Construction method of humanized immune system mouse with myeloid immune cells

An immune cell and construction method technology, applied in biochemical equipment and methods, botanical equipment and methods, animal husbandry, etc., can solve the difficulty of increasing the development of human myeloid immune cells, the inability of cytokine binding, Problems such as poor origin

Inactive Publication Date: 2020-09-25
澎立生物医药技术(上海)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the homology of these cytokines to humans and mice is poor, and the cytokine receptors on human hematopoietic stem cells cannot bind to mouse-derived cytokines
Further increasing the degree of difficulty in the development of human myeloid immune cells

Method used

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  • Construction method of humanized immune system mouse with myeloid immune cells
  • Construction method of humanized immune system mouse with myeloid immune cells
  • Construction method of humanized immune system mouse with myeloid immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The following uses AAV transfection as an example to illustrate.

[0074] 1. Production method

[0075] 1.1. Plasmid construction

[0076] Firstly, the human gene sequences of GM-CSF, IL-3 and SCF were obtained, and primers were designed. Amplify the target gene with high-fidelity PrimeSTAR enzyme, the reaction system and conditions are as follows:

[0077] Table 1: PCR reaction system

[0078]

[0079] PCR products were subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut from the gel after agarose gel electrophoresis, and TaKaRa MiniBESTAgarose Gel DNAExtraction KitVer.3.0 was used for gel recovery.

[0080] The expression vector was digested with restriction endonucleases. The enzyme digestion reaction system was: 2 μg of plasmid, 5 μL of 10x reaction buffer, 1 μL of each restriction enzyme, supplemented with 50 μL of water, and incubated in a 37°C water bath for more than 2 hours. The digested product w...

Embodiment 2

[0202] This embodiment is described by taking the injection of GM-CSF, IL-3, SCF and Fc fusion protein as an example.

[0203] Construction of GM-CSF, IL-3 and SCF and Fc Fusion Protein Expression Plasmids

[0204] 1. Cloning of functional genes of GM-CSF, IL-3 and SCF

[0205] Adherent mononuclear cells were isolated from peripheral blood of normal people, stimulated by adding LPS for 4 hours, total RNA was extracted by one-step method of guanidine isothiocyanate, the first strand of cDNA was synthesized by MMLV reverse transcriptase, and then used as a template Amplify the entire sequence of GM-CSF, IL-3 and SCF extracellular region, including the secretion signal peptide sequence, and use the downstream primer to change the 145th amino acid Cys to Ser, and the upstream and downstream primers respectively introduce NotI and BamHI sites.

[0206] The PCR reaction was carried out, and the size of the product was consistent with the expected size. The obtained gene product is...

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Abstract

The invention provides a construction method of a humanized immune system mouse with myeloid immune cells. The construction method is characterized by comprising the step of artificially supplementingexogenous cytokines GM-CSF, IL-3 and SCF. By the construction method, the humanized immune system mouse is further perfected, so that the humanized immune system mouse has a more complete immune system. The antigen presentation capability of the humanized immune system mouse is greatly improved by promoting development of myeloid immune cell subgroups including mononuclear-macrophages, DC cells and the like. On the basis of an original immune system mainly containing T and B lymphocytes, the capabilities of generating antibodies, performing specific cytotoxic killing and the like are increased. After the exogenous human cytokines are supplemented, the development level of the myeloid immune cells in the humanized immune system mouse is greatly improved, the level of the corresponding immune cell subgroups in normal human peripheral blood is achieved, and the corresponding functions are fulfilled.

Description

technical field [0001] The invention relates to a method for constructing a humanized immune line mouse with myeloid immune cells, belonging to the field of animal models. Background technique [0002] The human immune response needs to be carried out in the body, and due to ethical restrictions, it is not possible to directly use humans as experimental subjects. Rodents are small animal models widely used in experimental research. At present, most studies on the development, differentiation and function of the immune system come from these models. However, due to the existence of species specificity, the conclusions obtained from rodent models cannot be directly derived from human immune system. Human hematopoietic immune function research, infectious diseases, autoimmune diseases and tumor research are difficult to achieve dynamic observation in vivo. Some researchers use primates as substitutes, trying to make up for the huge species difference between humans and rodent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A01K67/027
CPCA01K67/0278A01K2217/206A01K2227/105A01K2267/0387C12N15/86C12N2750/14143
Inventor 仝莉杨楠陈洁罗艳段继峰
Owner 澎立生物医药技术(上海)股份有限公司
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