77 results about "Heparinase" patented technology

The present invention relates to immobilized biologically active entities that retain a significant biological activity following manipulation. The invention also comprises a medical substrate comprising a heparin entity bound onto a substrate via at least one heparin molecule, wherein said bound heparin entity is heparinase-1 sensitive.

The invention discloses a heparanase III fusion protein and a coding gene and an expression method thereof. The fusion protein of the invention (named as MBP-HepC) is a protein in a) or b): a) a protein consisting of amino acid sequences shown by the 1-1028th site of a sequence 2 in a sequence table; and b) a protein derived from a), provided with heparanase III activity and obtained by substituting and / or deleting and / or adding one or more amino acids in an amino acid sequence of the sequence 2 in the sequence table. The coding gene of the protein is also within the protection range of the invention. By introducing the gene into protein heparanase expressed in recombinant escherichia coli, the enzymatic activity can reach 1,776.3 IU per liter of fermentation liquid, the expression amount can reach 240 mg per liter of fermentation liquid, and the specific enzymatic activity can reach 7.39 IU / mg. The invention also can realize further purification of the fusion protein through affinity separation.

The present invention relates to immobilized biologically active entities that retain a significant biological activity following manipulation. The invention also comprises a medical substrate comprising a heparin entity bound onto a substrate via at least one heparin molecule, wherein said bound heparin entity is heparinase-1 sensitive.

The invention provides sulfated heparin oligosaccharide as well as a preparation method and application thereof. The sulfated heparin oligosaccharide is characterized in that a non-reducing end of a sulfated heparin oligosaccharide molecule contains an unsaturated double bond produced through enzymolysis of heparinase as well as uronic acid derivatives and glycosylamine derivatives; the sulfated heparin oligosaccharide has a structure represented by a formula I, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, Ra, Rb, Rc and Rd are independently SO3<-> or H; Rx', Ry' and Rz' are independently COCH3 or SO3<->, and n is 1-3. The sulfated heparin oligosaccharide with controllable sulfating degree prepared by virtue of the preparation method has very high activity for inhibiting heparanase in vitro, the activity of the sulfated heparin oligosaccharide for inhibiting cell adhesion and migration is 4-5 times higher than that of heparin, and the activity for inhibiting tumor metastasis in mice is 2-3 times higher than that of the heparin; the sulfated heparin oligosaccharide has relatively good tumor metastasis resistance and relatively high specificity.

Heparinase enzymes can be used as a medical treatment to reduce localized inflammatory responses. Treatment of activated endothelium with heparinase inhibits leukocyte rolling, adhesion and extravasation. Most of the heparin and heparan sulfate on endothelial cell surfaces and in basement membranes is degraded by exposure to heparinase. In addition, immobilized chemokines, which are attached to heparin / heparan sulfate on activated endothelium are solubilized by heparinase digestion. Heparinase can be infused into the vascular system to inhibit accumulation of leukocytes in inflamed tissue and decrease damage resulting from localized inflammations. Targeting of heparinase to activated endothelium can be accomplished through localized administration and / or use of genetically engineered heparinase containing endothelium ligand-binding domains.

The invention relates to the field of blood detection, in particular to a detection method for evaluating the curative effect and the residual condition of heparin drugs. The heparin drugs mainly comprise heparin, low-molecular-weight heparin and some heparinoid drugs. The detection method mainly comprises steps as follows: heparinase, a freeze-drying protective agent and an adhesive are mixed, then a mixed reagent in appropriate proportion is prepared from an obtained mixture by the aid of a buffer solution, the mixed reagent is enveloped in a test-cup and subjected to freezing and vacuum pumping treatment, and a heparinase cup is obtained. The detection method has a plurality of advantages that the operation is simple, the guarantee period is long, the stability is good and the like. Theheparinase cup and a common cup (a blank sample cup) are put in a thrombelastogram, an anticoagulant and a chelating agent are added to the cups respectively, endogenously activated whole blood samples are added to the test-cups, and thromboelastography is started for detection; values R of starting time of blood clot formation in the heparinase cup and the common cup are compared, and whether heparin exists in the blood can be judged; if heparin exists, the value R of the starting time of blood clot formation in the heparinase cup is smaller than that of the starting time of blood clot formation in the common cup. The method is simple to operate, a detection result is good in repeatability, and the accuracy is high.

The invention belongs to the technical field of biological medicine, and particularly relates to a method for preparing a protamine-deoxycholic acid conjugate with a heparin transfer function. The amidogen of protamine and the carboxyl of deoxycholic acid form an amide bond through teh effect of a cross-linking agent to prepare an amphipathic conjugate, and then the amphipathic conjugate and heparin form a self-assembled aggregation in order to conveniently release the heparin in cells. The hydrophobic modification of the conjugate can enhance the self-aggregation stability of nano-composites; the cationic performance of the conjugate entraps the heparin, so that the distribution capability of the heparin in the cells is increased, and the heparin is prevented from, degradation caused by the effect of heparinase and further the transfer of the heparin into cancer cells and the release and biological effect of the heparin in the cells are realized.

The invention discloses a method for producing low-molecular-weight heparin or ultralow-molecular-weight heparin. According to the method, heparinases selected from more than two of heparinases I, II and III are used for degrading heparin so as to produce the low-molecular-weight heparin or ultralow-molecular-weight heparin.

The invention discloses a method for preparing recombinant heparinase III by utilizing an SUMO fusion expression system and the heparinase III prepared by the method, the preparation method comprises the following steps: selecting a heparinase III sequence from Pedobacter heparinus, wherein the amino acid sequence of the heparinase III is shown as SEQ ID No.1, and the total number is 659aa; removing a signal peptide sequence to obtain a DNA sequence of the heparinase III, such as SEQ ID No.2; inserting the DNA sequence of the heparinase III into a pSMART vector plasmid with an N-terminal SUMO protein tag; transforming the correct plasmids into BL21 (DE3) escherichia coli competent cells, selecting monoclone, and obtaining fusion protein through fermentation and purification; and cutting the SUMO tag protein from the fusion protein by using the SUMO protease to obtain the heparinase III. The method has the advantages that the solubility of the target protein is improved, correct folding of the target protein is promoted, and inclusion bodies are prevented from being formed; the purification cost is reduced and the purification efficiency is improved; the molecular weight of the SUMO protein tag is small, the influence on the enzymatic activity of the heparinase III is small, and the purified heparinase III is obtained.

The invention relates to non-animal-derived LMW (low molecular weight) heparin, a preparation method therefor and application of the non-animal-derived LMW heparin. The preparation method disclosed bythe invention comprises the steps of performing a one-step chemical method and a variety of enzyme catalysis methods in order: extracting an exopolysaccharide K5CPS, subjecting the exopolysaccharideK5CPS to chemical-method N-deacetylation / N-sulfation modification, and then, carrying out partial depolymerization in a buffer solution through heparinase III; and subjecting obtained LMW products toenzyme-method C-5 epimerization / 2-O-sulfation modification, 6-O-sulfation modification and 3-O-sulfation modification sequentially, thereby obtaining a product with anti-FXa and anti-FIIa activity. The invention provides a novel method for preparing the LMW product with typical structure characteristics of the heparin and remarkable anticoagulation activity. According to the method, raw materialsare non-animal-derived, the quality is controllable, the risk of contamination is low, adopted reaction conditions are mild and efficient, the obtained product is easy to separate, and large-scale preparation of the LMW heparin with good structural homogeneity and high anticoagulation activity can be achieved.