Humanized Animal Model of CRBN Gene and Construction System, Construction Method and Application

Abstract

The present invention relates to the technical field of animal genetic engineering, and particularly relates to a humanized animal model of the CRBN gene, a construction system, a construction method and an application. C57BL / 6J strain mice are used, and C57BL / 6J is abbreviated as B6; specifically, B6.Fas<supgt;lpr / lpr< / supgt> strain, B6.Ncf1<supgt;m1j / m1j< / supgt> strain, B6.Crbn<supgt;tm1.1Ble< / supgt> strain, B6.Stat1<supgt;fl / fl< / supgt> strain, B6.Cd68<supgt;Cre / Cre< / supgt> strain, and B6.Lyz2<supgt;Akaluc< / supgt> strain mice are used to establish breeding cages and perform cross-breeding to finally obtain an animal model of four types of mice; the present invention combines the polymorphism of the Ncf1 gene and the macrophage tracing method of Lyz2 gene-targeted spontaneous fluorescence labeling. By mating a Cre-labeled mouse transcribed from the promoter position of the membrane protein Cd68-encoding gene with a Stat1-Floxp mouse, a conditional gene knockout CKO mouse is established, and a mouse model with multiple controls and a wide application range is established, which is helpful for the research of immune diseases and the development of drugs for the treatment of immune diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of animal genetic engineering, and particularly relates to a humanized animal model of the CRBN gene, a construction system, a construction method and an application thereof. Background Art

[0002] The only known molecular target of the immunomodulatory drugs thalidomide and related compounds is Cereblon (Crbn). However, thalidomide and related compounds lack activity in mouse models, resulting in the inability of mouse Crbn to respond. Recent studies have shown that the mutation of isoleucine at position 391 in the mouse Crbn gene to valine causes mouse Crbn to exhibit the degradation of the thalidomide-induced drug target identified in human cells. Establishing a humanized animal model of the CRBN gene is an urgent need for immunological research. Single nucleotide polymorphisms of the Ncf1 gene play an important role in mouse chronic inflammatory autoimmune diseases, and the lack of reactive oxygen species can lead to enhanced autoimmune responses and even exacerbation of the patient's condition. This effect plays a role in many different mouse diseases, such as lupus, systemic sclerosis and psoriasis. Downstream signaling pathways regulated by the Ncf1 gene include macrophages and the Stat1 gene.

[0003] A humanized animal model of a gene is an animal model that uses genetic technology to insert a human gene into the animal genome so that it carries a human gene. Mice rarely show exactly the same as functional genes, resulting in differences between human and mouse results. Therefore, in order to use the above genes to conduct research on the above immune diseases and develop related drugs, it is urgent to construct a humanized animal model with related genes, especially a mouse model. Summary of the Invention

[0004] To solve the above problems, the present invention proposes to construct a humanized animal model of the CRBN gene. The construction method of this animal model combines the polymorphism of the Ncf1 gene and the method of tracing macrophages by targeting the spontaneous fluorescence labeling of the Lyz2 gene. After mating a Cre-labeled mouse transcribed at the promoter position of the membrane protein Cd68-encoding gene with a Stat1-Floxp mouse, a conditional gene knockout CKO mouse is established, and a mouse model with multiple controls and a wide range of applications is established, which is helpful for immunological disease research and drug development.

[0005] The technical solution adopted is as follows:

[0006] A construction method of a humanized animal model of the CRBN gene, the specific steps are as follows:

[0007] Step Sa: In the construction method, C57BL / 6J strain mice are used. C57BL / 6J is abbreviated as B6. All mice are labeled with genotypes after B6, and the "+" marked in the superscript after the genotype represents the wild type; specifically, B6.Fas is usedlpr / lpr Strain, B6.Ncf1 m1j / m1j Strain, B6.Crbn tm1.1Ble Strain, B6.Stat1 fl / fl Strain, B6.Cd68 Cre / Cre Strain, B6.Lyz2 Akaluc Strain mice;

[0008] Step Sb: Use B6.Fas lpr / lpr Strain, B6.Ncf1 m1j / m1j Strain, B6.Lyz2 Akaluc Cross three pairs of the three strains of mice pairwise to establish breeding cages 1, 2, and 3, and obtain the filial generation double-gene heterozygous B6.Fas lpr / + .Ncf1 m1J / + mice, B6.Fas lpr / + .Lyz2 Akaluc / + mice and B6.Ncf1 m1j / + .Lyz2 Akaluc / + mice;

[0009] Step Sc: Use the filial generation double-gene heterozygous mice in the breeding cages 1, 2, and 3 to self-cross to establish breeding cages 4, 5, and 6, and conduct gene identification on the filial generation mice in the breeding cages 4, 5, and 6, which are respectively double-gene homozygous B6.Fas lpr / lpr .Ncf1 m1jm1j mice, B6.Fas lpr / lpr .Lyz2 Akaluc mice and B6.Ncf1 m1j / m1j .Lyz2 Akaluc mice;

[0010] Step Sd: Cross two kinds of the filial generation double-gene homozygous mice in the breeding cages 4 and 5 to establish breeding cage 7;

[0011] Step Se: Cross the filial generation B6.Fas lpr / lpr .Ncf1 m1J / +.Lyz2 Akaluc / + mice with the filial generation double-gene homozygous mice in the breeding cage 6 to establish breeding cage 8, and conduct gene identification on the filial generation mice in the breeding cage 8;

[0012] Step Sf: Use the filial generation mice in the breeding cage 8 to self-cross to establish breeding cage 9, and conduct gene identification on the mice in the breeding cage 9;

[0013] Step Sg: Cross the filial generation triple-gene homozygous mice in the breeding cage 9 with B6.Stat1 fl / fl strain mice to establish breeding cage 10;

[0014] Step Sh: Use the four-gene heterozygous mice in the breeding cage 10 to cross with the three-gene homozygous mice in the breeding cage 9 to establish the breeding cage 11, and perform gene identification on the offspring of the breeding cage 11;

[0015] Step Si: Use the offspring B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / + mice in the breeding cage 11 to inbreed to establish the breeding cage 12, and perform gene identification on the offspring of the breeding cage 12;

[0016] Step Sj: Use the four-gene homozygous mice in the breeding cage 12 to cross with the B6.Cd68 Cre / Cre strain mice to establish the breeding cage 13;

[0017] Step Sk: Use the five-gene heterozygous mice in the breeding cage 13 to cross with the four-gene homozygous mice in the breeding cage 12 to establish the breeding cage 14, and perform gene identification on the offspring of the breeding cage 14;

[0018] Step Sl: Use the offspring mice in the breeding cage 14 to inbreed to establish the breeding cage 15, and perform gene identification on the offspring of the breeding cage 15;

[0019] Step Sm: Use the five-gene homozygous mice in the breeding cage 15 to cross with the B6.Crbn tm1.1Ble strain mice to establish the breeding cage 16, and perform gene identification on the offspring of the breeding cage 16;

[0020] Step Sn: Use the six-gene heterozygous mice in the breeding cage 16 to cross with the five-gene homozygous mice in the breeding cage 16 to establish the breeding cage 17, and perform gene identification on the offspring of the breeding cage 17;

[0021] Step So: The offspring mice in the breeding cage 17 inbreed to establish the breeding cage 18, and perform gene identification on the offspring of the breeding cage 18.

[0022] Furthermore, when performing gene identification on the offspring genes, among them, the Ncf1 and Crbn genes are identified by pyrosequencing, and the other genes are identified by PCR sequencing.

[0023] Furthermore, by performing gene identification on the offspring of the breeding cage 18 obtained in the step So, the model: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble .

[0024] Further, in the steps Sa - So, the steps Sg - Sl are omitted, and in the step Sm, the offspring triple - gene homozygous mice in the breeding cage 9 obtained in the step Sf are directly used to hybridize with B6.Crbn tm1.1Ble mice to establish a breeding cage 16, and then finally, by performing gene identification on the offspring of the breeding cage 18 obtained in the step So, the model: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Crbn tm1.1Ble can be obtained.

[0025] Further, in the steps Sa - So, the steps Sd - Sf are omitted, and in the step Sg, the offspring B6.Fas lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + mice in the breeding cage 5 are directly used to hybridize with B6.Stat1 fl / fl mice to establish a breeding cage 10, and then finally, by performing gene identification on the offspring of the breeding cage 18 obtained in the step So, the model: B6.Fas lpr / lpr .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble can be obtained.

[0026] Further, each animal model is actually hybridized for more than ten generations.

[0027] Application of an animal model obtained by a method for constructing a CRBN - gene humanized animal model in immune disease research and drug development for treating immune diseases.

[0028] A construction system for implementing a method for constructing a CRBN - gene humanized animal model.

[0029] The beneficial effects of the present invention compared with the prior art:

[0030] 1. The present invention uses B6.Lyz2 Akaluc strain mice to hybridize with mice of different genotypes, making them show spontaneous fluorescence to achieve macrophage fluorescence tracing under different backgrounds;

[0031] 2. The present invention hybridizes macrophages fluorescence - tracing mice under the background of Ncf1 gene polymorphism with B6.Crbn tm1.1Ble strain mice to construct a CRBN - gene humanized Ncf1 - polymorphism spontaneous - fluorescence mouse model, which can be applied to various immune disease research;

[0032] 3. In the present invention, Stat1 conditional knockout mice are established by mating Cre-labeled mice transcribed at the Cd68 gene promoter position with Stat1-Floxp mice, which is conducive to the research of immune inflammation and the development of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is a flow chart of the method for constructing a humanized animal model of the CRBN gene of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0036] The present invention will be further described in detail below with reference to the drawings:

[0037] The mouse model in this embodiment: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble , that is, B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO .Crbn I391V / I391V , for example Figure 1 As shown, taking the acquisition of this mouse model as an example, the specific construction method of the Crbn gene humanized mouse model in the construction system will be described in detail:

[0038] Step Sa: During the construction of the Crbn gene humanized mouse model, the C57BL / 6J.Fas strain from The Jackson Laboratory in the United States (hereinafter, C57BL / 6J is abbreviated as B6, and all mice are labeled with genotypes after B6, and the "+" marked in the superscript after the genotype represents the wild type), B6.Ncf1 lpr / lpr strains (hereinafter, C57BL / 6J is abbreviated as B6, and all mice are labeled with genotypes after B6, and the "+" marked in the superscript after the genotype represents the wild type), B6.Ncf1m1j / m1j Strain, B6.Crbn tm1.1Ble (i.e., B6.Crbn I391V / I391V ) mice of the strain, and B6.Stat1 from Shanghai Model Organisms Center, Inc. fl / fl Strain, B6.Cd68 Cre / Cre Strain, B6.Lyz2 Akaluc mice of the strain to establish breeding cages;

[0039] After each generation of hybridization is completed, the genes of the offspring are identified. Among them, the Ncf1 and Crbn genes are identified by pyrosequencing, and other genes are identified by PCR sequencing.

[0040] Step Sb: Use B6.Fas lpr / lpr Strain, B6.Ncf1 m1j / m1j Strain, B6.Lyz2 Akaluc Three strains of mice are crossbred pairwise to establish breeding cages 1, 2, and 3;

[0041] The offspring double-gene heterozygous B6.Fas lpr / + .Ncf1 m1J / + mice, B6.Fas lpr / + .Lyz2 Akaluc / + mice, and B6.Ncf1 m1j / + .Lyz2 Akaluc / + mice are obtained respectively from breeding cages 1, 2, and 3; This step utilizes the Ncf1 gene polymorphism and the macrophage tracing method with Lyz2 gene-targeted spontaneous fluorescence labeling.

[0042] Step Sc: Use the offspring double-gene heterozygous mice from the above-mentioned breeding cages 1, 2, and 3 to inbreed to establish breeding cages 4, 5, and 6, and identify the genes of the offspring mice in the breeding cages 4, 5, and 6;

[0043] Identify the genes of the offspring mice in breeding cages 4, 5, and 6, and obtain double-gene homozygous B6.Fas lpr / lpr .Ncf1 m1jm1j mice, B6.Fas lpr / lpr .Lyz2 Akaluc mice, and B6.Ncf1 m1j / m1j .Lyz2 Akaluc mice respectively.

[0044] Step Sd: Use the offspring B6.Fas lpr / lpr .Ncf1 m1J / +.Lyz2 Akaluc / + mice in breeding cage 7 to cross with the offspring double-gene homozygous mice in breeding cage 6 to establish breeding cage 8, and identify the genes of the offspring mice in breeding cage 8;

[0045] Select homozygous B6.Fas lpr / lpr .Ncf1 m1J / m1J mice and B6.Fas lpr / lpr .Lyz2 Akaluc mice to establish breeding cage 5, and cross them to obtain B6.Fas lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + mice.

[0046] Step Se: Use the offspring B6.Faslpr / lpr.Ncf1m1J / +.Lyz2Akaluc / + mice in breeding cage 7 to cross with the offspring double-gene homozygous mice in breeding cage 6 to establish breeding cage 8, and identify the genes of the offspring mice in breeding cage 8.

[0047] Select B6.Fas lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + mice and cross them with B6.Ncf1 m1j / m1j .Lyz2 Akaluc mice to establish breeding cage 8, identify the genes of the offspring mice in breeding cage 8, and obtain B6.Fas lpr / + .Ncf1 m1J / m1J .Lyz2 Akaluc mice.

[0048] Step Sf: Use the offspring mice in the said breeding cage 8 to self-cross to establish breeding cage 9, and identify the genes of the mice in the said breeding cage 9;

[0049] Identify the genes of the offspring mice in breeding cage 9, and obtain B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc triple-gene homozygous mice.

[0050] Step Sg: Use the offspring triple-gene homozygous mice in the said breeding cage 9 to cross with B6.Stat1 fl / fl mice to establish breeding cage 10;

[0051] Select the offspring B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc mice and cross them with B6.Stat1 fl / fl mice to establish breeding cage 10, and obtain the offspring B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + quadruple-gene heterozygous mice.

[0052] Step Sh: Use the offspring four-gene heterozygous mice in the breeding cage 10 to cross with the offspring three-gene homozygous mice in the breeding cage 9 to establish the breeding cage 11, and conduct gene identification on the offspring of the breeding cage 11;

[0053] Use the offspring B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + mice in the breeding cage 10 to cross with the offspring B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc mice in the breeding cage 9 to establish the breeding cage 11, and identify the offspring genes to obtain B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / + mice.

[0054] Step Si: Use the offspring B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / + mice in the breeding cage 11 to self-cross to establish the breeding cage 12, and conduct gene identification on the offspring of the breeding cage 12;

[0055] Conduct gene identification on the offspring of the breeding cage 12: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl four-gene homozygous mice.

[0056] Step Sj: Use the offspring four-gene homozygous mice in the breeding cage 12 to cross with B6.Cd68 Cre / Cre mice to establish the breeding cage 13;

[0057] Select homozygous B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl mice to cross with B6.Cd68 Cre / Cre mice and B6.Cd68 Cre / Cre mice to establish the breeding cage 13, and cross to obtain B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + .Cd68 Cre / + five-gene heterozygous mice.

[0058] Step Sk: Establish breeding cage 14 by crossing the five-gene heterozygous mice in the breeding cage 13 with the four-gene homozygous mice in the breeding cage 12, and perform gene identification on the offspring in the breeding cage 14;

[0059] Select B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + .Cd68 Cre / + The five-gene heterozygous mice and B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl Establish breeding cage 14 with the four-gene homozygous mice, and identify the genes of the offspring to obtain B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl .Cd68 Cre / + mice.

[0060] Step Sl: Establish breeding cage 15 by self-crossing the offspring mice in the breeding cage 14, and perform gene identification on the offspring in the breeding cage 15;

[0061] Perform gene identification on the offspring mice in the above breeding cage 15 to obtain B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl .Cd68 Cre / Cre five-gene homozygous mice, namely the model: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO mice; In this step, conditional gene knockout mice, namely CKO mice, are established by mating Cre-labeled mice transcribed at the promoter position of the membrane protein Cd68-encoding gene with Stat1-Floxp mice.

[0062] Step Sm: Establish breeding cage 16 by crossing the offspring five-gene homozygous mice in the breeding cage 15 with B6.Crbn tm1.1Ble mice, and perform gene identification on the offspring in the breeding cage 16;

[0063] Select homozygous B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl .Cd68 Cre / CreMice were crossed with B6.Crbn mice to establish breeding cage 16, and the offspring of the breeding cage 16 were subjected to gene identification to obtain offspring B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + .Cd68 Cre / + .Crbn I391V / + Six-gene heterozygous mice.

[0064] Step Sn: The offspring six-gene heterozygous mice in the breeding cage 16 were crossed with the offspring five-gene homozygous mice in the breeding cage 16 to establish breeding cage 17, and the offspring of the breeding cage 17 were subjected to gene identification;

[0065] Select B6.Fas lpr / + .Ncf1 m1J / + .Lyz2 Akaluc / + .Stat1 fl / + .Cd68 Cre / + .Crbn I391V / + Six-gene heterozygous mice and B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl .Cd68 Cre / Cre Five-gene homozygous mice to establish breeding cage 17, and the offspring were gene-identified to obtain B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / fl .Cd68 Cre / Cre .Crbn I391V / + Mice.

[0066] Step So: The offspring mice in the breeding cage 17 were self-crossed to establish breeding cage 18, and the offspring of the breeding cage 18 were subjected to gene identification.

[0067] Based on the above steps Sa to So, a humanized animal model was finally obtained: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO .Crbn I391V / I391V Mice.

[0068] In the above steps Sa-So, steps Sg-Sl were omitted, and in step Sm, the offspring three-gene homozygous mice in breeding cage 9 obtained in step Sf were directly used and crossed with B6.Crbn tm1.1BleHybridize mice to establish breeding cage 16, and finally, by performing genetic identification on the offspring of the breeding cage 18 obtained in the above step So, the model: B6.Fas can be obtained. lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Crbn tm1.1Ble 。

[0069] In the above steps Sa - So, omit steps Sd - Sf, and directly use the offspring B6.Fas in breeding cage 5 in step Sg. lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + Hybridize mice with B6.Stat1 fl / fl Hybridize mice to establish breeding cage 10, and finally, by performing genetic identification on the offspring of the breeding cage 18 obtained in the above step So, the model: B6.Fas can be obtained. lpr / lpr .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble 。

[0070] The above three mouse animal models obtained can be applied in the research of immune diseases and the development of drugs for treating immune diseases.

[0071] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A method for constructing a humanized animal model of the CRBN gene, characterized in that, The specific steps are as follows: Step Sa: In the construction method, C57BL / 6J strain mice are used. C57BL / 6J is abbreviated as B6. All mice are labeled with genotypes after B6, and the "+" marked in the superscript after the genotype indicates the wild type. Specifically, B6.Fas lpr / lpr strain, B6.Ncf1 m1j / m1j strain, B6.Crbn tm1.1Ble strain, B6.Stat1 fl / fl strain, B6.Cd68 Cre / Cre strain, B6.Lyz2 Akaluc strain mice; Step Sb: Use B6.Fas lpr / lpr strain, B6.Ncf1 m1j / m1j strain, B6.Lyz2 Akaluc strain to cross-breed three pairs of mice pairwise to establish breeding cages one, two, and three, respectively obtaining offspring double-gene heterozygous B6.Fas lpr / + .Ncf1 m1J / + mice, B6.Fas lpr / + .Lyz2 Akaluc / + mice and B6.Ncf1 m1j / + .Lyz2 Akaluc / + mice; Step Sc: Use the offspring double-gene heterozygous mice in the first, second, and third breeding cages to self-cross and establish the fourth, fifth, and sixth breeding cages. Conduct gene identification on the offspring mice in the fourth, fifth, and sixth breeding cages, which are the double-gene homozygous B6.Fas lpr / lpr .Ncf1 m1jm1j mice, B6.Fas lpr / lpr .Lyz2 Akaluc mice, and B6.Ncf1 m1j / m1j .Lyz2 Akaluc mice; Step Sd: Establish breeding cage seven by crossing two kinds of offspring double-gene homozygous mice in breeding cages four and five; Step Se: Use the offspring B6.Fas lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + mice in breeding cage seven to cross with the offspring double-gene homozygous mice in breeding cage six to establish breeding cage eight, and perform gene identification on the offspring mice in breeding cage eight; Step Sf: Establish breeding cage nine by self-crossing the offspring mice in breeding cage eight, and conduct gene identification on the mice in breeding cage nine; Step Sg: Use the triple-gene homozygous mice of the offspring in the breeding cage nine to cross with the B6.Stat1 fl / fl strain mice to establish breeding cage ten; Step Sh: Establish breeding cage eleven by crossing the offspring four-gene heterozygous mice in breeding cage ten with the offspring three-gene homozygous mice in breeding cage nine, and conduct gene identification on the offspring of breeding cage eleven; Step Si: Use breeding cage eleven for the offspring B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 fl / + Self-cross the mice to establish breeding cage twelve, and conduct gene identification on the offspring of the breeding cage twelve; Step Sj: Use the twelve offspring four-gene homozygous mice in the breeding cage to cross with B6.Cd68 Cre / Cre strain mice to establish breeding cage thirteen; Step Sk: Establish breeding cage fourteen by crossing the offspring five-gene heterozygous mice in breeding cage thirteen with the four-gene homozygous mice in breeding cage twelve, and conduct gene identification on the offspring of breeding cage fourteen; Step Sl: Establish breeding cage fifteen by self-crossing the offspring mice in breeding cage fourteen, and conduct gene identification on the offspring of breeding cage fifteen; Step Sm: Use the offspring five-gene homozygous mice in the breeding cage fifteen to cross with B6.Crbn tm1.1Ble strain mice to establish breeding cage sixteen, and perform gene identification on the offspring of the breeding cage sixteen; Step Sn: Establish breeding cage seventeen by crossing the offspring six-gene heterozygous mice in breeding cage sixteen with the offspring five-gene homozygous mice in breeding cage sixteen, and conduct gene identification on the offspring of breeding cage seventeen; Step So: Establish breeding cage eighteen by self-crossing the offspring mice in breeding cage seventeen, and conduct gene identification on the offspring of breeding cage eighteen.

2. The method for constructing a humanized animal model of the CRBN gene according to claim 1, wherein When conducting gene identification on the offspring, among them, the Ncf1 and Crbn genes are identified by pyrosequencing, and other genes are identified by PCR sequencing.

3. Based on the method for constructing a humanized animal model of the CRBN gene as described in claim 2, it is characterized in that, Including: By performing gene identification on the offspring of the breeding cage No. 18 obtained in the step So, the model: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble .

4. The method for constructing a CRBN gene humanized animal model according to claim 3, wherein, Also including: Omit the steps Sg - Sl in the steps Sa - So, and directly use the triple - gene homozygous mice of the offspring in the breeding cage nine obtained in step Sf in step Sm to hybridize with B6.Crbn tm1.1Ble mice to establish breeding cage sixteen, and then finally, by performing gene identification on the offspring of the breeding cage eighteen obtained in step So, the model: B6.Fas lpr / lpr .Ncf1 m1J / m1J .Lyz2 Akaluc .Crbn tm1.1Ble .

5. The method for constructing a CRBN gene humanized animal model according to claim 4, wherein, Including: In the steps Sa - So, omit the steps Sd - Sf, and directly use the offspring B6.Fas in the fifth breeding cage in step Sg lpr / lpr .Ncf1 m1J / + .Lyz2 Akaluc / + mice are hybridized with B6.Stat1 fl / fl mice to establish the tenth breeding cage, and then finally, by performing gene identification on the offspring of the eighteenth breeding cage obtained in the step So, the model: B6.Fas lpr / lpr .Lyz2 Akaluc .Stat1 CKO .Crbn tm1.1Ble .

6. The method for constructing a CRBN gene humanized animal model according to any one of claims 3-5, characterized in that Each animal model is actually crossed for more than ten generations.

7. An application of an animal model obtained by the method for constructing a CRBN gene humanized animal model according to any one of claims 3-5 in immune disease research and drug development for treating immune diseases.

8. A construction system for implementing the method for constructing a CRBN gene humanized animal model according to claim 1 or 2.

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