14 results about "Transient transfection" patented technology

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 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 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.

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.

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 relates to the technical field of gene therapy, in particular to a method for knocking out a Bax gene on the basis of an HEK293 cell line by utilizing a CRISPR / Cas9 technology, constructing a stable cell line expressed by foreign protein and improving the AAV packaging efficiency. The invention discloses a Bax2.6 cell strain. A pro-apoptosis factor Bax gene is knocked out from an HEK293T cell. Compared with a wild type HEK293T cell, the Bax2.6 cell strain provided by the invention shows higher cell activity under various stress conditions, the cell survival rate of the Bax2.6 cell strain is improved by at least 20% under the treatment conditions of small molecule inhibitors ZV-FMK, Nec-1, puromycin (Puro), cis-platinum (CDDP) and rotenone, and the Bax2.6 cell strain shows excellent environmental tolerance and production robustness. When the Bax2.6 cell strain provided by the invention is used for transient transfection and packaging of an adeno-associated virus (AAV) vector, the copy number of the obtained AAV virus can be up to 2 * 10 copies / mL, and the AAV packaging efficiency is improved by more than 10 times compared with that of a conventional HEK293T cell line.