556 results about "Gene vector" patented technology

A group of modular cloning vector plasmids for the synthesis of a transgene or other complicated DNA construct, by providing a backbone having docking points therein, for the purpose of gene expression or analysis of gene expression. The invention is useful for assembling a variety of DNA fragments into a de novo DNA construct or transgene by using cloning vectors optimized to reduce the amount of manipulation frequently needed. The module vector contains at least one multiple cloning site (MCS) and multiple sets of rare restriction and / or unique homing endonuclease (“HE”) sites, arranged in a linear pattern. This arrangement defines a modular architecture that allows the user to place domain modules or inserts into a PE3 transgene vector construct without disturbing the integrity of DNA elements already incorporated into the PE3 vector in previous cloning steps. The PE3 transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

The invention discloses a CAR-T transgene vector based on replication defective recombinant lentivirus. The CAR-T transgene vector comprises an original nuclear replicon pUCOri sequence, a resistance gene AmpR sequence containing ampicillin, a virus replicon SV40 Ori sequence, a lentivirus packaging cis element, ZsGreen1 green fluorescent protein, an IRES ribosome binding sequence, a human EF1 alpha promoter , a chimeric antigen receptor of second-generation CAR or third-generation CAR and a regulating element, wherein the original nuclear replicon pUCOri sequence is used for plasmid replication; the resistance gene AmpR sequence is used for massively proliferating target strains; the virus replicon SV40 Ori sequence is used for enhancing replication in eukaryocyte; the lentivirus packaging cis element is used for lentivirus packaging; the ZsGreen1 green fluorescent protein is used for expressing green fluorescent for eukaryocyte; the IRES ribosome binding sequence is used for jointly transcribing and expressing protein; the human EF1 alpha promoter is used for conducting eukaryotic transcription on antigen receptor genes; the chimeric antigen receptor is used for forming the second-generation CAR or the third-generation CAR integrating recognition, transfer and start; the regulating element is used for enhancing expression efficiency of transgenes and used after eWPRE-enhanced type woodchuck hepatitis b virus is transcribed. Besides, the invention further discloses a construction method and application of the vector. By means of the CAR-T transgene vector and the construction method and application of the vector, secretion of cell factors and an in vitro killing effect of CAR-T cells can be remarkably improved, and the clinical treatment effect is remarkable.

The invention discloses a method for targetedly integrating an exogenous gene into a target gene of a mammal, which is implemented by targetedly integrating an exogenous gene into a target gene by utilizing a CRISPR / Cas9 system, and especially targetedly integrating a Cre gene to a first exon of a mouse Enpp1 gene. The technique has the advantages of high integration efficiency (20%) and simple operation step, only needs to construct one exogenous gene vector, and can not be influenced by the position effect.

The invention discloses construction and application of a CRISPR / Cas9 gene editing vector for microorganisms. The constructed CRISPR / Cas9 gene vector consists of a replication start site, a selection marker gene, a Cas9 protein gene, gRNA coding DNA, a homologous recombinant element and an operon. The CRISPR / Cas9 gene vector constructed by the invention is capable of editing (including performing such operations as knocking out, replacing, interpolating and the like on gene or DNA sequence) escherichia coli or corynebacterium glutamicum genome; and the gene vector has the advantages of being short in test cycle, time-saving and cost-saving, high in efficiency and the like.

The invention provides sgRNA and a gene vector for inhibiting bladder cancer by targeting human lncRNA-UCA1 and an application of the sgRNA; specifically, sgRNA sequences of an lncRNA-UCA1 gene and a PD-1 gene suitable for CRISPR-Cas9 targeted splicing are designed; by transforming plasmids of specific splicing lncRNA-UCA1 gene and CRISPR-Cas9 nuclease gene into human bladder cancer cells, the expression of the lncRNA-UCA1 gene drops so as to inhibit the growth of tumor cells; and the plasmid, together with a human PD-1 gene targeted knockout vector, is transformed into a humanized mouse model of bladder cancer transplanted tumor, so as to obviously inhibit the growth of tumors. According to the invention, the vector is simple in preparing steps, the sgRNA is good in targeting property and the CRISPR-Cas9 is high in knockout efficiency.

The invention discloses preparation of a double-function water-soluble perylene bisimide derivative and an application of the derivative to a mercury ion probe and a gene carrier. A perylene fluorescent core CHO-PDI carrying four formyl groups is utilized, and a protection reaction is carried out on the formyl groups by sulfydryl to obtain the double-function water-soluble perylene bisimide derivative. On basis of de-protection reaction of mercury ions to the formyl groups of the double-function water-soluble perylene bisimide derivative, the aim of highly-sensitively and highly-selectively rapidly detecting the trace mercury ions in water is realized; the double-function water-soluble perylene bisimide derivative has very good biocompatibility and very low cytotoxicity; an amino with a positive charge is carried at the periphery of a molecule, can enter a live body culture cell and can be combined with DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) with a negative charge to form a stable compound which is used as a carrier for carrying and protecting an exogenous nucleic acid to enter the live body culture cell. In combination with the two functions, the double-function water-soluble perylene bisimide derivative can be applied to biological cell imaging to detect the mercury ions in the cells.

Methods and apparatus are provided for delivering drugs, gene vectors, naturally-occurring or synthetic hormones or proteins or other bioactive agents within a patient's vasculature. In a preferred embodiment, the apparatus comprises a material that elutes or secretes a bioactive agent and is held in place within the patient's vessel by an anchor. The material may comprise a biocompatible, and optionally, absorbable matrix, or a culture medium that sustains and nourishes stem cells, spleen cells or pancreatic islets or other beneficial cells. The anchor and material are sized and / or collapsible from a delivery configuration, in which the anchor and material may be delivered into the patient's vasculature within a delivery sheath, to a deployed configuration, wherein the anchor engages an interior wall of the patient's vessel. The apparatus of the invention may be temporarily or permanently implanted, and may in addition self reconfigure after a predetermined period of residency.

Methods and apparatus are provided for delivering drugs, gene vectors, naturally-occurring or synthetic hormones or proteins or other bioactive agents within a patient's vasculature. In a preferred embodiment, the apparatus comprises a material that elutes or secretes a bioactive agent and is held in place within the patient's vessel by an anchor. The material may comprise a biocompatible, and optionally, absorbable matrix, or a culture medium that sustains and nourishes stem cells, spleen cells or pancreatic islets or other beneficial cells. The anchor and material are sized and / or collapsible from a delivery configuration, in which the anchor and material may be delivered into the patient's vasculature within a delivery sheath, to a deployed configuration, wherein the anchor engages an interior wall of the patient's vessel. The apparatus of the invention may be temporarily or permanently implanted, and may in addition self reconfigure after a predetermined period of residency.

A novel nano polycation transgenic vector relates to a nano polycation transgenic vector of a cyclodextrin-polyethyleneimine-folacin ternary assembly structure which takes the cyclodextrin as a skeleton material and polymerizes the cyclodextrin after activated with small molecular polyethyleneimine(PEI) as well as couples the decorative folacin on the cyclodextrin-polyethyleneimine to manufacture the nano polycation transgenic vector with tumor targeting. The material composing method of the vector is simple and effective with a high recycling rate; the material is shown to have the characteristics of low poison and high transfecting efficiency by the filtering of a plurality of tumor cell strains and experiences in the body of a rat.

The invention discloses a boron-containing nano-mesoporous and macroporous bioactive glass, a preparation method thereof and the application thereof to medical supports. The boron-containing mesoporous bioactive glass is prepared by substituting BO33- for part of SiO44- by a sol-gel method. According to the method, controllable porosity (78 to 90 percent) is obtained by adjusting immersion times (2 to 5 times) and aperture (300 to 500 microns). The composite glass material has pores suitable for cell growth; tissue growth and nutrition transmission are facilitated due to the porosity characteristic; and the structural integrity of the supports can be maintained in vitro. When the boron content is 0 to 10 percent, the boron-containing ordered mesoporous bioactive glass support has large surface area of 365 to 194 m<2> / g and is suitable for serving as vectors of medicines and growth factors. The biological material can slowly release boron ions, can serve as a tissue repair material applied to bone and periodontium engineering, and can serve as vectors of medicines or genes.

The present invention provides novel multi-functional methacrylic copolymers that exhibit cationic pH-sensitive behavior as well as good water solubility under acidic conditions. The copolymers are constructed from tertiary amine methacrylates and poly(ethylene glycol) containing methacrylates. The copolymers are useful as gene vectors, pharmaceutical carriers, and in protein separation applications.

The invention discloses a nucleic acid transfected cationic liposome, a nucleic acid medicinal preparation comprising a cationic liposome as well as a preparation method and use thereof. The cationic liposome comprises a cationic liposome, an auxiliary liposome and a polyethylene glycol modified neutral liposome. The cationic liposome nucleic acid medicinal preparation comprises the cationic liposome and the nucleic acid medicinal preparation. The cationic liposome disclosed by the invention serves as a non-virus gene vector and is low in cytotoxicity, strong in blood stability and high in gene silencing efficiency. The highest gene silencing efficiency on a HepG-2 cell which constantly expresses luciferase is 90.8%. The cationic liposome has an excellent anti-non-specific protein adsorptive property and long recyclability, thus improving the stability and the running efficiency of the liposome and effectively improving the effect of the nucleic acid medicine. The cationic liposome nucleic acid medicinal preparation can be used in the field of medicine and the like.

The invention discloses a cationic polymer gene vector having low cytotoxicity and high transfection efficiency, which takes multi aldehydesodium alginate (MASA) as a framework and Poly(ethylene imine) PEI as lateral chain, wherein the molecule amount of the PEI is smaller than 2K. The invention further discloses a preparation method and use of the cationic polymer gene vector. The graft copolymer has low cost, low cytotoxicity, low biodegradability, and high transfection efficiency not affected by serum, and is a cationic polymer gene vector having wide application range and good prospect.

The invention provide a kind of non-virogene transfection vector, by grafting chitose that average molecular weight is 1.5kD~51kD and C10~C22 fatty acid to forming the chitose-fatty acid grafting colloidal cluster, critical colloidal cluster concentration< 0.1mg / ml, particle size between 20 and 200 nm, colloidal cluster surface charge on positive charge, amidol degree of substitution of the chitose is 1%~50%. By synthesizing the chitose-fatty acid grafting substance to preparing the chitose-fatty acid grafting colloidal cluster of non-virogene transfection vector. The invention's synthetic colloidal cluster is low-toxic cationic polymer colloidal cluster, the colloidal cluster has characteristic of penetrate cell membrane fast, destroyed by intracellular lysosome rarely and the function of target nucleus, and by the cationic feature of itself joint seal the negative charge biomacromolecule to forming stable colloidal cluster / DNA complex administer drug system, it can apply in the field of medicine as highly effective and safety non-virogene transfection vector.

The invention belongs to the field of medicines, and in particular relates to DOTAP-mPEG-PLA nanoparticles, a solution of the nanoparticles, a medicine-carrying compound as well as a preparation method and an application. The invention aims at providing an amphiphilic mPEG-PLA diblock copolymer modified by DOTAP, and a novel biodegradable gene carrier, namely, the DOTAP-mPEG-PLA nanoparticles (DPP nanoparticles for short) is prepared through a self-assembly method. The DPP nanoparticles have good DNA binding capacity, gene plasmids can be effectively guided into cancer cells, and the advantages of high transfection efficiency and low cell toxicity are achieved.

The invention discloses a gene vector system of a redox sensitive shielding system having targeting function, its preparation and application in the field of gene therapy. The gene vector system disclosed herein comprises a redox sensitive shielding system having targeting function, a cationic high-molecular material and plasmid DNA; wherein the cationic high-molecular material and plasmid DNA form compound particles, the redox sensitive shielding system having targeting function shields the compound surface by electrostatic action, thus the toxicity of the vector can be reduced, the loaded genetic material is transferred into the cell successfully, the expression of the genetic material is realized, the transfection process is completed, the targeting of gene transfection can be raised, and simultaneously the gene transfection efficiency is raised.

The invention relates to PEI (Polyetherimide) modified chitosan triply compound gene vector coated with RGD (Arginyl-Glycyl-Aspartic acid)-chondroitin sulfate and a preparation method and application thereof. The triply compound gene vector is formed by mixing RGD polypeptide modified chondroitin sulfate, pDNA and PEI grafted chitosan in the mass ratio of 15:1:4. The transfection efficiency of a chitosan vector is improved by using a PEI grafting method; by using a coating method of electronegative chondroitin sulfate, red cells in blood plasma and plasma protein can be prevented from being adsorbed and reduced and the stability of blood circulation can be enhanced; in addition, by using the special targeted RGD polypeptide to modify the chondroitin sulfate, the transmembrane capability of compound particles can be enhanced. A triply gene delivery system can be constructed by taking the PEI grafted chitosan and DNA compound particles as the core and the RGD-modified chondroitin sulfate as the casing. The preparation method is simple and convenient and has mild conditions; the modified chitosan is used as a gene vector, so that the transfection efficiency is high and the cytotoxicity is low; in addition, the invention can effectively prevent the adsorption to the red cells and electronegative macromolecules, has favorable body fluid circulating stability and is hopeful to be used for gene treatment clinical experiments.

The present invention provides a hairpin ribozyme library having a randomized recognition sequence, packaged in a vector and operably linked to a promoter suitable for high level expression in a wide variety of cells. The invention comprises using the library in a variety of selection protocols for identifying, isolating and characterizing known or unknown target RNAs, to reveal the phenotypic effects of such cleavage, and to identify the gene products that produce those phenotypic effects.

The invention provides a halogenohydrin dehalogenation enzyme originating from Agromyces sp., an encoding gene and a vector, and provides application of the halogenohydrin dehalogenation enzyme, the encoding gene and the vector in the process of preparing epoxy chloropropane and (R)-4- cyano-cn-3- hydroxybutyric acid ethyl ester. The halogenohydrin dehalogenation enzyme amino acid sequence is shown in SEQ ID NO.2, and the encoding gene sequence is shown in SEQ ID NO.1. The halogenohydrin dehalogenation enzyme, the encoding gene, the vector and the application have the advantages that the halogenohydrin dehalogenation enzyme originating from Agromyces sp. CCTCC NO. M 2012299 and the encoding gene of the halogenohydrin dehalogenation enzyme are provided; and the encoding gene of the halogenohydrin dehalogenation enzyme can be connected and constructed with an expression vector to obtain expression recombinant plasmid pET28b-Deh of the encoding gene, then can be transformed to escherichia coli BL21, obtained and transformed to escherichia coli bacterial strain respectively and correspondingly to obtain recombinant escherichia coli, and the recombinant escherichia coli has the halogenohydrin dehalogenation enzyme and can be utilized for carrying out biotransformation and catalysis for an enzyme source.

The invention discloses a tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof. Plasmid T-GFP is constructed by using plasmid rDNA-GFP in the methods such as enzyme cutting and PCR enzyme cutting site mutation; thermophile tetrahymena genome DNA is used for constructing plasmid T-HSP702 in the methods such as PCR augmentation and clone; Plasmids T-GFP, T-HSP702 and pD5H8 are used for construction and finally obtaining plasmid HSP702-GFP-pD5H8 by enzyme cutting and connection. The carrier contains HSP702 promoter, thereby being capable of realizing high-efficient expression of exogenous gene; the carrier contains replaceable exogenous gene gfp so that an exogenous gene expression system is realized for tetrahymena by replacing different exogenous genes; with the sieving effect of paromomycin medicine, the carrier replaces most rDNAs originally in the tetrahymena so that the exogenous genes are largely increased in the tetrahymena so as to realize genetic transformation of target genes. The method in the invention is easy and has convenient operation, and the carrier can be used for detection of environmental tributyl tin in transfection in tetrahymena.

The invention belongs to the technical field of medicine, and relates to a guanidinylation SS-PAAs gene vector polymer as well as preparation and application thereof. The preparation method comprises the following steps: the guanidinylation SS-PAAs gene vector polymer is finally prepared from a cross-linking agent with a dual-acroloyl structure and a guanidinylation reagent through the processes of protection reaction performed on sulfonyl chlorides by sulfonyl chlorides, Michael addition polymerization and removal of protection of a benzene sulfonyl chloride guanidyl protecting agent in sequence. The polymer gene vector can be self-assembled with different kinds of gene segments to form a compound, so that the excellent biological membrane permeability is achieved, and the responsive degradation is realized in the reducible environment of cells, and the nuclear localization effect is achieved. The capability of loading the gene segments of the vector and the transport process can be regulated through controlling the guanidinylation reagent species, the proportion of the guanidinylation reagent in the polymer, and the molecular mass. The guanidinylation SS-PAAs gene vector can improve the transfection efficiency of genes, and reduce cytotoxicity, and is a novel gene vector adopted in the process of gene therapy.

The invention synthesizes a type of liver targeted, biodegradable high molecular MRI contrast medium with alpha,beta- poly [(2-aminoethyl) - L-asparagine] as fundamental chain, galactose as target gene, diethylenetriamine pentaacetic acid or L-tyrosine methyl ester derivatives as Gd3+ ligand. The contrast agent is less toxic, biocompatible, and has good relaxation rate than low molecular business contrast medium. Animal experiments showed that in liver organizations its enhancing effect is three times better than commercial and low molecular contrast medium (gadolinium sprayed acid cyelophosphate); the residence time in liver is more than 24 hours, which is 18 hours longer than low molecular business contrast medium. The feature is that Zeta potential for the contrast medium is negative and less than -10mV. We can achieve the MRI visible, the liver targeted, core-shell nanogene vector system with it as a gene vector system shell structure.

The invention belongs to the field of polymer modification, and particularly relates to an acid-sensitive hydrophobic modified polyethyleneimine and application thereof as a gene vector. The derivative contains an acid-sensitive acetal functional group; and specifically, the derivative is an acetal-molecular trimethoxy benzylacetal-trihydroxy ethylacetal (TMB-THME) modified polyethyleneimine derivative, named polyethyleneimine-(trimethoxy benzylacetal-trihydroxy ethane). The polyethyleneimine-(trimethoxy benzylacetal-trihydroxy ethane) provided by the invention enhances the DNA (deoxyribonucleic acid) compounding capability of PEI (polyetherimide) and the interaction with cells, can convert a hydrophobic group into a hydrophilic group in an endosome, and implements the dissociation of theDNA compound and the intracellular release of the DNA; and the cytotoxicity is low.

The invention belongs to the technical field of biological medicine, and designs a multifunctional graphene gene vector, a gene transfection reagent based on the gene vector and a preparation method thereof. The multifunctional graphene comprises the following three parts of graphene, cationic polymer, and an active reagent, wherein the active reagent comprises one or more than one of a surfactant, or cell-penetrating peptide, or nuclear localization signal peptide; the cationic polymer and the active reagent are connected to the graphene. According to the invention, through multi-functionalization, the stability, dispersibility, biocompatibility of the gene vector in the gene transfection process are improved; the toxicity is reduced; and a plurality of gene transfection promotion mechanisms are adopted to improve transfection efficiency. Gene transfection experiment results show that high transfection efficiency and low toxicity are realized in a plurality of cell strains. The main purpose of the invention is gene transfection, and is applicable to cytobiological research, gene product production, and gene therapy.

The invention discloses a series of low-toxicity and efficient cationic gene vectors with PGMA (polyglycidyl methacrylate) serving as a framework constructed by an ATRP (atom transfer radical polymerization) method, which belong to the technical field of nonviral gene vectors. The ATRP method is stable in polymerization reaction and easy in regulation and control, and can prepare various high-performance cationic gene vectors different in molecular weight and narrow molecular weight distribution according to needs. The prepared cationic gene vectors are high in storage stability, higher than gold mark PEI (polyethyleneimine) in transfection efficiency in cells such as Hepg2, C6, Cos7, HEK293 and the like and simple in use method, and have commercial potential.

The present invention discloses that an intratumoral injection of: i) glycolipids with α-gal epitope; ii) gene vectors comprising an α1,3galactosyltransferase gene; or iii) a mixture of α1,3galactosyltransferase, neuraminidase, and uridine diphosphate galactose results in tumor regression and / or destruction. Binding of the natural anti-Gal antibody to de novo expressed tumoral α-gal epitopes induces inflammation resulting in an anti-Gal antibody mediated opsonization of tumor cells and their uptake by antigen presenting cells. These antigen presenting cells migrate to draining lymph nodes and activate tumor specific T cells thereby converting the treated tumor lesions into in situ autologous tumor vaccines. This therapy can be applied to patients with multiple lesions and in neo-adjuvant therapy to patients before tumor resection. In addition to the regression and / or destruction of the treated tumor, such a vaccine will help in the immune mediated destruction of micrometastases that are not detectable during the removal of the treated tumor.

The invention provides a gene vector system. The gene vector system comprises a pH value sensitive material, wherein the pH value sensitive material is a hyperbranched polyethylenimine-polylysine-polyglutamic acid copolymer, a polyethylene glycol-hyperbranched polyethylenimine-polylysine-polyglutamic acid copolymer, low molecular weight sulfanilamide, or a polyethylene glycol-low molecular weightsulfanilamide condensation compound, and a complex comprising gene vectors and a gene substance, wherein a mass ratio of the gene vectors to the gene substance is in a range of (0.5: 1) to (50: 1); the pH value sensitive material is bonded to the surface of the complex; and a mass ratio of the pH value sensitive material to the gene vectors of the complex is in a range of (1: 1) to (80: 1). The gene vector system provided by the invention can guarantee effectively transportation of gene vectors and a gene substance in normal cells and promote the gene vectors and the gene substance to bond with tumor cells thereby improving transfection efficiency.

The invention discloses a genetic vector system of a nanoparticle with multiple oxidation-reduction stimulus response as well as a preparation method and application of the genetic vector system. The genetic vector system is composed of a shielding system, a cation material and foreign plasmid DNA (deoxyribonucleic acid); and the shielding system and the cation material both have oxidation-reduction stimulus response, so that a ternary complex nanoparticle with multiple oxidation-reduction stimulus response is formed. The genetic vector of the ternary complex nanoparticle is low in cytotoxicity, and capable of well compressing and compounding the plasmid DNA under physiological conditions, successfully transferring a supported genetic substance into a cell and entering the cell; and the reduction environment in the cell can be more rapidly broken to release the supported gene, so that the expression of gene substances is realized, the transfection process is finished, and the gene transfection efficiency can be remarkably increased.

The invention discloses a reducible and biodegradable comb type high polymer gene vector and a preparation method of the same. The structure of the vector is as shown in the following formula, wherein CP is a cationic polymer with low molecular weight between 600 and 30000; F1 and F2 are groups capable of interaction; F1 or F2 contains a disulfide bond; M is a main chain unit structure of the polymer; n equals to 10-500; m is smaller than n; m equals to 0-400; l equals to 0-800; and l, m and n are integers. In the invention, the comb type high polymer gene vector has small cytotoxicity, gene DNA can be well bound and compounded under physical conditions so as to enter cells; and the carried genes can be released as the reducing environment in the cells can rupture quickly. The result showthat the reducible and biodegradable comb type high polymer gene vector has higher transfection efficiency and lower cytotoxicity compared with 25kDa PEI (polyether imide); moreover, the preparation method is simple, and the structure is easy to control.