12results about "Leech-based protease inhibitors" patented technology

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 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 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 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 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 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 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 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 invention relates to a recombinant hirudin fusion protein, an intelligent response type anticoagulant material as well as a preparation method and application of the intelligent response type anticoagulant material. The intelligent response type anticoagulant material is formed by constructing the recombinant hirudin fusion protein with a carbohydrate binding domain (CBM) and combining the recombinant hirudin fusion protein with bacterial nanocellulose or a composite material of the bacterial nanocellulose. The recombinant hirudin fusion protein disclosed by the invention can specifically respond to blood coagulation formation related enzymes so as to recover the anticoagulant activity, and can be well combined with a bacterial nanocellulose material or a composite material thereof; the intelligent response type anticoagulant material is mild in preparation condition and green and environment-friendly in technology, and has great potential in the medical application aspect of membranous anticoagulant biological materials such as heart valves and the like and tubular anticoagulant biological materials such as artificial blood vessels and the like.

The invention relates to a reconstructed hirudin fusion protein and application thereof, and the reconstructed hirudin fusion protein comprises a first part of thrombus targeting peptide and a second part of hirudin or hirudin derivative mutant, wherein the first part of thrombus targeting peptide is connected with the second part of hirudin or hirudin derivative through a recognition sequence of thrombus site related enzyme. The anticoagulant is different from some traditional anticoagulants such as heparin and hirudin, and has important significance in preventing and treating thrombus and reducing side effects generated in prevention and treatment of thrombus.

The invention belongs to the technical field of polypeptide synthesis, and relates to a compound containing a 2-phenyl-2-trimethylsilyl ethanol structure as well as a preparation method and application thereof. The structural formula of the compound containing the 2-phenyl-2-trimethylsilylethanol structure is shown as a formula I. The carrier compound containing the 2-phenyl-2-trimethylsilylethanol structure is used as a protective carrier of amino acid or polypeptide C terminal; the amino acid or polypeptide protected by the carrier compound containing the 2-phenyl-2-trimethylsilyl ethanol structure can be dissolved in a proper solvent, so that the peptide grafting reaction is carried out in a homogeneous system, the reaction condition is mild, the energy consumption is low, the reaction is rapid and efficient, the reaction speed is high, the reagent utilization rate is high, and the reaction process can be quantitatively monitored in real time; after the reaction, rapid separation and purification are realized through sedimentation or extraction separation, the operation process is simple and efficient, and the universality is high.