35results about How to "High transfection efficiency" patented technology

The invention belongs to the field of biomedical materials, and particularly relates to a PBAE-DOPE-PEG2000 polymer-lipid nanocomposite and a preparation method and application thereof.The preparation method comprises the steps that a dimethyl sulfoxide-ethanol solution formed by mixing DOPE-PEG2000 lipid and PBAE serves as an oil phase, a sodium acetate / acetic acid buffer solution serves as a water phase, and the PBAE-DOPE-PEG2000 polymer-lipid nanocomposite is prepared through a microfluidic synthesis mode; the PBAE-DOPE-PEG2000 polymer-lipid nano composite material is prepared by the steps of carrying out ultrasonic treatment on the PBAE-DOPE-PEG The polymer-lipid nano composite material can efficiently load exogenous genes, the surface performance of the polymer-lipid nano composite material is changed and the stability of nano particles is improved by introducing DOPE-PEG2000 lipid, membrane fusion can be promoted, the risk of lysosome degradation is reduced, the transfection process of the exogenous genes in recipient cells is accelerated, and efficient expression in 293FT cells is realized. The efficient transfection of the polymer-lipid nanocomposite material is expected to show important application prospects in immune cell therapy or tumor cell therapy.

The invention discloses a yak intramuscular precursor fat immortalized cell line and a preparation method thereof. The method comprises the following steps: (1) obtaining yak primary intramuscular precursor fat cells; (2) performing SV40T lentivirus infection on the precursor fat cells in the yak muscle and screening cell strains: performing SV40T lentivirus infection by utilizing a 1 / 2 small-volume infection method to obtain immortalized cells after the precursor fat cells in the yak primary muscle are infected with SV40T, namely immortalized cells for short; puromycin is adopted to screen immortalized cells; and diluting the screened cells by using a limited dilution method, and carrying out enlarged culture to obtain the immortalized cell line. The yak intramuscular precursor fat immortalized cell line constructed by the invention has genetic homogeneity, the heterogeneity influence of cell populations is eliminated, and an in-vitro cell model is provided for subsequent research related to yak intramuscular fat cells.

The invention relates to the technical field of virus packaging. The invention provides a packaging method of a CAR (chimeric antigen receptor) virus. The packaging method comprises the following steps: mixing a plasmid pCMX-dR8.91, a plasmid pCMV-BaEV, a plasmid pAdVAnti and an opti-MEM culture medium, standing, mixing with a PEI transfection reagent, standing, mixing with a 293T cell, culturing, replacing the culture medium, continuously culturing, and harvesting the virus. A second-generation lentivirus packaging system is adopted, transfer plasmids are second-generation transfer plasmids needing to be activated by a Tat element, packaging plasmids are second-generation packaging plasmids pCMV-dR8.91 containing ag, pol, rev and tat, enveloping plasmids are second-generation enveloping plasmids pCMV-BaEV, plasmids pAdVAnti beneficial to improvement of transfection efficiency are added, and the packaging efficiency of the CAR virus is improved through the system.

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 invention discloses an anguilla japonica testis tissue cell AJTTC as well as an establishment method, a cryopreservation method, a resuscitation method and application of the anguilla japonica testis tissue cell AJTTC. The strain is preserved in the China Center for Type Culture Collection on January 22, 2024, and the preservation number is CCTCC NO: C202430. According to the method disclosed by the invention, the Japanese eel testis tissue is subjected to isolated culture by adopting a dry sticking method, then passage and cryopreservation are carried out, and the stable Japanese eel testis cell line is finally established, so that a new strategy mode and a research model are provided for researches on analysis of a Japanese eel germ cell differentiation mechanism, sex control, genetic breeding and the like.

The present invention relates to a PDRN derived from HEK293 cells and a preparation method thereof. The steps include: (1) centrifuging HEK293 cells, adding a lysate to obtain a lysate solution; (2) adding magnesium sulfate, protease and nuclease to the lysate solution, adding sodium dodecyl sulfate and sodium chloride after incubation, and obtaining a crude PDRN product after enzyme inactivation and alcohol precipitation; (3) dissolving the crude PDRN product in a sodium chloride solution, filtering, alcohol precipitation, washing, dehydration and drying to obtain PDRN. The PDRN preparation method provided by the present invention uses HEK293 cells as a source, uses a nuclease and a protease double enzyme one-pot method to prepare PDRN, has simple process control, does not have special environments such as high temperature, strong acid and alkali, and has relatively mild conditions. It does not need to add special equipment, and can be industrially produced on a large scale. In addition, the base composition ratio of the PDRN derived from HEK293 cells is completely consistent with that of the human body, has anti-inflammatory function and tissue repair activity, and has great application value in the fields of soft tissue repair, medical and aesthetic post-repair.

The invention provides a separated Actin5C promoter as well as a construction method and an expression vector thereof, the separated Actin5C promoter is a partial region of a wild Actin5C promoter, and the partial region comprises a region from the 94th site in the downstream direction of a transcription start site to 400bp-600bp in the upstream direction. The separated Actin5C promoter is short in length, so that the expression vector containing the Actin5C promoter is small in size and high in transfection efficiency. By utilizing the expression vector provided by the invention, the target protein yield of in-vitro insect S2 cells can be remarkably improved, and the scientific research and clinical application values are high.

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 invention discloses lentivirus transfection enhanced nanoparticles as well as a preparation method and application thereof, and belongs to the field of biomedical engineering. According to the invention, macromolecular polyethylene glycol with anions and a surfactant effect and micromolecular spermidine with cations and a cell phagocytosis enhancement inducing effect are used as raw materials for preparing the lentivirus transfection enhanced nanoparticles; nanoparticles with uniform particle size and high dispersity are prepared through electrostatic adsorption and low-temperature and low-speed self-assembly. After lentivirus is loaded on the nano-particles, lentivirus transfection enhanced nano-particles are obtained. The lentiviral transfection enhanced nanoparticles provided by the invention have very high transfection efficiency on tissue cells with relatively low cell membrane fluidity or relatively long growth cycle, have good biocompatibility, and can rapidly discharge cells after transfection, so that interference of empty carriers of the nanoparticles on subsequent research is reduced; therefore, the efficiency and accuracy of gene editing operation are greatly improved.

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 provides a CRISPR-Cas9 near-infrared two-region precise light-operated editing system delivery carrier based on an erythrocyte membrane, a preparation method and application, and particularly, the CRISPR-Cas9 gene editing system delivery carrier provided by the invention has the advantages of low cytotoxicity, efficient transfection, high yield, targeting property, space-time controllability, easiness in large-scale production and multifunctionality. Compared with a traditional liposome transfection reagent, the carrier shows lower cytotoxicity, protects the CRISPR / Cas9 compound from being influenced by the in-vivo environment, and ensures that the CRISPR / Cas9 compound is smoothly delivered to target cells. By embedding a specific targeting receptor, efficient targeting delivery is realized, and the pertinence and effectiveness of treatment are improved. And by combining a near-infrared two-region material, the time-space precise control of a CRISPR / Cas9 system is realized, and deeper tissue penetrating power and smaller biotoxicity are achieved. The preparation process is simple, the method is suitable for industrial production and clinical transformation, and new possibility is provided for gene therapy and personalized medical treatment.

Provided is a method for modifying a cell, which comprises the following steps: transfecting a target molecule and a screening marker molecule to a cell in a non-viral manner, so that the cell expresses the target molecule and the screening marker molecule. Also provided is a modified cell obtained using the method. The method can effectively enrich the desired cell and increase cell positivity with respect to the target molecule.

The invention discloses a Bac-to-Bac baculovirus expression system, a recombinant baculosome, a recombinant baculovirus and an application of the recombinant baculovirus. The Bac-to-Bac baculovirus expression system comprises a donor plasmid and a DH10Bac competent bacterium, and is characterized in that the donor plasmid is a recombinant plasmid; the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene segment expression cassette; the DH10Bac competent bacteria comprise a baculovirus genome plasmid bacmid and an auxiliary plasmid, and the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression cassette; the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different. The recombinant bacilli are prepared on the basis of the Bac-to-Bac baculovirus expression system. The recombinant baculovirus is obtained by transfecting the recombinant baculovirus into insect cells and culturing the recombinant baculovirus. The recombinant plasmid prepared by the invention is purer and is not polluted by helper plasmids and donor plasmids.

The invention provides a guanidyl-derived ionizable lipid material. The guanidyl-derived ionizable lipid material is a compound as shown in a formula (I), and a solvent crystal substance or an isomeride or a pharmaceutically acceptable salt of the compound as shown in the formula (I), in the imgabs0 #, R1 is a substituted or non-substituted linear alkyl alcohol of C2-C6, and R2 is a substituted or non-substituted linear alkyl alcohol of C2-C6; r < 2 >, R < 3 >, R < 4 > and R < 5 > are all C6-C18 substituted or unsubstituted alkyl, alkenyl or alkynyl, and comprise or do not comprise one or more heteroatoms; r < 2 >, R < 3 >, R < 4 > and R < 5 > are the same or different; n is a positive integer from 3 to 18; and m is a positive integer from 3 to 18. The guanidyl-derived ionizable lipid material disclosed by the invention has excellent biocompatibility, efficient drug delivery performance, efficient transfection efficiency, wide application potential and simplicity and convenience in preparation and can be produced on a large scale, and the stability, targeting and drug release control of an inner body membrane are effectively destroyed.

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 invention provides an SLCO1B1 overexpression cell strain as well as a construction method and application thereof, and belongs to the technical field of molecular biology. The cell strain constructed by the invention has a good probe substrate transfer function, and due to the characteristic, the cell strain has important application value in drug development and research. The invention not only provides a construction method, but also covers the application of the overexpression cell strains in a plurality of fields, including research on a drug liver uptake mechanism, a drug liver metabolic pathway, drug interaction, a disease mechanism, drug screening or optimization, drug toxicity evaluation and drug curative effect evaluation. The applications show wide potential of the SLCO1B1 overexpression cell strain in drug development and research, and a powerful tool is provided for researchers in related fields.

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.