126 results about "Plasmid dna" patented technology

The invention relates to the technical field of plasmid extraction, in particular to an automatic extraction method of endotoxin-free large-volume plasmid DNA and a kit. According to the kit provided by the invention, a magnetic bead adsorption technology and a buffer system with a unique separation effect are adopted, and specially modified protein is combined with magnetic beads, so that endotoxin and protein impurities can be adsorbed; an endotoxin removing solution is adopted, so that endotoxin can be efficiently removed; the method comprises the following steps: firstly, preparing plasmid DNA, then, uniquely embedded nucleic acid adsorption magnetic beads have very strong affinity to the plasmid DNA under a certain condition and are not easy to adsorb endotoxin, and when the condition is changed, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA, so that the purpose of rapidly separating and purifying the plasmid DNA is achieved. The plasmid DNA extracted by utilizing the kit and the extraction method disclosed by the invention is high in yield, good in purity and extremely low in endotoxin content, and can be suitable for transfecting various cells and various conventional operations including enzyme digestion, PCR (Polymerase Chain Reaction), sequencing, connection and other various downstream experiment requirements.

A method for producing extracellular vesicles (EV) in large quantities by using a peptide derived from Noxa protein, which plays a key role in apoptosis, and derivatives thereof, are described. Also described are the use of the peptide for promoting extracellular vesicle production and methods for producing extracellular vesicles by using the same. The methods of the present invention can efficiently and uniformly be used to mass-produce extracellular vesicles and can be used to produce extracellular vesicles which load drugs that pass poorly through cellular membranes, as well as recombinant proteins or plasmid DNA.

The invention relates to a process for producing three recombinases capable of hydrolyzing organophosphorus, carbamate and pyrethroid insecticides, and preparations containing these enzymes. The present invention provides recombinant plasmid DNA vectors in which the protein secretion system has been modified to improve the ability of recombinant proteins to secrete into the space around the plasmid or culture medium. A nucleotide sequence for coding a PelB (butyric acid lyase B) signal sequence is deleted and substituted by a nucleotide sequence for coding a LamB (maltodextrin or phage lambda receptor) signal sequence, so that the secretion efficiency of the recombinant protein is higher. The invention discloses three recombinant plasmid DNA (deoxyribonucleic acid) vectors which are constructed by utilizing the improved plasmid DNA vectors and are used for expressing recombinant proteins. The recombinant protein comprises (a) a recombinant organophosphate hydrolase having at least 80% sequence identity with the amino acid sequence of methyl parathion hydrolase (MPH) (Protein Database (PDB) ID: 1P9E) as shown in SEQ ID NO: 1, wherein the recombinant organophosphate hydrolase has hydrolytic activity on organophosphate insecticides; (b) a recombinant carbamate hydrolase having at least 80% sequence identity with the amino acid sequence of L-aminopeptidase-D-Ala-esterase / amidase (aryl amidase) (MAH) (PDB ID: 1B65), as shown in SEQ ID NO: 2, wherein the recombinant carbamate hydrolase has hydrolytic activity on carbamate insecticides; and (c) a recombinant pyrethroid hydrolase having at least 80% sequence identity with the amino acid sequence of the pyrethroid hydrolytic carboxylesterase (EstA) (PDB ID: 4CCY) as shown in SEQ ID NO: 3, wherein the recombinant pyrethroid hydrolase has hydrolytic activity on a pyrethroid insecticide. The embodiment of the invention further provides three recombinant bacterial strains, an escherichia coli strain BL21 (DE3) is used as a host cell, and each recombinant plasmid DNA vector is used for transformation. The invention also discloses culture conditions of the recombinant strain, wherein the culture conditions comprise culture medium composition, induction conditions and enzyme harvesting and enzyme activity detection methods. In addition, the embodiment of the invention also provides a preparation which contains the three recombinases as functional components.

The invention discloses a method for detecting a beta-lactamase drug-resistant gene. The method comprises the following steps: extracting bacterial plasmids; the method comprises the following steps of: downloading sequences of TEM-1 and ampC from GenBank, comparing nucleotide sequences by utilizing Megaalign, finding out a gene conserved region, and designing a primer by utilizing Primer expansion V5; and adding reactants of a PCR reaction system by taking the extracted whole-genome DNA of the bacteria and the extracted plasmid DNA of the bacteria as templates, and carrying out PCR amplification according to the reaction temperature required by the corresponding amplified substances. According to the invention, plasmid-mediated beta-lactam drug resistance detection and analysis are carried out on bovine-derived escherichia coli, so that drug resistance detection rates of TEM-1 type and ampC type genes of a whole genome are 76% and 95% (n is equal to 38); the drug resistance detection rates of the plasmid DNA are 98% and 89% (n = 44), and the drug resistance detection rates of the plasmid DNA are both 100%. The invention provides a scientific basis for further prevention and control of bovine-derived bacterial infectious diseases and reasonable use of antibacterial drugs, and also provides valuable data support and theoretical reference for guiding livestock farm medication and veterinary clinical scientific medication.

The invention discloses a CAR donor plasmid, a T cell as well as a preparation method and application of the CAR donor plasmid and the T cell, and belongs to the field of genetic engineering and immune cell therapy. An RNP compound and donor plasmid DNA are transferred into a T cell through electroporation, and a CAR expression unit is integrated into a T cell genome at a fixed point to prepare the CAR T cell. The donor plasmid DNA comprises a plasmid skeleton, a homologous arm sequence, an EF1 alpha core promoter, a CAR expression element and an SV40 poly A termination signal. The number of base pairs of the plasmid is 4544 bp, the toxicity of the plasmid to T cells after electroporation can be effectively reduced, and the T cell viability and the CAR integration efficiency are improved. Compared with dsDNA and ssDNA, the plasmid DNA is convenient to prepare and low in cost; therefore, the plasmid is taken as the donor template DNA, and the CRISPR / Cas9 technology is combined, so that the use of a virus vector can be avoided to improve the safety of the CAR T cell, and the production cost of the preparation of the CAR T cell can be effectively reduced.

The invention discloses a construction and identification method of an influenza B DNA vaccine, which comprises the following steps: step 1, downloading sequence information of an influenza B recommended strain B / Austria / 1359417 / 2021 in vaccine strain recommended components in 2022-2023, carrying out codon optimization on an HA gene of the strain, and then carrying out gene synthesis, and naming the gene as BHA; 2, highly conservative sequences of HA protein, M2e protein and NA protein are selected for series combination, gene synthesis is performed after codon optimization, and the gene is named as G3; 3, respectively extracting plasmid DNAs of the synthetic fragments for later use; 4, amplifying a target fragment by using a PCR technology so as to connect a carrier; and 5, carrying out a target gene and vector connection experiment. According to the present invention, the HA protein sequence of the DNA vaccine can be rapidly replaced by using the restriction enzyme cutting site, the preparation method is simple, the immunogenicity is good, the cross protection is good, the preparation cost is low, the preparation requirement of the large-scale influenza vaccine can be met, and the important scientific significance and the important social value are provided.

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 nuclear targeting peptide modified DNA nano-particle based on a microfluidic technology and a preparation method and application thereof, and relates to the technical field of biological medicine, a poly (beta-amino ester) cationic polymer is used as a gene delivery carrier, the gene delivery carrier comprises a microtubule related sequence MTAS and a nuclear localization signal NLS oligopeptide, the gene delivery carrier and the plasmid DNA carrying the target gene are mixed to prepare nano-particles, and the plasmid DNA carrying the target gene is uniformly dispersed in the nano-particles by adopting a microfluidic mixing process; the nuclear targeting peptide modified DNA nano-particle is ingenious in structural design, the mixing uniformity of a nano-carrier and plasmid DNA is improved by adopting a micro-fluidic mixing process, the performance of the nano-particle is further improved by optimizing dialysis conditions, potential cytotoxicity and adverse effects are reduced, and the nuclear targeting peptide modified DNA nano-particle is suitable for large-scale production. And after transduction of non-activated T cells, strong tumor killing ability and cytokine secretion can be generated.

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.

The present invention pertains to a method for generating a target-specific large serine recombinase (LSR), the method comprising the steps of: a) generating a plurality of first variants of an LSR by introducing one or more mutations into a sequence encoding the LSR; b) generating a library of expression vectors, wherein each expression vector comprises a first region encoding one of the first variants of an LSR, and a second region comprising at least two first target sites of the LSR, wherein the two first target sites are different from each other; c) introducing the library of expression vectors into host cells; d) culturing the host cells and expressing the first variant LSR; e) isolating plasmid DNA from the culture of host cells; f) determining whether a portion of the second region of the expression vector between the two first target sites has been excised by the variant LSR; g) generating a plurality of second LSR variants by introducing one or more mutations into the sequence encoding those first LSR variants that excised the portion of the second region of the expression vector between the two first target sites; and h) repeating steps b) to f) optionally with at least one second target site, wherein the at least one second target site differs from the first target sites in at least one nucleotide. The present invention further provides variant LSRs obtained by the method, a nucleic acid or group of nucleic acids encoding a variant LSR, a vector comprising said nucleic acid(s); a system for integrating a donor DNA into a target nucleic acid; and uses of the LSRs, the nucleic acid(s) or the vector.

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.

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.

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 invention discloses a single-tube nested PCR (Polymerase Chain Reaction) specific primer probe for detecting southern corn rust disease and a detection method of the single-tube nested PCR specific primer probe. The single-tube nested PCR specific detection primer probe is used as an inner primer pair of puccinia multiflora, the nucleotide sequence of an upstream primer of the inner primer pair is as shown in SEQ ID NO.3, and the nucleotide sequence of a downstream primer of the inner primer pair is as shown in SEQ ID NO.4. The method comprises the following steps: carrying out single-tube nested PCR (Polymerase Chain Reaction) amplification by taking puccinia multiflora genome DNA or positive plasmid DNA as a template, then carrying out immunochromatography nucleic acid test strip detection on an amplification product, and carrying out result visual judgment through a test strip. The method has high sensitivity, meanwhile, the identification time of puccinia multiflora can be shortened through the detection system, and the problems that a traditional identification method is tedious in step, long in identification period and the like are solved.

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 present invention relates to a method for isolating a covalently closed circular (ccc) DNA molecule from a microbial cell containing said ccc DNA molecule, the method comprising the steps of: contacting said microbial cell with a lysing agent to obtain a lysing composition and moving said composition through a tube system with a flow rate having a Reynolds number of 3000 or more; incubating said lysing composition to obtain a lysing product; contacting said lysing product with a neutralizing solution to obtain a neutralized lysing product; and further treating said neutralized lysing product to obtain said ccc DNA molecule.

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

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.