50 results about "Proteinase C" patented technology

The present invention provides a nucleic acid extraction kit, a preparation method and an extraction method for fecal samples, which relates to the field of biotechnology. The kit includes: proteinase K, primary lysis buffer and lysis-binding buffer; the concentration range of proteinase K is 10-30 mg / mL; the primary lysis buffer contains: 50-200 mM Tris-HCl with pH 6.0-8.5, 0.5-30% (w / v) SDS, 1-10 mM EDTA with pH 8.0-9.0; the lysis-binding buffer contains: 50-200 mM Tris-HCl, 1-15 M guanidine hydrochloride, 1-10 mM EDTA, 0.1-5% Triton X-100, 0.1-5% Tween-20, with pH 7.0-8.0. The DNA nucleic acid extracted and purified by the kit has a high yield and good purity, and solves the problem of short storage time of the DNA extracted from fecal samples.

The invention pertains to methods and kits that simplify and speed the purification and recovery of RNA and DNA and avoid the use of lytic and digestive agents such as proteinase K thus avoiding. These kits are stable at room temperature and reduce the costs and processing time for isolation purification of RNA or DNA.

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 present invention relates to the field of modified proteins, immunogenic compositions and vaccines comprising the modified proteins, their manufacture and the use of such compositions in medicine. More particularly, it relates to a modified Sap2 (Secreted Aspartyl Proteinase 2 of Candida albicans') protein. The modified Sap2 protein can be used as a carrier protein for other antigens, particularly saccharide antigens or other antigens lacking T cell epitopes.

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 discloses a construction method of a sequencing library for detecting high-resolution chromatin interaction of plants. The method comprises the following steps: mixing a purified to-be-detected plant cell nucleus with a dendritic high-molecular compound, irradiating for 30 minutes under ultraviolet light, crosslinking to obtain a crosslinked compound, digesting the crosslinked compound by using protease K, digesting for 30-45 minutes by using DNaseI, and connecting an enzyme-digested product by using a bridge linker to obtain a connecting product containing a target DNA fragment, and finally, enriching and purifying the connected product to obtain the interacting chromatin DNA captured by the dendritic high-molecular compound. By using the method disclosed by the invention, experimental data of high-resolution chromatin interaction in the plant can be captured, and the method can be used for identifying the interaction between gene promoter regions in a whole genome range of the plant.

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 discloses a rapid free DNA enrichment method based on a paramagnetic particle method, which comprises the following steps: (1) taking plasma, adding a protease K solution, fully and uniformly mixing, and incubating; (2) adding the magnetic beads and the binding liquid, oscillating, instantaneously centrifuging and standing until the magnetic beads are completely adhered to the wall; (3) absorbing and abandoning all supernatant; (4) adding an eluent, oscillating, instantaneously centrifuging, continuously placing on the magnetic frame, and standing for 5 minutes until the magnetic beads are completely adhered to the wall; and (5) taking the supernatant, and transferring the supernatant into a sample storage tube for enrichment. According to the method, the DNA purity is high, a QSep result shows that no impurity peak interference exists, the feasibility of the method is proved, and the corresponding magnetic beads carrying positive charges can be used for extraction of the method, so that the method has relatively high practicability. A high-quality template can be provided for downstream molecule detection and analysis (including NGS sequencing, digital PCR and the like), and particularly, important values are shown in clinical application scenes such as liquid biopsy and antenatal diagnosis.

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 relates to the technical field of immunodetection, and discloses an immunochromatography reagent for detecting an antiproteinase 3 antibody and application, which are used for detecting the antiproteinase 3 antibody in human serum, plasma or whole blood and assisting in diagnosing wegener granuloma. The sample pad is used for bearing a to-be-detected sample; the combination pad is coated with an anti-human immune globulin antibody or an anti-human IgG antibody marked by a marker; the detection line is coated with a recombinant protease 3 antigen, myeloperoxidase antigen fragments related to Wegner granuloma attack are added, and hydrophilic polysaccharides are adopted as spacer arms to be arranged at intervals; the quality control line is coated with a goat anti-mouse IgG antibody; the water absorption pad is used for absorbing liquid in the chromatography process and promoting the liquid to flow unidirectionally; the bottom plate is used for bearing all the components, and all the components are sequentially and mutually assembled on the bottom plate to form a detection test strip; the immunochromatography reagent is extremely simple and convenient to operate, professional laboratory equipment and complex operation skills are not needed, and a detection result can be obtained only by dropwise adding a sample onto the test strip.

The present invention provides a method for purifying recombinant proteinase K, comprising the following steps: S1, adding buffer A to the recombinant proteinase K fermentation broth to be purified, and performing ultrafiltration once after mixing to remove metal ions and miscellaneous proteins with a molecular weight less than 10kDa; S2, pumping the obtained ultrafiltrate into a cation exchange column balanced with buffer A, then rebalancing with buffer A, eluting with buffer B, and collecting the eluate; S3, adding buffer A to the obtained eluate, performing ultrafiltration and concentration, and obtaining a highly purified recombinant proteinase K; wherein, buffer A: 9-11mM HAc-NaAc, pH=5.0-5.5; buffer B: 9-11mM HAc-NaAc, 1.0-2.0M NaCl, pH=5.0-5.5. The method described in the present invention has simple process and high product yield and purity, and is suitable for industrial-grade production of products.

The present invention relates to methods and compositions for the dissociation of biological tissue with the aim of obtaining single cells for analysis. More particularly, the compositions of the present invention are enzyme compositions comprising proteinase K. The inventive compositions can be used in methods that effectively dissociate tissue at lower temperatures for application in high-throughput single cell omics research.

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 invention provides a universal method for automatically and rapidly extracting DNA (deoxyribonucleic acid) of a skeleton detection material and a conventional detection material, which comprises the following steps: grinding the skeleton detection material into powder, incubating with protease K and skeleton incubation liquid, digesting the conventional detection material with protease K and digestive juice, heating and digesting together, and subsequently, adopting the same adsorption system and washing system for the skeleton detection material and the conventional detection material. The method can be carried out by adopting an existing DNA extraction automatic workstation. The DNA extraction device can automatically and rapidly extract DNA of a skeleton detection material and DNA of a conventional detection material, overcomes the defects that DNA extraction of the skeleton detection material is tedious in operation and free of automatic operation, can perform DNA extraction and purification with the conventional detection material at the same time, and greatly saves extraction time.

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.

The invention provides a cytoplasmic DNA imaging method for reducing background interference by using protease K. The method comprises the steps of fixation, cleaning, permeation, cleaning, enzymolysis, cleaning, dyeing, cleaning, sheet sealing and the like. Protein is degraded by protease K, and background interference fluorescence generated when nucleic acid fluorescent dye is used is reduced, so that cytoplasmic DNA is accurately dyed, and a more accurate cytoplasmic DNA imaging result is obtained. According to the method, protease K enzymolysis is adopted to assist nucleic acid dye in cytoplasm DNA imaging, dynamic regulation and control of signal specificity and cell integrity are achieved, background fluorescence is successfully removed, and a good imaging effect is achieved. And a new strategy is provided for realizing accurate imaging of the cytoplasmic DNA.

The invention discloses a recombinant proteinase K industrial purification and freeze-drying method. After the recombinant proteinase K fermentation liquid expressed by Pichia pastoris is clarified and filtered, a diluent is added to dilute, and concentrated and dialyzed through an ultrafiltration step to obtain a proteinase K dialyzate containing trace metal ions and pigments, and the dialyzate is separated and purified by cation exchange chromatography to obtain an eluent containing recombinant proteinase K; the eluent containing recombinant proteinase K is placed in a room temperature crystallization precipitation for 4-6h, and poured into a positive pressure filtration device after stirring and mixing, and a proteinase K filter cake is obtained by filter pressing; after the filter cake is redissolved, a freeze-drying excipient is added, and freeze-dried according to an optimized freeze-drying process, and a proteinase K finished product is obtained after sub-packaging. The method has simple process steps, high yield, low cost, and is easy to industrially scale up production. The obtained proteinase K target has high yield and good activity.