71 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 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 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 discloses a construction, high-throughput sequencing and transcription intensity analysis method of a promoter random library. The invention establishes a process and an implementation method for rapidly researching the relative transcription intensity of a large number of promoters in a random promoter library, the promoter random library is rapidly constructed through a random primer method, and the transcription intensity of a large number of promoters is analyzed through deep sequencing of random tags carried by plasmid DNA and transcribed mRNA. According to the promoter library construction-library construction-sequencing-analysis process disclosed by the invention, the transcription intensity of promoters with different sequences can be quickly obtained with high throughput, and the promoter library with ideal promoter transcription intensity span is obtained. The method is rapid and efficient, and the transcription intensity of massive promoter sequences can be obtained.

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.

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 present invention relates to the field of biomedicine. Particularly provided are a high-bioactivity plasmid DNA, preparation and storage thereof, a vaccine, a kit, and a use thereof. The preparation method for the high-bioactivity plasmid DNA comprises: lysing bacterial cells after expansion culture and collecting a supernatant to obtain a crude solution containing a plasmid; purifying the crude solution by means of two-step liquid chromatography to obtain a purified plasmid DNA; precipitating the purified plasmid DNA by means of an alcohol and / or a polyol to concentrate and activate the plasmid DNA; and storing the plasmid DNA as a precipitate. The provided preparation method meets medical-grade standards, shortens the plasmid DNA purification time and reduces the material cost, improves the plasmid DNA yield and activity, and facilitates long-term storage. In addition, the plasmid DNA has higher in-vivo transfection efficiency and expression stability, thereby facilitating efficient introduction of a target gene into an organism. Thus, the plasmid DNA obtained by means of the method is suitable for use as an active component of a gene therapy drug or a DNA vaccine.

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.

Sample preparation compositions and methods for purifying plasmid DNA from biological samples is provided, are provided. The compositions and methods provided herein allow pDNA analysis to be carried out without centrifugation. The preparation process is amenable to high throughput processing using manual or robotic platforms.

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 present invention addresses the problem of providing a new replication initiation region capable of increasing the yield of plasmid DNA.　Provided is a double-strand circular DNA vector having a replication initiation region in which at least the base corresponding to position 22 in the base sequence represented by SEQ ID NO: 1 is an adenine base.

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 present invention relates to the field of biomedicine. Particularly provided are a high-bioactivity plasmid DNA, preparation and storage thereof, a vaccine, a kit, and a use thereof. The preparation method for the high-bioactivity plasmid DNA comprises: lysing bacterial cells after expansion culture and collecting a supernatant to obtain a crude solution containing a plasmid; purifying the crude solution by means of two-step liquid chromatography to obtain a purified plasmid DNA; precipitating the purified plasmid DNA by means of an alcohol and / or a polyol to concentrate and activate the plasmid DNA; and storing the plasmid DNA as a precipitate. The provided preparation method meets medical-grade standards, shortens the plasmid DNA purification time and reduces the material cost, improves the plasmid DNA yield and activity, and facilitates long-term storage. In addition, the plasmid DNA has higher in-vivo transfection efficiency and expression stability, thereby facilitating efficient introduction of a target gene into an organism. Thus, the plasmid DNA obtained by means of the method is suitable for use as an active component of a gene therapy drug or a DNA vaccine.

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 present invention provides tumor-targeting nanoplexes. Generally, a tumor-targeting nanoplex is a chemotherapeutic agent DNA conjugate with a first tumor-targeting agent and an optional second tumor-targeting agent linked thereto, such as a doxorubicin plasmid DNA conjugate and a linear histidine-lysine peptide with an optional cRGD-PEG-H3K4b second targeting agent. Also provided are methods for treating a cancer in a subject and for decreasing growth of a tumor in a cancer in a subject in need thereof in which the tumor targeting nanoplex is administered.

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.

Presented arc two new methods, single primer extension reaction (SSPER) and reduce recycle PCR (rrPCR). These methods have a step that easily removes the oligonucleotide primer(s) after the first reaction, thus, allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). A high efficiency of the methods is demonstrated by generating all ten site-directed mutations and six paired combinations of these mutations on four plasmid DNA templates ranging from 10 to 12 kb and 57 to 59% GC-content at a rate of 50-100%. The methods are i) highly accurate allowing for screening of plasmids by DNA sequencing, ii) streamlined to generate the mutations within a single day, iii) cost-effective in requiring only two primers and two enzymes (Dpnl and a proofreading DNA polymerase), iv) straightforward in primer design, and v) applicable for both large and small plasmids.

The subject technology relates generally to compositions and methods for producing plasmid DNA of a desired quality. In addition, it relates to the discovery of Escherichia coli (E. coli) bacteria with a constitutive methylase gene stably incorporated into the chromosomal DNA and uses thereof.

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 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.