Purification Process And Activity Analysis Of Recombinant Fibrinolytic Enzyme Of Sipunculus Nudus

BackgroundThromboembolic disease is a common cardiovascular and cerebrovascular disea-se,the incidence of which ranks first among all kinds of diseases.The global potential market for thrombolytic agents is about$2 billion per year.Marine active substances have gradually become the focus of research in recent years,because of its novel structure,high pharmacological activity and low molecular weight.Our research team has cloned the full-length cDNA sequence of novel fibrinolytic enzyme from the digestive tract of Sipunculus nudus through molecular biology techniques.This gene was named PL after codon optimization.After that,recombinant yeast strains GS115-pPIC9-PL and GS115-pPIC9-CYGB-PL were successfully constructed.ObjectiveThe purpose of this paper is to establish a set of purification technology of reco-mbinant plasmin PL and CYGB-PL,to study the enzyme properties and thrombolytic activity of them in vivo and in vitro.These studies can provide theoretical basis for the industrialized application of recombinant fibrinolytic enzyme from Sipunculus nudus.Method?1?PL and CYGB-PL were purified by column chromatography,tangential flow filtration and ammonium sulphate precipitation.The effect of different temperatures,pH,metal ions and inhibitors on enzyme activity was investigated by fiber plate method to study their enzymatic properties.?2?CYGB-PL-His was produced by genetic engineering in order to simplify the purification steps.?3?PL and CYGB-PL thrombolytic effect of fibrinogen in vitro was determined by the degradation of fibrinogen and the dissolution rate of blood clot.?4?The thrombolytic effect in vivo was verified by the carrageenan-induced mice tail thrombosis model.Results?1?PL was purified by tangential flow filtration,anion-exchange chromatograph-y and heparin affinity chromatography.From 1 liter fermentation media,we obtained5.04 mg of PL with a specific activity of 1324.98 U/mg protein and the final yield was 18.00%.PL has good thermal stability and the enzyme was stable at pH 5.0-9.0.The enzyme activity was strongly inhibited by Cu²⁺and PMSF.?2?By comparing the experimental results,it was shown that the expression of CYGB-PL in the fermentation broth was 46.33%higher than PL.CYGB-PL was prepared by precipitation of fermentation broths with ammonium sulfate and desalted by Sephadex G-25,and further fractionated by anion-exchange chromatography.From 1 liter fermentation media,we obtained 10.19 mg of CYGB-PL with a specific activity of 658.70 U/mg protein and the final yield was 14.65%.Through in vitro enzymatic experiments,it was proved that CYGB-PL also has good thermal stability,and the activity is stable at pH 5.0-9.0.The effect of metal ions and inhibitors on CYGB-PL is similar to that of PL.?3?The engineered strain GS115-pPIC9-CYGB-PL-His was successfully constr-ucted,but its enzyme activity was less than that of CYGB-PL.?4?In vivo and in vitro studies showed that PL and CYGB-PL have the ability to directly degrade fibrin and activate plasminogen into plasmin.PL and CYGB-PL degraded?chains of fibrinogen first and then?and?chain.They has the ability to dissolve fibrin in blood clots in vitro.They also can relieve thrombosis in mouse tails.ConclusionThe results in this thesis demonstrate that the expression of CYGB-PL from fermentation broth is higher than that of PL.We also have proved that PL and CYGB-PL have good thermal stability and good resilience to pH.PL and CYGB-PL purified by the above process have good thermal stability and good resilience to pH.And it has thrombolytic activity in vivo and in vitro.These studies provide a theoretical basis for the application of plasminogen in the starfish.