37 results about "Transient transfection" patented technology

The invention relates to the field of biomedicine, and discloses a method for screening KRAS mutant interacting proteins, an HEK293T cell line for continuously expressing an HA-Turbo ID-KRAS variant is established by utilizing lentivirus transduction, the stability of transgene expression is ensured through puromycin screening, and variation caused by transient transfection is reduced as much as possible. After a Turbo ID mediated proximity marker is activated by biotin treatment, KRAS proximity binding proteins are covalently biotinylated, captured by NeutrAvidin bead purification, and analyzed by quantitative mass spectrometry. According to the invention, two potential KRAS mutation treatment target proteins of LZTR1 and LAMTOR1 can simultaneously inhibit activation and efficacy exertion of three different mutants of G12C, G12D and G12V of the G12 mutant, compared with drugs on the market that a single target of G12C directly targets the KRAS mutant, a brand new KRAS mutation targeting intervention strategy is provided, the clinical curative effect can be further improved, and the occurrence rate of secondary drug resistance can be effectively reduced.

The invention belongs to the technical field of gene transfection, and discloses a transfection enhancement method for improving transient transfection efficiency of plasmids, which comprises the following steps: step 1, plasmid extraction: extracting plasmids by using an endotoxin-removing plasmid extraction kit; 2, preparing cells before transfection; and the cells are recovered and cultured, so that the density of the cells reaches 70-90% during transfection. Step 3, transfection: 1) adding a proper amount of the serum-free culture medium into one centrifugal tube under a sterile condition, then adding a transfection reagent, and fully and uniformly mixing; 2) adding the serum-free culture medium and a reinforcing agent into the other centrifugal tube, fully and uniformly mixing, and then adding plasmids. According to the scheme, the transient transfection efficiency and effect of plasmids are comprehensively improved from plasmid extraction, cell preparation, transfection compound formation to subsequent treatment through a series of optimization steps. Meanwhile, cytotoxicity and stress reaction are reduced, and the cell survival rate and the target protein expression quantity are improved, so that the transfection method becomes an efficient and reliable transfection method.

The invention belongs to the technical field of biology. The invention provides a recombinant protein, an amino acid sequence of the recombinant protein is formed by repeatedly connecting dominant epitopes of canine parvovirus VP2 protein in series, the amino acid sequence of the canine parvovirus recombinant protein is converted into a corresponding nucleotide sequence by adopting escherichia coli preferred codons, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed; therefore, the expression quantity of the recombinant protein in escherichia coli is improved. The invention also relates to a method for establishing a bacteriophage library by immunizing a mouse with the recombinant protein, obtaining a corresponding canine parvovirus VP2 protein single-chain antibody scfv sequence through panning screening, constructing a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, expressing a monoclonal antibody through transient HEK293F cells, and purifying the monoclonal antibody, thereby obtaining the canine parvovirus VP2 protein single-chain antibody. An optimal monoclonal antibody pairing combination is determined through an immunofluorescence orthogonal experiment.

Provided are a universal cell system, a construction method therefor, and use thereof. Universal mesenchymal stem cells are constructed by means of the combination of electroporation and CRISPRoff gene editing, and the cells can survive stably in a pulmonary inflammatory pathological environment to provide sustained treatment, significantly improve multiple indicators of acute pneumonia, solve immune rejection, and provide new cell therapies and ideas for acute pneumonia. CRISPRoff is introduced into universal CTL cells by means of electroporation technology, targeting a specific region and inhibiting the expression of HLA-I in T cells; thus, T cells with low immunogenicity are constructed, and the cells can avoid allogeneic immune rejection. The universal cell system combines the advantages of both transient transfection and CRISPRoff gene editing, has great significance in the field of cell therapy, provides a more comprehensive and effective solution for the treatment of various diseases, and has a wide application prospect.

The invention discloses siRNA (small interfering Ribonucleic Acid) and application thereof in preparation of a medicine for treating BPD-like lung injury, the siRNA is NR4A1-targeted siRNA and comprises a first chain and a second chain, the first chain and the second chain are complementary and jointly form an RNA dimer, and the nucleotide sequence of the first chain is shown as SEQ ID No.2. The invention further discloses a preparation method of the siRNA for treating the BPD-like lung injury and application of the siRNA to preparation of the medicine for treating the BPD-like lung injury. It is found for the first time that an overexpression transient transfection cell strain is constructed aiming at the NR4A1 gene, specific siRNA is designed, NR4A1 expression is specifically increased or reduced in lung epithelial cells, cell proliferation can be correspondingly inhibited by increasing the NR4A1 expression, and meanwhile the purpose of reversing BPD-like lung injury can be achieved by injecting an in-vivo biological agent interfering NR4A1 gene expression into an animal model. The possibility is provided for the clinical development of medicines for treating the BPD-like lung injury caused by the IUI based on the NR4A1 gene, and the application value and the prospect are relatively high.

The invention belongs to the technical field of gene engineering, and particularly relates to a CHO cell transient transfection method and application thereof.The CHO cell transient transfection method comprises the following steps that subcultured CHO cells are resuspended through a transfection culture medium, the cell density in CHO cell suspension is 0.8 * 10 < 7 >-1.5 * 10 < 7 > cells / mL, a PEI transfection reagent containing 40 kDa linear PEI is used, plasmid DNA and the PEI transfection reagent with the mass ratio being 1: (1-6) are added into the CHO cell suspension for transfection, and transfection is conducted after the PEI transfection reagent is added into the CHO cell suspension; the addition amount of plasmid DNA in each milliliter of CHO cell suspension is 3.0-7.0 [mu] g, transfecting for 20-24 h, then adding a cell suspension culture medium, transfecting for 48-72 h, then repeatedly transfecting, and culturing to obtain the recombinant CHO cell. According to the method, the transfection efficiency can be improved, more cells for expressing the recombinant protein can be obtained, and the transient expression of the target protein can be remarkably improved.

The invention discloses an instantaneous transfection method for high-yield CHO-K1 (CHO-K1) by taking linearized PEI (Polyetherimide) as a transfection reagent. The antibody titer reaches about 1g / L within 10 days by replacing a culture medium, zero incubation of a compound, a specific DNA proportion and temperature regulation, the dosage of an expression vector is remarkably reduced, the research and development period is shortened, and the method is suitable for early research and development of antibody drugs and sample preparation.

The invention relates to the technical field of antibodies, in particular to a humanized anti-beta2-GP1 monoclonal antibody as well as a preparation method and application thereof. The full-length IgG / IgM / IgA type antibody sequence is obtained by constructing a natural full human source library, adopting a phage display technology, screening out a high-affinity antigen binding fragment, and assembling a variable region sequence and an IgG / IgM / IgA constant region. An anti-beta2-GPI antibody is expressed through transient transfection, a protein A / L affinity purification method is adopted for preparation, and binding activity determination is carried out. Results show that the specific beta2-GPI antibody obtained by the method is high in antigen binding activity, high in specificity and good in affinity.

The application discloses a method for constructing a cell membrane fusion model induced by a novel coronavirus-like particle, and belongs to the technical field of biology. The method comprises the following steps: constructing a virus-like particle containing a SARS-CoV-2 spike protein gene and an RNA packaging sequence PS9, using HEK-293T cells as packaging cells, co-expressing structural proteins M, E, N and S protein, and realizing assembly of VLPs without replication ability. Subsequently, the VLPs are used to infect immortalized type II alveolar epithelial cells, S protein expression is induced and cell-cell membrane fusion is mediated without the need of BSL-3 laboratory conditions. The formation of fused giant cells can be directly observed through ZO-1 protein immunofluorescence staining, the model is stable, has high biological safety, and is repeatable in vitro. The application overcomes the safety risk of live virus infection and the authenticity problem of a transient transfection model, can be used for research on a SARS-CoV-2 virus membrane fusion mechanism, and has a good application prospect.

This invention relates to a detection kit and preparation method for enhancing the detection efficacy of autoimmune encephalitis (CBA) antibodies. The kit comprises genetically engineered 293T cells stably integrating and co-expressing the eIF2α S52A mutant and HSP70, and transiently transfected with CBA-associated antigens. The preparation method includes constructing a co-expression vector, establishing a stable transfection cell line, stress preconditioning treatment, antigen transfection, and kit assembly. A method for assessing antibody affinity maturation using this kit is also provided. By synergistically modifying detection cells at three levels—translational regulation, protein folding, and anti-apoptosis—the invention significantly increases antigen expression, reduces apoptosis, and improves detection sensitivity and specificity. Furthermore, it integrates antibody affinity assessment into CBA detection for the first time, providing auxiliary information for clinical staging.

The application discloses a transient transfection method for improving recombinant protein expression of CHO cells, and relates to the technical fields of eukaryotic cell gene transfection and animal cell culture. A cell suspension is obtained by mixing a CHO cell and a serum-free CHO cell basic culture medium; DNA and a transfection reagent are added into the cell suspension to mix a transfection culture, 5-azadeoxycytidine acid solution is added into the transfection culture at 0h-24h to form a transfection system and continue the transfection culture; the transfection system is added with a feeding culture medium at 1d-4d after the transfection culture to carry out suspension culture; the suspension culture is centrifuged at 7d to separate and harvest cell culture supernatant to complete recombinant protein expression. According to the application, 5-azadeoxycytidine acid is added into the transfection culture at a specific concentration, so that the expression of recombinant protein of the CHO cell is significantly improved during the transfection expression.

This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to a method for codon optimization of human coagulation factor IX and the recombinant human coagulation factor IX encoding gene and its applications. Based on RiboDecode, this invention optimizes relevant parameters to improve the codons of the target gene, thereby increasing the expression level and activity of the target gene in the host. The optimized FIX gene mRNA molecule exhibits good stability and high translation efficiency; further addition of GCCACC upstream of the start codon further enhances expression efficiency. This invention optimizes transfection-related operations, identifies optimal transfection conditions, and achieves maximum expression levels through transient transfection, effectively improving the yield of recombinant human coagulation factor IX. The results of the examples show that the yield of recombinant human coagulation factor IX can reach 1.58±0.10 mg / L, and the coagulation activity can reach 66.8±0.51%, with significant optimization of all key indicators.

The invention discloses siRNA for inhibiting ASGR1 expression and application of the siRNA, and belongs to the technical field of molecular biology. The preparation method comprises the following steps: firstly, designing siRNA according to an ASGR1 sequence, and carrying out transient transfection on the designed siRNA; according to the present invention, RT-qPCR (reverse transcription-quantitative polymerase chain reaction) and Western blotting are adopted to verify the ASGR1 expression inhibition effect; experiments prove that the siRNA molecule provided by the invention can be used for effectively inhibiting the expression of ASGR1; the protein is further delivered into a body through a carrier, the mRNA level is obviously reduced, protein expression is obviously inhibited, and a new method is provided for research and development of drugs for treating fatty liver, hyperlipidemia and high cholesterol.

The present invention relates to a method for rescue of Vesicular Stomatitis Virus (VSV) from DNA in a HEK293 cell line or a HEK293 cell line adapted to suspension growth comprising (a) providing cells from a HEK293 cell line or a HEK293 cell line adapted to suspension growth in cell culture, (b) transfecting the cells with at least one plasmid, wherein the at least one plasmid comprises (i) an expression cassette comprising a VSV genomic cDNA; (ii) at least one expression cassette encoding VSV nucleoprotein (N) protein, VSV phosphoprotein (P) protein, and VSV large (L) protein; and (iii) an expression cassette encoding SV40 Large T antigen; (c) culturing the transfected cells; and (d) harvesting the cell culture supernatant comprising the rescued VSV. Also provided is the use of a HEK293 cell line or a HEK293 cell line adapted to suspension growth for rescue of Vesicular Stomatitis Virus (VSV) or the use of a plasmid encoding SV40 Large T antigen for rescue of Vesicular Stomatitis Virus (VSV) in a HEK293 cell line or a HEK293 cell line adapted to suspension growth HEK293-F cells by means of transient transfection.

The application discloses a shark-derived synthetic nanobody targeting green fluorescent protein and an application thereof. The nucleotide sequence of the nanobody is as shown in SEQ ID NO:1-4, and the amino acid sequence is as shown in SEQ ID NO:5-8. The preparation method comprises the following steps: screening a green fluorescent protein specific nanobody sequence from a shark-derived synthetic antibody library, performing eukaryotic expression vector construction, then performing transient transfection in HEK 293F cells for expression, and finally obtaining the shark-derived synthetic nanobody targeting the green fluorescent protein. The shark-derived synthetic nanobody is used for affinity purification of the green fluorescent protein. The nanobody of the application is derived from Chlamydoselachus anguineus, has small molecular weight, strong stability, strong tissue penetration ability and can recognize hidden antigen epitopes, and can be used in the fields of affinity purification, immunological detection, molecular imaging, immunosensing probes and the like of the green fluorescent protein.

Nucleic acids comprising multifunctional double-stranded break reporter constructs for stable or transient transfection into a cell, as well as detecting a type of double-stranded break repair mechanism in a cell. Methods for detecting types of double-stranded break repair mechanism in a cell, as well as vectors and cells comprising nucleic acids comprising multifunctional double-stranded break reporter constructs.

The invention provides a composite enhancer for improving transient transfection expression quantity of mammalian cells and application thereof, and relates to the technical field of cell culture, the composite enhancer comprises six functional modules of an osmotic pressure protective agent, a functional amino acid combination, vitamins and coenzymes, an ROS alleviator, a shear force protective agent and anti-caking agent, and a cell growth promoting and Qp improving agent. All the modules form a synergistic system through accurate matching, data shows that the transient transfection expression quantity of mammalian cells can be increased to 1.5 g / L or above when the system is used for transient transfection culture of the mammalian cells, the cell viability maintenance rate is increased by 20% or above, meanwhile, the polymer content is controlled to be smaller than 7%, and the cell viability maintenance rate is increased by 20% or above. The technical problems that in the prior art, an additive used for improving the transient transfection expression quantity cannot simultaneously solve the problems that the cell viability attenuation is fast, the expression quantity is low and the product quality is unstable are solved.

The invention discloses a method for producing a target protein. The method comprises the following steps: providing a host cell, a vector containing a nucleic acid sequence for coding the target protein and transposase; transfecting the host cell using the vector and the transposase to obtain a transfected cell; culturing the transfected cells to form a culture; and harvesting the culture to obtain the protein of interest from the harvested culture, wherein the cells of the culture are not passaged prior to harvesting the culture.

To provide a method for producing a human embryonic kidney (HEK) cell line that does not express functional endogenous dihydrofolate reductase (DHFR) and does not express functional endogenous glutamine synthetase (GS).SOLUTION: The present invention provides a method comprising the steps of: (a) mutating or knocking out the endogenous DHFR gene and the GS gene in a HEK cell line; (b) stably or transiently transfecting the HEK cell line with an AAV vector genome comprising a first heterologous nucleic acid sequence encoding a therapeutic protein or an inhibitory nucleic acid sequence and a first selectable marker; and (c) stably or transiently transfecting the HEK cell line with a second heterologous nucleic acid sequence and a second selectable marker.SELECTED DRAWING: Figure 2

The application discloses a method for improving the transient transfection efficiency of HEK293 suspension cells, comprising the following steps: (1) strain activation: picking the E. coli strain preserved in a-80 DEG C low-temperature refrigerator to inoculate a solid culture medium slope which is sterilized, and culturing overnight at 37 DEG C to activate the strain; (2) E. coli 5 alpha shake flask culture: picking the colony on the slope to inoculate into liquid culture medium, and culturing overnight at 220 rpm and 37 DEG C; (3) plasmid extraction: centrifuging the E. coli in the step (2), using an endotoxin-free plasmid large extraction kit to extract the plasmid, and measuring the plasmid concentration and purity after extraction. The HEK293 suspension cells used in the experiment have the advantages of fast growth speed, strong cell activity, easy control, short experimental period, simple operation and good experimental repeatability.

This invention belongs to the field of biotechnology. It provides a recombinant protein whose amino acid sequence consists of two dominant epitopes of cardiac troponin I (cTnI) repeated in tandem. The amino acid sequence is converted into a corresponding nucleotide sequence using E. coli-preferred codons. The nucleotide sequence is then chemically synthesized and a recombinant expression vector is constructed, thereby increasing the expression level of the recombinant protein in E. coli. This invention also involves using this recombinant protein to immunize mice to establish a phage library, obtaining the corresponding cardiac troponin I single-chain antibody scfv sequence through panning and screening, constructing a complete mouse IgG1 antibody sequence expression vector from the obtained scfv sequence, expressing the monoclonal antibody via transient transfection of HEK293F cells, purifying the monoclonal antibody, and determining the optimal monoclonal antibody pairing using an immunofluorescence orthogonal experiment.

The invention discloses a chimeric antigen receptor targeting CD26 and an application of the chimeric antigen receptor. According to the present invention, the binding recognition sequence of the natural ligand Caveolin-1 of CD26 is adopted as the antigen binding region to be constructed in the CAR structure, and the mRNA-LNP particle is adopted to instantaneously transfect the T cell so as to obtain the instantaneous CAR-T cell with the targeting CD26 killing function, such that the safety is further improved; the CAR-T cell provided by the invention has a good effect of removing CD26 positive senescence cells and hepatic fibrosis cells, and is a novel CAR-T cell which is expected to be applied to clinical anti-aging and hepatic fibrosis treatment.

The invention belongs to the technical field of medicines, and particularly relates to an FRET (Fluorescence Resonance Energy Transfer)-based A-type and E-type botulinum toxin detection cell sensor and application thereof. The invention accidentally finds that EGFP / tDimer2 is used as a donor fluorophore and a receptor fluorophore, SNAP-25 is used as a connexon, and fusion expression of EGFP-SANP-25-tDimer2 is carried out; the method comprises the following steps: carrying out transient transfection on a Neuro-2a cell by using a plasmid for expressing EGFP-SANP-25-tDimer2, so as to construct a cell sensor; the cell sensor can be used for evaluating the biological activity of materials containing BoNT / A and BoNT / E in the environment and in the pharmaceutical production process. The in-vitro screening requirement of the botulinum toxin inhibitor can also be met, and the kit can also be used for diagnosing BoNT / A and BoNT / E botulinum toxin poisoning.

The invention provides a bovine herpesvirus type 1 (BHV-1) infectious clone as well as a construction method and application thereof, and belongs to the technical field of virus genetic manipulation. According to the invention, a group of cosmid combinations for constructing the BHV-1 infectious clone is screened based on a Fosmid library, and the cosmid combinations comprise FosBHV-1-a, FosBHV-1-b, FosBHV-1-c and FosBHV-1-d. According to the cosmid combination, BHV-1 can be efficiently rescued by infecting a susceptible cell line MDBK after transient transfection of HEK293F suspension cells. According to the method provided by the invention, the BHV-1 virus seed bank can be stably obtained, the subsequent large-scale production process of BHV-1 genetic engineering vaccines is facilitated, and the production cost can be effectively reduced.

Disclosed are T cell-specific membrane-fusogenic virus-like particles, a preparation method therefor and a use thereof. The T cell-specific membrane-fusogenic virus-like particles are prepared by transiently transfecting 293T cells with expression plasmids for human T cell-specific membrane-fusogenic protein, expression plasmids for mRNA-loaded protein, and expression plasmids for CAR mRNA; the molar ratio of the expression plasmids for the mRNA-loaded protein to the expression plasmids for the CAR mRNA is 1:2-4. In the present invention, by using a viral budding mechanism to produce T cell-specific membrane-fusogenic virus-like particles, a loaded CAR mRNA drug can be specifically delivered into cytoplasms of T cells, thereby generating CAR-T cells.

The invention provides a method for preparing a recombinant virus vector in eukaryotic cells. In particular, the present invention relates to a method for preparing a recombinant viral vector by transient transfection of eukaryotic cells, comprising the step of transient transfection of cells in a cell culture wherein the cell culture has an unexpectedly high cell density of at least 25 MVC / mL. After the transfection step, an optional cell recovery step may be performed according to the host cell type, the exogenous nucleic acid, and the transient transfection method used. After the transfection step, or after an optional cell recovery step, the preparation step of the recombinant viral vector begins at an unexpectedly high cell density of at least 20 MVC / mL, a limited dilution or no dilution being performed after the transfection step.

Methods and systems for improving the scalability and associated efficacy of continuous transfection processes, such as continuous transient transfection processes. The continuous transfection system includes at least a first input line and a second input line, wherein the first input line and the second input line are configured for sterile, fluid connection to a first reagent housing and a second reagent housing, respectively, a mixing chamber that is fluidly connected to the at least first and second input lines, a delivery line fluidly connected to the mixing chamber, wherein the delivery line is configured for sterile, fluid connection to a cell culture vessel, and a flow rate control component, such as a pump operable to control flow rate of liquid into the at least first and second input lines, throughout the mixing chamber, and into the cell culture vessel.

Application of adeno-associated virus (AAV) vectors in large animal studies and clinical trials often requires high-titer and high-potency vectors manufactured at a scale that can support large doses and / or large populations. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. Here we report novel methods and compositions for high-titer AAV production with several key improvements and advantages: (1) a one-step cloning of therapeutic AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines.

The present invention provides bicyclic heteroaryl BRAF negative allosteric modulators, their preparation methods and uses. The general structural formula of the bicyclic heteroaryl BRAF negative allosteric modulators or their pharmaceutically acceptable salts is shown as Ⅰ. The present invention synthesizes bicyclic heteroaryl BRAF negative allosteric modulators that are potent and have good drug-likeness and target the BRAF kinase, overcoming the drug resistance phenomenon caused by BRAF protein dimerization. Verified by test examples, the compounds synthesized by the present invention have significant cell proliferation inhibitory activity against BRAF WT , BRAF V600E and the p61-BRAF V600E tumor drug-resistant cell lines constructed by lentivirus transient transfection, and at the same time have significant kinase inhibitory activity, showing good application prospects; Ⅰ.

The invention discloses a method for improving the expression quantity of transient transfected recombinant protein of CHO-K1 cells. The method comprises the following steps: S1, recovering the CHO-K1 cells and subculturing the CHO-K1 cells; s2, adding the cells of which the density is adjusted into a recombinant protein DNA-PEI compound to carry out transient transfection; s3, adding an additive into the transfection system, wherein the additive is a mixture of a sodium butyrate solution and an EN3 solution. According to the method, the culture medium, the cell density before transfection and the proportion of recombinant protein DNA to PEI in a transfection system are optimized, and the additive is added after transfection, so that the antibody expression after cell transfection is promoted, and the expression level of the recombinant protein is effectively improved.