34 results about "Stable transfection" patented technology

The invention discloses a recombinant protein for detecting an anti-myelin sheath related glycoprotein MAG antibody and a detection reagent of the recombinant protein, and belongs to the technical field of neural immunodiagnostics, five amino acid sites in MAG protein are subjected to specific amino acid point mutation replacement to obtain an MAG protein mutation sequence, the MAG protein mutation sequence is fused with GlcATP enzyme and HNK-1ST enzyme full-length sequences to construct a lentiviral expression vector, and the recombinant protein and the detection reagent are used for detecting the anti-myelin sheath related glycoprotein MAG antibody. According to the present invention, three genes are constructed, and are stably transfected in a host cell so as to stably express the three genes in one cell at the same time, such that the constructed recombinant protein and the wild type MAG protein have similar anti-MAG IgM antibody binding domain, and on the premise of not using GlcATP enzyme and HNK-1ST enzyme small molecule agonists, the sensitivity and the specificity of the detection of the MAG autoimmune antibody by using the CBA method are improved; the method is suitable for preparing related detection reagents for in-vitro detection of the IgM type anti-MAG antibody.

The invention discloses a stably transfected cell strain for evaluating fibroblast autophagy flux and a construction method of the stably transfected cell strain. The construction method comprises the following step: infecting fibroblasts by using HBAD (Hepatitis B Antigen)-red fluorescent protein-EGFP-LC3 autophagy double-labeled adenovirus. The construction method can be combined with a super-resolution microscope to dynamically observe the autophagy intensity change in the primary fibroblasts through an imaging method.

The invention discloses a monoclonal cell strain screening method based on fluorescence intensity, which comprises the following steps: determining a cell line, and stably transfecting fluorescent protein to cells of the cell line; a microwell plate is selected, a datum hole is set, the cells stably transfected with the fluorescent protein are separated and put into the microwell plate, a plurality of cells are put into the datum hole, and only one cell is put into the other holes; screening out single-cell pores with only one cell from the rest of pores except the reference pore; screening out clone group holes capable of growing clone groups; respectively digesting cells in the cloning group pores and then transferring the cells into new pores for culturing; screening out fluorescent screening holes; splitting proliferation holes are screened out; and determining the cells in the division proliferation hole as the screened monoclonal cell strain. According to the monoclonal cell strain screening method provided by the invention, the monoclonal cell strain which is high in fluorescence intensity and can proliferate at a normal speed can be screened, compared with fluorescence microscope observation and imaging, the method is convenient and rapid, and errors possibly generated by comparing the fluorescence intensity with human eyes are reduced.

The invention belongs to the technical field of biomedicine, and particularly relates to application of SOX4 in preparation of a preparation for improving sensitivity of cervical cancer to chemotherapeutic drugs, and the amino acid sequence of the SOX4 is as shown in SEQ ID NO.1. An SOX4 knocked-down stable transfection cell line is constructed in a Hela cell, then carboplatin and oxaliplatin are respectively added into the SOX4 knocked-down Hela cell, and the IC50 of the cell to the carboplatin and the oxaliplatin is obviously reduced, which indicates that the sensitivity of the Hela cell to the carboplatin and the oxaliplatin is enhanced by SOX4 knock-down. By reducing the expression of SOX4, the sensitivity of Hela cells to chemotherapeutic drugs such as carboplatin and oxaliplatin is enhanced, so that the treatment effect of the chemotherapeutic drugs such as carboplatin and oxaliplatin is effectively improved. Results of the invention prove that inhibition of SOX4 is beneficial to recovery of chemosensitivity of cervical cancer cells to drugs such as carboplatin and oxaliplatin, and a new treatment strategy is provided for treatment of drug-resistant cervical cancer.

The invention relates to the technical field of bioengineering, and aims to provide a CHO stably transfected cell strain capable of expressing a recombinant antibody capable of specifically recognizing CSFV2 type Erns protein. The cell strain is preserved in the China Center for Type Culture Collection, the preservation name is Chinese hamster ovary cell gs weak strain CHO-9B1-C, and the preservation number is CCTCC NO: C2024393. The invention also provides application of the cell strain in preparation of a diagnostic kit for specifically detecting the CSFV2 type hog cholera virus. The CHO stably transfected cell strain provided by the invention has the advantages of short monoclonal antibody production cycle, high consistency and high repeatability, is suitable for large-scale production, and is easy to transform.

The invention discloses a Betti alkali derivative as well as a synthesis method and application thereof. According to the present invention, the substituent group on the molecular formula of the Betti base is regulated so as to obtain the Betti base derivative with high EGFR inhibition activity; the betati base derivative has a relatively strong inhibitory activity effect on an H3255 cell strain highly expressing EGFRL858R, a PC-9 cell strain of EGFRdel19, an H1975 cell strain of EGFRL858R / T790M or a stably transfected Ba / F3-EGFRL858R / T790M / C797S cell strain, can effectively inhibit the growth of lung cancer cells, and can be applied to preparation of drugs for treating, preventing, delaying, assisting in treating or treating diseases related to EGFR activity passing and drugs for treating tumor diseases.

The invention provides an organoid for simulating a dormancy and recurrence process of breast cancer cells as well as a construction method and application of the organoid. The construction method comprises the following steps: culturing breast cancer cells, and carrying out flow sorting on surface markers CD44 and ITGB4 to obtain an ITGB4 low-expression (ITGB4low) cell population; packaging a lentiviral vector, infecting a cell population with low expression of ITGB4, and carrying out flow sorting to obtain a stably transfected cell population; and carrying out induction treatment on the stably transfected cell population and culturing to obtain the organoid. The organoid provided by the invention provides powerful support for prevention and / or treatment of metastatic breast cancer, and shows extraordinary application prospects in accurate and efficient screening of drugs for targeting dormancy-recurrence process.

The present invention discloses a recombinant protein for detecting anti-myelin associated glycoprotein (MAG) antibody and a detection reagent thereof, belonging to the technical field of neuro-immunodiagnosis. By performing specific amino acid point mutation substitutions at five amino acid positions in the MAG protein, a MAG protein mutant sequence is obtained, and then it is fused with the full-length sequences of GlcATPase and HNK-1ST enzyme to construct a lentiviral expression vector, which is stably transfected into host cells, enabling the simultaneous stable expression of the three genes in one cell. The constructed recombinant protein has a similar anti-MAG IgM antibody binding domain to the wild-type MAG protein, and without using small molecule agonists of GlcATPase and HNK-1ST enzyme, it improves the sensitivity and specificity of detecting MAG autoantibodies by the CBA method, and is suitable for preparing related detection reagents for in vitro detection of IgM-type anti-MAG antibodies.

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 relates to a cell line stably expressing an orthogonal aminoacyl tRNA synthetase / tRNA pair, a HEK293T cell line capable of stably expressing an EcTyr orthogonal aminoacyl tRNA synthetase / BsTyr tRNA pair is constructed, and a construction method of the cell line is disclosed. The construction method is to stably integrate the orthogonal EcTyr aminoacyl tRNA synthetase and BsTyr tRNA genes into the HEK293T cell genome by means of a PiggyBac transposon system and plasmid stable transfection. The constructed cell line genome is integrated with the EcTyr orthogonal aminoacyl tRNA synthetase and BsTyr tRNA genes, and provides a stable platform for efficiently introducing unnatural amino acids at any site of a protein. The application has the effects of further laying an experimental foundation for the exploration of novel protein structures and functions and the research and development of protein drugs containing certain specific functions.

The present invention provides a transfection reagent based on blank lipid nanoparticles, a preparation method thereof and applications, belonging to the technical field of biomedicine. The transfection reagent based on blank lipid nanoparticles of the present invention comprises: (1) blank lipid nanoparticles; (2) biologically active components; the composition of the blank lipid nanoparticles includes: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids. The preparation processes of the blank lipid nanoparticles and the transfection reagent are simple and can be completed without the aid of equipment. The transfection reagent based on blank lipid nanoparticles is prepared from blank lipid nanoparticles and nucleic acids, can flexibly adjust the dosage according to the needs of users, has a stable transfection effect, can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7), has low cytotoxicity and good transfection effect.

The invention belongs to the technical field of medicine, and particularly relates to a preparation method and application of a recombinant human blood coagulation factor VII, and the preparation method comprises the following steps: transforming a nucleotide sequence of the recombinant human blood coagulation factor VII into a vector, and constructing a recombinant expression plasmid; transfecting the recombinant expression plasmid into a eukaryotic cell to obtain a recombinant expression cell; screening the recombinant expression cells to obtain stable transfected cells; domesticating the stably transfected cells to obtain monoclonal suspension engineered cells; and performing amplification culture on the monoclonal suspension engineered cells, collecting supernate, and extracting target protein in the supernate to obtain the recombinant human blood coagulation factor VII. The recombinant human blood coagulation factor VII produced by using the method disclosed by the invention is high in yield and good in procoagulant activity, so that the production efficiency and the application effect of the recombinant human blood coagulation factor VII can be improved by using the preparation method disclosed by the invention.

The invention provides a method for constructing a multi-copy eukaryotic expression vector based on a programmable enzyme Argonaute and application of the multi-copy eukaryotic expression vector based on the programmable enzyme Argonaute, and belongs to the technical field of biological medicines.The method comprises the following operation steps that DNA assembly is conducted on recombinant plasmids In-pc-CDs based on Argonaute and resistance gene reconstruction, and expression units in the plasmids obtained through assembly are transferred to plasmids Lamp; in R-Arm, the multi-copy expression vector is obtained. A set of in-vitro seamless DNA assembly technology which is low in mutation rate, convenient to operate, easy to screen, short in assembly period, wide in base sequence universality and low in both economic cost and time cost is developed by starting from a novel programmable enzyme Argonaute and combining a resistance reconstruction strategy, and the in-vitro seamless DNA assembly technology is used as an eukaryotic expression platform DNA assembly tool to realize the in-vitro seamless DNA assembly of the eukaryotic expression platform. The invention provides a method for rapidly constructing a multi-copy expression vector and a signal peptide expression plasmid library, so that the construction of the CHO stable transfection cell strain is realized.

The embodiment of the invention provides a library design and analysis method for high-throughput analysis of non-classical open reading frame coding capability and function, and belongs to the technical field of data analysis. The invention discloses a library design method for high-throughput analysis of non-classical open reading frame coding capability and function. The library design method comprises the following steps: acquiring target non-classical open reading frame (ncORFs) data; designing an overexpression library vector according to the ncORFs data of the target; and obtaining the overexpression library according to the overexpression library vector. The analysis method for analyzing the coding capability and function of the non-classical open reading frame at high throughput comprises the following steps: obtaining an overexpression library; obtaining stable transfected cells; according to the stably transfected cells, obtaining the translation ability information of the ncORFs; acquiring the influence information of the ncORFs on the survival of the cells; and acquiring information of the regulation effect of the ncORFs on the cells. According to the invention, the coding capability and function of ncORFs can be efficiently screened and evaluated, and novel functional protein products can be found.

The invention discloses an application of a construction method of a novel stably transfected cell pool in degradation research of a virulent pathogen. Belongs to the technical field of stable transfection cell pool application, and the application can be used for carrying out virulent pathogen research in a low-grade laboratory or an unprotected environment, so that the research and development cost is reduced, and the application value and strategic value are further improved.

The invention discloses a human renal tubular epithelial cell immortalized cell strain as well as a construction method and application thereof, and belongs to the technical field of cell biology. In the process of constructing the renal tubular epithelial cell immortalized cell strain, a high-quality renal cortex sample is firstly obtained, high-purity primary cells are separated out, the cell activity and proliferation are guaranteed in the culture process, the problem of cell heterogeneity caused by traditional manual operation is solved, and a foundation is laid for subsequent research. The infection efficiency is improved by optimizing virus infection conditions, and stable transfection is ensured in combination with optimal BSD concentration screening. According to the construction method disclosed by the invention, the virus infection efficiency and the gene integration stability are remarkably improved, the immortalized cell strain capable of infinitely proliferating is successfully obtained, the problem that primary cell amplification is limited is solved, and a cell source with stable functions is provided for the bioartificial renal tubule.

The invention discloses a cell line for stably expressing porcine delta coronavirus S protein, a porcine delta coronavirus IgA antibody detection antigen and a kit. The method comprises the following steps: based on an S protein sequence of a CH / XJYN / 2016 strain, selecting an extracellular domain part D20-N1077, adding a human source septoglobin family 1D meber 1 signal peptide sequence MRLSVCLLLTLALCCYRANA to an amino terminal of the S protein, adding a 6 * His tag sequence to a carboxyl terminal, carrying out codon optimization according to a CHO expression system, and constructing into a pCDNA3.1 (+) eukaryotic expression vector to obtain a recombinant eukaryotic expression plasmid pcDNA3.1 (+)-PDCoV-S; then transfecting CHO cells with the plasmid, and carrying out two rounds of heredity mycin G418 pressurized screening, limited dilution cloning and continuous subculture to obtain an expiCHO-PDCoV-S stably transfected cell line through screening; and finally, constructing the PDCoV IgA antibody detection kit based on the cell line. The kit has no cross reaction with other common porcine disease virus positive serum, has intra-batch and inter-batch variation coefficients of less than 10%, has good specificity, repeatability and sensitivity, and can be used for early prevention and control of PDCoV.

The invention relates to application of RNA (Ribonucleic Acid) binding protein SAMD4A or a coding gene thereof in differentiation from stem cells to myocardial cells. The importance of SAMD4A in the myocardial cell differentiation process is elaborated by inducing human embryonic stem cells to differentiate into myocardial cells, and in the myocardial directional differentiation process, SAMD4A expression is gradually up-regulated; an SAMD4A knock-down and overexpression plasmid is constructed, a human embryonic stem cell is infected by lentivirus to establish an SAMD4A knock-down and overexpression stable transfection cell line, and the key effect of the SAMD4A on myocardial differentiation is explored; the function of the FGF2mRNA specifically combined with the SAMD4A is explored; and a molecular mechanism that SAMD4A targets FGF2mRNA to influence myocardial cell differentiation is explored. The SAMD4A has important significance in specific high expression in myocardial cells and differentiation and function of the myocardial cells, and plays a key role in inducing stem cells to differentiate into the myocardial cells.

The application discloses a monoclonal cell strain screening method based on fluorescence intensity, and the method comprises the following steps: determining a cell line, and stably transfecting cells of the cell line with fluorescent proteins; selecting a microwell plate and setting a reference hole, and separating the cells stably transfected with the fluorescent proteins into the microwell plate, wherein the reference hole is filled with multiple cells, and the remaining holes are filled with only one cell; screening single-cell holes with only one cell in the remaining holes except the reference hole; screening clone group holes capable of growing clone groups; transferring the cells in the clone group holes to new holes for culture after being respectively digested; screening fluorescence screening holes; screening division and proliferation holes; and determining the cells in the division and proliferation holes as screened monoclonal cell strains. The monoclonal cell strain screening method provided by the application can screen monoclonal cell strains with strong fluorescence intensity and normal proliferation speed, is convenient and fast compared with fluorescence microscope observation and imaging, and also reduces errors possibly caused by human eye comparison of fluorescence intensity.

The present disclosure provide novel methods of large-scale production of recombinant proteins, e.g., therapeutic proteins such as antibodies, comprising concentrating an eukaryotic cell culture to a high density and transiently transfecting the eukaryotic cells with a polynucleotide encoding the recombinant protein using electroporation, e.g., flow electroporation. In some aspects, the culture is performed under perfusion conditions using, e.g., a tangential flow filtration method such as alternating tangential flow filtration. The proteins obtained using the disclosed methods are comparable to those produced using stable transfection. The methods disclosed herein can be used, for example, to accelerate therapeutic agent development, to reduce host cell toxicity, or for individualized therapeutics such as small scale manufacturing of treatments for rare or orphan diseases.

Provided herein, inter alia, are methods for making extracellular vesicles comprising small interfering RNA (siRNA) and cells comprising said extracellular vesicles. Provided herein are stably transfected cells comprising a nucleic acid encoding an Argonaute2 protein and a nucleic acid encoding an shRNA nucleic acid. The stably transfected cells are useful for methods of making extracellular vesicles comprising siRNA.

The invention belongs to the field of drug detection, and particularly discloses a method for detecting antibody-dependent cell-mediated cytotoxic biological activity of human immune globulin, which comprises the following steps: 1) adding a target cell suspension into a pore plate, respectively adding human immune globulin solutions with a series of concentrations for incubation, and finally adding an effector cell suspension for incubation; (2) detecting fluorescence of the incubated pore plate in the step (1), drawing a dose effect curve and calculating half effect concentration; the target cell is a CHO-K1 cell for expressing SARS-CoV-2 Spike protein, and the effector cell is a Jurkat cell which stably expresses CD16a on a cell membrane and is stably transfected with a firefly luciferase gene which is driven to express by an NFAT response element. The detection method disclosed by the invention can be used for accurately and reliably detecting the antibody-dependent cell-mediated cytotoxic biological activity of two different human immune globulin preparations, namely IVIG and SCIG.

The invention discloses an instrument and a method for screening a stable transfection cell line. The instrument comprises an image acquisition module, an image analysis and processing module and a laser scanning device, the image acquisition module is used for acquiring a cell developing image of the transfected cells in the culture plate or the culture dish under a fluorescence microscope; the image analyzing and processing module is used for receiving the cell developing image and marking an area with relatively weak fluorescence on the cell developing image; the culture plate or the culture dish is placed on a laser scanning device, the laser scanning device carries out laser irradiation on the area, marked as weak fluorescence, in the culture plate or the culture dish according to the marked cell development image, and non-transfected cells are killed. According to the principle that cells are damaged and die due to high temperature, local high temperature is generated, cells which are not successfully transfected are removed from the culture dish, space is provided for proliferation and division of remaining cells which are successfully transfected, and more reliable means and basis are provided for screening stable transfected cells.

The invention relates to the technical field of biology, and particularly discloses a construction method of a stably transfected cell strain based on a COS-7 cell LXR alpha reporter gene. An in-vitro monkey-derived kidney cell COS-7 is used as a host cell, a lentivirus transfection LUC reporter gene vector and a human LXR alpha expression vector are used to construct a stably-transfected recombinant cell, and the recombinant cell can be used for evaluating the activation or inhibition effect of an exogenous compound on LXR alpha, and can be used for preparing a human LXR alpha expression vector. Therefore, the method has a good application prospect in the aspects of LXR alpha gene function research, target verification, drug screening, endocrine disrupter (EDCs) screening and the like. The method for carrying out LXR alpha effector in-vitro screening by using the recombinant cell has the advantages of simplicity in operation, short experimental period, high sensitivity, high accuracy, suitability for high-throughput screening and the like.

The present invention belongs to the field of drug detection and specifically discloses a method for detecting the antibody-dependent cell-mediated cytotoxic biological activity of human immunoglobulin, comprising the following steps: 1) taking a target cell suspension and adding it to a well plate, then adding a series of concentrations of human immunoglobulin solutions for incubation, and finally adding an effector cell suspension for incubation; 2) taking the well plate incubated in step 1) to detect fluorescence, draw a dose-effect curve and calculate the half-maximal effective concentration; the target cells are CHO-K1 cells expressing the SARS-CoV-2 Spike protein, and the effector cells are Jurkat cells stably expressing CD16a on the cell membrane and stably transfected with the firefly luciferase gene driven by the NFAT response element. The detection method of the present invention can accurately and reliably detect the antibody-dependent cell-mediated cytotoxic biological activity of two different human immunoglobulin preparations, IVIG and SCIG.

The invention discloses a cell line and method for stably expressing porcine epidemic diarrhea virus S protein and application, the cell line is an IPEC-PEDV-S stably transfected cell line, and is a cell line for stably expressing porcine epidemic diarrhea virus S protein constructed by a bioengineering technology. The cell line can be used for research and development of PEDV replication-deficient vaccines and PEDV antibody detection reagents, overcomes the defects of a traditional IFA antibody detection method for PEDV, and is safer, simpler, more convenient and more accurate.

The present invention provides a method for preparing Syn-CAR-T cells, the cells thereof and applications. The method includes constructing a stably transfected SIN-γ-RV vector, i.e., a PSR vector, by using the PiggyBac transposon system to prepare a Syn CAR plasmid vector: expressing a SynNotch system with the PSR vector, combining a GAL4 induction vector and a UAS effector vector of the SynNotch system on one PSR vector, so that the SynNotch system is delivered by one PSR vector, and constructing a plasmid of a Syn CAR retroviral vector; and transducing T cells with the plasmid of the retroviral vector to prepare the Syn-CAR-T cells. The results of the present invention show that the induction efficiencies of the targets BCMA, CD38, and GPC3 are extremely low and are not suitable for the SynNotch system of the present invention; the target CD19 has the highest induction efficiency and is most suitable for application to the SynNotch system of the present invention.

The invention discloses a Vero cell model based on VZV neutralizing antibody detection. The lentivirus is used for stably transfecting Vero cells to obtain a cell strain capable of recognizing the VZV virus, and meanwhile, an RFP reporter gene is carried, so that the condition of a neutralizing antibody in serum is conveniently detected. According to the construction method of the stably transfected cell strain for detecting the chicken pox-herpes zoster virus neutralizing antibody, the high-titer stably transfected cell strain for detecting the chicken pox-herpes zoster virus neutralizing antibody is obtained; the stably transfected cell strain for detecting the chicken pox-herpes zoster virus neutralizing antibody can be prepared into a stably transfected cell model for detecting the chicken pox-herpes zoster virus neutralizing antibody, and the cell model can be used for evaluating the influence of a VZV vaccine (chicken pox-herpes zoster virus vaccine) and related drugs on the neutralizing antibody. And the value of the neutralizing antibody in the serum is objectively and quantitatively detected.

The present invention provides a method for detecting transient and stable transfection of autoimmunity encephalitis antibodies and its application. Specifically, the present invention provides a recombinant cell expressing mutant NMDAR, wherein the recombinant cell expresses a fusion protein of exogenous mutant NMDAR and a fluorescent protein, and has a high transfection survival rate. The recombinant cell of the present invention can be used to detect antibodies related to autoimmunity encephalitis, including NMDAR, AMPAR1, AMPAR2, LGI1, Caspr2, GABA B R. The present invention also provides a recombinant transfected cell line based on the present invention, and uses the transfected cell line to detect the method for anti-autoimmunity encephalitis by indirect immunofluorescence method. The method of the present invention has high sensitivity and specificity, and can accurately detect the types of pathogenic antibodies, so as to help patients implement targeted immunotherapy and recover health.

The invention provides a transfection reagent based on blank lipid nanoparticles as well as a preparation method and application of the transfection reagent, and belongs to the technical field of biological medicines. The transfection reagent based on the blank lipid nanoparticles comprises the following components: (1) the blank lipid nanoparticles; (2) a biologically active ingredient; the blank lipid nanoparticles comprise ionizable lipid, phospholipid, cholesterol and polyethylene glycol conjugated lipid. The preparation process of the blank lipid nanoparticles and the transfection reagent is simple, and the preparation can be completed without the help of equipment. The transfection reagent based on the blank lipid nanoparticles is prepared from the blank lipid nanoparticles and nucleic acid, the dosage can be flexibly adjusted according to the requirements of a user, the transfection effect is stable, various cells (such as 293T, Hela, HepG2, TWO3 and MCF7) can be transfected, the cytotoxicity is low, and the transfection effect is good.