206 results about "Recombinase" patented technology

The invention discloses a CRISPR / Cas12a-RPA (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 12a-recombinase polymerase amplification)-based sequence combination for rapidly detecting mouse hepatitis virus and a kit thereof, and belongs to the technical field of biological detection The sequence combination comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence; the kit comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence, the kit further comprises a mouse hepatitis virus negative control standard substance, RNase-free water, an RPA reaction buffer solution, an NEB buffer solution, Lba Cas12a nuclease, dry powder RPA reaction microspheres containing recombinase polymerase and magnesium acetate with the concentration of 280 mM. The kit can be used for rapidly detecting the mouse hepatitis virus. The primer sequence combination provided by the invention is good in conservative property and specificity and relatively high in sensitivity, and rapid detection of the mouse hepatitis virus can be realized.

The invention discloses a construction method and application of a mouse model for specifically tracing heart valve cells, and belongs to the technical field of animal model construction. The model construction method comprises the following steps: (1) constructing a Wid1-Cre hybrid mouse before inserting a Cre recombinase coding gene into a termination codon of a mouse Wid1 gene; and (2) hybridizing the Wid1-Cre hybrid mouse with the Rosa26-tdTomato homozygous report mouse, so as to obtain the double transgenic mouse which simultaneously carries the Wid1-Cre gene and the Rosa26-tdTomato gene. According to the invention, on the basis of the expression characteristic that Wif1 is only limited to a valve area in the heart, Wif1-Cre is constructed; according to the Rosa26-tdTomato mouse model, specific tracing of heart valve cells is achieved, and an important experimental tool is provided for mechanism research of heart valve diseases and development of therapeutic drugs.

The invention belongs to the technical field of biological medicine, and particularly relates to an engineered cell microvesicle and a delivery system based on the engineered cell microvesicle, the system realizes efficient preparation of 1-5 [mu] m cell microvesicles, and the cell microvesicles have a large space volume and can be used for preparing the cell microvesicles. The carrier can be used for loading and delivery of target protein, polypeptide and recombinase which are specifically expressed in mother cells. The system transfects mother cells through lentivirus transfection or plasmid transfection to further produce cell microvesicles, and the microvesicles can load more goods and inherit membrane proteins of the mother cells, and can also effectively load intracellular proteins to realize effective delivery. By virtue of good structural stability, high immunogenicity and excellent biocompatibility, the cell microvesicle reduces systematic toxic and side effects of a traditional carrier, is expected to become an effective drug delivery system, and has great application potential in the field of gene therapy.

SpRYc is a grafted ScCas9++-SpRY chimeric Cas9 possessing minimal 5′-NNN-3′ PAM specificity. SpRYc comprises the N-terminus (residues 1-1119) of ScCas9++ (Sc++), including the flexible loop, followed by the region of SpRY (residues 1111-1368) spanning its PAM-interacting domain mutations. Methods of altering gene expression include use of SpRYc in complex with guide RNA in a CRISPR-Cas9 system.

The invention discloses a preparation method of recombinant catechol-2, 3-dioxygenase and an application of the recombinant catechol-2, 3-dioxygenase. According to the invention, a novel catechol-2, 3-dioxygenase group C23O927 is identified from a constructed metagenome library, and the catechol-2, 3-dioxygenase group C23O927 is a key enzyme for catalyzing degradation of polycyclic aromatic hydrocarbon pollutants. A C23O927-pET32a (+) recombinant expression vector is constructed and converted into host bacteria, heterologous expression of target protein is achieved, and the prepared recombinase belongs to acid enzymes and has good resistance to high temperature, acid, alkali, high-concentration metal ions, organic solvents and surfactants. The recombinase provided by the invention can be used in an acidic environment, has very good enzymatic activity, can catalytically degrade various catechol compounds, can be better used for treating polycyclic aromatic hydrocarbon polluted environments, and has important significance for developing more products which can tolerate various extreme environments and efficiently catalyze degradation of polycyclic aromatic hydrocarbons.

The invention relates to the technical field of technical gene detection, in particular to a recombinase and recognition site screening method and system based on structure classification prediction.The method comprises the steps that to-be-matched recombinase is obtained for structure resolution prediction.The functional domain of the to-be-matched recombinase is obtained; searching in a first database according to the functional domain, searching candidate recombinase of which the similarity meets a preset condition, and screening to obtain optimal recombinase; determining an interaction region of the current large serine recombinase and the target DNA sequence from a second database, and sequentially obtaining interaction amino acid sequences corresponding to the preferable recombinase; and matching with the amino acid characteristic sequence of the current large serine recombinase in the second database to obtain the target large serine recombinase with the highest similarity in the preferred recombinase and the corresponding recognition site sequence. According to the method, the structure of the large protein is subjected to structure comparison mining and splitting, and recognition site prediction is performed on interaction region amino acid characteristics, so that the research and development period is shortened, and the research and development cost is saved.

The invention relates to the field of molecular diagnosis and nucleic acid amplification, in particular to a recombinase polymerization amplification method based on Twinkle helicase enhancement and application of the recombinase polymerization amplification method. According to the invention, Twinkle helicase is introduced into an RPA system for the first time, and primer combination and template unlinking are assisted by utilizing ATP-dependent unlinking activity and DNA annealing activity of the Twinkle helicase, so that the amplification efficiency and the detection sensitivity are remarkably improved. The method is suitable for plasmid DNA, a high GC template and a clinical nucleic acid sample, and can be used for constructing an RPA kit containing Twinkle, so that the application of the RPA kit in pathogen detection, molecular diagnosis and on-site rapid detection is expanded. According to the invention, the defects of the existing RPA technology in sensitivity and stability are overcome, and a new solution is provided for developing a novel nucleic acid self-detection and field detection tool.

The present disclosure relates to enzymes, compositions and methods for performing conditional homologous recombination of a targeted DNA molecule or genome by using modified proteins comprising nucleic acid DNA binding proteins with protease-conditional recombinase (hereinafter “ProRec”) activity. Namely a new cre recombinase pro enzyme, compositions / kit and sensors comprising the cre recombinase pro enzyme of the present disclosure. Namely, a cre recombinase pro-enzyme, for identifying and quantifying proteolytic activity in a disease or an infection, comprising intein mediated circularization, a flip-excision cassette comprising an output reporter and / or effector protein; a linker sequence cleavable by a protease.

The invention belongs to the technical field of biological engineering, and provides curcumin / dihydrocurcumin reductase MaCurA, a coding gene and application. The reductase can efficiently catalyze 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone to synthesize tetrahydrocurcumin, an efficient solution is provided for industrial preparation of tetrahydrocurcumin, and experiments prove that the recombinase can catalyze the synthesis of tetrahydrocurcumin in the catalysis of 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone. In a reaction system for preparing tetrahydrocurcumin from 2, 5-diketone, actually measured enzyme activity is stabilized at 3.38 U / mg level, and compared with a traditional chemical synthesis method, the production efficiency is remarkably improved.

The invention discloses Bxb1 recombinase with improved activity and application of the Bxb1 recombinase. The invention provides a recombinase, and the amino acid sequence of the recombinase comprises one or more amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 1. The recombinase provided by the invention can act on large-fragment DNA in a genome, so that the activity of a recombinase genome editing system is improved, and the application scene and selection of recombinase genome editing are enriched.

The application discloses a carbonyl reductase mutant and application thereof in preparation of (R)-8-chloro-6-hydroxyoctanoate compounds. The application relates to a mutant of carbonyl reductase with improved catalytic performance and stereoselectivity after rational molecular modification, a recombinant expression plasmid containing the mutant gene of the enzyme, a recombinant expression transformant, a preparation method of the recombinant enzyme, and application of the recombinant enzyme or the recombinant cell as a catalyst in asymmetric reduction of 8-chloro-6-carbonyloctanoate compounds (I) to prepare (R)-8-chloro-6-hydroxyoctanoate compounds (II), in particular, in an intermediate (R)-8-chloro-6-hydroxyoctanoate methyl ester of thioctic acid. The disclosed carbonyl reductase SsCR mutant has the advantages of high reaction efficiency and high catalytic activity, and exhibits the advantages of high substrate concentration, low catalyst consumption and short reaction time in the process of catalytic preparation of the compound (II).

The invention discloses polyethylene glycol terephthalate hydrolase PmPETase and application thereof.The hydrolase PmPETase is protein with the amino acid sequence shown in SEQ ID NO.1. An expression system suitable for the PmPETase is constructed, recombinase protein with catalytic activity is obtained, and the polyethylene glycol terephthalate hydrolase PmPETase can be used for preparing the polyethylene glycol terephthalate hydrolase PmPETase. The substrate specificity, the catalytic property and the potential plastic degradation capacity of the PET degrading enzyme are comprehensively analyzed, and a new theoretical basis and application reference are provided for development and utilization of the PET degrading enzyme and a high polymer material green treatment technology.

The invention relates to the technical field of water pollution and water body odor substance detection, in particular to a primer group, a kit and a method for detecting cyanobacteria generating 2-methylisoborneol in a water body based on a recombinase-mediated isothermal nucleic acid amplification (RAA) technology. The primer group is designed based on a mic gene conserved region of the 2-methylisoborneol-producing cyanobacteria, and comprises a forward primer, a reverse primer and a specific probe; the 5'end of the reverse primer is modified with a fluorescent marker, the 5 'end of the probe is modified with biotin, the 3' end of the probe is modified with a phosphorylation group, and nuclease cleavage sites are contained in the probe. The primer group disclosed by the invention can effectively amplify the mic gene of the 2-methylisoborneol, has relatively high specificity and sensitivity, has no cross reaction with cyanobacteria which does not generate the 2-methylisoborneol, and can be combined with lateral flow immunochromatography to be used for on-site rapid detection of the 2-methylisoborneol; the method is of great significance in early warning of excessive content of 2-methylisoborneol in the water body.

The present disclosure relates to production of cis-3-hexenol from unsaturated fatty acid, such as alpha linoleic acid and linoleic acid. The process of the present disclosure involves combinatorial and sequential action of recombinant enzymes, such as LOX, HPL and ADH for the production of cis-3-hexenol.

The present invention refers to a recombinant Ten Eleven Translocation 3 (TET3) enzyme, a nucleic acid molecule encoding the TET3 enzyme, a vector comprising the nucleic acid molecule and a host cell transformed or transfected with the vector, the use of the recombinant TET3 enzyme for sequencing of a nucleic acid substrate, a method for sequencing a nucleic acid substrate and a reagent for sequencing a nucleic acid substrate.

The invention relates to screening methods. In particular, it relates to a method of identifying a functional TCR from a library of particles, which displays a plurality of different T cell receptors (TCRs). The method may comprise a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen. Transfection of the T cells is such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain. The T cells do not express endogenous TCR. A T-cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR, is also claimed.

The invention relates to the related technical field of molecular biology and cell engineering, in particular to an FLP-IN-PIEC cell line and a construction method and application thereof, and the construction method comprises the following steps: step 1, constructing CRISPR / Cas9 site-specific cleavage plasmid containing a pig HIPP11 safety site sequence; step 2, constructing a donor plasmid Donor-FRT containing a homologous arm of a safety site of the HIPP11; 3, jointly transfecting plasmids in the step 1 and the step 2 into a porcine hip artery endothelial cell line (PIEC) to obtain an FLP-IN-PIEC cell line for expressing red fluorescence; and 4, under the action of Flp recombinase, transfecting an exogenous gene donor plasmid containing an FRT site to the FLP-IN-PIEC cell line obtained in the step 3, and driving the expression of a target gene through an optimized strong promoter to realize fixed-point transfer and efficient expression of the exogenous gene in the pig (PIEC) cell line. Different from an existing Flp-In system depending on a random integration strategy, the construction method disclosed by the invention emphasizes fixed-point integration and promoter optimization, and improves the homogeneity, stability and screening efficiency of exogenous gene expression.

The present invention pertains to a method for generating a spacer-specific DNA recombining enzyme (DRE), the method comprising the steps of: a) providing a library of expression vectors encoding a plurality of variants of a DRE (vDRE), wherein the amino acid sequences of the vDREs comprise one or more amino acid modifications in comparison to a non-variant DRE (nvDRE) from which the vDREs are derived, wherein the nvDRE binds to a first target site comprising in 5′ to 3′ direction a half-site A (HSA), a spacer (Spacer) and a half-site B (HSB), wherein each expression vector comprises: (i) a first region comprising a nucleotide sequence encoding one of the vDREs from among the plurality of vDREs operably linked to an expression control sequence, and (ii) a second region comprising a nucleotide sequence comprising in 5′ to 3′ direction a first target site, an insert nucleotide sequence (INS) of a length at least 1 nucleotide and a second target site, wherein each of the first and second target site comprises the HSA, a variant spacer (vSpacer) and the HSB, wherein the vSpacer differs by at least one nucleotide from the Spacer; b) introducing the library of expression vectors into host cells; c) expressing the plurality of vDREs in the host cells; d) optionally isolating DNA from the host cells; and e) determining whether the vDRE shows activity on the target sites of at least one expression vector.

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

The purpose of the present invention is to provide a novel method for targeting a neuron population that is a neural circuit element which has more limited functional localization. The purpose of the present invention is also to provide a method for treating a disease or the like by targeting a specific neuron population involved in a disease or the like by said novel method and controlling the neural activity thereof.　The present invention is a vector set used for targeting a specific neuron population in a neural circuit, said vector set being characterized by comprising first to third vectors, wherein: the first vector is an antegrade trans-synaptic virus vector that contains a base sequence which codes for a split-Cre recombinase consisting of a C-terminal-side sequence of a Cre recombinase; the second vector is a retrograde trans-synaptic virus vector that contains a base sequence which codes for a split-Cre recombinase consisting of an N-terminal-side sequence of a Cre recombinase; the third vector is a non-trans-synaptic virus vector that contains an inverted sequence of a gene sequence which codes for a desired protein and that has a loxP sequence and a loxP mutation sequence which face opposite to each other and which are at both ends of the inverted sequence; and a functional Cre recombinase is formed in a cell that has been infected with the first vector and the second vector.

The construct for site-specific insertion of DNA fragments and transgenes into the genome comprises a recombinant enzyme. This enzyme contains a modified HIV-1 integrase (HIV IN) tetramer with multiple mutations favoring a heterodimer structure in which the first and second outer monomers are bound to the first and second inner monomers, respectively. A transcription activator-like effector (TALE) is bound to the N-terminal region of the outer monomer via a polypeptide linker. The two TALEs confer sequence specificity to the enzyme and prevent non-specific interactions with the target DNA structure. The modified HIV IN monomer ensures that the TALE binds to the outermost monomer and that the TALE is positioned relative to the catalytic region of HIV IN. A polynucleotide is bound to the inner monomer of HIV IN. When the TALE is transported to the nucleus, either alone or within a lentiviral capsid, it interacts with the DNA to localize HIV IN, which then incorporates the polynucleotide.

The application discloses a kind of methyl aspartate lyase EcMAL mutant and its preparation method and application, belong to enzyme engineering technical field.The methyl aspartate lyase EcMAL mutant is any one of mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4.The application is obtained methyl aspartate lyase EcMAL mutant by the mutation of the 329th, 331st, 361st and 365th amino acid sites of methyl aspartate lyase combined with high-throughput screening method.The mutant changes the previous recombinant enzyme only L-aspartic acid production status, so that it is possible to produce D-aspartic acid with methyl aspartate lyase as biological catalyst, and it is expected to be used in pharmaceutical industry.

The invention belongs to the field of detection, and discloses a method for detecting staphylococcus aureus in bulk ready-to-eat food through semi-quantitative developing, which comprises the following steps: designing a specific primer aiming at a tuf gene of staphylococcus aureus; dNA is extracted from a bulk instant food sample to be detected to serve as a template; s2, template DNA is added into an isothermal amplification reaction system for a recombinase-mediated isothermal nucleic acid amplification reaction, wherein the isothermal amplification reaction system comprises the specific primer in the step S1 and SYBR Green I fluorescent dye; after the amplification reaction is completed, a color change judgment result of a reaction system is observed: yellow green fluorescence is judged to be positive under an ultraviolet lamp, orange is judged to be negative under the ultraviolet lamp, and DNA amplification only needs 15 minutes; amplification and detection are completed in one tube and one step at the same time, and the detection result can be accurately, simply, conveniently and rapidly judged according to the presence or absence of an S-shaped curve and the change of the color of a reaction system.

This invention discloses a reaction tube for recombinase polymerase amplification (RPA), belonging to the field of nucleic acid detection. The reaction tube of this invention includes a tube body and a cap separate from the tube body. The tube body contains lyophilized bulbs or lyophilized powder containing premixed nucleic acid detection reagents, and the cap contains lyophilized powder containing an activator. The tube body and cap are sealed with a thin film. The lyophilized bulbs or lyophilized powder containing the premixed nucleic acid detection reagents include: recombinase, single-stranded DNA-binding protein, strand displacement DNA polymerase, buffer components, ATP, ATP regeneration components, and dNTPs. The lyophilized powder containing the activator includes: potassium ions, magnesium ions, and polyethylene glycol. This invention enables magnesium acetate to stably adhere to the cap, solving the problem of easy spillage of the lyophilized activator in the cap. In use, only the nucleic acid of the sample to be tested needs to be added; the operation is simple, easy to promote and apply, and suitable for batch detection.

A process for producing three recombinant enzymes capable of hydrolyzing each class of organophosphate, carbamate, and pyrethroid insecticides, as well as a formulation containing these enzymes provides the recombinant plasmid DNA vector, in which the protein secretory system has been modified to enhance the secretion of recombinant protein into the periplasmic space or culture medium. A nucleotide sequence encoding PelB (pectate lyase B) signal sequence for periplasmic localization is deleted and replaced with a nucleotide sequence encoding LamB (maltoporin or phage lambda receptor) signal sequence to enable a more efficient secretory recombinant protein. Three recombinant plasmid DNA vectors for the expression of recombinant proteins that are constructed using the modified plasmid DNA vector described above. The recombinant proteins include a recombinant organophosphate-hydrolyzing enzyme; a recombinant carbamate-hydrolyzing enzyme; and a recombinant pyrethroid-hydrolyzing enzyme.

Provided is an RNA editing system activating a Wnt signaling pathway, relating to the field of gene editing. Provided is an RNA editing element, comprising an Axin1-targeting RNA fragment and an Axin2-targeting RNA fragment. A gRNA that targets both Axin1 and Axin2 is constructed, which, compared with gRNAs targeting Axin1 or Axin2 alone, can significantly up-regulate Wnt activity. An AAV6 vector is screened from three AAV vectors of different serotypes, and exhibits relatively high expression intensity and efficiency and high infection efficiency with respect to airway epithelial cells and alveolar epithelial cells while exhibiting very low infection efficiency with respect to immune cells and endothelial cells. The AAV vector is assembled with a Cre-LoxP recombinase system to together infect a host cell, thereby facilitating the measurement of Cas13d knockdown efficiency by means of fluorescent labeling.

This invention discloses a recombinant 2-O-sulfonyltransferase mutant and its preparation method, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (E28~N356) mutant of hamster-derived 2-O-sulfonyltransferase (L119V-R188E-A240I) with an N-terminal maltose-binding protein (MBP) tag. The recombinant 2-O-sulfonyltransferase mutant exhibits higher activity, 1.9 times that of the recombinant wild type, and greater stability, retaining 86.6% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant 2-O-sulfonyltransferase is increased by 5 times. This mutant, as a novel biocatalyst with high efficiency, stability, and controllable production costs, has significant application value and broad industrialization prospects in the industrial enzymatic synthesis of glycosaminoglycans such as heparin and heparan sulfate.

The invention belongs to the technical field of virus detection, and particularly relates to a nucleic acid detection primer, a probe, a detection kit, a detection method and application of an immature skin-adding virus. The primer, the probe and the reaction system designed by the invention are high in sensitivity, the lowest detection limit can reach 3 copies / mL, the accuracy is 100%, the specificity is high, and cross amplification with other 76 pathogens is avoided. The kit adopts a recombinase polymerase isothermal amplification technology, does not need a large-scale PCR instrument, can complete detection only in 25 minutes, saves about 2 hours of time compared with a traditional fluorescent PCR detection method, adopts an M13 bacteriophage probe as internal positive quality control, can prevent false negative conditions, and can prevent pollution by adding UNG enzyme. The kit can be used for laboratory detection, field detection and bedside detection, and is especially suitable for detection of the yearly skin-adding virus at quarantine ports, airports and epidemic disease outbreak sites.

The new alpharetrovirus-based particles are suitable for high efficiency of transiently transducing animal cells. e.g. human or murine cells, and which efficiently introduce coding and non-coding RNA contained in the alpharetrovirus-based particles into target cells The alpharetrovirus-based particles also provide for high efficiency of the activity and / or integrity of the RNA that is introduced into the animal cells, as the particles protect the incoming RNA from degradation during entry. The transferred RNA can be of non-coding nature (e.g. single guide (sg) RNA. short-hairpin (sh) RNA. micro RNA. or long non-coding (Inc) RNA. or the RNA may encode proteins or peptides. e.g. receptors. transcription factors. cellular enzymes, antigens for use in vaccination, gene / protein therapy and / or gene editing nucleases, recombinases and transposases.

The invention discloses a recombinant ribitol dehydrogenase strain and application thereof, and belongs to the technical field of bioengineering. According to the recombinant ribitol dehydrogenase strain disclosed by the invention, pichia pastoris GS115 is taken as a starting strain, a recombinant ribitol dehydrogenase carrier is transformed, and the ribitol dehydrogenase gene sequence is shown as SEQ ID. NO 1. After fermentation in a 50L fermentation tank, the fermentation liquor is centrifuged, the supernate contains recombinase RDH with the purity of 95% or above, and the enzyme activity can reach 1059U / mL. The recombinase catalyzes a redox reaction between D-psicose and D-psilol to produce D-psilol, and the conversion rate can reach 56% after the reaction is balanced. The enzyme has good heat resistance, does not need metal ions to participate in a catalytic reaction, is low in product separation and purification difficulty, can keep the maximum enzyme activity of 80% or above in a wide pH (6-9) range, and can be applied to large-scale industrial production of rare sugar D-psilol.