22results about How to "High transfection efficiency" patented technology

The invention discloses a method for transfecting a plant receptor by using a cell penetrating peptide. The method comprises the following steps: (1) constructing an expression vector of a cell penetrating peptide fusion protein CPPs-Cys-mCherry; (2) transforming an expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry into a protein expression system; (3) inducing expression of cell penetrating peptide fusion protein CPPs-Cys-mCherry by using IPTG (isopropyl-beta-d-thiogalactoside) to obtain a crude protein sample; (4) purifying the crude protein sample obtained in the step (3), and then adjusting the concentration of the cell penetrating peptide fusion protein CPPs-Cys-mCherry to 1-200 [mu] g / ml; and (5) transfecting different types of plant receptors by using a solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (4), and completing the transfection.

This invention relates to the field of gene vector technology, and more particularly to an ionizable comb-like polymer, its preparation method, and its applications. This comb-like polymer is a block polymer, comprising at least one block unit. Each block unit includes a first block, a second block, and a third block linked by covalent bonds. It exhibits high molecular flexibility and readily binds to nucleic acid-like substances to form polymer / nucleic acid complexes. Due to the presence of easily degradable intracellular glycosidic bonds in its structure, the polymer demonstrates high transfection efficiency. Furthermore, cell experiments show that the polymer / nucleic acid complex exhibits low toxicity to cells and significantly improves cell survival compared to the positive control. Therefore, it holds promise for widespread application in cell transfection. The preparation method provided by this invention utilizes a redox amination tandem reaction to directly prepare the target product, eliminating the need for intermediate product purification processes. This simple process facilitates large-scale production.

The application provides an isolated Actin5C promoter, a construction method and an expression vector thereof, wherein the isolated Actin5C promoter is a partial region of a wild-type Actin5C promoter, and the partial region comprises a region from the 94th site in the downstream direction to the 400bp-600bp in the upstream direction from a transcription initiation site. The length of the isolated Actin5C promoter is short, so that the size of the expression vector containing the same is small, and the transfection efficiency is high. The expression vector can significantly improve the target protein yield of insect S2 cells in vitro, and has high scientific research and clinical application values.

The application discloses a cationic lipid molecule and a preparation method of the cationic lipid molecule for a lipid nanoparticle system, and provides a compound of formula I or a pharmaceutically acceptable salt thereof. In the application, a PEG lipid component is not contained in a three-component LNP composition, so that biological toxicity caused by the PEG lipid component is avoided, and safety is improved. Four-component LNP is simplified, and transfection efficiency is improved.

This invention belongs to the field of bioengineering technology, specifically relating to an improved transient transfection method for CHO-S cells. This transfection method includes the following steps: passage CHO-S cells for 3 days prior to transient transfection, adjusting the cell density to 0.35 × 10⁻⁶ cells / year. 6 Cells / mL were cultured in a shaker; on the day of transient transfection, the CHO-S cell density was adjusted to 4 × 10⁶ cells / mL. 6 -6×10 6 The cells / mL concentration was used to obtain a CHO-S cell suspension. Before transfection, the prepared solution A and solution B were mixed and allowed to stand to obtain a mixed droplet. The mixed droplet was added to the CHO-S cell suspension for culture to obtain recombinant CHO-S cells. The transfection method of the present invention not only has high transfection efficiency, but also obtains higher expression levels of the target antibody in CHO-S cells, providing a new approach for the development of protein biotherapeutic agents.

The invention relates to HIF-1alpha siRNA lipid nanoparticles and a preparation method thereof, and belongs to the technical field of pharmaceutical preparations. The preparation method comprises the following steps: S1, dissolving ionizable lipid, neutral lipid, cholesterol and pegylated lipid in an organic solvent to obtain an oil phase; the method comprises the following steps: dissolving HIF-1alpha siRNA in a first buffer solution to obtain a water phase; s2, mixing the oil phase and the water phase by adopting a micro-fluidic technology, diluting with a second buffer solution, removing the organic solvent, and concentrating to obtain the HIF-1alpha siRNA lipid nanoparticles, the targeted delivery of the HIF-1alpha siRNA is realized, so that the HIF-1alpha siRNA is effectively delivered to the rheumatoid arthritis diseased region through the delivery system to play a therapeutic role.

The invention discloses a signal peptide, a polypeptide-nucleic acid conjugate and a synthesis method and application thereof, and belongs to the technical field of molecular biology. The signal peptide is one of NPXY, YXXphi, [DE] XXX [LI] and DXXLL; wherein X is any amino acid, phi is any large-volume hydrophobic side chain amino acid, [DE] represents one of D and E, and [LI] represents one of L and I. In addition, the invention also provides application of the signal peptide in preparation of a compound capable of penetrating through a cell membrane or a body tissue barrier by being connected with a nucleic acid fragment. The invention also provides a polypeptide-nucleic acid conjugate which is a conjugate of any one of the signal peptides and siRNA (small interfering Ribonucleic Acid). After several signal peptides provided by the invention are coupled with the siRNA, the siRNA can be promoted to escape into cytoplasm from an endosome, so that the siRNA drug transfection efficiency is improved.

The present application relates to the technical fields of biological medicine and gene therapy, and particularly relates to a protonatable cationic lipid, a nucleic acid delivery system and application thereof. The protonatable cationic lipid has a structure as shown in formula (I), and contains a protonatable group such as a hydroxyl group or an amino group at the end. The protonatable cationic lipid can form a lipid nanoparticle for loading various nucleic acid substances. The lipid nanoparticle nucleic acid delivery system has high cell transfection efficiency in vitro, and can be targeted to organs such as liver in vivo to achieve efficient gene editing. The lipid nanoparticle constructed by the protonatable cationic lipid provided by the present application has high encapsulation efficiency, good stability and excellent delivery efficiency, and has a wide application prospect in the fields of gene therapy and cell engineering.

The present application relates to the technical field of biomedical materials, in particular to a non-viral gene carrier and a preparation method and application thereof. The non-viral gene carrier prepared by EDC / NHS cross-linking reaction of baicalin and polyethyleneimine can be used for gene delivery and preparation of a gene therapy drug. The present application further provides a nano-complex composed of the non-viral gene carrier and nucleic acid, which can be used for gene delivery and preparation of a gene therapy drug. The nano-complex can not only effectively deliver target genes into cells, but also synergize miR-34a and baicalin to jointly exert an anti-tumor effect, greatly enhancing the anti-tumor treatment efficacy.

The invention discloses preparation and application of a hyperbranched polymer with high gene transfection capacity, and belongs to the technical field of chemical synthesis and modification. The preparation method of the hyperbranched polymer with high gene transfection capacity comprises the following steps: adding an acrylate compound and organic amine into an organic solvent, dissolving, carrying out addition reaction, cooling, adding an end-capping reagent, and continuously reacting to obtain the hyperbranched polymer. The hyperbranched polymer prepared by the invention can solve the problem that the transfection efficiency is low after the existing precursor is polymerized, the transfection capacity of nucleic acid drugs in cells is remarkably improved while the biological safety is ensured, and the hyperbranched polymer is expected to replace viral vectors to be used as mainstream nucleic acid drug delivery vectors in the market.

This invention relates to a type I collagen truncated peptide and its delivery system. The amino acid sequence of the α1 chain of the type I collagen truncated peptide includes the sequence shown in SEQ ID NO.1; the amino acid sequence of the α2 chain of the type I collagen truncated peptide includes the sequence shown in SEQ ID NO.2. This invention achieves in situ expression of the type I collagen truncated peptide in vivo through a specially designed expression method, enabling self-sufficient supplementation of type I collagen. The truncated type I collagen retains the first half of the type I collagen sequence and part of the C-terminal sequence, thereby preserving the main functional components and the ability to form a triple helix, improving synthesis efficiency and mRNA stability; the removal of some metalloprotein binding sites increases its stability, and the absence of amino acid mutations ensures safety.

The invention relates to the technical field of crossing of a stem cell engineering technology, a nano-drug delivery system and a gene therapy technology, in particular to a PEI-LNPmiR-21 delivery system, a preparation method and application thereof and an engineered long-acting stem cell preparation. According to the delivery system, a lipid nanoparticle core is prepared by adopting a microfluidic technology, and low-molecular-weight PEI is covalently grafted on the surface of a lipid layer by utilizing carbodiimide chemistry, so that a nano compound with a core-shell structure is formed. The compound can efficiently deliver miR-21 into stem cells, and by regulating and controlling a PTEN / PI3K / Akt signal channel, the expression of a cell aging marker is remarkably inhibited, the proliferation capacity of the stem cells is enhanced, and the stemness characteristic of the stem cells is maintained.

Provided in the present invention are a cationic lipid compound and a composition for delivery of a nucleic acid, and the use. The compound is represented by the following formula (I). Further provided in the present invention are the use of a nano-lipid particle using the compound as a key component in the delivery of a nucleic acid; a component containing a delivery carrier; a preparation method; and a usage method.

The invention belongs to the technical field of gene engineering, and discloses a plasmid targeting a ZDHHC5 gene in order to solve the problems of transient effect, low knock-down efficiency and high off-target rate of the existing siRNA technology. The plasmid comprises a DNA sequence for expressing shRNA, preferably a SEQ ID NO: 1 site, adopts a stem-loop structure 'CTCGAG' to improve stability, and integrates green fluorescent protein and puromycin resistance genes on the basis of a lentiviral vector to realize visual screening and long-term expression after transfection. And by optimizing the shRNA design and the special transfection process, the specificity and repeatability are remarkably enhanced. Experiments show that the plasmid can efficiently inhibit ZDHHC5 expression, effectively inhibit proliferation in Huh-7 cells and induce apoptosis.

The present application relates to a small nucleic acid combination microneedle preparation method and its skin light damage repair purposes, specifically discloses a microneedle composition, including the complex of SNAP25 specific siRNA and nano selenium and hyaluronic acid, wherein the nucleotide sequence of the sense strand of siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of siRNA is as shown in SEQ ID NO.2.The microneedle composition of the present application can realize the stable delivery of siRNA, inhibit the expression of UV-induced skin photoaging related genes, increase the repair of skin extracellular matrix, significantly improve the skin light damage, and is suitable for clinical transformation application.

The application discloses a cyanobacterial nanogene carrier and a preparation method thereof. By irradiating cyanobacteria under appropriate wavelength light, toxic active oxygen is generated in the cyanobacteria, lysosomes are destroyed, and the gene carrier trapped in the lysosomes is released into the cytoplasm, so that the biological function of the gene can be better exerted, and the problems of the gene carrier in the prior art, such as cationic lipids, cationic polymers, difficult lysosome escape and low gene transfection efficiency, are overcome. Meanwhile, the preparation process is complex, other substances are easily introduced, and the safety is low, and the like are overcome, so that the application is more suitable for efficient delivery of the gene carrier, an effective treatment method is provided for treatment of tumors and the like, and the treatment of cancer and the like is beneficial.

The invention discloses a preparation method for realizing safe and effective antibacterial gene therapy of colorectal cancer by combining antibiosis and anti-tumor, and belongs to the field of drug carrier materials. The preparation method comprises the following steps: modifying lauric acid (LA) resisting fusobacterium nucleatum (Fn) and a targeting molecule 4-carboxyphenylboronic acid (PBA) into low-molecular-weight polyethyleneimine (OEI), and self-assembling with LA to obtain LA-OLP (AFs); the AFs is compounded with an anti-angiogenesis gene (sFlt-1), and the AFs and distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-mPEG) are co-assembled to obtain the nano particle LA (at) OLP / sFlt-1 / DSPE-mPEG (AFGTs-PEG). The method has the advantages of low cost and simple and feasible process flow, can be widely applied to the fields of materials science, biology, medicine and the like, and cooperates with antibacterial and anti-angiogenic gene therapy to enhance gene therapy on colorectal cancer.

The application provides a structural lipid compound and a preparation method and application thereof. The structural lipid compound has a structure shown in formula I. The structural lipid compound provided by the application can replace traditional neutral phospholipids to prepare drug-loaded lipid nanoparticles. The drug-loaded lipid nanoparticles containing the structural lipid compound have small particle sizes, uniform particle size distribution, and good loading and delivery effects on nucleic acid drugs.

The invention relates to the technical field of cell culture, and discloses a tracheal epithelial cell and application thereof, the cell is a guinea pig tracheal epithelial immortalized cell, and the preservation number is CGMCC No. 46763. In the invention, the cell has good susceptibility to respiratory pathogens, and has excellent transfection efficiency. The cells can be used for separating or culturing respiratory pathogens, preparing respiratory pathogen vaccines, screening and / or preparing medicines for preventing and / or treating diseases caused by the respiratory pathogens, serving as host cells for researching the respiratory pathogens, detecting the respiratory pathogens for a non-diagnostic purpose and constructing organoids. The method has high scientific research value and practical application potential.

The invention discloses a culture medium containing a yolk extract as well as a preparation method and application of the culture medium, and belongs to the technical field of cell biology. The core of the culture medium is that a basic culture medium is combined with specifically prepared yolk extract and yolk small molecule peptide. Wherein the yolk small molecule peptide is prepared by the following steps: homogenizing yolk, performing alkaline protease enzymolysis, performing enzyme deactivation, centrifuging, performing 5kDa ultrafiltration on supernate, collecting filtrate, performing 500Da nanofiltration for further treatment, and drying; the yolk extract is prepared by centrifugally filtering and sterilizing membrane-removed yolk. The culture medium is used for polyethyleneimine transfection, the cytotoxicity can be remarkably reduced through the synergistic effect of the yolk extract and the specific yolk small molecule peptide, and the cell survival rate and the transfection efficiency after transfection are improved. Therefore, the invention provides a safe, efficient and serum-free culture medium formula.

The application discloses a construction method and application of a SOX18 lentivirus recombinant tag carrier stably overexpressed in sheep hair papilla cells, and the method comprises the following steps: amplifying a full-length CDS product of a sheep SOX18 gene, double-enzyme cutting a carrier, connecting the full-length CDS amplification product of the SOX18 gene with a linearized carrier, and expanding the recombinant carrier, mixing the SOX18 lentivirus recombinant tag carrier with a lentivirus packaging auxiliary plasmid and an auxiliary plasmid, and transfecting 293T cells to realize packaging of the SOX18 lentivirus recombinant tag carrier, and transfecting the SOX18 lentivirus recombinant tag carrier into sheep hair papilla cells, and it is found that overexpression of SOX18 can inhibit apoptosis of the sheep hair papilla cells. The application significantly improves the transfection efficiency of the hair papilla cells, greatly increases the probability of integration of the SOX18 gene into the genome of the hair papilla cells, constructs a high-level in-vitro expression model of SOX18, and proposes an application of the SOX18 gene in inhibiting apoptosis of the hair papilla cells.

The present application belongs to the field of plant biotechnology and bioengineering breeding, and particularly relates to a kind of agrobacterium-mediated maize commercial backbone inbred line embryogenic callus genetic transformation and seedling regeneration system and application.The present application constructs maize callus genetic transformation system with commercial maize breeding backbone inbred line as background material and not dependent on seasonal restriction, effectively overcomes the problems of low maize callus induction frequency, difficulty in long-term subculture of embryogenic callus, low transformation and seedling regeneration frequency of callus, can efficiently and quickly produce transgenic plants in batches, and has important value for genetic improvement and gene function research of maize.