1) could obviously prolong activated partial thromboplastin time(APTT) and thrombin time(TT) ;Middle dose of SSFE(5 mg?kg-1) could prolong plasma prothrombin time(PT) while high dose of SSFE(10 mg?kg-1) didn't prolong obviously.Conclusion SSFE has obvious thrombolytic effect and anticoagulant effect.