37 results about "Proteinase C" patented technology

The invention provides an in-vitro induction and purification method for Giardia lamblia cysts. The induction method is characterized in that Giardia lamblia trophozoite is cultured in a TYI-S-33 culture medium containing pulvis fellis suis to obtain the Giardia lamblia cysts. According to the in-vitro induction method for the Giardia lancea cysts, the pig gall powder is added into the TYI-S-33 culture medium to induce the Giardia lancea trophozoite, and it is found that compared with common ox gall powder, the proportion of converting the Giardia lancea trophozoite into the cysts can be remarkably increased, and the generation rate of the induced cysts stably reaches 50%; according to the giardia lamblia cyst purification method disclosed by the invention, protease K is utilized for purification, so that pure cysts can be obtained, long-term stability and activity can be maintained, and the cysts can be stably stored for several weeks to several months at 2-8 DEG C and can be prepared into giardia lamblia cyst standard substances; the method is used for method verification, quality control, disinfection efficiency research, filtration efficiency evaluation and the like.

This invention relates to a method and composition for dispersing biological tissues with the aim of obtaining single cells for analysis. More specifically, the composition of the present invention is an enzyme composition comprising proteinase K. The composition of the present invention can be used in a method for effectively dispersing tissues at low temperatures for applications in high-throughput single-cell omics research.

The invention discloses a kit and a method for extracting nucleic acid from an ophthalmic sample and application of the kit and the method in metagenome sequencing. A method of extracting nucleic acid from an ophthalmic sample includes extracting nucleic acid from the ophthalmic sample using a sample processing system comprising the ophthalmic sample, a carrier RNA, protease K, and a lysis reagent. According to the method, a carrier RNA is added to prevent loss of trace nucleic acid, high-concentration protease K is adopted to enhance digestion of protein, and a chemical reagent is adopted to lyse cells, so that excessive damage and degradation of nucleic acid and low-volume elution caused by severe oscillation of mechanical wall breaking are avoided, and the final concentration of nucleic acid is improved.

The application discloses a Cas protein PAM analysis method based on local geometric structure and machine learning, and belongs to the fields of biotechnology and artificial intelligence. The method comprises the following steps: constructing an initial structure of a Cas protein-DNA-RNA ternary complex by using a structure prediction tool, and generating a mutant structure data set containing different PAM sequences by calculation and optimization; extracting and embedding the local structure of a PAM site to obtain a structure feature vector, performing dimension reduction and cluster analysis by using an unsupervised machine learning method, realizing natural classification of the PAM sequence, and identifying the cluster result in combination with P prior biological knowledge. The application creatively combines the three-dimensional local geometric structure of a protein and machine learning, can predict and classify the PAM preference of a Cas protein from a structure source, has the advantages of objectivity, comprehensiveness, and the ability to reveal hidden patterns, and can be verified by experimental data.

The present invention relates to methods and compositions for dissociating biological tissue with the aim of obtaining single cells for analysis. More specifically, the compositions of the invention are enzyme compositions comprising protease K. The compositions of the invention are useful in methods for efficiently dissociating tissue at lower temperatures for application in high throughput unicellular omics studies.

The invention relates to the technical field of drug detection, in particular to immobilized protease K and application thereof in hair drug detection. The immobilized protease K comprises zirconia ceramic balls and protease K which is covalently connected with the zirconia ceramic balls through APTES amination; wherein the loading capacity of the protease K is 1-5mg / g. The specific detection method comprises the following steps: carrying out milling and enzyme digestion on to-be-detected hair and the immobilized protease K zirconia ceramic ball, and carrying out fluorescence immunochromatography on the obtained supernatant to detect the narcotics and metabolites thereof. According to the application, covalently solidified protease K is adopted, the operation stability is excellent, 50 DEG C high temperature and mechanical oscillation can be resisted, 85-95% efficient release of traditional difficult samples such as weak positive hair and dark hair can be realized, the detection rate and the detection accuracy of drugs and metabolites thereof are improved, and the false negative risk is reduced; the requirements of laboratory high-throughput detection and on-site rapid screening are met.

The invention relates to the field of astaxanthin synthesis, and discloses recombinant saccharomyces cerevisiae as well as a construction method and application thereof. The recombinant saccharomyces cerevisiae provided by the invention comprises the following exogenous genes: coding genes of beta-carotene hydroxylase CrtZ, beta-carotene ketolase CrtW, nicotinamide adenine dinucleotide kinase, ferredoxin-NADPH (nicotinamide adenine dinucleotide phosphate) oxidoreductase and ferredoxin; on the basis of the gene, other related genes are further introduced to realize that CrtZ and CrtW are positioned in lipid droplets by lipid droplet targeting peptide Olesion, so that the yield of astaxanthin is increased, finally geranyl-geranyl diphosphate synthase, phytoene dehydrogenase and phytoene synthase are targeted to endoplasmic reticulum through endoplasmic reticulum targeting peptide, and the yield of astaxanthin is increased. Therefore, the yield of the fermentation tank is up to 440mg / L. The recombinant saccharomyces cerevisiae provided by the invention is clear in genetic background, can stably and efficiently produce astaxanthin, and has a wide application prospect.

The application of BnaGSTU11 gene in regulating resistance to sclerotinia sclerotiorum of brassica napus, and the amino acid sequence is shown as SEQ ID NO: 2. A gene encoding glutathione sulfur transferase is cloned from brassica napus in the application BnaGSTU11 The proteinase encoded by the gene can enhance the resistance to sclerotinia sclerotiorum of brassica napus by removing active oxygen and free radicals. BnaGSTU11 The discovery of the antibacterial sclerotinia gene provides new genetic resources for breeding new germplasm of oilseed rape resistant to sclerotinia sclerotiorum, and provides a new idea for mining of sclerotinia resistance genes of other crops.

The present application relates to compost fermentation technical field, especially to a kind of compost fermentation device using duck blood protein enzyme.The technical scheme includes: support, jar body and top end top cover rotationally installed on support;Worm, worm wheel are arranged between jar body and support, worm engages worm wheel and one end is provided with hand wheel, can drive jar body rotation angle adjustment.The inner container of jar body is divided into two parts by drain plate;Top cover is provided with motor-driven stirring blade, air inlet pipe and air outlet pipe.The drain valve with elastic plugging element is arranged in the drain outlet of jar body bottom;It is also provided with air pressure control mechanism, including pressure valve on top cover and adjusting valve on air outlet pipe, can be raised by switching adjusting valve state The air pressure in jar, realize air pressure auxiliary drainage.The present application is through angle-adjustable jar body, stirring mixing, air pressure auxiliary drainage and ventilation heat preservation and other structures synergies, make duck blood protein enzyme and material fully contact, maintain stable fermentation environment, realize efficient enzymatic fermentation and percolate convenient discharge, improve compost efficiency and quality, reduce operating intensity.

This invention belongs to the field of biotechnology and discloses a plasma cell-free DNA extraction kit, extraction method, and applications. The kit includes magnetic beads, lysis buffer, proteinase K, binding buffer, washing buffer, and elution buffer. The extraction method involves mixing the plasma sample, lysis buffer, proteinase K, binding buffer, and magnetic beads. The lysed plasma cell-free DNA (cfDNA) is incubated with the magnetic beads to form a magnetic bead-cfDNA complex. Under a magnetic field, the complex is washed to remove various impurities, and the plasma cfDNA is obtained after elution. The method of this invention is simple and rapid, with short sample lysis time and stable quality. Replacing isopropanol with anhydrous ethanol during lysis results in high nucleic acid recovery, making it suitable for plasma cfDNA extraction. The purity is suitable for subsequent sequencing and PCR / Q-PCR. The magnetic bead method is beneficial for large-scale use and has significant implications for research and clinical applications.

The invention discloses a clonorchis sinensis recombinant protease inhibitor Cystatin-2 and a polarization effect of the clonorchis sinensis recombinant protease inhibitor Cystatin-2 on macrophages. The protease inhibitor Cystatin-2 is derived from clonorchis sinensis, can remarkably promote polarization of macrophages, and is an immunomodulatory molecule with application prospects.

The invention relates to the technical field of biology, in particular to a nucleic acid extraction reagent composition and application thereof. The invention provides a reagent combination simultaneously adaptive to common nucleic acid extraction and electrowetting platform adaptive nucleic acid extraction, according to the reagent combination provided by the invention, high-concentration guanidinium isothiocyanate is added into a lysis solution, heating is performed in a lysis step to assist lysis, lysis and nucleic acid adsorption are synchronously performed at the same time, and the extraction time is shortened; a surfactant with proper concentration is added into the lysis solution, the washing solution and the eluent, so that the reagent combination can be better driven on an electrowetting platform, the driving force is improved, and the uniform mixing effect is improved; meanwhile, protease K is added into the washing liquid, the common step of washing protein with high-concentration salt in a magnetic bead extraction method is omitted, a good washing effect can be achieved only through one-step washing, the extraction time is further shortened, meanwhile, residual protease K in the washing liquid is inactivated through high-temperature elution, follow-up amplification is not affected, and the method is suitable for application and popularization.

The invention discloses a protease K mutant with high catalytic activity and high thermal stability. The amino acid sequence of the protease K mutant is as shown in SEQ ID NO.1; the protease K mutant is used for hydrolyzing keratin, and oligopeptide or amino acid generated after hydrolysis can be used as a feed additive; after the protease K mutant is treated at 75 DEG C for 30 minutes, the residual enzyme activity is 55%; the specific activity of the protease K mutant is 324U / mg; reasonable design and molecular modification are carried out on protease K from Tritirachium album, site-directed mutagenesis and experimental verification are combined, a protease K mutant with improved thermal stability is obtained through screening, a G226F mutant enzyme is incubated for 30 min at 75 DEG C, the residual enzyme activity is 55%, and the residue of a wild type is 20% under the same treatment condition; in addition, the specific activity of the G226F mutant enzyme is 2.3 times that of a wild type, so that the problem of poor thermal stability of protease K is solved, and the catalytic activity is also improved.

The application provides a method for improving the detection rate of norovirus in aquatic products, which comprises the following steps: first, using a proteinase K solution to enzymolysis the digestive gland of shellfish, then adding a TGBE solution to elute, then adding enzymolysis corn starch to block activated carbon to remove the RT-qPCR inhibitor in the detection sample, taking the supernatant to extract nucleic acid, and using a fluorescent quantitative PCR instrument to detect. The detection method of the application reduces the inhibitor in the shellfish sample which interferes with the fluorescent quantitative PCR by using enzymolysis corn starch. In combination with the proteinase K enzymolysis, the TGBE elution and the RT-qRCR, the norovirus in the shellfish sample is detected, and the detection rate of the shellfish sample is increased by 10-20 times in 4h compared with the national standard method. The virus recovery rate and the detection sensitivity are greatly improved, and the method is suitable for shellfish samples, and has the characteristics of simple operation, high detection sensitivity and short time consumption.

The invention belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses a lysis solution composition and application thereof in preparation of a reagent for extracting genome DNA. The lysate composition comprises the following components: a pH regulator, 0.05-1.0% (v / v) of a nonionic surfactant, 0.1-2 mg / mL of bovine serum albumin, 0.5-5 mM of a reducing agent and 50-500 g / mL of protease K, and the pH value of the lysate composition is 7.0-8.0. The invention provides the genome DNA quick release lysate which is reasonable in composition and simple and convenient to operate, efficient release and stable storage of DNA in a complex biological sample are realized under the condition that a traditional purification step is not needed, and the obtained DNA can be directly used for nucleic acid amplification detection such as PCR (Polymerase Chain Reaction) and LAMP (Loop-Mediated Isothermal Amplification), so that the detection efficiency, repeatability and application convenience are improved; the actual requirements of rapid molecular diagnosis and on-site detection are met.

An automatic extraction device for large-volume free nucleic acid through a centrifugal column method comprises a back plate, a shifting mechanism and a reagent filling module, the shifting mechanism and the reagent filling module are sequentially and fixedly arranged on the back plate, and a centrifugal column filtering module with a negative-pressure liquid pumping module is arranged at the bottom of the reagent filling module. Through a centrifugal column specifically combined with nucleic acid and a special reagent filling module, after a sample is subjected to cracking and protease K digestion treatment, nucleic acid in the sample is rapidly adsorbed and rapidly washed through a filter membrane, so that protein, pigments, lipids and other impurities are removed to the maximum extent; the obtained total nucleic acid can be stored in an eluent for a long time to be used for a next experiment. The whole device can realize centrifugal column method large-volume free nucleic acid automatic extraction for extracting, enriching and purifying the free total nucleic acid from fresh or frozen serum, plasma, lymph and other cell-free body fluids.

This invention provides a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, comprising the following components: Tween 20, proteinase K, EDTA, and KOH. The rapid nucleic acid extraction and release agent of this invention, after incubation with the sample, completes the nucleic acid extraction and release process, improving extraction efficiency, reducing reagent and sample usage, simplifying operation steps, and lowering labor costs. It has advantages such as high efficiency, low cost, and high quality.

The invention discloses a cordyceps sinensis active peptide and application thereof in cosmetics. The cordyceps sinensis active peptide provided by the invention adopts a low-temperature freeze-drying process, so that the protein activity is protected to the greatest extent, and a loose structure is beneficial to subsequent crushing and extraction; the saccharomyces cerevisiae and the lactobacillus acidophilus are combined for fermentation, and the saccharomyces cerevisiae and the lactobacillus acidophilus have a synergistic effect, so that the content of target active substances such as polypeptide in a final product is remarkably increased; and then adopting a dialysis and ultrafiltration membrane combined method to obtain an active substance which has the molecular weight of 1000Da-10kDa and takes polypeptide as a main component. Tests prove that the obtained cordyceps sinensis active peptide has excellent capability of inhibiting the activity of elastinase, and can promote metabolism, maintain skin moisture and play a role in delaying skin aging when being applied to cosmetics.

This application relates to the field of biotechnology, specifically disclosing a method for extracting trace DNA from soil. This application utilizes extraction buffer and proteinase K to gently incubate and lyse soil samples, releasing DNA adsorbed on the soil and intracellular DNA into the extraction solution. Protein removal is performed using phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer, followed by centrifugation, filtration, and elution using ultrafiltration centrifuge tubes to obtain an extract containing the target DNA. This method can efficiently extract trace amounts of environmental DNA or metaDNA from soil, and is particularly suitable for DNA extraction in extreme environments where biological survival is difficult, providing technical support for DNA-based biological research in extreme habitats.

The invention relates to the technical field of cell culture and biological materials, and discloses a morphology-adjustable three-dimensional cell culture scaffold and a preparation method and application thereof.The preparation method comprises the following steps that oligopeptide Fmoc-FF is dissolved in an organic solvent to prepare an Fmoc-FF solution, the Fmoc-FF solution and a protease K aqueous solution are mixed, and a three-dimensional cell culture scaffold is obtained; the self-assembly process of the Fmoc-FF is initiated, and hydrogel is formed; after the Fmoc-FF forms hydrogel, protease K in a system is removed by utilizing the shear thinning characteristic of the hydrogel, and the Fmoc-FF nanofiber which is free of protease K residues and has a specific microstructure is obtained. According to the method, the diameter and length of the self-assembled nanofiber are adjusted by changing the adding concentration of protease K under the enzymatic hydrolysis effect of protease K on Fmoc-FF dipeptide. The method avoids the problem of high coupling of micro-morphology and macro-mechanical properties in a traditional collagen hydrogel system.

The invention discloses composite enzymolysis oat fermented milk and a preparation method thereof, and belongs to the technical field of dairy product processing. The fermented milk disclosed by the invention is prepared by taking cow milk as a substrate, adding oat enzymolysis powder subjected to compound enzymolysis of protease, cellulase and amylase, and fermenting at 42 DEG C by adopting a compound leavening agent of streptococcus salivarius subsp. Thermophilus, lactobacillus delbrueckii subsp. Bulgaricus, lactobacillus rhamnosus and lactobacillus plantarum. According to the oat fermented milk and the preparation method thereof, oat nutrition is fully released through a composite enzymolysis technology, product characteristics are optimized through synergistic fermentation of the four strains, the oat fermented milk has comprehensive nutrition, smooth taste and functional diversity, and the problems that existing oat fermented milk is insufficient in enzymolysis, poor in strain synergism and insufficient in product stability are solved.

The invention provides a magnetic bead method soil DNA extraction kit, and belongs to the technical field of molecular biology. The kit comprises protease K, a lysis buffer solution, a lysis strengthening solution, a precipitation solution, a lysis solution, a washing solution 1, a washing solution 2, a washing solution 3, an eluent and magnetic beads, according to the kit, DNA in soil can be rapidly extracted and purified, the extraction efficiency of pathogenic microorganism nucleic acid is improved, meanwhile, components in the kit can still keep the original activity and efficacy after long-term storage, and therefore the accuracy and reliability of experimental results are ensured, and a new thought is provided for extraction of the DNA in the soil.

The invention discloses an anti-protease K monoclonal antibody and application thereof. A heavy chain variable region of the antibody comprises the following complementarity determining regions: VH-CDR1 as shown in SEQ NO.2, VH-CDR2 as shown in SEQ NO.4 and VH-CDR3 as shown in SEQ NO.6; the light chain variable region comprises the following complementarity determining regions: VL-CDR1 as shown in SEQ NO.9, VL-CDR2 as shown in SEQ NO.11 and VL-CDR3 as shown in SEQ NO.13, and the VH-CDR1-3 and the VL-CDR1-3 jointly form a protease K binding site. The anti-proteinase K monoclonal antibody can be applied to various nucleic acid detection technologies such as PCR, qPCR and reverse transcription PCR, the use process is simple, the anti-proteinase K monoclonal antibody is directly added to a reaction system, the activity of the proteinase K can be effectively inhibited without a pretreatment step, the active site of the proteinase K can be accurately targeted through the specific inhibition effect, the activity of the proteinase K can be specifically blocked, and meanwhile, the application range of the anti-proteinase K monoclonal antibody is widened. The activity of common nucleic acid polymerase (Taq, reverse transcriptase and the like) is not obviously influenced, complete neutralization can be realized at a low dosage, and excellent high efficiency is shown.

The present application relates to the field of biotechnology, in particular to a blood cell pathogenic microorganism host nucleic acid extraction kit and method, comprising cell lysis solution, DNA enzyme, DNA enzyme buffer, proteinase K, lysozyme, cell wall lyase, glass beads and extraction lysis solution;The cell lysis solution is saponin and beta octyl thioglucoside solution;The DNA enzyme is Ultra Nuclease;The DNA enzyme buffer comprises MgCl2, Tris-HCl and BSA;Low concentration of saponin and low concentration of beta octyl thioglucoside are used, the detection of various pathogenic microorganisms is considered under the condition of ensuring good host removal effect. The chemical method and physical method are combined to break the wall of microbial cells, the positive detection rate of pathogenic microorganisms in clinic is improved, and the blank of pathogenic microorganism missed detection caused by the detection of plasma sample cfDNA at present is made up.

The invention belongs to the technical field of gene extraction, and particularly relates to a method for increasing the yield of cell genome DNA. Aiming at the technical problem of low yield in trace cell samples in the traditional DNA extraction method, the invention provides a method for washing cells by using a solution containing 1, 6-hexanediol and utilizing the characteristic of depolymerizing a protein aggregate, so that the chromatin structure is loose, and the release efficiency of protease K on genome DNA is remarkably improved. The method is simple and convenient to operate and suitable for adherent or suspension cells. Experiments prove that the DNA concentration is increased by about 67% compared with that of a traditional method, and the purity (A260 / A280) has no significant difference.

The application discloses an extractable nucleic acid trichomonad activity protection solution and relates to the technical field of biotechnology, which comprises two component reagents A and B, the reagent A comprises 0.8-1.0% NaCl, 0.1-5% Tris-Cl buffer, 0.1-1.5% SDS, 1.5-5% glucose, 5-15% serum, 0.5-5% betaine, and the balance is sterile deionized water, and the reagent B is a proteinase K solution. The application can not only dilute secretion, preserve trichomonas and culture trichomonas, but also be used for extracting trichomonad nucleic acid in PCR molecular experiment, so that the application can realize double application of trichomonad detection. The application not only helps to solve the health problems of women and improve fertility protection, but also is the first domestic invention to provide an example for new technology transformation service to the society.

This genetic analysis method comprises: isolating fixed maternal blood cells one by one; de-crosslinking DNA from protein in a proteinase-containing buffer for each of the isolated blood cells; extracting the de-crosslinked DNA; subjecting the extracted DNA to whole genome amplification; and amplifying a target sequence by using a plurality of specific markers from the amplified whole genome amplification product.

The present invention provides a gene extraction method for AAV third-generation sequencing. Specifically, a concise and efficient rAAV gene extraction method for third-generation sequencing is provided. First, the rAAV purified virus is decapped with proteinase K to obtain an initial reaction solution; the initial reaction solution is then subjected to alkaline denaturation, neutralized, and purified using a DNA product purification kit, eluted with nuclease-free water, to obtain denatured viral genomic DNA; finally, the denatured viral genomic DNA is incubated and annealed to ultimately form complete double-stranded DNA. Compared with traditional AAV genome extraction methods, the method provided by the present invention is simpler and more efficient, and the extracted DNA has higher integrity.

This invention belongs to the field of biomedical detection technology, specifically a lysis buffer, kit, and gene detection method for Candida albicans. This invention balances lysis efficiency and background interference by regulating the concentration of saponins; too much saponin micelles will encapsulate nucleic acids, while too little will result in incomplete lysis. Although citrate buffering to regulate the acidic environment may promote RNA degradation, the negative impact is offset by rapid lysis with high salt and proteinase K to clear nucleases. Pre-activated proteinase K synchronizes with membrane rupture, ensuring that the peak enzyme activity matches the exposure time of the target protein, thus improving degradation efficiency. This invention develops a lysis technology that can simultaneously achieve high yield, high-functionality nucleic acid production, and is adapted to rapid detection procedures.