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Construction method of humanized immune system mouse with NK cell and ADCC capacity

An immune system and NK cell technology, applied in the field of biological models, can solve the problems of increasing the difficulty of NK cell development, poor proliferation ability of NK cells, and inability to combine cytokines, so as to enhance the ability of specific cytotoxicity, Enhance the effect of ADCC ability and complete immune system

Inactive Publication Date: 2020-08-04
澎立生物医药技术(上海)股份有限公司
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  • Summary
  • 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
After the above-mentioned mice were inoculated with human hematopoietic stem cells, due to the poor proliferation ability of NK cells and the rejection and phagocytosis of foreign bodies from the mouse myeloid immune system, it is difficult for NK cells to develop in their bodies
Further increasing the difficulty of human NK cell development

Method used

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  • Construction method of humanized immune system mouse with NK cell and ADCC capacity
  • Construction method of humanized immune system mouse with NK cell and ADCC capacity
  • Construction method of humanized immune system mouse with NK cell and ADCC capacity

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Experimental program
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Embodiment 1

[0070] The following uses the AAV vector as an example to illustrate:

[0071] 2.1 Plasmid construction

[0072] Firstly, the human gene sequence of IL-15 was obtained and primers were designed. Amplify the target gene with high-fidelity PrimeSTAR enzyme, the reaction system and conditions are as follows:

[0073] Table 1: PCR reaction system

[0074]

[0075] PCR products were subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut out from the gel after agarose gel electrophoresis, and TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 was used for gel recovery.

[0076] 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 was subjected to agarose gel electrophoresis t...

Embodiment 2

[0189] This embodiment is described by taking the injection of IL-15 and Fc fusion protein as an example.

[0190] Construction of IL-15 and Fc Fusion Protein Expression Plasmid

[0191] 1. Cloning of IL-15 functional gene

[0192] 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 the extracellular region of IL-15, including the secretion signal peptide sequence.

[0193] The PCR reaction was carried out, and the size of the product was consistent with the expected size. The obtained gene product is recovered and purified. The pRc-CMV-IL15 plasmid obtained by screening and extraction was sequenced and compared, which was completely consistent with the expected sequence.

[0194] 2. Cloning of human IgG1 Fc...

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Abstract

The invention provides a construction method of a humanized immune system mouse with NK cell and ADCC capacity, which is characterized by comprising the step of artificially supplementing exogenous cytokines IL-15. After exogenous human cytokines are supplemented, the development level of NK cells in a mouse body of a humanized immune system is greatly improved, the level of a corresponding NK cell subset in peripheral blood of a normal person is achieved, and corresponding functions are achieved.

Description

technical field [0001] The invention relates to a method for constructing a humanized immune system mouse with NK cell and ADCC capabilities, belonging to the field of biological models. Background technique [0002] The human immune response needs to be carried out in vivo. Due to ethical restrictions, humans cannot be directly used 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 humans. 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 rodents,...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/24C12N15/62A01K67/027A61K49/00
CPCA01K67/0271A01K67/0278A01K2207/05A01K2207/12A01K2207/15A01K2227/105A01K2267/03A61K49/0008C07K14/5443C07K2319/30C12N15/86C12N2750/14143C12N2800/107
Inventor 杨楠陈洁傅秋华罗艳段继峰
Owner 澎立生物医药技术(上海)股份有限公司
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