The Expression Of Nattokinase In Pichia Pastoris And Its Thrombolytic Effects In Vivo Of Nattokinase In A Rat Model Of Thrombosis

Thrombosis is a kind of disease that involves blood circulation system,which has high disabled rate and case fatality rate,seriously endangering the life and health of human beings.With the excessive consumption of high fat and high protein dietary intake and the arrival of an aging society,thrombotic disease has exceeded cancer and diabetes becomes the first big health killer.Dissolve thrombus is an important mean to cure this kind of disease,thrombolytic drugs mainly through dissolving thrombus directly or through the activation of plasminogen into fibrinoclase to play a role.Nattokinase is a kind of alkaline serine protease produced by the Bacillus subtills which can dissolve thrombosis significantly in vivo and in vitro;it is a kind of potential thrombolytic drugs.Currently approved thrombolytic drugs in clinical practice are Streptokinase(SK)and Urokinase(UK)and so on.However,those drugs are easily resulting in bleeding and have a short lasting effect.Compared with the existing thrombolytic drugs,nattokinase come from traditional food with the advantages of low cost and safety,and can be absorbed by the intestinal,it has long half-life in the body and not easy to cause bleeding.Therefore,nattokinase has attracted the attention of biological medicine and food field.The traditional method of producing nattokinase is mainly by siolid fermentationt.Both fermentation and extraction process are complicated,furthermore,low yield of product is not suitable for large-scale production.The nattokinase extracted from solide fermentation is mixed with purine,vitamin K2,which has side effects.There were several reports about the nattokinase gene was expressed in engineered strains,but the yield is low,and cannot satisfy the requirement of production.A serine protease gene from Bacillus subtilis was optimized to match the codon usage preference of P.pastoris,the modified genes were cloned into pichia expression vectors pHBM905A,and then transformed into E.coli.Preliminary screening of recombinant plasmid by colony PCR,the positive clones were sequenced,the right recombinant plasmid was named pHBM905A-Natto.The recombinant plasmids were transformed into P.pastoris GS115 via electroporation.P.pastoris-Natto was used as the strain for the high-density fermentation,and the fermentation condition was optizmed in this study.The optimum pH and temperature for the growth of strain accumulation were 5.0 and 28?,and the 26? and pH 4.3 for inducing expression.After 96 h methanol induction,the maximum yield of total protein was 9.5 g/L with specific activity of 955.4 U/mg.Through technical research,this study found an effective method to fast purify recombinant nattokinase.The purity of protein was about 90%and the purity of target protein yield is about 56%,The purification method is more simple and efficient than traditional extracted from natto,and the recombination nattokinase of the production is more safe and reliable.This project studied the enzymatic properties of nattokinase.The optimum pH and temperature for the recombinant NATTO reaction were 9.0 and 65 ?,respectively.And the study found that the enzyme performed a tolerance to most organic solvents and metal ions,and the addition of Tween exhibited an activation of it.So it could be potentially applied in organic synthesis and detergent production.This subject has studied both the effect of nattokinase on thrombolysis in vivo and in vitro.The results show that the nattokinase has significant effect of thrombolysis in vitro.To establish a rat model of thrombosis,?-carrageenan was subcutaneously injected into the toes of Sprague-Dawley(SD)rats,and then the rats were treated with varying doses of nattokinase by gavage.Finally,the ear tissue and the plasma were respectively taken for subsequent experiment.ELISA kit was used to determine the levels of the fibrin/fibrinogen degradation products(FDPs)and D-dimer in plasma,which are sensitive indices of fibrinolytic activity.Both the histological and physiological evidence from this study indicate that nattokinase exerts thrombolytic effects in vivo.