55 results about "Humanized mouse" patented technology

The invention belongs to the technical field of biotechnology and animal models, and discloses a double-person-derived mouse model for simulating a tumor immune microenvironment and a construction method and application thereof. The method comprises the following steps: firstly, pretreating NSG immunodeficient mice by adopting low-dose whole-body irradiation in combination with double-antibody targeted bone marrow depletion, and transplanting CD34 + hematopoietic stem cells from the same human donor to complete human immune system reconstruction; separating tumor primary cells, tumor-related fibroblasts and tumor vascular endothelial cells of the same donor, performing three-dimensional co-culture to obtain homologous human tumor organs, and performing in-situ inoculation to immune reconstruction mice to obtain a target model. The core defects of MHC mismatching, low immune reconstruction efficiency, poor tumor immune microenvironment simulation degree, low clinical consistency and the like of an existing model are overcome, and the method can be used for tumor immune treatment drug screening, microenvironment mechanism research and personalized tumor treatment scheme verification.

The invention belongs to the field of antibody development, and discloses a screening method and application of a human-mouse protein high homologous target antibody based on a fully humanized antibody mouse. Aiming at the problem of weak antibody response caused by immune tolerance of human-mouse high homologous targets (protein homology is greater than or equal to 95%), the following scheme is provided: in embryonic stem cells (ES cells) of HUGO-Mabfully humanized antibody transgenic mice, a mouse target gene (such as ACVR2A) is knocked out through a Turbo Knockout technology, and homozygous knockout ES clones are screened; carrying out microinjection on the clones to the whitened B6 mouse blastocyst, and transplanting a pregnant mouse to obtain a Founder mouse; the Founder mouse is subjected to target antigen immunization for more than or equal to 4 times (the Freund's complete adjuvant is used for the first time), and the titer of the serum antibody is detected. According to the invention, 100% homozygous knockout chimeric efficiency is realized in the Founder stage, the mouse construction period is shortened from traditional 8-10 months to 3-4 months, and the diversity and affinity of the antibody are significantly improved (titer reaches 1: 729,000). The obtained antibody can be used for preparing medicines for treating tumors or autoimmune diseases.

The present disclosure provides transgenic mice for use as mouse models, and methods related thereto. The transgenic mice described herein comprise a humanized mouse telomerase (hmTert) gene included in a background mouse germline that provides shortened telomeres capable of reproducing humans and various human disease states.

The invention relates to an immune system humanized mouse biological system for preparing a fully humanized monoclonal antibody and a method for preparing the fully humanized monoclonal antibody. The method comprises the following steps: using a constructed immune system humanized mouse and a VLP chimeric antigen; the HSC immune system humanized mouse is an immunodeficient mouse transplanted with human immune cells, the human immune cells are reconstructed, and antigen-specific B cells and fully humanized antibodies can be generated in the immune system humanized mouse by using a VLP chimeric antigen without firstly activating DC and antigen-specific T cells. In addition, by coupling a VLP antigen and a target antigen, a specific B cell and a fully human antibody of any target can be generated.

The invention provides a construction method and application of a humanized MECP2 gene mouse model, and relates to the field of gene engineering. The model animal with the humanized hMECP2 gene is successfully prepared, the humanized hMECP2 protein can be normally expressed in the body of the model, and the model can be used for MECP2 gene function research and screening and evaluation of human MECP2 targeted drugs and therapies. The animal model prepared by the invention can be used for rapidly establishing more different MECP2 mutation humanized disease mouse models, and is applied to drug screening, drug effect research, related nervous system disease and tumor treatment and the like aiming at human MECP2 target sites, so that the research and development process of new drugs is accelerated, the time and the cost are saved, and the drug development risk is reduced. And a powerful tool is provided for researching the functions of the MECP2 protein and screening drugs.

The application provides a construction method of a humanized keloid mouse model, comprising the following steps: BAC plasmid construction and preparation; superovulation of experimental mice and collection of zygotes; pronuclear microinjection of zygotes; post-injection embryo transplantation; genotype identification; breeding and genetic analysis; and verification of construction results of the humanized immune system by immunohistochemistry and immunofluorescence. In the application, peripheral blood mononuclear cells treated by sCD27 and skin around keloids (homologous cells and tissues) are transplanted into NSG-MHC-DKO immune-deficient mice with overexpression of CD70 genes, so that HLA rejection of different homologous immune cells and tissues can be avoided, the internal microenvironment of keloids and the interaction between the internal microenvironment and the immune system can be restored to the maximum extent, and the influence of immune factors on the occurrence and development of keloids can be realized in vitro. The application first discovers and verifies that the activation of the CD27-CD70 axis can promote the occurrence and development of keloids, and provides a suitable animal model for the research and development of anti-keloid drugs, especially immunotherapy.

Humanized mouse models and methods are provided for determining whether administration of an immunomodulatory drug likely elicits a severe cytokine release syndrome in a human. Humanized mouse models and methods are also provided for determining the immunotoxicity in a human of a drug candidate or of drug combinations.

The application discloses a method for constructing a CRBN gene humanized mouse model, which comprises replacing a knockout region of a mouse CRBN gene with a knock-in region of a human CRBN gene, so that the CRBN gene humanized mouse model is constructed, and the CRBN gene of the CRBN gene humanized mouse model is subjected to amino acid substitution of S369C, V380E, I391V and E431D. The application replaces a key functional domain of a mouse endogenous CRBN gene with a corresponding sequence of a human CRBN gene, so that the mouse can express a functional human CRBN protein, thereby solving the problem of lack of sensitivity of a wild-type mouse to a CRBN-dependent drug. The model provides an ideal preclinical experimental platform for studying the efficacy, toxicity and mechanism of action of a CRBN-related drug.

The invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises five structural domains, namely an antigen specific binding domain, a hinge domain, a transmembrane domain, a co-stimulation domain and a CD3 zeta signal transduction domain, the antigen specific binding structural domain can be specifically bound with an antigen comprising immune globulin IgE; the invention also provides an application of the immunoglobulin E in preparation of a long-acting medicine for treating IgE-mediated allergic diseases. Compared with the prior art, the application has the advantages that the anti-IgE monoclonal antibody is combined with a CAR-T technology, IgE protein generated in a targeting manner is converted into B cells generating IgE in a targeting manner, the killing effect of IgE CAR-T on mIgE + B cells is proved in in-vitro and in-vivo experiments, and the problems that the half-life period of a monoclonal antibody drug is short, and high-frequency medication is needed are solved. In addition, the invention also provides a scheme for carrying out an in-vivo experiment by utilizing the humanized mouse model, and successfully verifies the treatment effect of the CAR-T.

The invention described herein provides non-HLA matched humanized mouse model (e.g., NSG mouse model) with patient-derived xenograft (PDX), as well as methods of making and using the same.

The invention relates to a construction method of a peripheral red blood cell humanized mouse, which comprises the following steps: transplanting human hematopoietic stem cells into the mouse and then applying a red blood cell inducer to the mouse, or applying a red blood cell inducer to the mouse and then infusing human red blood cells into the mouse. The red blood cell inducer comprises an antibody targeting mouse macrophage, dexamethasone or a derivative thereof, and a human macrophage colony stimulating factor. In a mouse transplanted with human hematopoietic stem cells, one or more of an antibody targeting mouse macrophages, dexamethasone and a human macrophage colony stimulating factor can be used in the red blood cell induced maturation process, and high-level human red blood cell reconstruction can be quickly realized within three weeks; the maturity and the denucleation ratio of peripheral human erythrocytes reconstructed in the mouse are completely consistent with those of normal human blood; in a mouse transplanted with human erythrocytes, the erythrocyte induction process can be single treatment of an antibody or dexamethasone, or combined use of the antibody and dexamethasone. The invention also provides application of the peripheral erythrocyte humanized mouse.

The invention relates to a method for screening and identifying metabolites and florae with a human immune regulation effect, which comprises the following steps: (1) establishing immune system humanized mice with multiple intestinal types, and analyzing the change of the florae, the metabolites and the immune system through intestinal flora sequencing, serum metabolome sequencing, flow cytometry and transcriptome sequencing; (2) determining the correlation and sequence between flora related metabolites and the change of the immune system through bioinformatics analysis; and (3) confirming the regulation and control relationship between the candidate metabolites and the immune subgroups in the step (2) through biological experiments. By means of the screening method, the chemical small molecule metabolites for regulating and controlling the human immune cells in the normal physiological state can be screened on a large scale, and the chemical small molecule metabolites can be developed into immunotherapy drugs or used as cell therapy drug culture system components. The screened propionic acid can promote cytotoxicity of T cells and CAR-T and secretion of related cytokines in the aspect of function regulation of the T cells, and the metabolite 2-hydroxybutyric acid is highly positively correlated with the Treg cells and can promote generation and functions of the Treg cells.

Provided herein are humanized mouse models generated using T cell-negative fractions or peripheral blood mononuclear cells obtained from T cell-negative fractions, and methods of using the mouse models to assess the efficacy and / or side effects of a therapeutic agent. Immune cell therapies require a large number of cells. Most commonly, the cells are collected using a process referred to as apheresis. Apheresis collection of the mononuclear cell (MNC) layer has been shown to be a safe and efficient method of collecting the large number of T cells.

The present application relates to a kind of construction method of atherosclerosis humanized mouse model, belong to genetic engineering and genetic modification technical field.The present application is first in ApoE and Ldlr double gene knockout mouse background and obtains NOD-4G gene knockout mouse by knockouting Prkdc and Il2rg gene in mouse, humanized atherosclerosis model is constructed by injecting human umbilical cord blood CD133+stem cell and high-fat feed feeding.Through to atherosclerosis degree analysis, it is found that the aorta and aortic root of the humanized atherosclerosis model constructed have a large number of plaque formation, and contain human CD45 + Leukocyte.This atherosclerosis humanized mouse model provides better animal genetic model for the analysis of the role of human immune cells in the occurrence and development of atherosclerosis disease, and has important significance for the research of human disease and the development of new therapy.

The invention discloses a method for implanting a cryopreserved human tissue into a mouse body, which comprises the following steps of: resuscitating the cryopreserved human tissue, implanting the cryopreserved human tissue into an immunodeficient mouse body, continuously feeding the mouse, and adding bFGF (basic fibroblast growth factor) and VEGFA (vascular endothelial growth factor A) when the cryopreserved human tissue is implanted. The invention further discloses a construction method of the cryopreserved tissue and immune system double-person-derived mouse model. The survival rate and structural integrity of the cryopreserved human tissue are remarkably improved by optimizing the size of a transplantation block, performing combined treatment on growth factors and other key technologies, so that the cryopreserved human tissue can be more effectively reconstructed in an immunodeficient mouse body, and a higher level of structural maturity is shown in histology. The two-person-derived mouse model constructed by combining cryopreserved tissues and an immune system can provide a better research platform for vaccine and efficacy evaluation.

The invention described herein provides non-HLA matched humanized mouse model (e.g., NSG mouse model) with patient-derived xenograft (PDX), as well as methods of making and using the same.

The present application relates to a kind of CD69 humanization mouse model construction method, the construction method: (1) the targeting vector of expression humanization CD69 gene is constructed;(2) design and obtain sgRNA for mouse Cd69 gene;(3) the targeting vector, sgRNA and Cas9 protein are co-injected or co-electrotransferred to mouse fertilized egg cytoplasm or nucleus, and the fertilized egg is transplanted to pseudopregnant mouse, and genotype identification is carried out to pseudopregnant litter mouse, and the positive F0 mouse of correct human source fragment successfully inserted is screened;(4)F0 mouse is mated with background mouse to obtain F1 mouse, and CD69 humanization mouse model is screened out.The CD69 humanization mouse constructed in the present application has application value in immunology and other fields.

The application discloses a kind of targeted exosome chimeric antigen receptor molecules for treating HIV infection and its application, belong to the field of biological medicine technology.The application is packaged into targeted exosome by coding anti-HIV CAR mRNA directionally.The nano antibody sequence of targeted CD3, CD4, CD8+T receptor on T cell is fused to the N-terminal of LAMP-2B to construct specific targeting plasmid, so that the targeted peptide is expressed on the surface of exosome membrane, the expression plasmid containing RNA binding protein L7A and exosome tag protein CD63 is constructed, and the exosome loaded with CAR mRNA and targeted to T cell is obtained by three-plasmid co-transfection of HEK293F cells containing C / D box CAR plasmid.The exosome can construct human CAR-T cell when incubated with PBMC in vitro or back into humanized mouse in vivo, and has significant killing virus infected cell activity.

This application relates to humanized monoclonal antibodies against monkeypox virus and their applications. By screening humanized mouse anti-B6R specific binding memory B cells with fully human antibodies, humanized high-neutralizing activity monkeypox virus antibodies B6R-16A1, B6R-21G7, B6R-22D12, B6R-22F9, and B6R-22H1 were obtained. These antibodies have the characteristics of strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and in vivo protective effect.

The invention discloses a construction method of a humanized diffuse malignant peritoneal mesothelioma mouse model, the mouse model obtained by the construction method and application of the mouse model. The construction method comprises the following steps: constructing a DMPM subcutaneous tumor NOG mouse model by utilizing tumor blocks from human malignant peritoneal mesothelioma; the method comprises the following steps: constructing a huDMPM PBMC-NOG-dko mouse model by utilizing a tumor tissue from a DMPM subcutaneous tumor NOG mouse model; the obtained humanized diffuse malignant peritoneal mesothelioma mouse model completely retains histopathologic characteristics and molecular phenotypes of primary tumors, has a humanized immune system, and can successfully reproduce tumor-immune system interaction characteristics. The method can be used for simulating a tumor microenvironment regulation mechanism of the DMPM, evaluating drugs or therapy of the DMPM and the like.

The invention discloses a three-fruit soup anti-inflammatory component high screening method based on an intestinal flora metabolism model, and belongs to the technical field of traditional Chinese medicine active component screening. Comprising the following steps: constructing a special intestinal flora metabolism model (containing an in-vitro SHIME dynamic simulation system and an in-vivo flora humanized mouse model) of the three-fruit soup, firstly preparing a total extract of the three-fruit soup, different polar parts and a single prototype component, and co-culturing the in-vitro flora to obtain a metabolite; then locking high-activity prototype components and metabolites through a three-level anti-inflammatory activity screening system, and carrying out structure identification, anti-inflammatory mechanism verification, content and metabolic efficiency detection and in-vivo effectiveness confirmation, and carrying out repeated optimization to form a standardized method. The method solves the problems that traditional screening does not consider intestinal metabolism and results are incomplete, direct and indirect anti-inflammatory activity is considered, the screening result is precise and clinically fit, operation is controllable, repeatability is good, and the method can be used for quality control of the three-fruit soup, dosage form improvement and development of anti-inflammatory innovative drugs.

This invention belongs to the field of biotechnology and relates to a method for tracking and assessing the metabolic distribution of platelets in organs based on mitochondrial DNA. Specifically, this invention constructs a humanized mouse model, injects exogenous platelets into the humanized mouse model, and sequences the specific SNVs of the exogenous platelet mitochondrial DNA to trace the metabolic distribution of human platelets or human drug-loaded platelets. The safety is assessed by analyzing the metabolic distribution when human platelets are used as a delivery system.

The invention belongs to the technical field of antibodies, particularly discloses an antibody for treating ulcerative colitis and application of the antibody, and particularly relates to a fully human single-chain antibody (ScFv) specifically combined with human IL-23 and application of the fully human single-chain antibody in treatment of ulcerative colitis. According to the antibody, a fully human bacteriophage ScFv antibody library is constructed, high-affinity IL-23 specific ScFv clone is obtained through three rounds of panning, and the antibody is named as P23A. ELISA detection shows that EC50 of the P23A to IL-23 is 10 nM, and the P23A has high affinity. In a TNBS-induced humanized mouse IBD model, the P23A can significantly improve weight loss of a mouse, reduce the score of a disease activity index (DAI) and recover the length of damaged intestinal tissues, and the treatment effect is equivalent to that of a positive control drug Guselkumab. The antibody provided by the invention has the advantages of high specificity, low immunogenicity and the like, and can be used for preparing medicines for treating ulcerative colitis and other IL-23 related diseases.

This invention discloses a method for constructing a PBMC-derived humanized mouse scleroderma model. Human peripheral blood mononuclear cells are transplanted into immunodeficient mice, and a human immune system is reconstructed in the mice. Bleomycin is injected into the local skin of the mice in a round-point manner to induce fibrosis and form a scleroderma model. This application can more comprehensively simulate the complex pathological process of immune abnormalities and tissue fibrosis coexisting in human scleroderma, and provides a more realistic and systematic experimental platform for in-depth exploration of the immune mechanism of this disease.

The application discloses a construction method and application of an NLRP3 humanized mouse model, and belongs to the technical field of genetic engineering. The construction method of the NLRP3 humanized mouse model comprises the step of mutating the 708th amino acid of mouse Nlrp3-201 protein into a non-acidic amino acid; the Ensembl ID of the mouse Nlrp3-201 is ENSMUST00000079476.10. It is verified that the 708th amino acid of the mouse NLRP3 protein is mutated into a non-acidic amino acid (for example, alanine), the NLRP3 of the obtained mutant transgenic mouse is similar to human NLRP3, and cannot be cut by caspase-3, and humanization is realized. The mutant mouse model can better reflect the biological processes such as human inflammasome activation, cell pyroptosis and inflammatory reaction, and provides a more accurate animal model for understanding human diseases and drug screening.

The present application relates to the field of biological medicine, and more particularly to a kind of anti-NMDAR encephalitis drug, curative effect analysis and mouse model establishment method.Mouse model has anti-GluN1 autoantibody, blood-brain barrier damage, IL-1β produced by endothelial cell;Whether mouse blood-brain barrier damage can be improved, the activity of endothelial cell IL-1β receptor is determined to determine the therapeutic effect by mouse model.Anakinra (Anakinra) is the drug for treating anti-NMDAR encephalitis.The present application can more accurately analyze and evaluate the cause and treatment effect of encephalitis by establishing humanized mouse model with anti-GluN1 (GluN1 is one subunit of NMDAR) antibody, blood-brain barrier damage and IL-1β produced by endothelial cell.

The invention discloses a construction method and application of an hMRGPRX3 humanized mouse model and an atopic dermatitis model, and relates to the technical field of disease model construction. The whole gene sequence of the human MRGPRX3 or a vector containing the gene sequence of the hMRGPRX3 is transferred into mouse genome DNA (Deoxyribonucleic Acid) by utilizing a transgenic technology, and a functional human hMRGPRX3 receptor can be simulated and expressed in a mouse model more truly by transferring upstream and downstream regulatory sequences of the hMRGPRX3 gene. The pruritus signal transduction mediated by the receptor in human dorsal root ganglion sensory neurons can be accurately simulated, and signal deviation caused by species difference of a traditional mouse model is overcome.

A method for inducing and activating a conventional dendritic cell subset, the method comprising the following steps: A. seeding human peripheral blood stem cells or umbilical cord blood stem cells in a plate, and performing expansion culture using a basal medium supplemented with cytokine 1; B. performing in vitro induced differentiation culture on the cells after the expansion culture using a basal medium supplemented with cytokine 2; C. sorting the differentiated cells to obtain pure cDC1 cells; and D. performing activation culture on the pure cDC1 cells using a basal medium supplemented with a stimulator to obtain activated cDC1 cells. A large number of human primary cDC1 are obtained by means of induction from umbilical cord blood stem cells, and the anti-tumor ability of cDC1 is then activated by means of a combination of stimulators. Moreover, the feasibility and efficacy of cDC1 for tumor therapy are verified for the first time by means of a humanized mouse tumor model.

The application provides a chimeric antigen receptor comprising five domain structures of antigen-specific binding, hinge, transmembrane, costimulation and CD3 zeta signaling; specifically binds to an antigen comprising an immunoglobulin IgE EMPD domain through the antigen-specific binding domain; and provides its long-term therapeutic application for IgE-mediated allergic diseases. Compared with the prior art, the application combines anti-IgE monoclonal antibody technology and CAR technology, changes from targeting the produced IgE protein to targeting the IgE-producing B cells, and the IgE CAR-T has been proved to have a killing effect on mIgE + B cells in vitro and in vivo experiments, solving the problems of short half-life of monoclonal antibody drugs and high drug frequency. In addition, the CAR of the application can effectively avoid the interference of free sIgE, and the therapeutic effect of CAR-T is successfully verified through in vivo experiments by constructing a humanized mouse model.