46 results about "Superhelix" patented technology

The present invention is directed to protocells for specific targeting of hepatocellular and other cancer cells which comprise a nanoporous silica core with a supported lipid bilayer; at least one agent which facilitates cancer cell death (such as a traditional small molecule, a macromolecular cargo (e.g. siRNA or a protein toxin such as ricin toxin A-chain or diphtheria toxin A-chain) and / or a histone-packaged plasmid DNA disposed within the nanoporous silica core (preferably supercoiled in order to more efficiently package the DNA into protocells) which is optionally modified with a nuclear localization sequence to assist in localizing protocells within the nucleus of the cancer cell and the ability to express peptides involved in therapy (apoptosis / cell death) of the cancer cell or as a reporter, a targeting peptide which targets cancer cells in tissue to be treated such that binding of the protocell to the targeted cells is specific and enhanced and a fusogenic peptide that promotes endosomal escape of protocells and encapsulated DNA. Protocells according to the present invention may be used to treat cancer, especially including hepatocellular (liver) cancer using novel binding peptides (c-MET peptides) which selectively bind to hepatocellular tissue or to function in diagnosis of cancer, including cancer treatment and drug discovery.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The invention discloses a heavy-load locomotive slip form extreme value search optimum adhesion control system and method. Heavy-load locomotive adhesion control can be achieved. A heavy-load locomotive adhesion control module comprises a superhelix slip form controller, a differential tracker, a load rotating torque and an adhesion coefficient observer and a slip form extreme value search unit so as to achieve dynamic closed-loop control over the rotating torque of a heavy-load locomotive traction motor. On the basis of locomotive motor model vector control, on the basis of the slip form extreme value search method, the adhesion coefficient observer is designed, a wheeltrack adhesion coefficient is estimated in real time, the locomotive is made to run near the best adhesion point under the external complex environment, the power of a traction motor is effectively brought into play, the optimum adhesion control over the heavy-load locomotive is achieved, and the heavy-load locomotive slip form extreme value search optimum adhesion control system and method can be widely applied to the best adhesion control occasion of the heavy-load locomotive.

The present invention is directed to protocells for specific targeting of hepatocellular and other cancer cells which comprise a nanoporous silica core with a supported lipid bilayer; at least one agent which facilitates cancer cell death (such as a traditional small molecule, a macromolecular cargo (e.g. siRNA or a protein toxin such as ricin toxin A-chain or diphtheria toxin A-chain) and / or a histone-packaged plasmid DNA disposed within the nanoporous silica core (preferably supercoiled in order to more efficiently package the DNA into protocells) which is optionally modified with a nuclear localization sequence to assist in localizing protocells within the nucleus of the cancer cell and the ability to express peptides involved in therapy (apoptosis / cell death) of the cancer cell or as a reporter, a targeting peptide which targets cancer cells in tissue to be treated such that binding of the protocell to the targeted cells is specific and enhanced and a fusogenic peptide that promotes endosomal escape of protocells and encapsulated DNA. Protocells according to the present invention may be used to treat cancer, especially including hepatocellular (liver) cancer using novel binding peptides (c-MET peptides) which selectively bind to hepatocellular tissue or to function in diagnosis of cancer, including cancer treatment and drug discovery.

In accordance with the invention, there is provided a method for preparing plasmid from host cells which contain the plasmid, comprising the steps: (a) preparing a cleared lysate of the host cells, wherein the cleared lysate comprises unligatable open circular plasmid, wherein the open circular plasmid is not 3'-hydroxyl, 5-phosphate nicked plasmid; (b) incubating the unligatable open circular plasmid with one or more enzymes in the presence of their appropriate nucleotide cofactors, whereby the unligatable open circular plasmid is converted to 3'-hydroxyl, 5'-phosphate nicked plasmid; (c) incubating the 3'-hydroxyl, 5'-phosphate nicked plasmid with DNA ligase in the presence of DNA ligase nucleotide cofactor, whereby 3'-hydroxyl, 5'-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) incubating the relaxed covalently closed circular plasmid with DNA gyrase in the presence of DNA gyrase nucleotide cofactor, whereby relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. Preferably, the enzymatic steps (b), (c), and (d) are performed in a single step using an enzyme mixture comprising DNA polymerase, DNA ligase, and DNA gyrase. Preferably, the mixture further comprises a 3' terminus deblocking enzyme, such as exonuclease III or 3'-phosphatase. Preferably, the mixture further comprises one or more regenerating enzymes and a high energy phosphate donor, whereby the nucleotide by-products of the nucleotide cofactors generated by DNA ligase and DNA gyrase are converted to back to nucleotide cofactor. Preferably, the enzyme mixture further comprises one or more exonucleases, such as ATP dependent exonuclease, whereby linear chromosomal DNA is selectively degraded.

The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications- The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence. Following amplification, concatamers are subsequently processed according to their intended use and may include: restriction enzyme digestion for the production of short expression cassettes (SECs); ligation steps to circularize the SEC (CNAs); and / or supercoiling steps to produce sCNAs. The final product contains nearly non-detectable levels of bacterial endotoxin.

The invention discloses a rubber plant translationally controlled tumor protein, an encoding gene thereof and an application of the rubber plant translationally controlled tumor protein. The translationally controlled tumor protein named HbTCTP (Hevea brasiliensis translationally controlled tumor protein) comes from Hevea brasiliensis which belongs to euphorbiaceae rubber category. The translationally controlled tumor protein is a protein (a) or a protein (b). The protein (a) is a protein which is composed of amino acid sequences showed in a second sequence of a sequence table. The protein (b) is a derivative protein, which is provided with antioxidant activity, of the second sequence by using one or more amino acid residues to substitute and / or delete and / or add the amino acid sequences. Experiments show that the rubber plant translationally controlled tumor protein can inhibit active oxygen cutting supercoiled plasmid DNA (deoxyribonucleic acid) generated by a metal catalytic oxidation system, so that the rubber plant translationally controlled tumor protein has antioxidant activity and can eliminate active oxygen in rubber plants.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.

The present application relates to detection units and methods for detecting one or more target analytes in a sample. The detection unit provides a first and second surface connected by a filament which is capable of binding the target analyte in the sample. Double-stranded DNA molecules are provided having a continuous strand and a discontinuous strand, and an active segment that is designed to hybridize to a target nucleic acid of interest, where the continuous strand has between 0 and 100 unpaired nucleotides in the active segment and the discontinuous strand has between 5 and 100 unpaired nucleotides at its 3′ end and / or its 5′ end. The unpaired nucleotides in the continuous strand can form a secondary structure, such as a loop. The methods provide for the detection of the target analyte through the generation of a detectable signal, such as supercoiling, following the binding of the target analyte to the filament and can be used to detect nucleic acids of interest including single nucleotide polymorphisms or somatic mutations.

The invention relates to a DNA and nano lamellar hydroxylapatite compound as well as a preparation method thereof. Lamellar hydroxylapatite and salmon sperm DNA are used as raw materials to be mixed in percentage by mass of 1:5-100 of DNA and lamellar hydroxylapatite. The preparation method comprises the following steps: intermittently shaking the suspension of lamellar hydroxylapatite and DNA solution at 37 DEG C to react for 5-7 times and drying at normal temperature. The compound has an interlayer type structure and the layer distance of 3.3-4.8nm and realizes the storage, protection and release of DNA with a superhelix structure. The preparation method of the compound is relatively easy and significant for applying the hydroxylapatite in aspects of polygene medicine treatment and protein / gene composite medicine treatment and has low cost.

The invention discloses an adaptive multivariable generalized superhelix method. The adaptive multivariable generalized superhelix method comprises the steps of determining a multivariable system containing internal perturbation and external disturbance, constructing a control input of the multivariable system, constructing an adaptive law of the multivariable system, and then checking the stability of the multivariable system. By adoption of the method, the derivative bounded interference and system uncertainty can be handled at the same time, meanwhile the information of interference does not need to be known in advance, and the method can be applied to the multivariable system.

The invention discloses a method for measuring content of superhelix DNA. The method comprises the following steps: performing first detection and separation treatment on a sample to be measured by adopting an AKTAexplorerTM10 detection instrument and utilizing a molecular sieve chromatography column so as to obtain total DNA samples, and calculating the mass content of the total DNA samples; and performing second detection and separation treatment on the total DNA samples by adopting a thiophilic aromatic chromatographic column, and calculating to obtain the mass content of the superhelix DNA in the sample to be measured. According to the method, the content of the superhelix DNA in plasmid DNA can be quantitatively and effectively measured, the analytical accuracy is equivalent to that of capillary gel electrophoresis, and the method is obviously superior to agarose gel electrophoresis and suitable for each step in DNA production.

In one embodiment of the invention, a method is provided for preparing plasmid from host cells which contain the plasmid, comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cofactors, such that unligatable open circular plasmid is converted to 3′-hydroxyl, 5′-phosphate nicked plasmid; (c) reacting the 3′-hydroxyl, 5′-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that 3′-hydroxyl, 5′-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that relaxed covalently closed circular plasmid is converted to negatively supercoiled plasmid. In other embodiments, DNA gyrase is replaced with reverse DNA gyrase or reaction (d) is not performed.

The invention discloses an extraction method for DNA of plasmids of escherichia coli. The extraction method comprises the following steps: (1) purification and cultivation; (2) amplified cultivation;(3) preparation of a premixed solution; (4) splitting of escherichia coli; (5) separation of DNA of plasmids; and (6) collection of the DNA of plasmids. The invention provides the extraction method for DNA of plasmids of escherichia coli. The whole process is scientific and reasonable. The contents of plasmids in a unit bacterial solution and DNA of superhelix plasmids in unit mass plasmids are improved. The time consumed is short, and the market popularizing applicability of the DNA of plasmids is promoted well.

The invention relates to the technical field of gene therapy drug analysis, in particular to a high-separation-degree detection method for the content of plasmid superspiral DNA. The invention discloses an analysis method for simultaneously separating four topological structures of plasmid DNA (deoxyribonucleic acid) and evaluating the homogenization condition of superspiral DNA. The analysis method comprises the following steps: preparing an analysis solution; with an anion exchange chromatographic column and a mixed solution of Tris-HCl-NaCl as a mobile phase, using a gradient elution method; and measuring the prepared analysis solution on a machine. According to the analysis method, four topological structures of plasmid DNA can be effectively and accurately separated and determined, the homogenization condition of the super-helix DNA can be evaluated, the stability of the super-helix DNA is further evaluated, so that the index quality of a product is accurately detected.

The invention provides a preparation and purification method of a lung cancer resistant plasmid T-VISA (VP16-Gal4-WPRE integrated systemic amplifier)-Bik (bcl-2interacting killer) DD. The method comprises the steps as follows: seed amplification culture, fermented cultivation, centrifugal collection of thalli, thallus pyrolysis, supernatant collection and vacuum filtration, sieve chromatography, affinity chromatography, ion chromatography, isopropyl alcohol precipitation, PBS (phosphate buffer) dissolution and the like. With the adoption of the method, the fermentation content of the finally obtained lung cancer resistant plasmid T-VISA-BikDD is 150-200 mg / L, and the superhelix proportion of the plasmid after purification is higher than 90%.

The invention belongs to the field of biological separation and purification, and particularly relates to a purification method suitable for large-scale plasmid DNA production. The method comprises the following steps: carrying out ion exchange chromatography by taking an anion exchange chromatography medium as a stationary phase to remove impurities such as RNA, endotoxin and Escherichia coli host protein, and retaining plasmids; and carrying out affinity chromatography to remove and adsorb the superhelix plasmids and other trace impurities so as to obtain purified DNA plasmids. According to the method, ion exchange chromatography and affinity chromatography are combined for plasmid purification, and compared with a traditional purification process, the time is short, and the cost is low; a gel filtration step is not needed, the volume of a sample subjected to direct chromatography treatment is not limited by the column volume and is only related to the quantity of plasmids to be purified, feed liquid with the same volume is purified, the dosage of a chromatography medium is only 1 / 10 or less of that of a traditional gel filtration chromatography medium, the diameter of a chromatography column needed during process amplification is small, and the cost can be saved.

The present invention relates to nucleic acid molecule compositions comprising minivectors encoding a nucleic acid sequence and methods of gene therapy and prophylaxis against infection using minivectors encoding a nucleic acid sequence.

The invention discloses a cold-adapted peroxidase as well as a coding gene and application thereof, and aims to provide the cold-adapted peroxiredoxin as well as the coding gene and application thereof. According to the invention, peroxiredoxin gene PsPrx is firstly cloned from Antarctic sea ice microorganism psychrobacter sp., and the amino acid residue sequence of the peroxiredoxin is shown in SEQ ID No.2. An expression method of the peroxiredoxin comprises the steps of constructing a recombinant expression vector containing the peroxiredoxin gene PsPrx, introducing the constructed recombinant expression vector into a host cell escherichia coli, and performing induction to realize the peroxiredoxin gene PsPrx gene expression. The expression product PsPrx provided by the invention has outstanding cold adaptability, better stability, and good ability to protect supercoiled DNA against oxidative damage, and can be applied to the related fields such as biomedicine, cosmetics and food.

The invention discloses a biological insecticide screening mode, which comprises the following steps: integrating acceptor EcR with ecdysone GAP with gene expressing box Promoter-EcR-AOX1 TT and super spiral protein USP gene with gene expressing box TEF1 Promoter-USP-CYC1 TT on the colorant layer of Pasteur pichia; integrating DNA fragment with fruit fly 5XEcRE-HSP27 Promoter-GFP on the colorant layer yeast; getting the product. To co-culture screening drug with this product, we can get stopping or reinforcing GFP expression biological insecticide.

A method for fixing a supercoiled DNA consists in deposing a supercoiled DNA sample on the surface of a porous polymer film, in fixing said supercoiled DNA therein by a passive diffusion, in obtaining a support by the inventive method and in using said support for analysing the DNA distribution.

The invention discloses a binding solution and a superhelix plasmid large extraction kit based on the binding solution. The binding solution comprises 1-5 M of guanidine isothiocyanate, 10-30% (V / V) polyethylene glycol 8000, 60-90 mM of Tris-HCl, 20-40 mM of EDTA and 0.5-2 M of NaCl, and the PH is 4-7. According to the superhelical plasmid large extraction kit based on the binding liquid, firstly, escherichia coli containing target plasmids is subjected to resuspension, splitting decomposition and neutralization through an alkali splitting decomposition method; adding a binding solution, uniformly mixing, and standing at room temperature to form a mixed solution; centrifuging, filtering the mixed solution through a centrifugal column, and discarding the filtrate; and finally, washing and eluting the plasmids adsorbed on the centrifugal column to obtain the target plasmids. The special binding liquid can enhance the binding efficiency of the plasmids and the centrifugal column and improve the content of the extracted superhelical plasmids.

The invention relates to an interactive parameterized three-dimensional DNA conformation simulation method for secondary school teaching, a double linked list is used as an underlayer data structure to generate a DNA base sequence fragment single chain 1 # and another single chain 2 # which is matched with the DNA base sequence fragment single chain 1 # but contains errors, the polarity directionof the single chain 2 # and the base complementary pairing error are interactively corrected , and a DNA primary structure is simulated; on the basis, the type of DNA is interactively selected, a local reference frame of a double-helix secondary structure is calculated, the dynamic process of spiraling while stacking of DNA base pairs is simulated according to the reference frame, and each secondary structure parameter is displayed on a three-dimensional model; and the local reference frame is updated by interactively editing a space curve, so that linear DNA double-chains are bent and wound in the space, and various superhelix high-level structures of the DNA from a linear shape and a bent shape to any zigzagging wound shape are simulated. According to the interactive parameterized three-dimensional DNA conformation simulation method for secondary school teaching in the invention, the DNA conformation can be simulated and displayed in an interactive mode, and the understanding and recognition of students on the DNA conformation can be accelerated and deepened.

A method for quantitative assessment of base excision repair (BER) and nucleotide excision repair (NER) DNA capacities of one or more cellular extracts using supercoiled plasmid DNA by characterizing lesions on the plasmids relative to a control.

The invention discloses adenosine monophosphate enzyme as well as a preparation method and an application thereof, including an expression vector of adenosine monophosphate enzyme, a preparation method of the adenosine monophosphate enzyme, an application of the adenosine monophosphate enzyme in in-vitro adenosine monophosphate modification of subunit GyrB and ParE proteins of DNA topoisomerase, and an application of the adenosine monophosphate enzyme in in-vitro inhibition of ATP hydrolysis activity of the DNA topoisomerase, and in inhibition of DNA topoisomerase negative superhelix and relaxation of DNA activity. The preparation method of the adenosine monophosphate enzyme provided by the invention is simple, short in fermentation period, low in purification cost and large in preparation amount; by utilizing the adenosine monophosphate enzyme provided by the invention, adenosine monophosphate modification of the DNA topoisomerase can be realized under an in-vitro condition; the adenosine monophosphate enzyme provided by the invention is expressed, the activity of DNA topoisomerase can be inhibited.

The invention relates to a DNA and nano lamellar hydroxylapatite compound as well as a preparation method thereof. Lamellar hydroxylapatite and salmon sperm DNA are used as raw materials to be mixed in percentage by mass of 1:5-100 of DNA and lamellar hydroxylapatite. The preparation method comprises the following steps: intermittently shaking the suspension of lamellar hydroxylapatite and DNA solution at 37 DEG C to react for 5-7 times and drying at normal temperature. The compound has an interlayer type structure and the layer distance of 3.3-4.8nm and realizes the storage, protection and release of DNA with a superhelix structure. The preparation method of the compound is relatively easy and significant for applying the hydroxylapatite in aspects of polygene medicine treatment and protein / gene composite medicine treatment and has low cost.

The invention relates to a magnetic nano lamellar hydroxyapatite and DNA composite and a preparation method thereof. Magnetic nano lamellar hydroxyapatite and salmon sperm DNA, used as raw materials, are blended according to the mass ratio of the DNA to the magnetic lamellar hydroxyapatite of 1:(5-200). The preparation method comprises the following steps of: shaking the suspension of the magnetic lamellar hydroxyapatite and a DNA solution at 37 DEG C for reacting for 0.5-3.5 hours; and after centrifuging at high speed, drying at normal temperature. The composite provided by the invention has an inserting and stripping type structure and realizes the bulk storage and effective protection of superhelix structure DNA. The preparation method of the composite is easier and has low cost and superparamagnetism. The invention has great significance for the magnetic lamellar hydroxyapatite in aspects of polygene medicament treatment and protein / gene composite medicament treatment.

Provided are methods, such as high throughput methods, of assessing or measuring the ability of an enzyme to modify the topology (e.g. supercoil topology) of a target nucleic acid, the methods comprising the steps of: (a) providing a solid support to which a capture nucleic acid is or may be immobilized, which capture nucleic acid is capable of binding the target nucleic acid in a manner that is proportional to the supercoil topology of said target nucleic acid (e.g. by triplex formation); (b) incubating a test mixture comprising (i) the enzyme, (ii) the target nucleic acid, (iii) capture nucleic acid, in the presence of (iv) said solid support, such that supercoiled target nucleic acid bound by the capture nucleic acid is immobilized to the solid support, (c) determining the amount of target nucleic acid bound by said capture nucleic acid in step (b) e.g. by use of a detectable label. The method may be used to screen for modulators of the enzyme activity.