145 results about "Enzyme digestion" patented technology

The invention provides a construction method, system and product of a false positive judgment model of a mutation site obtained by sequencing after enzyme digestion fragmentation and related application. Specifically, the false positive judgment model is constructed on the basis of a plurality of characteristic parameters associated with mutation sites obtained by enzyme digestion fragmentation sequencing; the characteristic parameters comprise scores obtained by performing reverse complementary sequence alignment on the front and back extension 30bp of the mutation site, the sequence proportion of 15 basic groups at the tail end of the mutation site, the Bayesian conditional probability of the extracted mutation site, and the KS test statistic of the extracted mutation site. Furthermore, the invention also provides a method for constructing the model by utilizing the crowd detection rate and the corresponding false positive. According to the method, the data characteristics of the false positive mutation sites are introduced by utilizing digestion fragmentation, so that the reliability of the digestion false positive mutation sites is objectively evaluated under the condition that high depth and large data volume are not needed, and the detection accuracy of the mutation sites can be remarkably improved.

The invention discloses a bubble self-stratification digital detection method and system for miRNA quantification of extracellular vesicles, and belongs to the technical field of biomedical engineering and molecular diagnosis. The technical problem to be solved is to provide a simple and sensitive tumor extracellular vesicle miRNA detection method capable of simultaneously detecting various miRNAs. According to the scheme, a to-be-detected sample and immunocapture bubbles coupled with an anti-EpCAM antibody are incubated to enrich tumor extracellular vesicles, a to-be-detected miRNA sample is obtained through in-situ ultrasonic lysis, then the to-be-detected miRNA sample, a fluorescence / DNA double-coding magnetic bead conjugate and the like are used for constructing an enzyme digestion system for enzyme digestion, multifunctional click glass microbubbles are added to capture unreacted magnetic beads, standing is conducted, self-stratification is conducted, bottom magnetic beads are collected, and the EpCAM antibody is obtained. After fluorescence imaging, an image is input into the AI automatic fluorescence counting and decoding module for counting to obtain a result. The method is used for detecting miR-21 and miR-155 in tumor extracellular vesicles, and is suitable for noninvasive early diagnosis of tumors.

The invention provides a method for improving the expression level of a meat toxin light chain. The method comprises the following steps: connecting a dissolution-promoting tag to an N end or a C end of meat toxin light chain protein to form soluble meat toxin light chain recombinant protein; the dissolution-promoting tag is a wild dissolution-promoting tag NT11 or a mutant protein mut7 of the wild dissolution-promoting tag NT11, enzyme digestion removal is not needed, the activity of a meat toxin light chain is hardly influenced and interfered, and the fusion protein can keep 93% of substrate enzyme digestion activity. The method further comprises overexpressing a molecular chaperone htpG in the genome of the host bacterium. According to the invention, a BoNT / A-LC high-efficiency expression system is constructed through multi-strategy optimization, so that the yield of BoNT / A-LC is increased by nearly 20% compared with that of wild type NT11-Lc, the yield reaches 647 mg / L through overexpression of a molecular chaperone htpG, and after combinatorial optimization, the yield is 8.5 times that of only expressed BoNT / A Lc, 1.79 times that of NT11-Lc and 1.43 times that of Mut7-Lc.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The invention relates to the technical field of crop breeding science, in particular to a molecular marker closely linked with a wheat powdery mildew resistance gene PmNJ3930 and application of the molecular marker. The marker closely linked with the PmNJ3930 is a dCAPS molecular marker, and an upstream primer sequence and a downstream primer sequence for amplifying the molecular marker are provided. A product amplified by the primer is 354 bp after being subjected to NcoI enzyme digestion, which indicates that the wheat to be detected contains the PmNJ3930 gene and is a wheat variety with high powdery mildew resistance. When the primer pair provided by the invention is used for detecting the PmNJ3930 gene, whether the PmNJ3930 gene exists or not and the existence state of the PmNJ3930 gene can be determined, and the powdery mildew resistance of wheat is predicted, so that plants carrying the PmNJ3930 gene are quickly screened and are used for breeding disease-resistant varieties.

The invention discloses construction of a Guangdong small-ear spotted pig spermatogonial stem cell in-vitro culture system and screening of surface markers, and belongs to the technical field of cell culture.The technical scheme includes that testis are broken firstly, then enzyme digestion, density gradient centrifugal purification and differential adherent purification are conducted, and purified spermatogonial stem cells are obtained; inoculating the obtained spermatogonial stem cells to feeder layer cells and culturing in a culture medium; the culture medium is a DMEM culture medium added with FBS and growth factors; the mass fraction of the FBS in the DMEM culture medium is 8-12%, an enzyme digestion method, a density gradient centrifugation method and a differential adhesion method are combined for application, the Guangdong small-ear spotted pig spermatogonia stem cells with certain purity are obtained, cells enriched and purified in vitro can proliferate massively, and an in-vitro culture system of the Guangdong small-ear spotted pig spermatogonia stem cells is established.

Provided in the present application are a cyclic peptide mutant and use and product thereof for the preparation of a coronavirus inhibitor, belonging to the technical field of cyclic peptide drugs. The present application is optimized on the basis of the cyclic peptide 6L3-3P11R. The obtained cyclic peptide mutant can resist the enzyme digestion effect of pancreatic enzymes and exhibits further improved antiviral activity, which is of great significance for expanding antiviral applications of cyclic peptide drugs.

A primer pair, method, and use for rapidly identifying newborn nude mice. On the basis that a deletion mutation occurs in the base G at position 337 of a coding region of the Foxn1 gene in nude mice, a primer pair introducing an enzyme digestion site is designed according to sequence characteristics. Using the genome of a mouse to be tested as a template, the Foxn1 gene is amplified according to a PCR-RFLP method. The PCR product is then digested with a SmaI restriction endonuclease, and the digested product is genotyped by agarose gel electrophoresis. In addition, a reaction system that can be combined with lateral flow technology is screened, thereby achieving instant detection of the newborn nude mice. The method has the characteristics of simple operation, short time consumption, low cost, instant detection, etc.

The present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

The invention discloses a preparation method of a fibrous protein / bacterial cellulose efficient hemostatic patch. The preparation method comprises the following steps: (1) in-situ preparation of large-aperture bacterial cellulose; (2) infiltration of fibrinogen in the large-aperture bacterial cellulose; and (3) enzyme digestion and freeze-drying treatment of fibrinogen. The problems that fibrinogen is fast in degradation and poor in stability and mechanical property are solved, the prepared fibrous protein / bacterial cellulose efficient hemostatic patch can prevent secondary bleeding and avoid thrombus to a certain extent, the preparation process is simple and easy to implement, and large-scale preparation and storage can be achieved.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

This application relates to a method, apparatus, computer device, and storage medium for identifying enzyme substrates. The method for identifying enzyme substrates analyzes the sequence information of substrate peptides before and after enzyme digestion by quantitative detection of control samples and enzyme inhibitor-treated samples, thereby identifying potential enzyme substrates. It effectively identifies enzyme cleavage sites, detects the sequences of substrate peptides before and after enzyme digestion, and promotes enzyme substrate identification and site analysis. This plays an important role in understanding biological processes and identifying biomarkers for diseases.

The invention discloses a cell crude sample sex determination primer suitable for chickens, ducks or pigeons and a PCR detection method, and belongs to the technical field of biological detection. A pair of gender-specific universal primers for various poultry such as chicken, duck and pigeon is designed for the first time, a cell crude sample is adopted as a PCR template, stable amplification can be realized without DNA extraction, the operation process is greatly simplified, the detection time is shortened, and the detection cost is reduced. Meanwhile, TrypLE enzyme digestion is very mild to cells, and the method is also suitable for preparation of single-cell suspensions related to single-cell omics. The method is suitable for the fields of early sex identification of poultry, breeding poultry breeding, molecular breeding and the like, and has wide application prospects.

This invention provides a high-throughput method for constructing dual sgRNA libraries and its applications. The method utilizes high-throughput microarray synthesis technology to prepare a set of DNA fragments containing multiple dual sgRNA expression cassettes in a single step. After amplification, these fragments are assembled with a target vector containing a first promoter and a second gRNA backbone sequence in a first round of directed assembly to obtain a preliminary recombinant plasmid set. Then, linearized enzyme digestion and homologous recombination technology are used to insert a fragment containing a transcription termination sequence and a complete second promoter to complete the construction of the dual sgRNA expression unit. Finally, transformation and amplification yield the dual sgRNA plasmid library. This invention avoids the high error rate and high cost of long-chain oligonucleotide synthesis by utilizing microarray synthesis and simplifies the operation process through two rounds of directed assembly, significantly improving the throughput, fidelity, and efficiency of library construction. It is applicable to the construction of genome-wide dual sgRNA libraries, providing an efficient and reliable technical platform for high-throughput gene function screening, drug target discovery, and gene interaction research based on CRISPR.

This invention belongs to the fields of biotechnology and genomics, and relates to a method and kit for capturing plant chromatin conformation. The method includes the following steps: (1) cross-linking and fixing the chromatin of a plant sample; (2) digesting the fixed chromatin using an enzyme digestion combination containing restriction endonuclease AluI and restriction endonuclease HypCh4V; (3) ligating the enzyme fragments generated in step (2) at spatially adjacent locations to form ligation products; (4) enriching the ligation products and constructing sequencing libraries using the enriched ligation products. This invention uses a combination of AluI and HypCh4V enzymes to replace traditional single-enzyme digestion, which significantly improves data resolution while reducing technical bias. It can achieve chromatin interaction mapping at a resolution of 500 bp in plants and has broad application prospects in the study of fine tissue characteristics at the single-gene level in plants.

This invention relates to an identification method and kit for overlapping PCR with enzyme digestion. The method includes selecting a corresponding restriction endonuclease using N4N5X or N4N5Y as the last three bases of the introduced restriction site; if no corresponding restriction endonuclease is available, selecting a corresponding restriction endonuclease using N5X or N5Y as the last two bases of the introduced restriction site; amplifying to obtain a universal fragment primary PCR product; designing PCR amplification primers based on the restriction endonuclease and universal primers to obtain the primary PCR product of the template to be tested; and performing enzyme digestion and genotyping on the final product of the overlapping PCR amplification. This invention utilizes the DOPCR method to identify SNPs / InDels. Compared with existing methods that create restriction sites, it adds an overlapping PCR step, resulting in significant differences in the digested fragments. It eliminates the need for PAGE electrophoresis; detection can be completed using ordinary electrophoresis, making it low-cost and rapid.

A genome-wide R-loop detection method, in particular, a genome-wide R-loop detection method independent of affinity enrichment and a use. According to the genome-wide R-loop detection method, nucleases are used to perform enzyme digestion on genomic DNA fragments, and R-loop detection can be achieved by performing one-step enzyme digestion simply using a system of Nuclease P1, T5 exonuclease, and Lambda exonuclease. The whole R-loop detection process does not require affinity enrichment or labeling treatment. The method is not only simple and easy to operate, but also can avoid the loss of samples and fragments, thereby reducing the cost and risk of detection, and improving the applicability.

The application provides a method for constructing an immortalized bovine nasopharyngeal tonsil epithelial cell line and application, and belongs to the technical field of cell engineering. The healthy yellow bovine nasopharyngeal tonsil is used as a material, and the primary epithelial cells are separated by enzyme digestion. The 3rd to 4th generation cells are taken, infected with SV40 T lentivirus (MOI=80), and screened by 1.5 μg / mL puromycin to obtain a stable immortalized cell line. The cell line is continuously passaged to the 50th generation and still stably expresses SV40LT protein and epithelial specific markers CD326, CD324 and KRT8, maintains a typical pavement stone-like morphology, normal proliferation and no malignant transformation. Infection experiments show that the cell is highly sensitive to FMDV, supports efficient replication, and the virus titer can reach 10 7.5 TCID 50 / mL. The application overcomes the defects of the existing immortalized bovine nasopharyngeal tonsil epithelial cell model, such as limited source, large batch difference, low sensitivity and poor persistence, and provides a stable and reliable in vitro model for the research of foot-and-mouth disease prevention and control related mechanisms, drug screening and vaccine development.

The invention belongs to the technical field of biology, and particularly relates to a nucleus pulposus cell separation and culture method. According to the method, a step-by-step enzyme digestion method is adopted, NB6 collagenase is firstly used for primary digestion, and then compound enzyme liquid composed of trypsin and hyaluronidase is adopted for subsequent digestion; and culturing in combination with a culture vessel coated with a specific concentration of extracellular matrix component and a special culture medium. According to the method, on the premise of not depending on an immortalization technology, the primary nucleus pulposus cells with good activity can be efficiently obtained, stable amplification and long-term passage of the primary nucleus pulposus cells in vitro are realized, and specific marker expression and biological functions of the nucleus pulposus cells can be effectively maintained. The invention provides a stable cell source and technical platform for the development of cell therapy products and the research of spinal degenerative diseases.

The invention discloses a long single-stranded DNA (deoxyribonucleic acid) preparation method based on hybridization chain reaction, which comprises the following steps: carrying out hybridization chain reaction on an initiation chain I0, a hairpin structure monomer H1 and a hairpin structure monomer H2 to obtain a long double-stranded DNA product; and sequentially connecting 5 ends and 3 ends of adjacent H1 chains in the obtained long double-chain DNA product by adopting T4 ligase, annealing at 95 DEG C, and separating an H1 series long single chain, an H2 short chain and an initiation chain I0, so that the H1 series long single chain is the long single-chain DNA. The invention develops a new method for preparing the long single-stranded DNA, rapid separation of the long single-stranded DNA and the connecting fragment can be realized in the reaction liquid only through simple annealing at 95 DEG C without enzyme digestion or other additional treatment steps, the generated long single-stranded DNA is a circulating sequence, the sequence accuracy is high, and the total length can reach 2240 nt.

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of recombinant A-type botulinum toxin. According to the method disclosed by the invention, after the recombinantly expressed protoxin is combined with cation exchange filler, enzyme digestion and activation are realized by utilizing single protease, and then purification is carried out. The preparation method disclosed by the invention has the advantages of good convenience and small batch-to-batch difference, and the prepared recombinant A-type botulinum toxin also has obvious advantages in protein effectiveness and biological activity.

Methods for performing non-reduced peptide mapping analysis of a protein of interest can include alkylating a protein of interest with N-ethyl maleimide (NEM) or an NEM analog under denaturing conditions to form an alkylated protein of interest; digesting the alkylated protein of interest with at least one digestive enzyme to form a peptide digest, and subjecting the peptide digest to liquid-chromatography-mass spectrometry analysis. The methods can be performed under mildly alkaline conditions.

This application relates to the biomedical field, specifically to a method for expressing tandem recombinant proteins using *E. coli*. The tandem recombinant protein has the general formula leader peptide-(cleavage site 1-spacer peptide-cleavage site 2-target protein)n, wherein the leader peptide is selected from any sequence of SEQ ID NO:1-4; cleavage sites 1 and 2 may be the same or different, and are double cleavage sites of Kex2 and CPB enzymes selected from dipeptides KR or RR; the spacer peptide is any sequence selected from tripeptide EDG, tetrapeptide SEQ ID NO:5-7, or heptapeptide SEQ ID NO:8; the target protein is GLP-1 or an analogue thereof; and n is a tandem repeat number of 2-10. The inclusion bodies obtained by this method do not require denaturation, and the target polypeptide is obtained through double enzyme digestion. The expression level of the fusion protein can reach 32 g / L, and the yield of the target polypeptide can reach 25.3 mg / g wet cells or 10 g / L fermentation broth.

The invention relates to the technical field of saccharomyces cerevisiae expression foreign protein, and discloses a saccharomyces cerevisiae strain with ASFV p14.5 displayed on the surface and a construction method and application thereof, and the method comprises the following steps: (1) synthesizing a delta site homologous arm sequence; (2) seamlessly connecting the homologous arm sequence of the delta site to a carrier pET23a, connecting a selective marker tryptophan biosynthetic gene TRP1 to the 5'end of the upstream homologous arm sequence of the delta site, and sequentially connecting an Aga2 gene, an E120R gene, a T2A peptide gene and an mCherry gene to the 5 'end; (3) carrying out enzyme digestion on two sides of the delta site through restriction endonuclease salI, and recovering a linearized fragment through agarose gel; and (4) integrating the recovered fragments into a host saccharomyces cerevisiae genome through a chemical conversion method, and culturing until bacterial colonies grow. The surface display type saccharomyces cerevisiae strain obtained by the invention not only has the stability of integrative saccharomyces cerevisiae for expressing foreign protein, but also can be used for more easily and more intuitively screening a high-expression quantity saccharomyces cerevisiae strain by combining the T2A peptide with mCherry.

The invention provides a fusion protein containing an SUMO tag and a connecting sequence and a preparation method of the fusion protein. Specifically, the invention provides a CCL22 fusion protein containing an SUMO tag and a long flexible connection sequence. The fusion protein is subjected to enzyme digestion, so that the enzyme digestion rate and completeness can be remarkably improved, and the loss rate of the recovered protein after enzyme digestion is less than 5%. By constructing the fusion protein disclosed by the invention and adopting the method disclosed by the invention, not only are the advantages of the SUMO tag reserved, but also the purification process can be simplified, and the method is suitable for the purification process of the protein which is difficult to efficiently express and purify, and has broad spectrum.

The invention belongs to the field of forensic detection, and particularly relates to a method for detecting allergens in drug-induced allergic shock. Based on a type I hypersensitivity reaction mechanism, carboxylated microspheres are used for cross-linking an anti-IgE antibody, an IgE-allergen compound in a sensitization stage of a detection sample is combined and captured, interference of residual components of the sample is removed through multiple times of cleaning, the antibody and drug binding protein are digested by enzyme to dissociate drug components, and the immunogenicity of the IgE-allergen compound in the sensitization stage of the detection sample is improved. And the LC-MS is used for qualitatively detecting medicine components so as to achieve the purpose of detecting the medicine allergen.

The invention relates to an automatic liquid treatment platform system with an anti-pollution function, and the system comprises a housing which is internally provided with a workbench; the liquid transfer module is arranged above the workbench in a sliding manner and is used for transferring nucleic acid liquid and adding the nucleic acid liquid into the pore plate; the PCR amplification module is arranged on the workbench and is used for carrying out amplification, enzyme digestion and marking on nucleic acid; the nucleic acid extraction / purification module is arranged on the workbench and is used for extracting and purifying the nucleic acid treated by the PCR amplification module; the nucleic acid concentration detection module is also arranged on the workbench and is used for detecting the concentration of extracted and purified nucleic acid; and the control unit is used for controlling each module to act so as to realize the automatic nucleic acid processing flow. The system solves the problem of low efficiency caused by respective operation of a nucleic acid extraction / purification module set, a PCR amplification module set, a nucleic acid concentration detection module set and the like at present.

The invention discloses a recombinant human lactalbumin plant expression cassette and construction and application thereof, and belongs to the technical field of genetic engineering and biological manufacturing. The invention aims to solve the problems of low heterologous expression efficiency and non-ideal expression quantity caused by mismatching of mammal-derived human lactalbumin genes and plant host codon use preferences. The method comprises the following steps: firstly obtaining an amino acid sequence of human lactalbumin, then optimizing a coding nucleotide sequence according to the codon use frequency of a target plant host species, and finally inserting the optimized gene into a linearized vector skeleton containing a plant specific promoter and a plant recognizable terminator through enzyme digestion connection or homologous recombination. The optimized gene is located downstream of the promoter and upstream of the terminator. The method provides support for high-efficiency expression of the recombinant human lactalbumin in plant host cells, and assists application of the recombinant human lactalbumin in food, nutritional health-care products or pharmaceutical preparations.

The invention discloses application of transgenic zebrafish in preparation of an animal model capable of visually monitoring mast cells. The transgenic zebrafish is prepared by the following method: respectively carrying out XhoI and BamHI double enzyme digestion on a cpa5 gene promoter sequence (SEQ ID NO: 1) and plasmids with fluorescent protein genes and Tol2 transposon recognition sites, and then connecting to obtain recombinant plasmids; and then introducing the recombinant plasmid and Tol2 transposase mRNA into a zebra fish embryo in a microinjection mode in a 1-cell period of zebra fish embryo development, and culturing at constant temperature to obtain the transgenic zebra fish. The cpa5 promoter is constructed to drive an expression framework of the fluorescent protein, so that the mast cells naturally emit fluorescence in the zebra fish body, and an effective animal model is provided for mechanism research and drug screening of mast cell related diseases.

This invention discloses a method for constructing an immune animal model for preparing biofusion enzyme antibodies and its application. The method includes the following steps: designing and screening sgRNAs with high cleavage efficiency based on signal protein genes, and constructing an sgRNA-Cas9 expression vector; linearizing the plasmid by double enzyme digestion, and then ligating it with a signal protein gene fragment containing left and right homologous arms to obtain the Donor plasmid; co-transfecting the sgRNA-Cas9 expression vector and the Donor plasmid into target animal somatic cells, and screening to obtain positive somatic cells that stably integrate the target gene; using the positive somatic cells as nuclear donors for nuclear transfer to construct recombinant embryos, and transferring the recombinant embryos into recipient female animals; after delivery, identifying transgenic animal individuals carrying biofusion enzyme antibodies by genomic PCR. Based on this transgenic animal model, different target antibodies with clinical value can be developed.