33 results about "Recombinase" patented technology

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.

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.

This invention discloses a recombinant glucuronide C5 epimerase mutant, expression vector, recombinant expression engineered bacteria, and their culture and induction methods, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (N29–N617) mutation (V173R–V197K–A239V–N269E) with an N-terminal maltose-binding protein (MBP) tag to a human glucuronide C5 epimerase (C5-epi). The recombinant glucuronide C5 epimerase mutant exhibits higher activity, 2.1 times that of the recombinant wild-type, and greater stability, retaining 72.5% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant glucuronide C5 epimerase is increased by 4-fold.

This invention discloses a first adeno-associated virus (AAV) vector, a composition comprising a first AAV vector and a second AAV vector, and the application thereof. The first AAV vector contains a nucleotide sequence encoding a fusion protein encoding a Magnet photosensitizing protein and a Dre recombinase fragment, wherein the Magnet photosensitizing protein comprises nMag and pMag, and the Dre recombinase fragment comprises Dre… 1‑246 Fragments and Dre 247‑342 The Magnet photosensitive protein and the Dre recombinase fragment are linked by linkers L4 and T2A. The structure of the fusion protein is shown below: Dre 1‑246 Fragment - L4 - nMag - T2A - pMag - L4 - Dre 247‑342 The first fragment; the second AAV vector carries the Rox sequence and the target protein gene sequence. The AAV vector expression system of this invention is stable and has high expression efficiency. By controlling light conditions, it can achieve precise control of target protein expression within specific time and space, and is widely used in gene editing technology development, gene function research, and the development of gene therapy drugs.

This invention discloses a polyethylene terephthalate (PET) hydrolase, PmPETase, and its applications. The hydrolase PmPETase is a protein with the amino acid sequence shown in SEQ ID NO:1. This invention constructs an expression system suitable for PmPETase, obtains a recombinant enzyme protein with catalytic activity, and comprehensively analyzes its substrate specificity, catalytic properties, and potential plastic degradation ability. This provides a new theoretical basis and application reference for the development and utilization of PET degrading enzymes and green treatment technology for polymer materials.

This invention discloses a phospholipase D and its application in the preparation of ω-3 PUFAs-PA, belonging to the field of functional enzyme technology. The amino acid sequence of the phospholipase D is shown in SEQ ID NO. 9. The application of phospholipase D in the preparation of ω-3 PUFAs-PA is also disclosed. This invention also discloses a recombinant engineered bacterium expressing OUC-nPLD. The phospholipase D of this invention—OUC-nPLD—is a recombinant enzyme obtained by functional domain recombination based on the Non-HKD phospholipase D of *Streptomyces aquamarine*, and its PLD hydrolytic activity is 221.73 U / mg. This invention constructs a recombinant engineered bacterium expressing OUC-nPLD. The OUC-nPLD of this invention can be used for the efficient preparation of ω-3 PUFAs-PA, which is of great significance for the large-scale production of ω-3 PUFAs-PA.

The present application relates to a kind of detection methods of strong putrefaction Pseudomonas lundensis, comprising extracting nucleic acid in the sample to be measured;Then with nucleic acid as template, using specific RAA primer pair is recombined enzyme-mediated amplification, and obtains amplification product;RAA primer pair targets the aprX gene of Pseudomonas lundensis comprising single nucleotide polymorphism site associated with strong putrefaction phenotype;Finally, the amplification product is added to CRISPR / Cas detection system, incubated at constant temperature, then real-time detects its fluorescence signal;Detection system includes Cas protein, specific crRNA and fluorescent reporter probe, and crRNA can recognize single nucleotide polymorphism site associated with strong putrefaction phenotype in Pseudomonas lundensis aprX gene.The present application improves the detection accuracy, and detection process is simple to operate, and reaction system is simple, not only improves detection efficiency, also reduces detection cost.

This disclosure relates to lipid-based formulations such as lipid nanoparticle (LNP) formulations for delivery of an mRNA encoding a large serine recombinase (LSR), a DNA comprising a donor attachment site (AttD) and a sequence of interest, a targeting moiety directed to the immune cell, and wherein the formulations comprise an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor, wherein the formulation is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune cell, and optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA encoding the LSR, or (iii) the formulation comprises an additional mRNA encoding the activation factor.

The present application relates to the technical field of biological detection, and a double-path apple codling moth identification system integrating RPA-LFS rapid screening and TaqMan qPCR real-time quantification, a locked nucleic acid probe primer set, a kit and a method thereof. COI Through gene screening and primer screening, the present application designs recombinase-mediated isothermal amplification (RPA) and TaqMan qPCR primers and probes for the barcode of the cytochrome oxidase subunit I (COI) gene of apple codling moth, and establishes an RPA-LFS method in combination with immunolateral flow. The RPA-LFS method can complete amplification and visual judgment within 20 minutes at 39 DEG C, and is suitable for on-site rapid detection. The TaqMan qPCR method can realize high-sensitivity quantitative analysis under the condition of crude extract. Through double-path complementary design, the consistency of the results of on-site screening and laboratory confirmation detection is realized, and the method is simple to operate and suitable for pest quarantine at ports, epidemic monitoring in agricultural production areas and quarantine at storage and transportation links.

This invention belongs to the fields of microbiology and molecular biology, specifically relating to a homologous recombinase combination in myxobacteria and its application in gene editing. Specifically, this invention provides four pairs of homologous recombinase systems capable of efficiently mediating in vivo recombination in myxobacteria. Each pair includes a protein YqaJ with exonuclease function and a protein RecT with single-strand annealing function. This invention provides a method for myxobacterial gene editing: under the action of homologous recombinase pairs, mutant strains are obtained through positive screening using resistance tags. The resistance tags are then successfully removed through the combined action of the Cre / loxP system and the reverse screening gene galK. This method achieves a gene editing efficiency of up to 100%, has a short cycle time, requires no purification, and can be used for iterative deletion of multiple genes to achieve genome simplification in myxobacteria, or for inserting functional promoters and other elements to activate silent gene clusters. This method is of great significance for the bioexploration and functional genomics research of myxobacteria.

Embodiments of the present invention provides recombinant systems, methods, and compositions for the biological production of Testosteronan (Testan), a linear α-(1→4)-linked glucuronic acid–N-acetylglucosamine polysaccharide. Microbial hosts engineered to express full-length CtTS, N-terminal truncation variants (including d64-CtTS), or the Pseudomonas CIPTS homolog synthesize high–molecular-weight Testan (≥800 kDa) with low polydispersity. Structural analysis by ¹H NMR and 2D HSQC demonstrates that all recombinant enzymes produce Testan that is chemically indistinguishable from native polymer. Purification using ultrafiltration and strong-anion exchange chromatography yields highly defined molecular-weight fractions, including monodisperse populations (PDI ~1.02). The invention further provides sulfated derivatives of Testan produced via aqueous sulfation methods, as well as Testan and functionalized Testan compositions useful for chromatographic stationary phases, biomaterial scaffolds, and chemically modifiable polymer platforms. These systems provide the first scalable, fermentation-based production methods for Testan and Testan-derived materials.

The present application relates to the technical field of biological detection, in particular to a one-tube pathogen detection method based on PAM-Disruptor regulated CRISPR-Cas12a system, the detection method comprising: constructing a PAM regulated CRISPR-Cas system; mixing and incubating PAM-Disruptor with LbCas12a effector protein, guide RNA, fluorescent reporter, F-primer, R-primer, recombinase, single-stranded DNA binding protein (SSB), DNA polymerase and reaction buffer solution to prepare a premixed reaction solution, and then performing reaction and judgment. The present application realizes light control and non-light control dual-mode one-tube detection, effectively avoids the problems of complicated operation, cross contamination, aerosol pollution and the like caused by step-by-step reaction, and provides a new strategy for one-tube detection of pathogens.

This invention discloses a PLA-degrading lipase mutant and its application. First, the lipase PaPlaA gene with the amino acid sequence shown in SEQ ID NO.1 was selected. Highly efficient heterologous expression was achieved by constructing a PgapA-T7 dual promoter system and optimizing the RBS sequence through ribosome binding site design, resulting in a recombinant enzyme activity yield of 6.25 U / mL, approximately 2.5 times higher than the original strain. Then, combining molecular dynamics simulations and rational design, the substrate pocket and flexible region were structurally modified to obtain the PLA-degrading lipase mutant. The obtained mutant exhibited excellent catalytic performance when applied to PLA degradation. This invention also provides a composite enzyme preparation obtained by combining the PLA-degrading lipase mutant with the protease nprT612. This composite enzyme preparation can achieve a degradation rate of up to 78.2% for PLA nonwoven fabrics. This provides a stable enzyme catalyst and a feasible synergistic degradation process for the efficient biodegradation of PLA at high temperatures.

This invention discloses a VIGS vector system based on the RNA virus SsOLV4 and its applications. The application of the RNA virus SsOLV4 in constructing the VIGS vector system is described. The VIGS vector for silencing target genes in *Sclerotinia sclerotiorum* is obtained by inserting a 99 bp target gene fragment in reverse between the CDS stop codon and the 3'UTR of SsOLV4 in the vector pMD18-P1 under the action of homologous recombinase. The vector pMD18-P1 contains the full-length cDNA sequence of the virus SsOLV4. Based on SsOLV4, this invention establishes a VIGS vector, overcoming the complexity, time-consuming nature of *Sclerotinia sclerotiorum* genetic transformation technology and the difficulties caused by the purification of multinucleated *Sclerotinia sclerotiorum* transformants. It can rapidly silence exogenous and endogenous genes in *Sclerotinia sclerotiorum*, making it suitable for rapid and efficient gene silencing and functional studies in *Sclerotinia sclerotiorum*.

The disclosure relates to gene editing systems comprising serine recombinases and methods of using such serine recombinases for integration of nucleic acid sequences. More specifically, the disclosure relates to sequence-defined serine recombinases having attachment sites such as a bacterial genomic recombination sequence (attB). Methods are also provided for recombinant pro-duction of said recombinases.

This invention discloses the application of a glutamine-depleting engineered bacterium and its pharmaceutical composition. The engineered bacteria use attenuated bacteria with tumor-targeting capabilities as vector strains. The genome or plasmid of the vector strain integrates a therapeutic gene encoding glutaminase. This method achieves deep and continuous depletion of glutamine in the tumor, avoiding drug resistance caused by tumor metabolic compensation. The inhibition rate against c-Myc-overexpressing soft tissue sarcomas is significantly higher than that of small molecule inhibitors. Enzyme production is only carried out locally in the tumor, with no effect on normal tissues and no systemic toxicity. The engineered bacteria can penetrate the dense tumor matrix and colonize the tumor, solving the problem of poor tissue penetration of small molecule inhibitors. The engineered bacteria continuously multiply and produce enzymes in the tumor, achieving continuous release of glutaminase. The half-life is significantly longer than that of recombinant enzymes, eliminating the need for frequent dosing. The glutaminase optimized by protein engineering has significantly reduced immunogenicity.

The application discloses a VIGS carrier for preventing and treating Sclerotinia sclerotiorum and application thereof. The VIGS carrier is obtained by reversely inserting a fragment of a gene which plays an important function in the growth, pathogenicity or sclerotia formation of Sclerotinia sclerotiorum into a CDS stop codon and 3'UTR of a VIGS carrier containing an RNA virus SsOLV4 under the action of a homologous recombinase, the VIGS carrier containing the RNA virus SsOLV4 has Amp resistance, and the full-length cDNA sequence of the RNA virus SsOLV4 has a similarity of greater than or equal to 85% to the sequence shown in SEQ ID NO:1. The application realizes the application of an asymptomatic fungal virus in biological prevention and treatment of plant diseases for the first time, and provides an innovative technical path for green prevention and control of crop sclerotinia.

Disclosed herein are fusion polypeptides and nucleic acid encoding the same, wherein the fusion polypeptide comprises a large serine recombinase (LSR) portion and a DNA binding domain (DBD) portion.

This invention relates to a method for antibody reprogramming and screening based on engineered cells and its applications. This method achieves efficient in vitro rearrangement and diversity generation of antibody genes in non-lymphocytes by constructing engineered human cells containing the RAG1 / 2 recombinase system and programmable recombination signal sequence (RSS) elements. Utilizing this invention, the V(D)J rearrangement mechanism of B cells can be simulated in engineered cells, allowing for the targeted introduction of insertions, deletions, and point mutations in the antibody complementarity-determining region (CDR), resulting in a structurally diverse antibody library. Combined with piggyBac transposase-mediated genome integration, mammalian cell surface display technology, and flow cytometry sorting, high-throughput antibody screening and functional identification are achieved. This invention is simple to operate, has high antibody gene rearrangement efficiency, and produces a rich diversity of antibody libraries, making it suitable for rapid optimization of antibody drugs and discovery of new antibodies.

The present invention relates to an RPA primer set for identifying Hanwoo, a composition including the primer set, a kit including the composition, and a method for identifying Hanwoo using the primer set. According to one aspect of the present invention, the primer set, the composition including the primer set, the kit including the composition, and the method for identifying Hanwoo using the primer set can rapidly amplify specific nucleotide sequences using a single-strand DNA binding (SSB) and a recombinase as an RPA method. Since the reaction is possible under isothermal conditions, it is possible to specifically distinguish between Hanwoo and imported cattle (non-Hanwoo) simply by the presence or absence of a gene amplification product without the need for temperature-circulating equipment such as a PCR machine. Furthermore, it has excellent effects that allow for rapid and accurate on-site identification with only a small amount of sample, for example, about 25 mg, which can be utilized for origin labeling management.

This invention discloses a method for constructing a TrpA1-3×FLAG-P2A-iCre gene knock-in mouse. A fragment containing a 3×FLAG tag, a P2A sequence, and an iCre recombinase sequence is inserted into the 27th exon region of the mouse TrpA1 gene using a CRISPR / Cas9 system, achieving genetic labeling and Cre functionalization in TrpA1-expressing cells. The donor vector provided by this invention contains a 5' homologous arm and a 3' homologous arm that are homologous to the TrpA1 genome sequence. Stably inherited knock-in mice are obtained by microinjecting the CRISPR / Cas9 system and the donor vector into fertilized eggs.

This invention discloses a method for constructing a zebrafish model of fatty liver based on glucokinase overexpression and its application. The method includes the following steps: using cDNA as a template, a zebrafish GCK gene fragment is obtained by PCR amplification; the vector fragment and the GCK gene fragment are homologously recombined using a homologous recombinase to obtain a recombinant plasmid; the recombinant plasmid is transformed into competent cells, and an endotoxin-free plasmid is extracted as a zebrafish GCK expression vector; a microinjection system is used to inject the plasmid into one-cell-stage fertilized eggs; after the fertilized eggs hatch into juveniles, fluorescent juveniles are selected for further culture until sexual maturity; sexually mature males and females are paired to spawn, and a stably heritable GCK-overexpressing zebrafish strain is selected from the offspring. This invention can obtain a zebrafish strain with stable high GCK expression, which can be used as a zebrafish model of fatty liver based on GCK overexpression.

This invention pertains to genetic engineering and relates to an orally administered engineered microbial system and its applications. Through genetic manipulation techniques such as genome integration, a dominant mutant of the nicotine-degrading enzyme Nox is displayed on the surface of Bacillus subtilis spores. This mutant is then coupled with catalase or peroxidase, and genes related to spore germination and the system's own recombinant enzymes are knocked out to construct a strain with self-eliminating resistance genes. This invention utilizes orally administered engineered bacteria to colonize the intestines. The "molecular degradation machine and metabolic siphon" effect produced by the engineered bacteria continuously degrades nicotine diffused from the bloodstream, thereby significantly reducing the concentration of nicotine binding to central nicotine receptors. This fundamentally weakens the dopamine reward effect induced by smoking, effectively breaking the positive feedback loop of "smoking-pleasure," and providing a new method for treating nicotine dependence. As a "plug-and-play" platform technology, displaying different degradation enzymes on the surface of Bacillus subtilis spores can also degrade other harmful substances.

This invention relates to the field of biological detection technology, specifically to a method for detecting tomato leafminer based on recombinase-mediated isothermal nucleic acid amplification (RAA) technology. Tuta absoluta = Phthorimaea absoluta Animal mitochondria COI This invention discloses a primer set, kit, and method for detecting tomato leafminer. The forward primer sequence for detecting tomato leafminer designed in this invention is shown in SEQ ID No. 1, the reverse primer sequence is shown in SEQ ID No. 2, and the probe primer for detecting tomato leafminer is shown in SEQ ID No. 3. The primer set of this invention can effectively amplify a portion of the tomato leafminer. COⅠ and COⅡ The gene and primer set have high specificity and sensitivity, and show no cross-reactivity with other leafminer insects that coexist with the tomato leafminer. When used in conjunction with lateral flow immunochromatography, it can be used for rapid on-site identification of the tomato leafminer at various stages, which is of great significance for the effective prevention and control of the tomato leafminer.

The application belongs to the technical field of biological detection, and particularly relates to a RPA primer for detecting Escherichia coli, a kit and application thereof. The target sequence is a new target DNA sequence which is broad-spectrum and specific in Escherichia coli. The RPA primer designed based on the target sequence can be used in recombinase polymerase isothermal amplification reaction of Escherichia coli. On this basis, the application provides a RPA detection method for detecting Escherichia coli by using the RPA primer. Compared with the existing PCR detection technology, the isothermal amplification detection method of the application can rapidly detect Escherichia coli at room temperature or under slight heating, and the detection can be completed within 30 min, thereby significantly improving the detection efficiency. Moreover, the detection method has high specificity, and the detection sensitivity can detect Escherichia coli as low as 10 1 copies / µL, and the specificity and sensitivity are much higher than those of the traditional PCR detection technology.

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

This invention discloses a chitin deacetylase product and its application in agricultural disease prevention. Through codon optimization, the *Rhodococcus* chitin deacetylase gene (SEQ ID NO:1) was cloned into the *Pichia pastoris* expression vector pGAPZa A, constructing the recombinant vector pGAPZaA-CDA-His, which was then transformed into the *Pichia pastoris* SMD1168 host, achieving efficient secretory expression of the enzyme. The resulting recombinant enzyme exhibits high activity and good thermostability. This invention includes a universal enzymatic deacetylation process characterized by deacetylation efficiency, applicable to chitin deacetylases from various sources. This invention also provides a method for further enzymatic hydrolysis to produce N-acetylglucosamine monomers or their oligosaccharides, and the application of the recombinant enzyme and its product in the preparation of biopesticides and the treatment of chitin-containing biomass materials.

This invention discloses nox variants and their application in simultaneous integration of multiple targets and fragments into the genome. The invention provides a set of DNA molecules composed of all or some of the following: SEQ ID No. 5 (denoted as nox4); SEQ ID No. 6 (denoted as nox5); SEQ ID No. 9 (denoted as nox8); SEQ ID No. 10 (denoted as nox9); SEQ ID No. 11 (denoted as nox10); SEQ ID No. 12 (denoted as nox11); SEQ ID No. 1 (denoted as nox). All seven nox sites can be recognized by Nigri recombinase and mediate recombination between two identical sites; however, each nox site is independent and does not undergo recombination. This invention enables rapid, continuous, multi-site genome integration using simple molecular biology manipulations, and also allows for CRISPR-mediated integration.