47 results about "Enzyme specificity" patented technology

The invention discloses an EV membrane protein single molecule detection method based on droplet microfluidics and application thereof. The method comprises the following steps: firstly, constructing an Ab-oligo compound which is formed by sequentially connecting an antibody, Linker, Linker connection DNA, RNA capable of being specifically cut by RNA enzyme and template DNA for starting signal amplification; then capturing the extracellular vesicles in the plasma by using the CD9 / CD63 / CD81 functionalized magnetic beads; the method comprises the following steps: adding an Ab-oligo compound and a sealing buffer solution into extracellular vesicles, incubating, removing the uncombined Ab-oligo compound, adding RNA enzyme for incubating and enzyme digestion, collecting template DNA for starting signal amplification, and performing microdroplet digital PCR analysis to realize single molecule analysis of EV membrane protein. According to the invention, the one-to-one correspondence relationship between the antibody and oligonucleotide is established through Linker for the first time, single protein information is converted into single nucleic acid chain information, and EV membrane protein single molecule detection is realized. The method is simple to operate, can qualitatively and quantitatively detect the single EV membrane protein, and is beneficial to further research on the relationship between the EV membrane protein and diseases.

The invention discloses a nanofiber based on a polypeptide derivative and an enzymatic self-assembly method and application of the nanofiber based on the polypeptide derivative, the sequence of the polypeptide derivative is Nap-Gly-Phe-Phe-pTyr-Lys-Trp-Tyr-Gln-Asn-Met-Ile-Arg, and the Gly-Phe-Phe-pTyr is of an L configuration or a D configuration. The self-assembled polypeptide derivative disclosed by the invention contains an alkaline phosphatase restriction enzyme cutting site, and the self-assembled polypeptide derivative is self-assembled at a specific part with high enzyme specificity expression through enzyme catalysis to form nanofibers. The nanofiber hydrogel obtained through enzymatic treatment is carried out under physiological conditions, high temperature or ultraviolet irradiation is not needed, damage of high temperature to bioactive substances is avoided, the nanofiber hydrogel is more suitable for a temperature-sensitive in-vivo environment, toxic chemical cross-linking agents or photoinitiators do not need to be introduced, the cytotoxicity of the material is remarkably reduced, active enzymes or drugs can be embedded in situ, and the nanofiber hydrogel can be used for in-situ treatment. The biological function is maintained.

The invention discloses a modified 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), which has higher activity and stability in the synthesis process of ursodesoxycholic acid (UDCA), and takes NAD (P) H as a coenzyme. According to the 7beta-hydroxysteroid dehydrogenase mutant, the V104 site in the 7beta-hydroxysteroid dehydrogenase is subjected to saturated mutation modification, and the mutant with enhanced activity and changed coenzyme specificity is screened out. The mutant enzyme can keep high catalytic efficiency under the condition of taking NADH as a coenzyme, so that the synthesis yield of UDCA is remarkably increased, and the production cost is reduced. The enzyme mutant disclosed by the invention has a wide application prospect in industrial production, is particularly suitable for the production process of large-scale biosynthesis of UDCA, can effectively reduce the production cost, improve the product yield and reduce the environmental pollution and the production cost, and provides a technical support for wide application of UDCA.

The invention belongs to the field of biomolecule detection, and discloses a polysaccharide high-throughput detection method and application. The method can be used for detecting the heparin precursor, a sample does not need to be purified, the heparin precursor in a complex sample can be directly recognized and degraded by enzyme specificity to generate disaccharide, and then the content of the disaccharide of the heparin precursor is detected through high-throughput EchoMS, so that the heparin precursor is subjected to rapid, high-throughput, qualitative and quantitative analysis; the method is used for screening and evaluating elements influencing heparin precursor synthesis, screening and evaluating heparin precursor synthesis strains, optimizing the heparin precursor fermentation process and the like. The method can also be used for high-throughput detection of glycosaminoglycans such as hyaluronic acid, heparin and chondroitin sulfate and other polysaccharides such as pullulan, curdlan, xanthan gum and glucan, and can be used for structural analysis of polysaccharides.

The invention discloses a lysosome targeted degradation system based on a DNA phase separation aggregate as well as a preparation method and application of the lysosome targeted degradation system, and belongs to the technical field of biological medicine and nanotechnology. The lysosome targeted degradation system based on the DNA phase separation aggregate comprises an RNA-DNA tetrahedral framework with a cohesive end, the RNA-DNA tetrahedral framework is formed by self-assembly of a core chain and an edge chain through complementary base pairing, the edge chain is a DNA-RNA chimeric oligonucleotide chain, and the DNA-RNA chimeric oligonucleotide chain is a DNA-RNA chimeric oligonucleotide chain. One or more sections of RNA ribonucleotide sequences which can be specifically recognized and cut by RNase H enzyme are embedded in the sequence, and the tail end of the RNA ribonucleotide sequence is modified with a cohesive tail end for driving phase separation and a cell membrane anchoring group; the RNA-DNA tetrahedral framework is modified with an aptamer of a targeted membrane protein. After entering cells, the system is subjected to liquid-liquid phase separation in a lysosome acid environment through interaction of cohesive ends, a micron-sized large-size aggregate is formed in situ, the residence time of a nano-drug in the lysosome is prolonged, and efficient degradation of target membrane protein is realized.

The application discloses a kind of cell membrane chromatographic column based on biotin ligase directional covalent fixation and its preparation method and application, belong to chromatography science and technology field. By bonding 2-imino biotin to silica gel, then using biotin ligase specific recognition with Avi-Tag cell membrane fragments, and stable covalent bonding occurs with 2-imino biotin silica gel, so that cell membrane is fixed on the surface of silica gel to obtain Avi-Tag bridged cell membrane chromatographic stationary phase, finally using wet column packing, obtain cell membrane chromatographic column based on biotin ligase directional covalent fixation. The cell membrane chromatographic column can prolong the service life of cell membrane chromatographic column, improve the stability of cell membrane chromatographic column during use, and the short peptide tag (Avi-Tag) used can improve the non-specific adsorption and poor specificity of the cell membrane chromatographic column prepared based on protein tag technology to a certain extent, providing technical support for the wide application of cell membrane chromatographic column.

The invention provides an extraction method of high-content alpha-linolenic acid, and relates to the technical field of biological extraction. The extraction method of the high-content alpha-linolenic acid comprises the following steps: pretreating raw materials, destroying a plant cell wall structure through related operations of crushing and degreasing, releasing grease components, improving the subsequent extraction efficiency, removing phospholipid and free fat, and avoiding impurities influencing the purification effect; the method comprises the following steps: carrying out directional hydrolysis on lipase, cutting off sn-1 and 3 site ester bonds by utilizing lipase specificity, converting triglyceride into free fatty acid, reserving the structural integrity of sn-2 site alpha-linolenic acid, and enabling free alpha-linolenic acid to be more easily separated through subsequent distillation / chromatography compared with glyceride form. Through a silver ion complexing chromatography technology, alpha-linolenic acid and other unsaturated fatty acids with similar structures can be accurately separated, the purity of the final product stably reaches 98% or above, the pharmaceutical grade application standard is met, and the technical bottleneck that a traditional purification method is difficult to break through the purity of 90% is solved.

This invention relates to the field of antibodies, and more particularly to TAT antibodies and their applications. The invention provides coated antibodies comprising a heavy chain variable region and a light chain variable region, wherein: the CDR1, CDR2, and CDR3 regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID No:1 to SEQ ID No:3; and the CDR1, CDR2, and CDR3 regions of the light chain variable region have amino acid sequences as shown in SEQ ID No:4 to SEQ ID No:6. This invention successfully prepared highly specific monoclonal antibodies 7E10 and 2B4 against the thrombin-antithrombin III complex (TAT), wherein 7E10 specifically binds to thrombin (THR), and 2B4 specifically binds to antithrombin III (AT-III). Using these two antibodies in combination enables highly sensitive and specific detection of the TAT complex.

The present invention relates to the fields of fatty acid- and polyketide synthesis, and protein engineering. In particular, it relates to methods of using altered acyltransferases having an altered coenzyme specificity for acyl group transfer and polyketide synthesis.

This invention relates to the field of biomedical technology, specifically disclosing an anti-inflammatory and bone-promoting rhodioloside composite hydrogel and its preparation method. The thermosensitive carrier is a triple-response system, which enhances the drug release rate only in a specific microenvironment at 37°C, with a pH of 5.5-6.5 at the site of inflammation, and in which MMPs are overexpressed. MMPs are highly expressed at the implant periodontitis site, and MMP-sensitive peptides can be specifically degraded by this enzyme. This process can trigger the precise release of rhodioloside and quercetin active ingredients in the gel, avoiding systemic drug diffusion that could lead to drug waste or side effects at other sites. Rhodioloside and quercetin synergistically downregulate the expression levels of inflammatory factors such as IL-6 and TNF-α, while nisin and chitosan-ε-polylysine grafts inhibit the growth of Porphyromonas gingivalis. Runx2 / CON expression is increased, achieving a closed-loop treatment of anti-inflammatory, antibacterial, and bone-promoting effects.

The invention relates to the technical field of genetic engineering, in particular to recombinant acidic pectinase GsPG3 and application thereof. The amino acid sequence of the recombinant acidic pectinase GsPG3 is as shown in SEQ ID NO: 4, the optimum pH of the recombinant acidic pectinase GsPG3 is 3.5, the optimum temperature of the recombinant acidic pectinase GsPG3 is 65 DEG C, and the recombinant acidic pectinase GsPG3 has excellent stability under an acidic condition (pH is 3-5) and can be activated by Ca < 2 + >. The enzyme can be efficiently expressed in pichia pastoris, the shake flask enzyme activity reaches 7235.4 U / ml, and the fermentation activity in a 30L fermentation tank reaches 91,150.3 U / ml. According to the method, the GsPG3 enzyme is specifically applied to beet pulp enzymolysis for the first time, the optimal process conditions are obtained through system optimization: the pH is 3.0, the temperature is 50-55 DEG C, the enzyme concentration is 2,000 U / ml, the substrate concentration is 0.25 g / ml, the time is 3h, and the pectin degradation rate of the beet pulp can reach 45.4% under the conditions.

The invention discloses an enzyme response magnetic resonance imaging nanoprobe as well as a preparation method and application thereof. The enzyme response magnetic resonance imaging nanoprobe comprises a metal nanoparticle with a surface functional group A as an imaging basic unit, and a polypeptide sequence with functional groups B at two ends as a cross-linking agent and an enzyme response unit, the enzyme response magnetic resonance imaging nano-probe is a metal nano-particle cross-linked body formed by cross-linking the metal nano-particles through a polypeptide sequence. The enzyme response magnetic resonance imaging nanoprobe disclosed by the invention can be specifically cut by a high-expression enzyme in a cell differentiation process to form monodispersed metal nanoparticles, so that signal transformation is presented on magnetic resonance imaging; meanwhile, the enzyme response magnetic resonance imaging nanoprobe can establish effective association among the probe existence form, the cell state and the imaging signal, and is used for tracking the directional differentiation process of mesenchymal stem cells, neural stem cells and promyelocytic leukemia cells in real time.

The present invention relates to the fields of fatty acid- and polyketide synthesis, and protein engineering. In particular, it relates to methods of using altered acyltransferases having an altered coenzyme specificity for acyl group transfer and polyketide synthesis.

The application discloses an enzyme-responsive magnetic resonance imaging nano probe and a preparation method and application thereof. The enzyme-responsive magnetic resonance imaging nano probe comprises a surface functional group A metal nanoparticle as an imaging basic unit, and a functional group B polypeptide sequence as a crosslinking agent and an enzyme-responsive unit; and the enzyme-responsive magnetic resonance imaging nano probe is a metal nanoparticle crosslinking body formed by crosslinking the metal nanoparticle through the polypeptide sequence. The enzyme-responsive magnetic resonance imaging nano probe can be specifically cut by an enzyme highly expressed in a cell differentiation process, and forms a monodispersed metal nanoparticle, so that a signal change is presented on magnetic resonance imaging. Meanwhile, the enzyme-responsive magnetic resonance imaging nano probe can establish an effective correlation among a probe existing form, a cell state and an imaging signal, and is used for real-time tracking of a directional differentiation process of mesenchymal stem cells, neural stem cells and promyelocytic leukemia cells.

The invention discloses a method for detecting the activity of skin flap endonuclease 1 based on double cascade signal amplification. Belongs to the technical field of enzyme activity detection. According to the invention, the specific dumbbell probe is designed; when the skin flap endonuclease 1 exists in the sample to be detected, the skin flap endonuclease 1 can be detected; a flap endonuclease 1 specifically recognizes and cuts off a 5'overhanging flap in the dumbbell probe to generate a single-stranded DNA with a 3 '-OH terminal and a dumbbell-shaped DNA with a notch, two products generated by cutting respectively trigger rolling circle amplification and strand displacement amplification reactions, specific signals are amplified, the purpose of sensitive detection is achieved, the substrate utilization rate is 100%, no probe is wasted, and the detection cost is reduced. The method is carried out under isothermal and homogeneous conditions, does not need high-precision thermal circulation and separation steps, does not need to prepare a functional nano material, does not relate to a biological coupling procedure, effectively reduces the analysis cost and complexity, and provides a technical support for the detection of the activity of the skin flap endonuclease 1.

The invention discloses a small molecule peptide with cytotoxicity to breast cancer cells and application, and relates to the technical field of establishment and preparation of a small molecule peptide nano-drug delivery system. The small molecule peptide is a targeted peptide modified alkaline phosphatase reaction peptide, responds to ALP enzyme, is specifically combined with an integrin receptor, is self-assembled into spherical nanoparticles and serves as a nano drug carrier, and the structural formula is shown in figure 1. The research successfully designs and synthesizes a small molecule peptide with cytotoxicity to breast cancer cells, the small molecule peptide can be self-assembled into a nano NP drug delivery system, and an in-vitro experiment result shows that the self-assembled nano NP drug delivery system can quickly release chemotherapeutic drugs in an ALP overexpression environment, has small damage to healthy cells, is relatively low in cytotoxicity, and can be used for preparing a chemotherapeutic drug for treating breast cancer. However, the damage to cancer cells is obvious, and the action time of the medicine can be prolonged. An in-vivo anti-tumor experiment shows that compared with a non-deformable control peptide, the small molecule peptide drug loading system has a remarkable anti-tumor effect.

The application discloses a Turn-Off type cell in-vivo tracing probe and a preparation method and application thereof, and the probe structure is composed of three parts, including a membrane anchoring part, an enzyme specificity response part and a fluorescent group. The probe can mark cells through the hydrophobic tail chain of the membrane anchoring part, at this time, the fluorescent group part emits near-infrared fluorescence, and shows a Turn state. When the adoptive cells are apoptotic in the body, the probe falls off from the cells. The free probe falling off is phagocytosed by macrophages in the body, in the intracellular macrophages, the enzyme specificity response part in the probe structure responds to enzyme bond breaking, the fluorescent group is dissociated, does not emit light, and shows an Off state. Therefore, the fluorescent probe can not only trace the in-vivo living cells and monitor the death and life of the cells in the body, but also can effectively overcome the fluorescent "pollution" phenomenon caused by the persistent existence of the fluorescent of the free probe falling off, and provides a new visual tool for monitoring the in-vivo fate of the living cell drugs.

To provide a method for efficiently and accurately distinguishing two similar diseases that differ in the presence or absence of a mutant allele in the same gene region.SOLUTION: A method for distinguishing between a genetic disease caused by a codon mutation in a gene and another disease includes the steps of: performing an amplification reaction to amplify a genetic region containing the codon, the amplification reaction including adding a DNA primer for amplifying the genetic region containing the codon and a PNA that inhibits the amplification of a normal allele containing the codon; and treating a product obtained by the amplification reaction with a restriction enzyme that specifically recognizes and cleaves either a base sequence containing the codon with the mutation or a base sequence containing the codon without the mutation, thereby obtaining a restriction enzyme-treated product.SELECTED DRAWING: Figure 3

This invention belongs to the field of pharmaceutical quality testing technology. It discloses a high-throughput detection method for trace components in pharmaceuticals based on enzyme-specific reactions. The method includes: obtaining the initial sample addition timestamps of each well in a microplate that triggers the enzyme-specific reaction, as well as the dead time of mechanical flow; scanning and analyzing the high-frequency fluorescence signals in each well within an observation window after the dead time ends to obtain the fluorescence signal evolution matrix of the microplate; judging and labeling the state of each well by analyzing the obtained nonlinear distortion trend; reconstructing and extrapolating the dead time of the rapidly saturated wells to obtain the concentration values ​​of trace components exceeding the standard; analyzing the subthreshold fluctuations of the latent wells to obtain the subthreshold concentration values ​​of trace components; analyzing the photoelectric information of the normal wells to obtain the concentration values ​​of normal trace components; and combining the concentration values ​​of trace components exceeding the standard and the subthreshold concentration values ​​to form a pharmaceutical trace component detection report, thus reducing the blind zone of the detection range.

The invention discloses a 2-(2-phenethyl) chromone compound with COX-2 and 5-LOX dual-targeting inhibitory activity as well as a preparation method and application of the 2-(2-phenethyl) chromone compound, and belongs to the technical field of natural pharmaceutical chemistry. According to the compound, 2-(2-phenethyl) chromone is taken as a skeleton, and a functional group capable of being specifically combined with COX-2 and 5-LOX enzymes is introduced through chemical modification, so that double-target synergistic inhibition on two inflammatory mediators is realized. The invention also provides a synthesis method of the compound, which comprises the following steps: selecting high-purity 2-(2-phenethyl) chromone as an initial raw material, carrying out structural modification under the action of a catalyst, introducing key functional groups such as acrolein and acrylic acid into specific positions of a chromone ring, synthesizing a target compound through a multi-step reaction, and carrying out purification treatment on a reaction product. An in-vitro experiment result shows that IC50 (half maximal inhibitory concentration) of COX-2 is 1.12 [mu] M, and IC50 of 5-LOX is 1.18 [mu] M; and an acute toxicity experiment LD50 is greater than 2000mg / kg.

The invention belongs to the technical field of plant biology and agricultural biology, and relates to development and application of an FER kinase specific inhibitor FRV. Specifically, the invention provides an agricultural composition containing Forovicin, and provides an application of the agricultural composition in regulating and controlling an FER mediated signal channel and improving plants by combining the agricultural composition with an ATP binding pocket of FER to inhibit kinase activity. The invention provides an important tool for analyzing an FER signal mechanism and promoting agricultural application research.

The invention discloses an activated double-locking fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of new material technology and biomedical detection. The activated double-lock fluorescent probe is formed by covalently connecting a fluorescent parent nucleus, an acid-sensitive response unit and an enzyme specific recognition response unit, and precise tumor recognition is realized through a cascade activation mechanism: firstly, cracking an acid-sensitive linking group in a tumor acidic microenvironment to expose an amino acid sequence capable of being specifically recognized by aminopeptidase N; and releasing the fluorescent parent nucleus after enzyme digestion reaction. The double-lock fluorescent probe overcomes the false positive problem caused by response of a single biomarker, and has the remarkable advantages of hypersensitive signal response, rapid dynamic response, multi-modal imaging compatibility, broad-spectrum applicability, specificity and the like.

The present invention relates to the fields of fatty acid- and polyketide synthesis, and protein engineering. In particular, it relates to methods of using altered acyltransferases having an altered coenzyme specificity for acyl group transfer and polyketide synthesis.

This invention discloses a method for preparing BI8626, a specific antagonist of the ubiquitin ligase HUWE1, comprising the following steps: deprotecting compound G to obtain compound BI8626; obtaining compound G through the preparation method of the intermediate G; this invention reduces the difficulty of obtaining raw materials, simplifies purification, and increases yield, thereby reducing production costs; and finally, the production steps are simple and easy to produce and promote.

The application provides an Argonaute-mediated chain displacement exponential amplification method and application. First, TtAgo enzyme is specifically recognized and cut under the guidance of gDNA, the cutting site is located in the complementary fragment formed by gDNA and a target gene or a DNA fragment, a pair of amplification primers are extended to form a double strand under the action of a polymerase, the double strand is continuously recognized and cut by the protein due to the existence of the cutting site, and the single strand replaced can be used as a substrate of cycle two; the sequence replaced in the extension process of cycle one can be combined with the amplification primer in a complementary manner and is extended under the action of the polymerase, the product extended also has a cutting site, and a single strand formed under the action of TtAgo enzyme can continue to be used as a substrate of cycle two to participate in the cycle. The application realizes efficient amplification, significantly shortens the time required for the amplification reaction, and gradually detects in the amplification process, thereby realizing significant distinction of single-base differences on DNA.

This invention discloses a bio-depolymerase composite system for polymer deblocking in oilfields, its preparation method, and its application. For the first time, a ternary synergistic combination of bio-depolymerase, a sugar coenzyme, and an organic acid salt synergist is established. The bio-depolymerase specifically cleaves the main chain or side chain amide groups of polyacrylamide causing blockage in the formation; the sugar coenzyme participates in the enzymatic reaction and stabilizes the enzyme's active conformation, preventing enzyme inactivation under high temperature and high salt conditions; the organic acid salt synergist regulates the reaction microenvironment, chelates metal ions, and enhances the enzymatic reaction rate. The synergistic effect of these three components achieves mild, efficient, and low-residue degradation of various polymers during fracturing oil recovery, providing a green, low-damage, and efficient new technological approach for deep deblocking and increased production in polymer-driven oilfields.

The invention discloses a targeted antibacterial tooth paste based on an enzyme specific response mechanism as well as a preparation method and application thereof. The targeted antibacterial tooth paste consists of the following components in percentage by weight: 30-80% of a film-forming agent, 2-40% of a humectant, 0.2-2% of a thickening agent, 0.2-5% of a flavoring agent, 0.1-5.0% of an active medicine and 0.1-20.0% of an enzyme sensitive unit, according to the invention, a wrapping technology is innovatively introduced, effective components are wrapped in the sensitive unit with enzyme specific response, the sensitive unit has an intelligent response and active targeting mechanism, after the specific pathogenic bacterium characteristic enzyme is triggered, the drug is accurately released at the infected part, the frequency of drug use is lower, the side effect is smaller, the normal flora in the oral cavity is protected, and the safety is higher. According to the dental patch, the defect that a traditional dental patch releases drugs indiscriminately is overcome, intelligent response to a dental plaque biofilm is achieved, and the dental patch has wide application prospects in the aspect of precise prevention and treatment of periodontitis, caries and other oral diseases.

An antibody selectively activated in a target cell or tissue for treating one of symptoms. The antibody comprises a functional antibody which contains at least one light chain and at least one heavy chain and can be used for treating a condition in an activated state; at least one peptide, which can bind to an interferon receptor as required and activate a signaling pathway of the peptide; and at least one cleavable polypeptide linker, wherein each cleavable polypeptide linker contains a substrate peptide that can be cleaved by means of an enzyme, the enzyme is specifically or highly expressed in a target cell or tissue, and the cleavable polypeptide linker links one of the peptides to the N terminal of one of the at least one light chain and the at least one heavy chain of the functional antibody.

The invention belongs to the technical field of DNA modification site detection, and particularly discloses a sequencing method for identifying a zooxanthellae genome 5-hydroxymethyl uracil based on double-enzyme cascade. The method comprises the following steps: specifically recognizing and cutting 5-hydroxymethyl uracil through human single-chain selective single-function uracil glycosylase to generate a base removal site, efficiently cutting an AP site by combining with cutting mismatched nuclease optimized by a catalytic structural domain, and directionally generating a 200-300bp DNA fragment containing an adhesive tail end; and analyzing the cleavage site by using next-generation sequencing to realize single-base resolution positioning. According to the method, cross interference and DNA damage of a traditional chemical oxidation method to 5hmC / 5fC are avoided through a double-enzyme cascade reaction, and single-base resolution positioning can be achieved; meanwhile, according to the method, the experimental process is simplified, 5hmU recognition, AP site generation and cutting are integrated into one-step reaction, and the library building period is shortened. Accurate positioning and quantitative analysis of the single base level of 5hmU are realized in a zooxanthellae genome, and epigenetic regulation and environmental stress response of the zooxanthellae are disclosed.