Expression,Purification And Application Of Heparinase

Heparinase is a type of polysaccharide lyase that can specifically lyse heparin or heparan sulfate biological macromolecules.It has a wide range of applications in the preparation of low molecular weight heparins,analysis of the molecular structure of heparins,and medicine.Among them,the thromboelastogram heparinase comparative test is widely used in clinical practice,which can guide doctors to use anticoagulant drugs rationally.Therefore,heparinase has broad application prospects in thromboelastography.However,the currently commercialized heparinase has a relatively single source,resulting in a relatively high price for heparinase in clinical use,which cannot be accepted by the general public.Therefore,reducing the production cost of Flavobacterium heparinum or researching and constructing new heparinase recombinant bacteria is of great significance.This subject aims at the above problems and studies the production of heparinase from two aspects at the same time.On the one hand,the process of producing heparinase by the fermentation of Flavobacterium heparinum is optimized.On the other hand,the Pichia pastoris expression system and the Bacillus subtilis expression system were selected to construct a genetically engineered strain expressing heparinase.The main research results of this subject are as follows:1.Construction of three expression systems for recombinant heparinase: The encoding c DNA sequence of heparinase I was optimized using gene designer 2.0 software,which was successfully cloned into yeast extracellular and intracellular recombinant expression vectors,and Bacillus subtilis secretory expression vectors to construct p PIC9K-He E,p PIC3.5K-He E and p HT43-He E recombinant plasmids using full gene synthesis technology and traditional cloning technology.Plasmid p PIC9K-He E and p PIC3.5K-He E were linearized by restriction enzyme Sal I,and transferred into Pichia pastoris GS115 by electric shock method;p HT43-He E was directly transferred into Bacillus subtilis WB800 N by electric shock method.Yeast and Bacillus subtilis expressing recombinant heparinase were obtained.It is a pity that the expression protein was not detected in recombinant yeast and Bacillus subtilis through induced expression.2.Study on the fermentation process of Flavobacterium heparinum heparinase: The Flavobacterium heparinum was cultured at the stage of recovery-culture-fermentation bacteria growth-fermentation induction.The fermentation process of Flavobacterium heparinum was optimized from four aspects: fermentation medium,inoculation fermentation form,three-stage fermentation broth fermentation density and heparin sodium induction time.It was found that the fermentation and cultivation of Flavobacterium heparinum in the form of monoclonal in LB medium is more conducive for enzyme production;when the OD600 of the tertiary bacterial solution is diluted by 5times,heparin sodium is added between 2.5-3.1 and induced for 8 hours.The expression level of Flavobacterium heparinum is high,and the highest amount can reach 183IU/g.3.Separation and purification of heparinase: the crude enzyme solution was optimized for Q HP and SP FF column conditions,and a complete purification process was explored.First,heparinase penetrates the Q HP chromatography column in a p H 9.0environment,and secondly,the Q HP penetrating solution passes through the SP FF chromatography column in a p H 7.0 environment.At this time,about 90% of the heparinase is suspended.In the column state,0.3M Na Cl buffer is used to elute the heparinase,and finally the SP FF elution is loaded to the Cellufine Sulfate column,and the same concentration salt is used to elute the target protein.The final electrophoresis purity can be achieved,and its specific enzyme activity is 625 times that of the crude enzyme solution.4.Application of heparinase in thromboelastography: The heparinase after three steps of purification is dialysis,ultrafiltration concentration and lyophilized to prepare a thromboelastogram heparinase detection cup.The blood test of heparin treatment shows that the self-made thromboelastogram heparinase test cup has the ability to neutralize heparin to a certain extent.