89 results about "Enzymatic digestion" patented technology

The invention relates to antarctic krill peptide with umami and an antarctic krill peptide mixture as well as a preparation method and application thereof. Specifically, the amino acid sequence of the euphausia superba peptide is shown as any one of SEQ ID NO: 1-5. The euphausia superba peptide mixture with delicate flavor is obtained by carrying out enzyme digestion treatment on an euphausia superba peptide solution by using incision enzyme, excision enzyme and freshness enhancing enzyme and harvesting supernate. Molecular docking simulation shows that the euphausia superba peptide disclosed by the invention can be combined with a delicate flavor receptor T1R1 / T1R3. The Antarctic krill peptide and the Antarctic krill peptide mixture can be used for development of umami seasonings, and a new idea is provided for development of marine seasonings.

The invention discloses a preparation method and application of yak blood peptide with anti-hypoxia and anti-fatigue functions. The method comprises the steps of raw material pretreatment, composite enzymolysis (adopting a mixture of enzyme digestion modified lumbrukinase and nattokinase), enzyme deactivation, separation and purification, ferrous chelation, Maillard reaction, concentration and drying and the like. The enzymolysis efficiency and the active peptide yield are remarkably improved through ultrasonic treatment and a composite enzymolysis technology, the stability and the anti-hypoxia capacity of peptide fragments are enhanced through ferrous chelation, and the antioxidant activity and the anti-fatigue activity are further improved through the Maillard reaction. Experiments show that the yak blood peptide prepared by the method can significantly improve the antioxidant enzyme activity, prolong the normal-pressure hypoxia survival time, prolong the swimming exhaustion time and significantly reduce the MDA content of brain tissue and myocardial tissue. The obtained product can be applied to anti-hypoxia and anti-fatigue medicines or health-care foods, and has the advantages of high process efficiency, remarkable activity, high safety and the like.

The present invention provides a method for preparing exosomes derived from antler periosteal mesenchymal stem cells and their applications. After obtaining antler periosteal tissue, the tissue is placed in a DPBS culture dish containing antibiotics for further processing. The periosteal tissue is then minced and washed with DPBS until it is free of blood. The minced tissue is treated with digestive enzymes and incubated in a carbon dioxide (CO2) incubator to facilitate enzymatic digestion. Subsequently, alpha-MEM complete culture medium is added to further process the tissue. The digested tissue is filtered and centrifuged to isolate the primary periosteal cells, which are then counted and cultured in a suitable medium to obtain mesenchymal stem cells (MSCs) derived from antler periosteum. The cultured periosteal MSCs are further processed to isolate exosomes, which can be applied to promote the proliferation and repair of human fibroblasts, enhance skin quality, and stimulate hair growth.

The invention belongs to the cross technical field of veterinary medicine and biomedicine, and relates to a construction and evaluation method of a dog spontaneous breast cancer organoid model, which specifically comprises the following steps: (1) obtaining dog spontaneous breast cancer tissues through operation or biopsy, separating tumor tissues under sterile conditions, and carrying out fine enzymolysis digestion treatment to obtain dog spontaneous breast cancer tissues; obtaining a single-cell suspension; and (2) inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with a growth factor, promoting the growth and amplification of the breast cancer organs, and carrying out timely passage according to the growth condition so as to maintain the activity of the model. The model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of primary tumors, does not need gene editing, is simple and convenient to operate and low in cost, is particularly suitable for individual drug sensitivity detection of pet dogs, and provides an efficient and high-simulation experimental platform for dog breast cancer mechanism research and clinical precise medication.

This invention discloses a method for extracting high-purity PDRN from salmon testes and its application in improving skin condition, belonging to the field of bioactive ingredient extraction technology. Frozen salmon testis tissue is cleaned of blood vessels and fatty impurities, then cryogenically pulverized at -20~-25℃. The pulverized tissue is added to a pre-treatment dispersion buffer and homogenized under high pressure to obtain a DNA fragmentation solution. The precipitate is then separated by centrifugation. The precipitate is dispersed in a dedicated enzyme digestion buffer, and enzymatic digestion with protease and RNase A is performed to inactivate the enzyme. DNase I is added to the digestion solution for enzymatic cleavage. After the reaction, a stop solution is added to terminate the reaction, and the supernatant is separated by centrifugation. Ethanol is added to the supernatant for precipitation, and centrifugation yields PDRN from salmon testes. This invention, through specific buffer systems and enzymatic digestion conditions, increases the PDRN content by 40~60 kDa, allowing for better absorption and utilization by the skin, improving facial hydration and elasticity, effectively reducing facial wrinkles, and improving facial skin condition. It can be used to prepare functional cosmetics.

This invention discloses a method for inducing and culturing burdock stem cells and stem cells, belonging to the field of plant biotechnology. Using burdock roots as explants, the cambium is determined by staining, and the cambium is obtained using enzymatic digestion. The cambium is then inoculated into a specific ratio of induction solid medium and cultured in the dark to efficiently induce primary stem cells. These primary stem cells are then transferred to a proliferation solid medium for subculture and selection to obtain and maintain a vigorous, soft stem cell line. The solid stem cell line is further transferred to a liquid medium for shaking culture to establish a suspension stem cell line. This invention provides high induction efficiency, stable stem cell characteristics, and good reproducibility, offering a high-quality starting material and technical platform for in vitro preservation of burdock germplasm, large-scale production of secondary metabolites, and genetic transformation.

FISH PROTEIN HYDROLYSATE AND APPLICATIONS IN FARM ANIMALS The present invention relates to a protein hydrolysate originating from fish tissues or cartilages rich in protein, in particular collagen, and its applications in the feeding and welfare of farm animals, in particular pigs and poultry. In particular, the invention relates to a hydrolysate obtained by enzymatic digestion using an endopeptidase enzyme, said hydrolysate being characterized in that it has a protein fraction having a molecular profile with the following distribution: between 15 and 60% of peptides with a molecular weight of between 1000 and 5000 Da, between 20 and 40% of peptides with a molecular weight of between 500 and 1000 Da, between 10 and 40% of peptides with a molecular weight of between 150 and 500 Da, between 1 and 10% of peptides with a molecular weight of between 1000 and 50 ... molecular weight less than 150 Da. Fig. 2

The invention discloses a high-throughput extraction method and application of hair stem sample protein. The invention belongs to the field of biological detection, and particularly relates to a high-throughput extraction method and application of hair stem sample protein. The preparation method of the hair stem protein peptide fragment for mass spectrometry comprises the following steps: extracting protein from an in-vitro hair stem by adopting a pressure cycle sample preparation technology to obtain hair stem protein; and carrying out enzymolysis treatment on the hair stem protein to obtain the hair stem protein peptide fragment for mass spectrometry. The method established by the invention has simple steps, can effectively remove lipid and impurity interference on the hair stem sample, can extract the protein in the hair stem at a high recovery rate, can efficiently carry out protein enzymolysis, and realizes high-sensitivity coverage depth analysis of the hair stem proteome.

The present invention discloses a method for capturing chromatin and / or chromosome conformation and reagents used therein, and belongs to the field of three-dimensional genomics. The method comprises: subjecting target cells to in vitro cross-linking, lysis, enzymatic digestion with blunt-end restriction endonucleases to add A bases at the ends, and connecting adjacent DNA ends with BP Linker; after labeling the target protein with a specific antibody, using Tn5 fusion protein to target and enrich DNA interactions mediated by the target protein, using biotin labeling to enrich the target fragments of "DNA-tag-DNA" to obtain adjacent ligated DNA fragments, sequencing and analyzing the adjacent ligated DNA fragments, and determining the adjacent ligated DNA bound to the target protein. Compared with traditional methods, the present invention has the advantages of using conventional experimental equipment and a small number of cells, high efficiency, short cycle, low data signal-to-noise ratio, and low cost.

The present invention discloses a recombinant humanized type XVII collagen, its expression vector and genetically engineered bacteria. In the present invention, 4 functional fragments with high activity are obtained through screening. After optimizing one of the fragments and combining them in a certain order, recombinant humanized type XVII collagen JYC1703 is obtained. Or after optimizing one of the fragments, combining them in a certain order and respectively fusing GPP sequences at the amino terminus and carboxyl terminus, recombinant humanized type XVII collagens JYC1701 and JYC1702 are obtained. After codon optimization, they are amplified and cloned into an expression vector. After enzymatic digestion and linearization, they are transformed into Pichia pastoris competent cells. High-copy recombinants are screened through G418 resistance gradient to obtain Pichia pastoris genetically engineered bacteria expressing recombinant humanized type XVII collagen. The recombinant humanized type XVII collagens JYC1701, JYC1702 and JYC1703 of the present invention exhibit excellent functions of promoting cell adhesion, cell proliferation and migration, and are suitable for preparing products such as skin care or medical devices.

The invention belongs to a proteomics analysis technology in the technical field of biology, and particularly relates to a single-cell-level proteomics analysis system and analysis method for a mass spectrum system. The analysis system comprises a sample pretreatment optimization subsystem and a chromatography-mass spectrometry acquisition optimization subsystem, the purpose of the sample pretreatment optimization subsystem is to efficiently and stably prepare a peptide fragment mixture capable of being used on a mass spectrum machine from a trace cell sample. The sample loss is reduced by improving the protein extraction and enzymolysis efficiency of trace (nanogram-level) and single-cell-level protein samples. By optimizing mass spectrum data acquisition parameters, the identification depth and quantity of proteins are improved while the analysis time is shortened (the flux is improved). By adopting the analysis system disclosed by the invention, the accuracy and reproducibility of quantitative analysis of the micro sample proteome can be remarkably improved.

The invention discloses a method for identifying aedes albopictus and aedes aegypti based on a micro-well array and gene bar code technology, which is characterized in that a specific probe is designed aiming at a variation fragment of a 620bp target gene of a mitochondrial gene COI sequence of aedes albopictus and aedes aegypti, and the probe is a single-chain sequence rich in G basic groups; the method comprises the following steps of: preparing a probe, performing sulfydryl modification on the 5'end of the probe, preparing a hydrophilic micro-well array on a glass sheet by utilizing amination derivation, fixing by using a cross-linking agent to form a specific capture probe, hybridizing a sample and the capture probe, performing enzyme digestion, adding a silver nano-cluster aqueous solution, incubating, and developing, thereby realizing efficient, accurate and rapid detection on the aedes albopictus and aedes aegypti types. And the practical application value is very high.

This invention discloses a PEG-mediated genetic transformation method for *A. anthracnose* protoplasts. Through the preparation, transformation, and transformation of *A. anthracnose* protoplasts, and the screening of transformants, the hygromycin B resistance gene fragment was successfully transferred into the *A. anthracnose* genome, achieving successful genetic transformation of *A. anthracnose* protoplasts. More importantly, this invention establishes a new enzymatic digestion system and improves the culture mechanism in protoplast preparation. During the culture and screening of transformants, a more suitable resistance concentration is used, balancing the stringency of screening with the survival of transformants. This improves transformation efficiency, shortens the overall transformation process, and establishes a more efficient and convenient genetic transformation system for *A. anthracnose* protoplasts.

The invention discloses a nucleotide sequence of PDRN / PN and a preparation method of PDRN / PN based on genetic engineering and microbial fermentation, and belongs to the technical field of biology. According to the method, the vibrio is adopted as a production chassis strain, and methods of pyrolysis crude extraction, enzyme digestion and magnetic bead selective separation are combined, so that the preparation of the multi-source PDRN / PN is realized, and the application range of the product is expanded. Compared with an escherichia coli system, the method has the advantages that the endotoxin pollution risk can be remarkably reduced, and the product safety is improved; compared with a yeast system, the production efficiency of PDRN / PN can be improved. Meanwhile, the method is simple and convenient to operate, is suitable for laboratory and pilot scale amplification, and has obvious industrial application potential.

The present invention relates to the field of pharmaceutical analysis technology, in particular to IPC G01N30, and more specifically to a method and application for detecting polyA tails in mRNA vaccines. The present invention provides a time-saving, labor-saving, and high-resolution method for detecting polyA tails in mRNA vaccines, comprising the following steps: S1: performing enzymatic digestion, magnetic bead purification, and elution recovery on mRNA in an mRNA vaccine solution to obtain polyA tail fragments; S2: detecting the poly A tail fragments using a liquid chromatography-mass spectrometer (LC-MS); the detection method of the present invention can detect single nucleotides and multiple oligonucleotide sequences, thereby accurately determining the polyA distribution range with high resolution; and the pretreatment method is simple, the detection time is short, the stability is greatly improved, and time is saved, and it can be used as a common characterization of mRNA vaccines.

This invention discloses a method for protein extraction and mass spectrometry pretreatment from methanogenic bacteria, belonging to the field of protein extraction technology. The method involves pretreating a methanogenic bacteria enrichment culture to remove inorganic salt ions and other impurities, resulting in a pretreated culture. Then, a lysis buffer composed of sodium dodecyl sulfate, Tris-HCl buffer, and a protease inhibitor is added to the pretreated culture, followed by ultrasonic disruption to obtain a cell lysate, from which a crude protein extract solution is obtained. Finally, the protein extract is extracted from the crude protein extract solution. This invention significantly improves protein extraction efficiency and peptide purity by optimizing the extraction process and subsequent enzymatic digestion, while preserving the methanogenic activity of the extracted protein as much as possible, providing more accurate and reliable sample support for downstream protein mass spectrometry analysis.

This invention belongs to the field of renal pathology testing technology and discloses a method for detecting type IV collagen expression in renal biopsy tissue, comprising the following steps: dewaxing and hydrating paraffin sections of renal biopsy tissue to obtain processed tissue sections; adding the processed tissue sections to a repair solution, heating to boiling, and then cooling to room temperature to obtain repaired tissue sections; adding protease to the repaired tissue sections for enzymatic digestion to obtain enzymatically digested tissue sections; adding a primary antibody to the enzymatically digested tissue sections for preliminary incubation, followed by adding a fluorescein-labeled secondary antibody against the primary antibody for further incubation. This method for detecting type IV collagen expression in renal biopsy tissue is applicable to cases where renal biopsy tissue lacks glomeruli or has too few glomeruli to detect type IV collagen expression. It has high specificity and can provide a new detection method for Alport syndrome in cases where renal biopsy tissue lacks glomeruli.

The application belongs to the technical field of genetic engineering. The application provides application of a thiol-oxidoreductase in promoting keratin enzymolysis and a method for promoting keratin enzymolysis. The thiol-oxidoreductase is applied to a keratin enzymolysis system, and the enzymolysis effect is obviously improved. Through testing, the enzymolysis effect of the thiol-oxidoreductase combined with a protease on keratin is not only obviously higher than the enzymolysis effect of a single protease, but also is better than the hydrolysis of keratin by a chemical reducing agent with the same concentration. The application retains the advantages of enzyme method "green chemistry" and is friendly to the environment.

This invention relates to the field of genetic engineering technology, and more particularly to a method for prokaryotic expression of antimicrobial peptides. The method provided by this invention involves inserting an antimicrobial peptide gene into the 3' or 5' end of a fusion tag gene for fusion expression. The protein, forming numerous inclusion bodies, eliminates the original antimicrobial activity of the antimicrobial peptide, thus preventing the killing of host bacteria. The method provided by this invention allows for the large-scale preparation of antimicrobial peptides in a prokaryotic expression system. The method is simple and easy to implement, and the obtained antimicrobial peptides exhibit significant in vitro antibacterial effects after enzymatic digestion and purification.

The present invention discloses an enzymatic digestion method for gently reducing agent to digest tissue samples and its mass spectrometry detection method. The present invention provides a lysis solution, which comprises: a detergent with a final concentration of 1-4% by mass fraction; the detergent is sodium deoxycholate and / or ammonium bicarbonate; 3-(2-carboxy)phosphine hydrochloride with a final concentration of 5-20 μM; 5-chloro-anthranilic acid with a final concentration of 20-60 mM; tris(hydroxymethyl)aminomethane-hydrochloride with a final concentration of 50-200 mM; phenylmethylsulfonyl fluoride with a final concentration of 0.1-2 mM; and water. After the tissue samples are lysed with the lysis solution of the present invention, tissue proteins and peptides can be obtained with a high yield.

This invention discloses a method and application for detecting N-glycans based on in-gel enzymatic digestion and electrophoresis. The method includes the following steps: separating glycoprotein samples and cutting target bands by gel electrophoresis; decolorizing and dehydrating the target glycoprotein particles; adding enzyme solution for direct enzymatic release of N-glycans within the gel; transferring the enzyme digestion solution after sonication, drying, and labeling the N-glycan sample; and finally, separating, detecting, and analyzing the N-glycans using capillary electrophoresis. This invention innovatively combines in-gel enzymatic digestion with capillary electrophoresis, avoiding the complex sample transfer and pretreatment steps of traditional methods, significantly simplifying the operation process, reducing sample loss, and making it particularly suitable for the efficient detection of low-abundance N-glycans. This method features high sensitivity, high resolution, and high throughput, and can accurately analyze N-glycans in complex biological samples, with wide applications in disease biomarker screening, clinical diagnosis, and glycomics research.

The invention discloses a liquid chromatography-tandem mass spectrometry determination method for altrenogest in pig tissue, which comprises the following steps: sample pretreatment: enzymolysis treatment: weighing 1.0 g of pig tissue sample which is pig liver, pig muscle or pig skin adipose tissue; a protease K solution is added into the pig tissue sample, pig liver or pig muscle tissue is subjected to enzymolysis for 1 h in a dark water bath at the temperature of 37 DEG C, and pig skin adipose tissue is subjected to enzymolysis for 30 min in a dark water bath at the temperature of 50 DEG C; performing ultrasonic treatment for 2 minutes after enzymolysis, and cooling to room temperature; according to the method, the purification efficiency and the specificity are greatly improved, and the problem of matrix interference is effectively solved. The MIP-MNPs prepared by taking altrenogest as a template molecule can specifically adsorb altrenogest and metabolites thereof through spatial structure matching and functional group action, so that non-specific interference of protein, fat and steroid impurities in pig tissues is eliminated, and the ionization inhibition effect of a matrix on mass spectrometry detection is remarkably reduced.

[Problem] To detect abalone, which may cause an allergic reaction, with high sensitivity even when trace amounts of abalone is contained in a food raw material, a product or the like. [Solution] Provided is a method for detecting abalone, the method comprising a step for extracting a protein from a sample, and a step for allowing the extracted protein to react with a protease to produce an enzymatically digested product, and the method involving analyzing the enzymatically digested product, and detecting at least one peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:2 by using a mass spectrometer to qualitatively or quantitatively determine whether or not an abalone protein is present in the sample.

The application discloses a DNA / RNA reagent kit based on a magnetic bead method and free of enzyme digestion, and belongs to the technical field of nucleic acid extraction and purification. The reagent kit comprises a lysis solution AL, protease K, a specific binding buffer CLB, amino magnetic bead suspension, silicon hydroxyl magnetic bead suspension and other components. The application solves the problems of nucleic acid degradation, incomplete removal of cross contamination and complicated process caused by enzyme digestion in the prior art, is suitable for automatic high-throughput processing, and has a wide application prospect in scientific research and clinical application.

This invention relates to the field of biodetection technology, specifically to a non-isolated digital multiplex detection method based on a DNA walker and TdT enzyme amplification. The method first utilizes coding microspheres with capture elements coupled to their surface to specifically bind to target miRNAs or proteins in the sample; then, specific guide DNA (gDNA) corresponding to the target molecule is released through enzymatic digestion or thermal denaturation; the gDNA then... Pf Under the action of Ago endonuclease, the DNA walker on the surface of the encoded microspheres is activated, cyclically cutting the orbital strand to generate multiple DNA fragments with 3'-hydroxyl ends. Finally, TdT enzyme is used to extend fluorescently labeled nucleotides at the hydroxyl ends, achieving in-situ signal amplification, and digital quantitative analysis is performed on the fluorescence signal of the encoded microspheres. This invention achieves high-sensitivity detection of multiple targets in a single reaction without chamber isolation, with detection limits for each target molecule reaching the fM or fg / mL level, providing an effective technical means for the early screening of major diseases such as Alzheimer's.

The invention discloses a construction and evaluation method of an Rb1 and Trp53 (p53) double-gene mutation mouse spontaneous lung cancer organoid model, and the construction method comprises the following steps: separating lung cancer tissues under a sterile condition, and carrying out fine enzymolysis digestion treatment to obtain a single-cell suspension; inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with growth factors, promoting the growth and amplification of lung cancer organs, and carrying out timely passage according to the growth condition to maintain the activity of the model; the model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of the primary tumor, in-vitro gene editing is not needed, the operation is simple and convenient, the cost is low, and an efficient and high-simulation experimental platform is provided for lung cancer mechanism research and individualized drug screening.

The invention discloses lung glial cells as well as an in-vitro culture method and application thereof. The in-vitro culture method comprises the steps that in-vitro mouse lung tissue is subjected to mechanical separation and enzymolysis digestion, a single-cell suspension is prepared, enzymolysis digestion is conducted by adding a mixed digestive enzyme solution, and the mixed digestive enzyme solution comprises type I collagenase and deoxyribonuclease I; the single cells are placed in a DMEM complete medium for subculture, the pulmonary glial cells are obtained, and the DMEM complete medium contains the heparin-binding epidermal growth factors. According to the in-vitro culture method, the lung glial cells can be successfully obtained, and the lung glial cells are high in purity, good in cell morphology and state, normal in function and suitable for follow-up cell experiments.

The present disclosure relates to a method of digesting a sample comprising a protein, the method comprising: adding the sample to a device, the device containing a buffer and a solid support surface comprising a surface coating, wherein the surface coating immobilizes an enzyme while reducing undesired interactions between the sample and the solid support surface; immobilizing the enzyme on the surface coating for digesting a protein of the sample; and heating the sample to complete a heat-assisted digestion of the protein.

Provided herein are methods of purifying messenger RNA (mRNA) by subjecting a preparation comprising in vitro synthesized mRNA to one or more steps of enzymatic digestion with a proteinase, optionally with a further oligo dT affinity chromatography step. Also provided are mRNA purified by the methods described herein.

This application belongs to the field of genetic engineering technology and discloses human enterokinase light chain mutants and their applications. This application involves single-point mutations or multiple rounds of combined mutations based on the wild-type human enterokinase light chain -C112S mutant sequence. The obtained variants have higher specific activity compared to the parent, which is expected to increase unit yield, reduce the residual amount of protease in the final product, and lower product costs. Furthermore, these mutants exhibit high stability in the enzymatic digestion of peptides / proteases, such as liraglutide and smegglutide, GLP-1 analogs, reducing enzyme usage, increasing digestion efficiency, and decreasing the proportion of non-specific products, thereby lowering the production cost of peptide drugs and promoting the rapid development of related industries.