46 results about "Plasmid dna" patented technology

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 invention discloses a preparation method of a novel composite carbon source. The preparation method comprises the following steps: S1, preparing a solution; s2, preparing a culture medium; s3, extracting a genome; s4, extraction of plasmid DNA (deoxyribonucleic acid); s5, preparation of agarose gel electrophoresis; s6, constructing a linearized vector plasmid; s7, preparing an electrotransfection competent cell; and S8, electric conversion treatment. According to the preparation method of the novel composite carbon source, pfkA and pfkB can be respectively, independently and simultaneously knocked out when the carbon source is prepared by utilizing a gene editing technology, the consumption of a glucosamine precursor 6-fructose phosphate in an EMP pathway can be reduced, more glucose is used for synthesizing a target product, and by optimizing the preparation process of the composite carbon source, the yield of the target product is improved. The shake-flask fermentation yield of the glucosamine can be obviously improved, the conversion rate of the composite carbon source is also improved, and cell growth and synthesis of the target product glucosamine can be reasonably balanced, so that the yield of the glucosamine is improved.

The disclosure is directed to compositions that comprise a plasmid DNA construct having a DNA sequencing encoding a therapeutic protein, or a fragment thereof, in vivo, along with methods of generating and manufacturing the antibody or therapeutic protein, as well as methods for preventing and / or treating a disease in a patient.

The application discloses a device for extracting plasmid DNA in bacteria and relates to the technical field of biological medicine. The device comprises a first mixing assembly and a second mixing assembly; the first mixing assembly is connected with the second mixing assembly through a lysis spiral pipe; at least one liquid inlet is arranged on a connecting pipeline of the lysis spiral pipe and the second mixing assembly; the device has the advantages that the mixing assembly is connected in series, the lysis and neutralization processes are carried out in a closed environment, the probability of polluting the environment is reduced, the device is convenient to clean after use, continuous processing is realized, and the production efficiency is improved; and the device is simple, and the design and production cost are small.

The present application belongs to the technical field of bioengineering, and particularly relates to a Claudin18.2 gene engineering antibody based on DNA immunization and a preparation method and application thereof. The Claudin18.2 gene engineering antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO. 15-16; and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO. 19-20. The Claudin18.2 gene engineering antibody is obtained by immunizing Balb / c mice with full-length plasmid DNA and full-length plasmid DNA+extracellular segment polypeptide as immunogens respectively, obtaining plasma cells capable of secreting specific antibodies against Claudin18.2, and then preparing specific mouse-derived gene engineering antibodies through single-cell optical guidance technology and genetic engineering technology. The gene engineering antibodies have high titer and strong specificity, and have specific recognition ability for natural protein Claudin18.2.

A nonviral gene therapy delivers DNA that allows cells to produce proteins such as their own growth factors in culture media. Lipid nanoparticles provide an effective delivery mechanism into a target cell. By encapsulating plasmid DNA encoding the growth factors with inducible promoters into lipid nanoparticles (LNPs), target cells create their own growth factors reducing a need for expensive growth factors from external sources. DNA expression lasts for several days reducing the amount of reagent needed. The plasmid DNA is more stable than mRNA and proteins, therefore reducing logistical challenges.

The invention relates to genetic and protein engineering and can be used in medicine and the pharmaceutical industry. Recombinant plasmid DNA pET23b-CBD-HS-ES-GLP1, pET-parB-CBD-HS-ES-GLP1 and pET23bKanR-CBD-HS-ES-GLP1 were constructed to provide the synthesis of a hybrid protein containing the GLP-1 polypeptide sequence in Escherichia coli cells. By transforming the E. coli BL21 (DE3) strain with plasmids pET23b-CBD-HS-ES-GLP1, pET-parB-CBD-HS-ES-GLP1 and pET23bKanR-CBD-HS-ES-GLP1, the CBD-HS-ES-GLP1 hybrid protein has been produced. A method of producing polypeptide GLP-1 has been developed, which involves preparing and cultivating a producer of a hybrid protein containing polypeptide GLP-1, followed by isolation and cleavage of the said hybrid protein. The invention enables the production of polypeptide GLP-1 with high yield and simplified technology.

The invention relates to the technical field of gene detection, in particular to a gene detection object and application of the gene detection object in preparation of products for detecting plasmid DNA residues in induced pluripotent stem cells, the induced pluripotent stem cells are obtained by transduction of somatic cells through four plasmids pCXWB-EBNA1, pCXLE-hOCT4-shp53, pCXLE-hSK2 and pCXLE-hUL, and the gene detection object can be used for detecting the plasmid DNA residues in the induced pluripotent stem cells. A target sequence of the gene detection object comprises a sequence as shown in SEQ ID NO. 7. The gene detection substance provided by the invention can efficiently detect the plasmid residue in the induced pluripotent stem cells, and the corresponding detection method has high specificity and high sensitivity, and can effectively meet the detection requirements of the plasmid residue in the safety quality control link of the induced pluripotent stem cells in the development and production process of cell treatment products.

Lipid nanoparticle formulations with cell type specific transfection activity and capable of producing Th1 and / or Th2 response in vivo and their use for plasmid DNA or mRNA delivery is disclosed.

Disclosed herein are methods and kits for purifying therapeutic-grade plasmid DNA using magnetic particles. In particular, the technology of the present disclosure relates to methods and kits for purifying therapeutic-grade plasmid DNA at a high degree of purity using magnetic particles with a siliceous oxide surface layer and a superparamagnetic core.

The application provides a double-strand DNA vaccine for late Edwardsiella tarda disease, which can significantly induce Th1 type immune bias, enhance fish cell immune response, improve the resistance to intracellular bacterial infection, and has the advantages of safety and high efficiency, easy preparation, strong adaptability for on-site promotion and the like. The double-strand DNA vaccine uses a fusion fragment of an immune adjuvant IL-12, which is constructed by connecting a nucleic acid fragment coding a subunit 12p35 with an amino acid sequence of SEQ ID NO:1 and a nucleic acid fragment coding a subunit 12p40 with an amino acid sequence of SEQ ID NO:3 through a flexible connecting peptide linker. The double-strand DNA vaccine realizes the synergistic expression of OmpC antigen and IL-12 adjuvant, enhances immunogenicity, and solves the problem of insufficient immune effect of a traditional DNA vaccine; and the vaccine has no obvious toxic side effect on fish bodies, is not dependent on the use of antibiotics, is green and environmentally friendly, and has a good industrialization prospect.

The invention discloses an improved plasmid site-directed mutagenesis method based on PCR (Polymerase Chain Reaction). The method comprises the following steps: introducing at least two mutations on a plasmid DNA (Deoxyribose Nucleic Acid) fragment through site-directed mutagenesis; wherein one mutation is expected mutation, and the other mutation changes the function of the mutant plasmid, so that the function of the mutant plasmid is different from that of a template plasmid. The change in function makes it possible to reduce the detention of unintegrated template DNA even without digesting the wild-type template with DpnI.

The present application relates to the field of molecular diagnosis and nucleic acid amplification, and particularly relates to a recombinase polymerase amplification method based on Twinkle helicase enhancement and application thereof. The present application first introduces Twinkle helicase into the RPA system, utilizes the ATP-dependent strand separation activity and DNA annealing activity of the Twinkle helicase, assists primer binding and template strand separation, and thus significantly improves amplification efficiency and detection sensitivity. The present application is suitable for plasmid DNA, high GC template and clinical nucleic acid sample, and can construct an RPA kit containing Twinkle, and expand the application of the RPA kit in pathogen detection, molecular diagnosis and on-site rapid detection. The present application overcomes the shortcomings of the existing RPA technology in sensitivity and stability, and provides a new solution for developing a new type of nucleic acid self-detection and on-site detection tool.

The invention provides a PEG (Polyethylene Glycol)-mediated powdery parasitic spore protoplast genetic transformation method and application thereof, and belongs to the technical field of biology. The method comprises the following steps: performing enzymolysis on tender hyphae of powdery parasitic spore by adopting a combination of driselase and lyase to efficiently prepare protoplast; transforming the protoplast and plasmid DNA (deoxyribonucleic acid) under the mediation of a PEG4000 (polyethylene glycol 4000) solution; a stable genetic transformant is screened and obtained through a culture medium containing G418. According to the invention, a genetic transformation system of the sphaerospora fuliginea is established for the first time, and the transformant can stably express an exogenous gene with high strength. The method disclosed by the invention is simple and convenient to operate, high in transformation efficiency and good in repeatability, provides a key technical platform for researching gene functions and parasitic mechanisms of the sphaerospora fuliginea and constructing efficient engineering biocontrol strains, and has important application value in the technical field of biological prevention and control of powdery mildew.

The invention belongs to the technical field of gene editing, and particularly discloses a Crimson fluorescent protein reporting system and application thereof in gene editing efficiency multi-scene evaluation. According to the Crimson fluorescent protein reporting system, an equidirectional repetitive sequence which can be targeted by sgAlb and contains a termination codon is introduced into a Crimson gene sequence, a frame-shift mutant fluorescent reporting module is successfully constructed, and the module can recover fluorescent expression through an SSA (homologous sequence annealing repair) repair mechanism after being cut in a Cas9 / sgAlb system. The developed Crimson report cell system can be used for accurately evaluating the stability and editing efficiency of different sgRNA skeletons; the method is suitable for optimizing a virus transduction system and screening a guiding aid for efficient gene delivery; plasmid pollution in a virus vector system can be rapidly detected, and accurate distinguishing of a functional RNP compound and residual plasmid DNA is realized by comparing the proportion of Crimson positive cells before and after DNase I treatment and heat treatment; and small molecular compounds for promoting homologous directional repair can be efficiently screened.

The present invention relates to circular RNA platforms, their manufacturing processes from an engineered parental circular covalently closed synthetic plasmid DNA and, uses thereof. Novel engineered circular covalently closed plasmids comprising sequences capable of aiding in RNA circularization either autonomously or when processed with an engineered ligase are provided. The circular RNA platforms of the current invention have improved stability and increased half-life, display exceptional and stable protein production, while avoiding double-stranded intramolecular self-pairing RNA segments.

The present disclosure provides a method for creating, by mutating a gene group, a plasmid that exhibits desired properties such as high production and by-product suppression. The present disclosure provides: a method for producing a combinatorial library; or a method for producing a combinatorial library (including Golden Gate), the method comprising 1) a step for providing source plasmid DNA (single type or multiple types), 2) a step for introducing mutations into the source plasmid DNA and generating a plasmid DNA population (seed plasmid population) (seed plasmids) including mutation-introduced plasmid DNA (seed plasmids) into which the mutations have been introduced, and 3) a step for subjecting the plasmid DNA population to a combinatorial library formation procedure.

The presently claimed and described technology provides chromatography processes for purifying plasmid DNA (pDNA) using elution buffers containing at least one basic amino acid and / or at least one denaturant.

A scalable method for producing DNA / polycation particles having an optimal, defined particle size with multiple virus assembly plasmids for efficient transfection of viral production cells in suspension cultures. The presently disclosed DNA / polycation particles yield superior and reproducible transfection activity and shelf stability in the suspension form and can be used as an off-the-shelf product. The presently disclosed DNA / polycation particle formulation can potentially simplify and streamline the viral manufacturing process and improve production quality and consistency.

The invention relates to the technical field of biology, in particular to a plasmid DNA purification method based on membrane chromatography. According to the method, a membrane chromatography technology is used, a packing experiment is not needed, the working flow speed is high, plasmid purification only needs 1-2 h once, the recovery rate of obtained DNA is 73% or above, the recovery rate of super-spiral DNA is 97% or above, and the proportion of the super-spiral DNA is 95% or above.

A composition for the production of plasmid nucleic acids, as well as a method for preparing and using the same, is provided. [Solution] Engineered bacterial strains and vectors for enhanced plasmid DNA production are provided herein.

Provided is a self-selection DNA editing system. A subject self-selection DNA editing system includes: (a) a gene editing tool, and (b) a donor DNA comprising a bacteriocin unit, which includes a nucleotide sequence encoding a bacteriocin protein and / or a nucleotide sequence encoding a bacteriocin immunity protein. In some cases, (a) and (b) are present on the same nucleic acid (e.g., plasmid DNA). When a bacteriocin unit of the subject disclosure is present on (as part of) a donor DNA, the bacteriocin unit is configured for integration into a target DNA such as a genomic DNA. The present disclosure also provides methods of modifying a microbial community and methods of editing a prokaryotic cell to facilitate selection. The targeted prokaryotic cell(s) can be in culture or can be in situ (e.g., part of a microbial community).

The present invention relates to a liquid dosage form pharmaceutical composition containing plasmid DNA. The liquid dosage form containing plasmid DNA according to the present invention exhibits excellent efficacy in terms of storage stability of the plasmid DNA, which is a pharma- ceutical active ingredient, and can be usefully used as a pharmaceutical composition with excellent product stability and safety.

The present invention relates to a process for purifying nucleic acids, particularly plasmid DNA, from a liquid sample, which contains endotoxins in addition to nucleic acids. The invention involves a process for purifying nucleic acids from a biological sample containing nucleic acids, preferably plasmid DNA, comprising the following steps: a) providing a liquid sample comprising nucleic acids and endotoxins; b) precipitating the nucleic acids and the endotoxins or binding the nucleic acids and the endotoxins on a solid carrier and thereby separating them from the remaining components of the liquid sample; c) optionally washing the precipitate or solid carrier from step b) at least once to further remove remaining components; d) treating the nucleic acids and endotoxins separated in step b), optionally after washing in step c), using a purifying buffer for at least partial removal of the endotoxin; e) optionally washing the precipitate or the solid carrier in step d) at least once with at least one washing composition, and f) optionally collecting the nucleic acids, optionally by eluting from the solid carrier or dissolving the precipitated nucleic acid; wherein the purifying buffer in step d) comprises an amino compound selected from the group consisting of triethylamine, triethanolamine, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol, 1,3-bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2-(hydroxymethyl)-propane-1,3-diol, and individual salts thereof, said amino compound having a total concentration in the range of 1.1 to 5.0 mol / L, an alcohol capable of dissolving the amino compound in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer, and water, wherein the purifying buffer has a pH in the range from 3.0 to 8.0.

The present invention relates to a method of isolating covalently closed circular (ccc) DNA molecules from microbial cells containing the ccc DNA molecules, comprising the steps of: contacting the microbial cells with a lysing agent and moving the composition through a tube system with a flow having a Reynolds number of at least 3000 to obtain a lysing composition, incubating the lysing composition to obtain a lysate, contacting the lysate with a neutralizing solution to obtain a neutralized lysate, and further processing the neutralized lysate to obtain the ccc DNA molecules.

The invention relates to a method for chromatographic purification of plasmid DNA. To this end, a phosphate is added to a wash buffer and / or an elution buffer in anion exchange chromatographic purification of plasmid DNA (pDNA).

The invention relates to the field of bioengineering, in particular to a chromatography-free plasmid DNA large-scale purification method which does not use a chromatography method for purification and comprises the following specific steps: (1) resuspending escherichia coli in a resuspending buffer solution; (2) thallus cracking: carrying out continuous cracking on the resuspended bacterial liquid; (3) clarification: carrying out clarification and filtration on the split bacterial liquid; (4) ultrafiltration concentration: the clarified filtrate is subjected to ultrafiltration concentration, and the ultrafiltration concentration comprises two stages, namely a concentration stage and a filter washing stage; and (5) sterilizing and filtering: sterilizing and filtering the sample after ultrafiltration. The plasmid DNA purification method provided by the invention is strong in operability and good in repeatability, and the purified plasmid DNA is high in total recovery rate, meets the medicinal standard and can meet the requirements of industrial large-scale production.

To provide plasmid DNA with a novel replication origin that can stably introduce foreign genes into actinomycetes. [Solution] The plasmid vector according to the present disclosure comprises (a) a nucleotide sequence having a specific sequence, or (b) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted in the nucleotide sequence having the specific sequence.