22 results about "Transient transfection" patented technology

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 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 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.

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.

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 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.

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 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.

The present application relates to the technical field of antibodies, in particular to humanized anti-beta2-GP1 monoclonal antibody, preparation method and application thereof. The present application constructs a natural full human source library, adopts phage display technology, screens out a high affinity antigen binding fragment, assembles a variable region sequence with an IgG / IgM / IgA constant region to obtain a full-length IgG / IgM / IgA type antibody sequence. The anti-beta2-GPI antibody is expressed by transient transfection, prepared by protein A / L affinity purification method and subjected to binding activity determination. The results show that the specific beta2-GPI antibody obtained by the above method has strong antigen binding activity, strong specificity and good affinity.

The invention discloses an efficient transient transfection method for human primary chondrocytes based on electroporation, and belongs to the field of cell transfection, and the method comprises the following specific steps: resuspending the human primary chondrocytes in an electrotransfection mixed solution, sucking a resuspension solution with a set volume, carrying out primary electroporation under the conditions that the pulse intensity is 550V and the pulse duration is 20ms, and carrying out secondary electroporation to obtain the human primary chondrocytes. Then, transferring the electroporated cells into a preheated culture medium for culturing, so that efficient instantaneous transfection can be realized; as a rapid, efficient, stable and convenient primary cell transient transfection method, the problems of low cell transfection efficiency, unknown electroporation parameters, low cell survival rate and the like caused by a cartilage extracellular matrix barrier effect in the prior art are solved, and the most basic conditions are provided for carrying out functional experiments in primary cells.

The invention discloses application of DMA in preparation of a preparation for improving transient expression quantity of CHO-S cells. In the transient transfection process of CHO-S cells, 0.25% N, N-dimethylacetamide solution (DMA) is added and optimized plasmids are used: a section of DNA sequence as shown in SEQ ID No.1 is additionally added behind a promoter of conventionally used pCMV plasmids, so that the expression level of the transient transfected CHO cells can be improved, and the performance of an antibody is not changed. From the aspect of economic value, the production cost of antibody raw materials can be greatly reduced, and the cost performance is relatively high.