39results about "Leech-based protease inhibitors" patented technology

The invention provides an extraction method of hirudin, an oral preparation containing the hirudin and a preparation method of the oral preparation, and belongs to the technical field of leech processing and traditional Chinese medicine. The hirudin extraction method comprises the following steps: S1, soaking fresh leeches in a freeze-drying protective agent, taking out the leeches, wiping the surfaces of the leeches, pre-freezing the leeches at the temperature of 40 DEG C below zero to 50 DEG C below zero for 1-2 hours, and freeze-drying the leeches at the temperature of 20 DEG C below zero to 30 DEG C below zero for 4-6 hours to obtain freeze-dried leeches; s2, crushing the freeze-dried leeches, adding the crushed leeches into water, adding enzyme and lecithin, performing enzymolysis for 4-6 hours under the conditions that the temperature is 35-40 DEG C and the pH value is 7.5-8.5, and cooling to room temperature to obtain an enzymolysis product; s3, sterilization, ultrafiltration and drying are conducted, and hirudin is obtained. The hirudin extracted by the method is high in activity and high in recovery rate.

The invention discloses a fusion protein rHTA and application thereof, the fusion protein rHTA is sequentially composed of a Hirudin protein, a thrombin cleavage site and an AnxV protein from the N terminal to the C terminal, and the amino acid sequence is as shown in SEQ ID NO: 4 from the 1st site to the 393th site from the N terminal. Experiments prove that the fusion protein rHTA is specifically activated by thrombin at the local part of thrombus and releases AnxV protein and Hirudin protein, and the AnxV protein can specifically recognize and combine with PS, so that the fusion protein rHTA is enriched at the thrombosis part containing platelets in a targeted manner, the Hirudin protein is locally released under the action of thrombin, local precise anticoagulation is realized, and the anticoagulation efficiency is further enhanced. Therefore, the fusion protein rHTA provided by the invention not only has high anticoagulant activity, but also realizes enrichment and release at the thrombus formation part, and reduces the bleeding risk. The method has an important application value.

The invention relates to a recombinant hirudin fusion protein, a coding gene and a prokaryotic expression method and application of the recombinant hirudin fusion protein. The recombinant hirudin fusion protein comprises an HMg variant amino acid sequence and a fusion tag. The recombinant hirudin fusion protein has the advantages that the hirudin variant in poecilobdella manillensis is adopted to construct an efficient prokaryotic expression system, the hirudin yield and activity are improved under the condition that post-translational modification is not needed, the anticoagulant efficiency and the production process of the recombinant hirudin fusion protein provide an important basis for development of a new generation of antithrombotic drugs, and the recombinant hirudin fusion protein has wide application prospects. The progress of cardiovascular disease treatment and hirudin production is expected to be promoted.

The invention discloses a hirudin escherichia coli engineering bacterium with controllable production generations as well as a construction method and application thereof, and relates to the technical field of biological medicines. The invention discloses a construction method of hirudin escherichia coli engineering bacteria with controllable production generations, which comprises the following steps: constructing a hokC protein gene as shown in the sequence of SEQ ID NO.1 and a constitutive promoter trxA as shown in the sequence of SEQ ID NO.2 into a recombinant expression vector pET28a (+)-ptrxA-hokC, and then transforming the recombinant expression vector into a hirudin synthetic strain E. coli Hirudin HV1-3 to obtain the hirudin escherichia coli engineering bacteria with controllable production generations. Through multiple rounds of culture, the hirudin yield of the engineering strain is reduced to 0 in the tenth round. The method is suitable for various hirudin types, and has the characteristics of reliable operation and high safety.

The present invention relates to a polypeptide derived from natural tridegin by structural modification, which may serve as an inhibitor of plasma coagulation factor XIIIa, and to pharmaceutical compositions comprising such polypeptide. The present invention further refers to such polypeptide or pharmaceutical composition for use in a method for treating or preventing a thrombotic event in a patient. Moreover, the invention relates to an endoprosthesis or an enzyme-linked immunosorbent assay (ELISA) plate coated with a polypeptide of the present invention. Furthermore, the invention also encompasses a recombinant fusion protein comprising this polypeptide linked to a fibrinolytic enzyme such as streptokinase, nattokinase or tPAs via a flexible peptide linker containing a thrombin-cleavable site. This construct allows selective inhibition of FXIIIa within a thrombus, while enabling thrombin-triggered release of the fibrinolytic enzyme to induce localized fibrinolysis, thereby achieving targeted dual-action thrombolytic therapy.

The invention discloses a recombinant leech polypeptide with high anticoagulant activity as well as a preparation method and application thereof. A PelB-T2Y-N47K-N52Y mutant is constructed by carrying out structural optimization on a hirudin HV2 sequence, a polypeptide product with relatively high anticoagulant activity is obtained after induced expression and nickel affinity chromatography purification are carried out in a prokaryotic expression system, and the anticoagulant activity of the polypeptide product reaches 9135.8 ATU / mg and is remarkably superior to that of an initial sequence HV2. Meanwhile, the polypeptide shows a lower IC50 value in a hydroxyl radical scavenging experiment, and has the activity of inhibiting generation of AGEs in a non-enzymatic saccharification model. Molecular docking and binding free energy analysis show that the combination of the mutant and thrombin (PDB ID: 4HTC) is more stable. The leech polypeptide disclosed by the invention has the potential of being developed into a multifunctional bioactive peptide preparation, and is suitable for the related fields of anticoagulation, antioxidation and anti-saccharification.

The present invention belongs to the fields of polypeptide synthesis and biomedicine technology, and specifically relates to a polypeptide PM7 and its application in preventing and treating thrombosis. The present invention obtains an original polypeptide from the transcriptome of the salivary gland of the leech Philippine cattle leech, and truncates the original polypeptide to bind to the fibrinogen binding site of thrombin, thereby interfering with the activity of thrombin. Furthermore, {d-Phe}-PRP is added to the N-terminus of the truncated polypeptide, and {d-Phe}-PRP and the truncated polypeptide are connected with a connecting polypeptide to obtain the polypeptide PM7. The polypeptide PM7 of the present invention has the activity of inhibiting thrombin, has a shorter half-life in the body, and is highly safe, thereby achieving the effect of preventing and treating thrombosis.

The invention provides a hirudin separation and purification method and system. The method comprises the following steps: grinding leeches into slurry; adding trypsin into the serous fluid, and then carrying out hydrolysis; performing counter-current extraction on the enzymatic hydrolysate obtained after hydrolysis; carrying out centrifugal separation on extract obtained by counter-current extraction, and collecting supernate; performing ultrafiltration concentration on the supernate by using an ultrafiltration membrane to obtain a concentrated solution; adsorbing hirudin in the concentrated solution by using DEAE-Sepharose FF resin, eluting, and then collecting an eluent; and carrying out vacuum freeze drying on the eluent to obtain freeze-dried powder. The system comprises a purification tank, a grinding device, an enzymolysis tank, a counter-current extraction device, a centrifugal machine, an ultrafiltration concentrator, an ion exchange chromatography instrument and a freeze dryer. In the hirudin production process, conditions are mild, damage to heat-sensitive components is small, high-toxicity organic solvents are not used, green and environment-friendly effects are achieved, and the prepared hirudin is high in activity retention rate and purity.

The present application relates to the technical field of genetic engineering, in particular to a kind of tyrosine sulfated recombinant hirudin, preparation method and purposes thereof.In order to improve the safety of recombinant hirudin, synthesis yield, reduce production cost, the present application successfully prepares tyrosine sulfated recombinant hirudin by algal expression vector.

The invention belongs to the field of genetic engineering and synthetic biology, and particularly relates to tyrosine sulfation modified high-activity recombinant hirudin, saccharomyces cerevisiae engineering bacteria and application. According to the invention, saccharomyces cerevisiae is taken as a chassis cell, tyrosine protein sulfotransferase and related enzymes and transport systems are introduced through genetic engineering modification, efficient sulfation modification of the hirudin Tyr63 site is realized, and the anticoagulant activity of the hirudin Tyr63 site is remarkably improved. The method overcomes the limitations of high extraction cost of natural hirudin and lack of modification function in the existing recombination technology. In addition, the tyrosine sulfation modified high-activity recombinant hirudin provided by the invention shows an excellent antioxidant effect in the field of beauty and skin care, and experiments prove that the antioxidant activity of the tyrosine sulfation modified high-activity recombinant hirudin is equivalent to that of natural hirudin. The invention provides an innovative solution for low-cost and large-scale production of high-activity hirudin, and has the application potentials of medicines and cosmetics.

Provided are a method for extracting a hirudin mutant, HV2-Lys47, from a fermentation broth of the hirudin mutant HV2-Lys47 produced by the fermentation of Escherichia coli, and purifying same, the use of the hirudin mutant HV2-Lys47 in an anticoagulated blood collection tube, and an anticoagulated blood collection tube containing the hirudin mutant HV2-Lys47. In the purification method, a membrane technology and a primary column chromatography technology are used to obtain the hirudin mutant HV2-Lys47.

The invention relates to the technical field of biological extraction, in particular to a method for extracting and purifying high-activity hirudin based on combination of composite enzymolysis and chromatography, which comprises the following steps: sample pretreatment: dissecting leeches to take salivary gland tissues at the heads of the leeches, and freeze-drying and crushing the salivary gland tissues to obtain salivary gland tissue powder; performing extraction: performing enzymolysis extraction on the salivary gland tissue powder by adopting a compound enzyme to obtain a crude extracting solution; the method comprises the following steps: carrying out primary enrichment by using macroporous adsorption resin to obtain a first purified solution, carrying out cation exchange chromatography refining to obtain a second purified solution, carrying out affinity chromatography high purification to obtain affinity chromatography high purification, and carrying out concentration and freeze-drying on the purified product to obtain the high-purity hirudin product. Compared with the prior art, the method has the advantages that the salivary gland tissue powder is subjected to enzymolysis with the compound enzyme at low temperature and purified, so that the purity of the hirudin is 95% or above, and the antithrombin activity of a product determined by a thrombin inhibition test is higher than 15000 ATU / mg.

The present application belongs to the technical field of biological medicine separation and purification, and particularly relates to a leech-derived hirudin separation and purification method based on thrombin affinity magnetic microspheres and application thereof. The present application uses self-prepared leaching liquor to coarsely extract hirudin from leech powder, then selects carboxyl magnetic microspheres to prepare thrombin affinity magnetic microspheres, and further separates and purifies by using the thrombin affinity magnetic microspheres, so as to finally obtain natural hirudin with an activity of 17000 ATU / g or more. The self-prepared leaching liquor can destroy the cell wall of leech, so that the hirudin in the leech is fully dissolved, thereby providing higher efficiency for the coarse extraction of hirudin in leech powder. The prepared thrombin affinity magnetic microspheres can fully utilize the sample enrichment effect thereof, and compared with agarose microspheres and polymer microspheres, can separate and purify hirudin with higher activity and purity, has a long storage period and stable quality, can provide safe and high-quality raw materials for health food, medicine or cosmetics, and is beneficial to industrialized production and application.

The invention discloses a natural hirudin extraction method and application, and relates to the technical field of natural hirudin extraction.The method is characterized by comprising the steps that a tissue structure capable of being subjected to salivary gland single cell combined gland culture is selected; washing away bloodstain in the tissue structure by using PBS, then washing for three times by using the antibacterial liquid, and finally washing away the residual antibacterial liquid by using PBS; inoculating the tissue structure to a cell culture plate, placing the cell culture plate in a 28 DEG C incubator for inverted culture for 2 hours, then adding a DMEM / F-12 complete medium containing a growth additive and not containing serum, and placing the cell culture plate in the 28 DEG C incubator; when the tissue structure generates a large number of secretory vesicles, collecting the culture solution, adding a new culture solution, taking the collected culture solution as a natural hirudin crude product, and separating and purifying the natural hirudin crude product to obtain the natural hirudin. The method can achieve the effects of long-term survival in vitro and generation of a large amount of secretions.

The invention provides a high-activity hirudin freeze-drying preparation as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) introducing a hirudin gene to a chassis cell to construct a recombinant engineering strain; (2) inoculating the engineering strain into a prepared culture medium, carrying out fermentation culture at a set temperature, after the thallus density reaches a target density, switching to methanol induced expression, and after continuous induction is carried out for a preset time, extracting a fermentation liquid; (3) performing ultrafiltration concentration and chromatographic purification on the obtained fermentation liquor to obtain a purified hirudin solution; and (4) adding a stabilizer into the obtained hirudin solution, pre-freezing to completely cure the hirudin solution, and drying to obtain the target hirudin freeze-dried preparation. According to the invention, the high-activity, high-purity, high-stability and low-cost industrial production of the hirudin freeze-drying preparation is realized through collaborative optimization of genetic engineering strain construction, high-density fermentation, multi-step purification and freeze-drying processes.

The present invention belongs to the field of genetic engineering and synthetic biology, and specifically relates to a tyrosine sulfated highly active recombinant hirudin, brewer's yeast engineered bacteria and applications. The present invention uses brewer's yeast as a chassis cell, and through genetic engineering modification, introduces tyrosine protein sulfonate transferase and related enzymes and transport systems to achieve efficient sulfate modification of the Tyr63 site of hirudin, significantly improving its anticoagulant activity. This method overcomes the limitations of the high cost of extracting natural hirudin and the lack of modification function of existing recombinant technologies. In addition, the tyrosine sulfated highly active recombinant hirudin provided by the present invention exhibits excellent antioxidant efficacy in the field of beauty and skin care, and experiments have shown that its antioxidant activity is equivalent to that of natural hirudin. The present invention provides an innovative solution for the low-cost, large-scale production of highly active hirudin, and has potential for application in both medicine and cosmetics.

The present invention relates to the technical field of leech saliva extraction, specifically a device for collecting leech saliva extract, comprising an outer frame, a saliva extraction part, a separation part, a negative pressure part and a shaking part, wherein the outer wall of the outer frame is penetrated with four negative pressure ports distributed at equal angles, and the saliva extraction part is fixedly mounted on the top of the outer frame and extends to the inside of the outer frame. The leech body is squeezed by the inner wall of the anti-reverse tube, and the leech continues to spit out saliva after being pressed. Since the conical extrusion cylinder and the anti-reverse tube are circularly arranged, the conical extrusion cylinder and the inner wall of the anti-reverse tube squeeze the leech body in an embracing manner during the downward movement of the water droplets, so that the leech body is more evenly stressed, and the leech saliva effect is more ideal. Compared with the existing means, it also avoids the situation that the leech is easily crushed due to local squeezing, and the inner wall of the anti-reverse tube is provided with an anti-reverse ring groove inclined downward, so that the leech cannot crawl back after entering the anti-reverse tube.

The utility model belongs to the technical field of hirudin extraction, and particularly relates to an extraction device for hirudin, which comprises an extraction box. Living leeches are placed in a filter box, then a top cover is fixed, a driving motor drives an eccentric wheel to rotate, the eccentric wheel extrudes a ball when rotating for the first half circle, the ball drives an L-shaped plate to move rightwards, the L-shaped plate drives a connecting plate to slide on a guide rod and compress a first spring, and the connecting plate drives the filter box to move rightwards. When the eccentric wheel rotates to the rear half circle, extrusion on the ball is gradually released, the first spring is reset, the first spring drives the filter box to move leftwards through the connecting plate, the filter box shakes left and right in a reciprocating mode, then the leeches are stimulated to automatically spit saliva, and then the saliva of the leeches is flushed to the bottom of the extraction box through the filter box by spraying water through a spray head; when hirudin is separated out in water, shaking of the filtering box is achieved, the leeches are stimulated to secrete saliva more actively, and therefore the extraction period is shortened.

The application provides an extraction method of hirudin, an oral preparation containing the hirudin and a preparation method of the oral preparation, and belongs to the technical fields of hirudo processing and traditional Chinese medicine. The extraction method of the hirudin comprises the following steps: S1, soaking fresh hirudo in a freeze-drying protective agent, wiping the surface after taking out, pre-freezing at-40 to-50 DEG C for 1 to 2 hours, and then freeze-drying at-20 to-30 DEG C for 4 to 6 hours to obtain freeze-dried hirudo; S2, crushing the freeze-dried hirudo, adding water, adding enzymes and lecithin, and enzymolysis at 35 to 40 DEG C and pH=7.5 to 8.5 for 4 to 6 hours, and then cooling to room temperature to obtain an enzymolysis product; and S3, sterilizing, ultrafiltering and drying to obtain the hirudin. The hirudin obtained by the method has high activity and high recovery rate.

The invention provides a recombinant bifunctional hirudin variant 2 fusion protein as well as a preparation method and application thereof, and belongs to the technical field of biotechnology and protein engineering. The recombinant bifunctional hirudin variant 2 fusion protein is obtained by introducing an RGD sequence into a mutation site in a hirudin variant 2, and introducing a His tag, a protease cleavage site and an FXa recognition sequence. After being activated, the recombinant bifunctional hirudin variant 2 fusion protein has obvious antithrombin effect, targeting characteristic and anti-platelet aggregation activity, and can achieve good effect of inhibiting and resisting platelet aggregation under the condition of low concentration. Besides, the expression quantity of the recombinant bifunctional hirudin variant 2 fusion protein is remarkably improved by overexpressing at least one of molecular chaperone genes, antioxidant and stress reaction genes, secretion pathway related genes and bZIP transcription factor genes, and the recombinant bifunctional hirudin variant 2 fusion protein is stable in production process, high in efficiency and suitable for large-scale industrial production and application.

The invention discloses a method for efficiently extracting hirudin from poecilobdella manillensis living bodies, and belongs to the technical field of hirudin separation and extraction. The method comprises the following steps: after hunger treatment of poecilobdella manillensis, inducing secretion by combining an induction solution containing malic acid, citric acid, calcium racemic ketoisoleucine and other components with a pig blood extract, and centrifuging to obtain a crude extract; the method comprises the following steps: preparing an immobilized microsphere carrier through L-cysteine functional modification and amino cationic starch crosslinking, immobilizing thrombin, mixing with a crude extract, extracting, eluting, and freeze-drying to obtain a hirudin product. By optimizing an induced secretion system and an immobilized adsorption purification process, the problems of low secretion induction efficiency, complex purification and insufficient activity retention rate in a traditional method are solved, in-vivo repeated extraction of hirudin is realized, and the method has the advantages of high extraction efficiency, high product purity, high specific activity, reutilization of leech living bodies and the like, and is suitable for industrial production. The large-scale production requirement can be met.

The invention discloses a hirudin separation and purification method based on affinity chromatography, and belongs to the technical field of hirudin separation and purification. Aiming at the characteristics of high viscosity and many impure proteins of a hirudin crude extract, an affinity chromatography medium polystyrene microsphere is modified: modified methoxy polyethylene glycol-polyaspartic acid containing 3, 5-di-tert-butyl-4-hydroxycinnamic acid is grafted, so that the hydrophilicity is improved, the non-specific adsorption is reduced, and the affinity chromatography efficiency is improved; meanwhile, free radicals are removed by utilizing a hindered phenol structure so as to protect the activity of thrombin; after hydroformylation treatment, fixing thrombin as a ligand to form a stable affinity medium; and combining with thrombin affinity chromatography separation, and then carrying out ultrafiltration concentration, desalination and freeze drying to obtain hirudin. A macroporous structure is reserved through a modified medium, the mass transfer resistance is reduced, and the binding efficiency of hirudin and ligands is improved; the activity loss of the hirudin is reduced in a subsequent process. The method can efficiently separate and purify hirudin, has good adsorption selectivity and stability, and is suitable for large-scale separation.

The present application relates to the field of living extraction technology, in particular to a leech living body collection and anticoagulant activity substance synchronous extraction process, which comprises the following steps: a large number of living leeches are poured into a shunt box and are shunted by a shunt channel and then fall into a row of a plurality of object cavities; a motor drives a transfer cylinder to rotate intermittently to pick up the living leeches, and the transfer cylinder adsorbs the leeches; when the leeches fall into a plurality of collection cavities of a collection box, the leeches are attracted by a blood-containing artificial membrane and bite, and the saliva is secreted by the current stimulation of an electrode sheet; the saliva flows into a liquid collecting box and is filtered by a filter membrane in the liquid collecting box, the filtered saliva is directly introduced into a low-temperature container through a bottom port connecting pipeline of the liquid collecting box for collection, and the leech anticoagulant is obtained by centrifugal impurity removal. The funnel structure of the shunt box is combined with the flow guide sleeve to increase the single processing capacity, the air pressure adsorption and the intermittent rotation mechanism of the transfer cylinder are used to complete individual transfer, and the electrode sheet and the artificial membrane integrated stimulation system are used to achieve directional saliva collection.

The invention discloses a supramolecular anticoagulant peptide and application thereof, and belongs to the technical field of medicines. The supramolecular anticoagulant peptide is constructed by adopting a peptide thrombin inhibitor, a self-assembly motif and a response motif of thrombosis related enzyme. The supramolecular anticoagulant peptide can form a semi-solid reservoir hydrogel in a physiological environment, and the supramolecular anticoagulant hydrogel not only has a long-acting anticoagulant characteristic, but also can realize accurate drug release according to the in-vivo thrombus formation level, and can significantly reduce the bleeding risk while ensuring the anticoagulant curative effect, thereby greatly improving the clinical medication safety, and having good clinical application prospects. And a new thought is developed for treatment of thromboembolic diseases.

The invention belongs to the field of daily chemicals, and discloses application of chlamydomonas reinhardtii recombinant hirudin in preparation of cosmetics with an oil control effect. The chlamydomonas reinhardtii recombinant hirudin has a good oil control effect, and can be used as an active ingredient in skin and scalp oil control products. Meanwhile, the invention further discloses a cosmetic containing the chlamydomonas reinhardtii recombinant hirudin.

The utility model discloses a hirudin extraction device convenient to clean, and relates to the technical field of hirudin extraction. Comprising a collecting box, a separating frame and a box cover, a first clip edge is arranged at the bottom end of the separating frame, the separating frame is clamped on the collecting box through the first clip edge, a stainless steel screen is installed in the separating frame, and a second clip edge is arranged at the bottom end of the box cover. According to the hirudin extraction device convenient to clean, living leeches are placed on a stainless steel screen, an electric heating plate is used for heating, the environment temperature in the box is kept within a suitable range, under irradiation of an ultraviolet induction lamp, an inducer is sprayed to the body surfaces of the leeches through a spraying pipe, and the leeches are stimulated to secrete secretory fluid in a warm white light flickering mode; due to the fact that the collecting box, the separating frame and the box cover are designed in a split mode, the living leeches can be conveniently cleaned while the living leeches are conveniently separated from the secretory fluid, follow-up cultivation and reutilization are facilitated, and operation is easy.

The invention relates to a polypeptide derived from natural tridegin by structural modification, which may serve as an inhibitor of plasma coagulation factor XIIIa, and to pharmaceutical compositions comprising such polypeptide. The present invention further refers to such polypeptide or pharmaceutical composition for use in a method for treating or preventing a thrombotic event in a patient. Moreover, the invention relates to an endoprosthesis or an enzyme-linked immunosorbent assay (ELISA) plate coated with a polypeptide of the present invention.

The present invention belongs to the technical field of genetic engineering. Provided are a recombinant hirudin with tyrosine sulfation, a preparation method therefor and the use thereof. In order to improve the safety and synthesis yield of recombinant hirudin, and reduce the production cost, a recombinant hirudin with tyrosine sulfation is prepared by means of an algae expression vector.

The present invention relates to the field of fermentation production. Provided is a method for acquiring chlamydomonas reinhardtii source recombinant hirudin algae powder by means of a fermentation culture method. The method can effectively multiply the algae source recombinant hirudin.

The present application belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to an antithrombotic polypeptide PM4 and application thereof. The present application is based on an original polypeptide obtained from a transcriptome of a leech salivary gland and an amino acid fragment cut from the original polypeptide, increases {d-Phe}-PRP at an N terminal of the cut amino acid fragment through a synthesis strategy of a hirudin analogue drug bivalirudin, and finally connects the {d-Phe}-PRP and the cut amino acid fragment through a connecting polypeptide to obtain the antithrombotic polypeptide PM4. The antithrombotic polypeptide PM4 can inhibit the activity of thrombin, has a small risk of bleeding, a shorter half-life in the body, higher safety, and significant resistance to thrombosis including arterial thrombosis. Meanwhile, the sequence of the antithrombotic polypeptide PM4 is shorter than the original sequence and is easier to synthesize.