CN114600837A - Animal model of agranulocytosis, construction method thereof and application of ikzf1 and cmyb in construction of model - Google Patents

Abstract

The invention relates to the technical field of disease animal models, in particular to a granulocytopenia animal model, a construction method thereof and application of ikzf1 and cmyb in model construction. The zebra fish model construction method comprises the following steps of: obtaining F0 zebra fish; the F0 generation zebra fish comprises ikzf1^+/‑Line and cmyb^+/‑Strain; mating the two strains in the F0 generation to obtain a F1 generation zebra fish double-gene mutant heterozygous strain; then selfing the heterozygous strain to obtain F2-generation zebra fish; the F2 generation zebrafish included three lines of granulocyte deficient models, and the extent of granulocyte depletion correlated with the in vivo dose of ikzf1 and cmyb. This schemeCan solve the technical problem that a disease model related to severe congenital granulocytopenia in the prior art is lacked, can be used for researching the pathogenesis of diseases related to granulocytopenia, and can also be used for drug screening and drug effect testing.

Description

Animal model for agranulocytosis, construction method thereof and application of ikzf1 and cmyb in construction of model

Technical Field

The invention relates to the technical field of disease animal models, in particular to a granulocytopenia animal model, a construction method thereof and application of ikzf1 and cmyb in model construction.

Background

Severe congenital agranulocytosis is a disease that affects bone marrow cell production, resulting in severe congenital neutropenia. The development of the granulocyte is stopped at the stage of the promyelocyte or the mesogranulocyte, so that the number of mature granulocytes in peripheral blood is reduced, the mature granulocytes are usually not accompanied by other organ malformations and are autosomal dominant or recessive inheritance, and 60-80 percent of the mature granulocytes have various mutations such as neutrophil elastase gene ELA2 and the like. Although severe congenital granulocytopenia has been listed in the first list of rare diseases, and is receiving extensive attention from society, the lack of animal models of severe congenital granulocytopenia in the prior art has led to the intensive study of the pathogenic mechanism and molecular mechanism of the disease, as well as the screening of drugs and testing of drug effects on the disease, which has not been effectively performed.

Zebrafish (Danio rerio) is a common tropical fish, and a zebrafish cell marker technology, a tissue transplantation technology, a mutation technology, a haploid breeding technology, a transgenic technology, a gene activity inhibition technology and the like are mature, and thousands of zebrafish embryonic mutants are good resources for researching embryonic development molecular mechanisms, and some zebrafish embryonic mutants can also be used as human disease models. Because the similarity of the zebra fish gene and the human gene reaches 87 percent, the result obtained by performing a drug experiment on the zebra fish gene is also suitable for the human body in most cases; the zebra fish embryo is transparent, so biologists can easily observe the influence of the medicine on the internal organs of the zebra fish; female zebra fish can lay eggs for 200, and embryos can grow and form within 24 hours, so that biologists can perform different experiments on the same generation of fish, further study the pathological evolution process and find out the causes of diseases. How to establish an animal model of severe congenital granulocytopenia by using the advantages of zebra fish as an animal model of diseases, further deeply research the pathogenic mechanism of the diseases and perform drug screening and drug effect testing is a problem to be solved at present.

Disclosure of Invention

The invention aims to provide a method for constructing an animal model of agranulocytosis, so as to solve the technical problem that a disease model related to severe agranulocytosis is lacked in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for constructing an animal model with agranulocytosis comprises the following steps which are carried out in sequence:

s1: obtaining F0 zebra fish; the F0 generation zebra fish comprises a heterozygote ikzf1 with single gene mutation^+/-Heterozygote cmyb for strain and monogenic mutation^+/-Strain;

s2: single gene mutated ikzf1^+/-Lines and single gene mutant cmyb^+/-Mating the strains to obtain F1-generation zebra fish; the F1 generation zebra fish comprises a heterozygote ikzf1 with double gene mutation^+/-cmyb^+/-Strain;

S3：ikzf1^+/-cmyb^+/-selfing the strain to obtain F3-generation zebra fish; the F3-generation zebra fish comprises kzf1^+/-cmyb^-/-Strain, ikzf1^-/-cmyb^+/-Lines and ikzf1^-/-cmyb^-/-Strain;

wherein, + represents a wild-type allele, -represents a mutant allele; the mutant allele is obtained by gene knockout of the wild type allele.

The scheme also provides a zebra fish model obtained according to the construction method of the granulocyte deficiency animal model.

The scheme also provides application of an ikzf1 gene and a cmyb gene in constructing a granulocyte deficiency zebra fish model.

The principle and the advantages of the scheme are as follows:

zebra fish heterozygote by using ikzf1 gene mutant (ikzf 1)^+/-Strain) and mutant of cmyb gene (cmyb) in zebrafish^+/-Strain) to obtain zebra fish mutant heterozygous strain (ikzf 1) with simultaneously defective ikzf1 gene and cmyb gene^+/-cmyb^+/-Strain) of the same species. Then obtaining kzf1 by inbreeding in the line^+/-cmyb^-/-Strain, ikzf1^-/-cmyb^+/-Lines and ikzf1^-/-cmyb^-/-The zebra fish of F3 generation including the strain shows the appearance of massive or complete disappearance of granulocytes in the juvenile fish of the strain, and can be used as a model for researching granulocytopenia in the practical operation such as pathogenic mechanism research, drug screening and the like. ikzf1^-/-cmyb^-/-The granulocyte depletion phenomenon of the strain is most remarkable. At ikzf1^-/-In the background, the cmyb function is missing half, or cmyb^-/-In the background, half of the ikzf1 function is missing, and a more severe agranulocytosis phenomenon also occurs.

The inventor finds that the Ikzf1 gene and the cmyb gene have the function of synergistically regulating the development, differentiation and function of granulocytes, which is not reported in the existing research. Wherein, the ikzf1 protein and the cmyb protein can synergistically form a complex to regulate the production of granulocytes, and when both factors are deleted simultaneously, granulocytes cannot be produced. The degree of granulocytic depletion (severity of the disease) correlates with the in vivo dose of these two factors. Therefore, by hybridizing the ikzf1 gene and the mutant of the cmyb gene, a series of zebrafish juvenile fishes with the two factors simultaneously deficient and with different degrees of deficiency can be obtained, so that an animal model with diseases of severe congenital granulocytopenia with different diseases is created to adapt to various research requirements.

In the prior art, disease models related to severe congenital agranulocytosis are few, the technical scheme fills the defects in the field, solves the technical problem that related disease models are lacking in the prior art, can be used for researching the pathogenesis of the agranulocytosis related diseases, and can also be used for drug screening and drug effect testing.

Wherein "+" and "-" in the upper right corner of the gene represent wild-type allele and mutant allele, respectively; mutant alleles are obtained by gene knockout of wild-type alleles. In a specific embodiment, the heterozygote for the single gene mutation, ikzf1^+/-The product is ikzf1^+/Δ4+3Strain; heterozygote cmyb with single gene mutation^+/-Product is cmyb^+/hkz3Strain; heterozygote ikzf1 for double gene mutation^+/-cmyb^+/-The product is ikzf1^+/Δ4+3cmyb^+/hkz3Strain; kzf1^+/-cmyb^-/-Strain, ikzf1^-/-cmyb^+/-Lines and ikzf1^-/-cmyb^-/-The strains are respectively kzf1^+/Δ4+3cmyb^hkz3/hkz3Strain, ikzf1^{Δ4+3/Δ4+ 3}cmyb^+/hkz3Lines and ikzf1^Δ4+3/Δ4+3cmyb^hkz3/hkz3And (5) strain.

Further, the F3-generation zebra fish is ikzf1^-/-cmyb^-/-And (5) strain.

ikzf1^-/-cmyb^-/-The line was a two-gene mutant homozygous line showing complete loss of granulocytes from juvenile fish starting 1.5 days after fertilization.

Further, the Ikzf1 gene wild type allele cDNA sequence is shown in SEQ ID NO.1, the CMyb gene wild type allele cDNA sequence is shown in SEQ ID NO. 2.

The ikzf1 gene and the cmyb gene are both two of the related regulatory genes in the process of granulocyte development. Through a co-localization experiment and a co-immunoprecipitation experiment, the protein products of the two genes have strong interaction and can be combined with each other in the same cell to form a complex to ensure the occurrence of the granulocyte, and the defect degree of the two genes directly influences the defect degree of the granulocyte.

Further, the Ikzf1 gene mutant allele cDNA sequence is shown in SEQ ID NO.3, the CMyb gene mutant allele cDNA sequence is shown in SEQ ID NO. 4.

In a specific embodiment of this protocol, the mutant allele of the ikzf1 gene and the mutant allele of the cmyb gene were obtained by conventional CRISPR/Cas9 techniques and ENU chemical induced mutation, respectively. The two mutant genes both express truncated proteins without biological activity, thereby realizing gene knockout. Using the above two techniques and screening can achieve the target gene knockout, but does not mean that only the two kinds of gene mutation specific forms can achieve the target of ikzf1 gene and cmyb gene expression product (protein) inactivation.

Further, one or both alleles of the ikzf1 gene were knocked out, and both alleles of the cmyb gene were knocked out; or one or both alleles of the cmyb gene are knocked out and both alleles of the ikzf1 gene are knocked out.

Using the above-described protocol, a double mutant (ikzf 1)^+/Δ4+3；cmyb^hkz3/hkz3)、(ikzf1^Δ4+3/Δ4+3；cmyb^+/hkz3) And (ikzf 1)^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) The embryo development is normal, the number of the granulocyte is in a low level state, and the construction of the animal model of the agranulocytosis is successful.

Further, all alleles of the ikzf1 gene and all alleles of the cmyb gene were knocked out.

The ikzf1 gene and the cmyb gene are both two of the related regulatory genes in the process of granulocyte development. After the inventors tried various granulocyte-developmental related genes, they found that it was possible to realize an experimental result in which granulocytes were not detected at all without constructing a double mutant homozygote of any granulocyte-developmental related gene. Some double-gene mutant homozygotes can not normally develop, and some double-gene mutant homozygotes can still detect the granulocyte positive signal. Only in the case of complete knock-out of ikzf1 gene and cmyb gene, granulocytes in zebrafish were completely disappeared and embryonic development was normal. Clinical diagnosis of severe congenital neutropenia SCN usually involves neutropenia with mononucleosis, without other appearance abnormalities, and hemoglobin and platelet counts are usually normal. In the double mutation (ikzf 1)^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) With an increase in macrophage/monocyte numbers, the phenotype is highly similar to that of severe congenital granulocytopenia.

Drawings

FIG. 1 is a typical image of the Sudan black B staining experiment at 3dpf of example 1.

FIG. 2 is the statistics of the positive cells of Sudan black B staining experiment at 3dpf of example 1.

FIG. 3 is a typical image of the 3dpf mpx in situ hybridization experiment of example 1.

FIG. 4 is the statistics of positive cells from the 3dpf mpx in situ hybridization experiment of example 1.

FIG. 5 is a typical image of the 36hpf Sudan black B staining experiment of example 1.

FIG. 6 shows the statistics of the cells positive to the 36hpf Sudan black B staining experiment in example 1.

FIG. 7 is a typical image of the 36hpf mpx in situ hybridization experiment of example 1.

FIG. 8 shows the statistics of positive cells from 36hpf mpx in situ hybridization experiments of example 1.

FIG. 9 is a typical image of the 28hpf csf1r in situ hybridization experiment of example 1.

FIG. 10 shows the statistics of positive cells from the 28hpf csf1r in situ hybridization experiment in example 1.

FIG. 11 shows the results of experiments on co-localization of the transcript of ikzf1 gene and the transcript of cmyb gene in example 2.

FIG. 12 shows the co-immunoprecipitation experiment results of Ikzf1 protein and cMyb protein of example 2.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional (including gene knock-out based on CRISPR/Cas9 technology, chemical mutagenesis with N-ethyl-N-nitrosourea (ENU) and screening for the inactivated target gene), and can be carried out according to the recombination, mutagenesis techniques described (e.g., molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.).

Example 1:

the construction process of the granulocyte deficiency disease zebra fish model is as follows:

(1) obtaining mutant heterozygote of ikzf1 (ikzf 1)^+/Δ4+3) And mutant heterozygotes of cmyb (cmyb)^+/hkz3)

Ikzf1(IKAROS family precise finger 1, Gene ID:30177, wild-type Gene cDNA sequence see SEQ ID NO.1, protein sequence see SEQ ID NO.12) mutant preparation methods are described in (Youkui Huang, Ikzf1 derivatives fermentation T-lymphoid via Ccr9& Irf4 in zebraphish, Journal of Biological Chemistry 2019, Nov 1; 294(44): 16152. 16163.), cMyb (Gene ID 30519, wild-type Gene cDNA sequence see SEQ ID NO.2, protein sequence see SEQ ID NO.13) mutant preparation methods are described in (Zhang Y, cMyb regulation peptide/protein synthesis peptide synthesis 101.201113; Oakkutsu 19).

Among them, the ikzf1 mutant was constructed using the conventional CRISPR/Cas9 technique of the prior art (Chang Nannan, Genome editing with RNA-guided Cas9 nucleic in zebraphish elements. cell Res.2013, 23,465-472). Knocking out the ikzf1 gene by CRISPR/Cas9 technology construction, obtaining the mutant allele of the ikzf1 gene by screening, wherein the mutant allele causes 1bp deletion (specifically, the deletion of the original 4bp nucleotide and the insertion of the additional 3bp nucleotide) in the third exon of the wild allele, and causes the stop codon appearing in advance on the coding sequence of the gene by frame shift mutation, namely, the ikzf1 gene^Δ4+3The mutant gene encodes a non-functional truncated ikzf1 protein (see the text in the sections Youkui Huang,2019, Figure 1A, 1B, Figure S1D-F, and "Embryonic T lymphopoesis is apolysed in ikzf1 mutets"). Mutant heterozygotes of ikzf1 (ik)zf1^+/Δ4+3) Refers to two alleles of the ikzf1 gene of zebrafish, one is wild-type gene and the other is ikzf1^Δ4+3Mutant genes (see SEQ ID NO.3 for sequence and SEQ ID NO.14 for protein sequence). In the case of obtaining the mutant gene of ikzf1 gene, stably inherited ikzf1 mutant heterozygote (ikzf 1) can be obtained by conventional hybridization means of the prior art^+/Δ4+3)。

The cmyb mutants were obtained by forward genetics methods using chemically induced mutations in N-ethyl-N-nitrosourea (ENU)^hkz3The strain, the mutation is due to the fact that a base A is inserted into the eighth intron in the genome of the cmyb, the mutation of the splicing site between the eighth exon and the ninth exon from the base A to the base T causes a competitive splicing acceptor, the eighth exon and the ninth exon are inserted into 13bp nucleotides due to the existence of the competitive splicing acceptor in the transcription process of the cmyb, a stop codon appears in advance in the coding sequence of the gene, and the cmyb^hkz3The protein structure in the mutant is truncated in the trans-activation domain (TAD) and translated to form a non-functional truncated cMyb protein (see Zhang Y,2011, Figure S1-S2 and "Failure of large and adult defined heterologous hematology in the cMyb^hkz3Described in part text "zebrafish mutant line"). Mutant heterozygotes of cmyb (cmyb)^+/hkz3) Means that two alleles of the zebrafish cmyb gene are wild-type gene, one is cmyb^hkz3Mutant genes (see SEQ ID NO.4 for sequence and SEQ ID NO.15 for sequence of protein, which has no biological function and is equivalent to the wild-type cmyb gene being knocked out). In the case of obtaining mutant gene of the cmyb gene, stable genetic cmyb mutant heterozygote (cmyb) can be obtained by conventional hybridization means in the prior art^+/hkz3)。

In order to generally describe the method for obtaining two F0 generation single mutants, gene knockout by using the CRISPR/Cas9 technology and the mutant for obtaining the target gene (protein product) inactivation by using the ENU chemical induction technology are conventional means in the prior art, and the target gene knockout can be realized by using the two technologies and screening. ikzf1^Δ4+3Mutant Gene and cmyb^hkz3Mutant genes have been obtained in the prior art literature, so in subsequent experiments we used ikzf1 mutant heterozygotes (ikzf 1)^+/Δ4+3) And mutant heterozygotes of cmyb (cmyb)^+/hkz3) However, it is not intended that only specific forms of mutations of these two genes could achieve the purpose of inactivating the expression products (proteins) of ikzf1 gene and cmyb gene.

The ikzf1 mutant heterozygote is prepared and screened by the following method:

firstly, inquiring a genome DNA sequence of zebra fish ikzf1 gene at https:// asia. ensemble. org/Danio _ reio/Info/Index website, designing a target site at the third exon of ikzf1 according to the CRISPR/Cas9 knockout principle, and designing a pair of SNP primers around the target site: f-ikzf 1: 5'-CACTATGGCATATGGTGTAG-3' (SEQ ID NO.5), R-ikzf 1: 5'-GCAAAAGCTGAAGATGCGAGT-3' (SEQ ID NO. 6).

Secondly, PCR amplifies the in vitro transcription template of gRNA, and a forward primer comprises three parts, namely a T7 promoter sequence (17bp), a target point sequence (20bp) and a gRNA scaffold sequence, namely F-gRNA: 5'-TAATACGACTCACTATAGGATGCTAATGAAGGCGGAGGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.7), the reverse primer is a universal primer: R-gRNA: 5'-AGCACCGACTCGGTGCCACT-3' (SEQ ID NO. 8).

③ in vitro transcribing the gRNA with an RNA in vitro transcription kit (10. mu.L), purifying with 75% precooled ethanol, and adding a proper amount of ultrapure water for resuspension.

Fourthly, mixed solution 1 of the purified gRNA and the Cas9 protein: 1, the final concentration of gRNA is 50 ng/. mu. L, Cas9 protein mixed solution is formed by mixing 1. mu.L of Cas9 protein and 0.5. mu.L of 10 xBuffer, and adding 3.5. mu.L of ddH₂And O, standing at normal temperature for 5min for later use, and performing microinjection at the zebra fish embryonic cell stage, wherein the injection amount is 1 nL.

F0 screening and identifying zebra fish, breeding the embryo with target spot work to adult, and F0 screening. Adult F0 generations were mated with AB wild type zebrafish, the resulting embryos were lysed at 1.5dpf, and the lysed templates were PCR amplified with SNP primers for sequencing. Offspring with detected spikes were bred to adults to yield adult F1.

Sixthly, screening and identifying F1 zebra fish mutants, shearing tail fins of each F1 generation adult zebra fish, cracking in proteinase K, sequencing by SNP primer PCR amplification cracking template, and obtaining the adult fish with double peaks at the target point, namely the ikzf1 mutant heterozygote (ikzf 1)^+/Δ4+3)。

The hybrid of the cmyb mutant is prepared and screened by the following method:

hybrid of (i) cmyb mutant (cmyb)^+/hkz3) Obtained by screening N-Ethyl-N-nitrosourea (ENU), cmyb^hkz3The mutant is one of the mutants existing in the prior art and is described in detail in the foregoing.

Identification of heterozygote adult fish of the cmyb mutant: for the specific detection of the aberrant spliced forms of cmyb WT or hkz3, the following primer sets were used: common forward primer: f-cmyb: 5'-CACTGCTGCTATCCAGAGACACTAC-3' (814_ FP, SEQ ID NO. 9); reverse primer R-WT: 5'-AGCCGTTCATGGAGATTGGTAG-3' (947_ wt _ RP, SEQ ID NO.10) and R-hkz 3: 5'-CATGGAGATCTGGGTGGGGG-3' (947_ hkz3_ RP, SEQ ID NO.11) are specific for the cmyb WT form and the hkz3 aberrantly spliced form, respectively. Amplification was performed according to the following PCR conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; then 10min at 72 ℃. And carrying out gel electrophoresis identification on the PCR product.

(2) Breeding the ikzf1 mutant heterozygote and the cmyb mutant heterozygote to adult fish according to the conventional zebra fish breeding method of the prior art, hybridizing the ikzf1 mutant heterozygote adult fish and the cmyb mutant heterozygote adult fish, and screening to obtain the double mutant heterozygote (ikzf 1)^+/Δ4+3；cmyb^+/hkz3) Is named as ikzf1^+/Δ4+3cmyb^+/hkz3Line (generation F1).

(3) Heterozygote for double mutation (ikzf 1)^+/Δ4+3；cmyb^+/hkz3) The conventional zebra fish breeding method in the prior art is used for breeding adult fish, and then the selfing of heterozygote adult fish is carried out to obtain different mutation types of zebra fish strains (F2 generation), which comprise the following steps: double mutant (kzf 1)^+/Δ4+3；cmyb^hkz3/hkz3)、(ikzf1^Δ4+3/Δ4+3；cmyb^+/hkz3)、(ikzf1^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) Are respectively named as kzf1^+/Δ4+3cmyb^hkz3/hkz3Strain, ikzf1^Δ4+3/Δ4+3cmyb^+/hkz3Lines and ikzf1^Δ4+3/Δ4+3cmyb^{hkz3 /hkz3}And (5) strain. The granulocytes were labeled with mpx (myeloid-specific peroxidase), sudan black b (sb), etc. by in situ hybridization and staining, respectively, and the results are shown in fig. 1. The positive products of sudan black B staining were black or blue-black particles localized in the cytoplasm. Except for the myeloblast, the granulocytes can be seen as positive granules, and lymphocytes, erythroblasts, megakaryocytes, platelets and the like of other blood cells are in negative reaction.

Figure 1 is a typical sudan black B staining micrograph (including zebrafish whole body, head and tail hematopoietic tissues (CHT)) at 3dpf (3 days post fertilization). FIG. 2 is a statistical chart of the number of Sudan black B positive cells at 3dpf per line (statistical analysis of positive signals of zebrafish whole body, Mean + -SEM, n ═ 7, 23, 3, 6, 7, from left to right, ikzf1^+/+cmyb^+/+Strain, ikzf1^+/Δ4+3cmyb^+/hkz3Strain, ikzf1^+/+cmyb^hkz3/hkz3Strain, ikzf1^Δ4+3/Δ4+3cmyb^+/+Strain, ikzf1^Δ4+3/Δ4+3cmyb^hkz3/hkz3Strain) of the same species. FIG. 3 is a typical mpx in situ hybridization micrograph (CHT) at 3 dpf. FIG. 4 shows the number of mpx positive cells at 3dpf for each strain (statistical analysis of positive signals for the whole zebrafish, Mean. + -. SEM, n: 6, 26, 5, 8, 7, from left to right, ikzf1^+/+cmyb^+/+Strain, ikzf1^+/Δ4+3cmyb^+/hkz3Strain, ikzf1^+/+cmyb^hkz3/hkz3Strain, ikzf1^Δ4+3/Δ4+3cmyb^+/+Strain, ikzf1^Δ4+3/Δ4+3cmyb^hkz3/hkz3Strain) of the same species. In FIGS. 1 and 3, the image in the lower left corner of the left image represents ikzf1^+/+cmyb^hkz3/hkz3Lines and ikzf1^Δ4+3/Δ4+3cmyb^+/+Lines are represented by a single graph, since the numbers of granulocytes are at similar levels for both genotypes.

As can be seen from the results of the experiments in FIGS. 1 to 4, parentsThis ikzf1^+/Δ4+3cmyb^+/hkz3In the inbred filial generation of the line, the filial generation ikzf1^+/+cmyb^+/+The lines (i.e., wild-type AB lines) contained a large number of granulocytes throughout their bodies, ikzf1^+/Δ4+3cmyb^+/hkz3The number of granulocytes in the line (double gene mutation heterozygote) is reduced relative to the wild type, and ikzf1^+/+cmyb^hkz3/hkz3Lines and ikzf1^Δ4+3/Δ4+3cmyb^+/+Both lines are homozygous for a single gene mutation, and the number of granulocytes in both lines is further reduced relative to a double gene mutation heterozygote. Furthermore, in the homozygote of the double gene mutation (ikzf 1)^Δ4+3/Δ4+3cmyb^hkz3/hkz3Line), granulocytes were completely eliminated at 3 dpf. The two genes are completely knocked out simultaneously, so that the development process of the granulocyte is remarkably influenced, the embryo-derived granulocyte precursor cell can be ensured not to be developed into mature granulocyte any more, and the success of constructing the granulocyte deficiency animal model is realized. However, this effect cannot be achieved by completely knocking out only the ikzf1 gene or the cmyb gene. The inventor finds that the experimental result that the granulocyte cannot be detected completely can be realized without constructing a double-mutant homozygote of the granulocyte development related gene after trying a plurality of granulocyte development related genes. Some double-gene mutant homozygotes can not normally develop, and some double-gene mutant homozygotes can still detect the granulocyte positive signal. Only in the homozygous case of the double gene mutation (ikzf 1)^{Δ4+3/Δ4+ 3}cmyb^hkz3/hkz3Strain) shows phenomena of complete undetectable granulocytes and normal embryonic development, and homozygote of double gene mutation (ikzf 1)^Δ4+3/Δ4+3cmyb^hkz3/hkz3Strain) can normally survive for 1-2 weeks, and the model can be used for the study of disease mechanisms and the screening of drugs.

Clinical diagnosis of severe congenital neutropenia SCN usually involves neutropenia with mononucleosis, without other appearance abnormalities, and hemoglobin and platelet counts are usually normal. Double mutant (ikzf 1)^+/Δ4+3；cmyb^hkz3/hkz3)、(ikzf1^Δ4+3/Δ4+3；cmyb^+/hkz3) And (ikzf 1)^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) The embryo develops normally, the number of granulocytes is low, and the double mutation (ikzf 1)^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) With an increase in macrophage/monocyte numbers, the phenotype is highly similar to that of severe congenital granulocytopenia. The double homozygote was shown to be a complete loss of granulocytes in young fish starting 1.5 days (36hpf) after fertilization, and the experimental results are shown in fig. 5, 6, 7 and 8. FIG. 5 is a photomicrograph of a typical mpx in situ hybridization of 36hpf (whole body); figure 6 is a statistical plot of the number of mpx positive cells at 36hpf for each line (statistical positive signals for zebrafish whole body, Mean ± SEM, n ═ 10, 26, 7, 8, 17, 8); FIG. 7 is a typical Sudan black B staining micrograph (including head and CHT) of 36 hpf; fig. 8 is a statistical plot of the number of sudan black B positive cells for each line of 36hpf (statistical positive signals for zebrafish whole body, Mean ± SEM, n ═ 13, 28, 12, 11, 21, 10). The results of the macrophage/monocyte assay are shown in FIGS. 9 and 10. CSF1R is a one-way tyrosine kinase transmembrane receptor belonging to the type iii family of protein tyrosine kinase receptors, which is expressed predominantly on the surface of monocytes and macrophages and controls macrophage growth, function and differentiation through its activation. The scheme detects the number of macrophages/monocytes in the zebra fish body by carrying out in-situ hybridization on the csf1 r. Fig. 9 is a typical micrograph of csf1r in situ hybridization (head and CHT) at 28 hours post fertilization (28hpf), and fig. 10 is a statistical plot of the number of csf1r positive cells at 28hpf for each line (statistical statistics for positive signals for zebrafish whole body, Mean ± SEM, n ═ 8, 25, 9, 13). From the experimental results of FIGS. 9 and 10, it can be seen that the double mutation (ikzf 1)^Δ4+3/Δ4+3；cmyb^hkz3/hkz3) In the group, the number of macrophages/monocytes is increased, the clinical diagnosis and adjustment of severe congenital neutrophilic agranulocytosis are met, and the model construction is successful.

In conclusion, by adopting the technical scheme, the severity of the agranulocytosis depends on the severity of functional defects of the ikzf1 gene and the cmyb gene. The double-gene mutant zebra fish model can be used as a disease model and is also helpful for researching disease occurrence mechanisms.

Example 2: study on the cooperative relationship between ikzf1 gene and cmyb gene

The double-gene mutation of the ikzf1 gene and the cmyb gene can be used for constructing a zebra fish model with agranulocytosis, and the inventor further researches the situation of the synergistic interaction of the ikzf1 gene and the cmyb gene in the zebra fish granulocyte development process. By RNA staining of the transcription product of ikzf1 gene and the transcription product of cmyb gene, it was suggested that these two factors were co-localized in the granulocyte precursor at the same time (see FIG. 11 for experimental results). Co-immunoprecipitation (Co-IP) was performed by transfecting cells, and the results suggest that these two factors interact strongly, and the results are shown in FIG. 12. FIG. 12A is an experiment of the interaction between the fusion proteins GFP-Ikzf1 and Flag-c-Myb in HEK293T cell line, which shows the detection of the band of interest (Flag-c-Myb) by precipitation of GFP-Ikzf1, indicated by the arrow. FIG. 12B shows the detection of the band of interest (GFP-Ikzf1) by precipitating Flag-c-Myb, and the band of interest is indicated by an arrow. This shows that there is strong interaction between Ikzf1 protein and cMyb protein, and they can combine with each other in the same cell to form complex to ensure the generation of granulocytes. Therefore, the defect degree of the two directly influences the granulocyte defect degree, so that the ikzf1/cmyb double mutant zebrafish juvenile fish can be used as a disease animal model of severe congenital granulocytopenia.

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Runkang biopharmaceutical (Suzhou) Co., Ltd

<120> an animal model of agranulocytosis and its construction method, and ikzf1 and cmyb in the construction model

Applications of the invention

<130> 2022.4.12

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 1614

<212> DNA

<213> Danio rerio

<400> 1

atggagactg aggaggcaca ggaaatgtcc cagataacag gaagagacag cccgatgaat 60

gctaatgaag gcggagagga tcaagatgag gccatgcctg ttcctgaaga cttgtcagca 120

agcactggcc tccaacacaa caatcgcaca gataaaccac tggcctgtaa tataaaagtt 180

gaggctcgga gtgacgagga aaacggtctg tcctgtgaga tgaatggaga ggcagaggaa 240

tgtgcagctg aggacttgcg catactcgat ggctctgggg ccaaagtgaa cggctcccac 300

gcaggccccg acagcaagcc ggccgcctac cccacagccg ggggcatccg cctccccaac 360

gggaagctga agtgcgatat ctgtgggata gtttgcattg ggcccaatgt gttgatggtt 420

cacaagcgaa gtcacactga agaaagaaag tcagttttgg aacaacaaaa aggtgaaagg 480

ccattccagt gcaatcaatg tggtgcttca ttcactcaga agggtaacct gctccgacac 540

atcaaacttc actctggcga gaaacctttc aaatgtcacc tgtgcaacta tgcttgccgc 600

cgcagagacg ctctcactgg acatctgcgc actcattcgg ttggaaagcc ccataagtgt 660

gcatattgcg gacgcagtta caagcagcgg agctcactgg aggaacataa ggagagatgt 720

cacaactact tgcagtgcat gggccttcag aacagcattt atacagtgaa ggaagagaac 780

agccagaatg agcagaggga ggacatgcct gcatctgaga gggccttggt gctagacagg 840

atagctaaca atgtagctaa gcgtaagagc tctatgccac agaggtttgt gggagagaat 900

cgtctgtcag agctatcttt cgagagtggc tcaggtgagc tgatgcagcc ccatgtgatt 960

gatcaggcca tcaacagtgc aattagctat ctgggtgcag agtccttgcg gcctctggtt 1020

cagacctctc ctgggtccgc cgacatggtg gtcagccctc tatacaacct gcacaagtca 1080

caaacagctg aaggcaatgg cgtttctgct aaagacagcg ccgcagagca ccttctctta 1140

ctctctaagt ccaaatccgc ctctgttgac aaagacggtt cccccagtcc cagcgggcag 1200

gattccactg acactgagag caacaatgag gagcgttcag ccggggtaag cggaacagca 1260

gccacaggtg gtctcatcta cctgaccaac cacatggctc caggtatgag aaatggaggc 1320

ctgccagggg tgaaggaaga acaacagcgg cattttgagg ctttgcgagc agcaggaatg 1380

gatttgagta tagcgtcatc agaaggattt aaggtgctga gtggagatgg agaagaactg 1440

agggcgtacc gctgtatcca ctgcagagtt ttgttcctgg atcatgtcat gtacaccatc 1500

cacatgggct gtcatggctt ccgagacccc tttgagtgca acctatgcgg gtaccgcagt 1560

caggaccgtt atgagttctc atcgcacatc acacgtggag agcaccgcat ctga 1614

<210> 2

<211> 1926

<212> DNA

<213> Danio rerio

<400> 2

atggcgaggc ggcacagaca cagtgtttac agtagcgacg aagatgatga tgatgtggag 60

atttatgatc atgactatga tggtcctcat gccaagacag gaaaacgcca cctcggcaag 120

acacgctgga cccgtgagga ggatgaaaag ttaaagaggc tggtagagca tcatggttct 180

gaagactgga aagtcatcgc cagctttcta ccgaatcgaa cagatgttca atgccagcat 240

cgttggcaga aagtcctcaa ccctgaactc attaaaggac catggactaa agaagaagac 300

caacgggtga ttgagttggt gcagaagtac ggccccaaac gttggtcagt aatcgcaaag 360

catttaaagg ggcgaatcgg aaagcagtgt agagagcgct ggcacaacca tctaaaccct 420

gaggtgaaga agacgtcctg gactgaggaa gaagatcaaa tcatttacca ggcgcacgag 480

aaacttggaa accgatgggc cgagattgcc aagttacttc cgggaagaac cgataatgcc 540

atcaagaatc actggaactc cactatgcgg cggaaagtgg agcaagaagg ttatttgcag 600

cacgctgcta aagtcagccc aactccgcta aacaacagct attctaaacc tcacctcctg 660

aactacaatc acacaccaag caacacatcc atgcctgcct cctccatgag caatcagtat 720

ccatactaca ctgaatcatc acgggtgcca tttccacttg ctctccagtt aaacatcttg 780

aactttccac aacatggcac tgctgctatc cagagacact acagcgacga ggaccccgaa 840

aaagaaaaga gggtgaaaga aatcgagatg ctgttgatgt caacagaaaa cgagctgaag 900

ggacaacagg cactaccaat ctccatgaac ggctacggtg gctggaacag aggctctttg 960

gcggacagtt cggttggcat tgttgtttca gtcccggctc catccctaga gcaaggatgc 1020

cttcctgaag aaagcgcaca tggcaacaca aacagcccaa ataacgactc cagctccact 1080

ttaccagagt ttatcgatgc tattgattcg gccccatcag tctgggccaa caaacaccct 1140

aaaacggagt gccaaaccgg tcagggttcg cctgcaaaca acggcaacag aagtgccccc 1200

acttttcttt gctcaacgcc caaatattca tccgtcagac acctgccttt ttctcccact 1260

cagttcttta atgcttcagt gagtcccgat tcagacagtc aaaatatacc agtcacgcca 1320

tcgccggtct gctcccagaa agcactgcag caagaccttg ctcttcgacc tcagaaggaa 1380

aatgagctgt tccgaactcc caacctgagg cgttcgatca tggagtgttc tcctcgcaca 1440

cccactccat tcaaatccac actgacaatg caggacagca aatacggacc actgaagagg 1500

gtccacagtc cttcgctgga ctctggagtg gtcggcacga ttaaacaaga gccccaggag 1560

tgtgagatca gtgttggagg ggtgcatcta gaccagcccc ctctgaaaaa gatcaaacag 1620

gaggtggagt cagtgtgtct tcagtgggag gggcaagatc tccacactca gctgttccct 1680

tctaatggcc ccgcccacga catgcctgat ctgctgacta gctctgtgct gatgcttccc 1740

aacacagaga agactgagga cggacacaaa gctgcccagt tgccccgcag acccatagga 1800

agtcctttgc agcagttgaa cacatgggaa caggtgcttt gtgggaagac ggaagagcag 1860

acgattccct ctgaagccac gcacaaatac ctcagcaatt attcttcgcg agctctggtc 1920

atatga 1926

<210> 3

<211> 1613

<212> DNA

<213> Danio rerio

<400> 3

atggagactg aggaggcaca ggaaatgtcc cagataacag gaagagacag cccgatgaat 60

gctaatgaag gcggagagga tggcatgagg ccatgcctgt tcctgaagac ttgtcagcaa 120

gcactggcct ccaacacaac aatcgcacag ataaaccact ggcctgtaat ataaaagttg 180

aggctcggag tgacgaggaa aacggtctgt cctgtgagat gaatggagag gcagaggaat 240

gtgcagctga ggacttgcgc atactcgatg gctctggggc caaagtgaac ggctcccacg 300

caggccccga cagcaagccg gccgcctacc ccacagccgg gggcatccgc ctccccaacg 360

ggaagctgaa gtgcgatatc tgtgggatag tttgcattgg gcccaatgtg ttgatggttc 420

acaagcgaag tcacactgaa gaaagaaagt cagttttgga acaacaaaaa ggtgaaaggc 480

cattccagtg caatcaatgt ggtgcttcat tcactcagaa gggtaacctg ctccgacaca 540

tcaaacttca ctctggcgag aaacctttca aatgtcacct gtgcaactat gcttgccgcc 600

gcagagacgc tctcactgga catctgcgca ctcattcggt tggaaagccc cataagtgtg 660

catattgcgg acgcagttac aagcagcgga gctcactgga ggaacataag gagagatgtc 720

acaactactt gcagtgcatg ggccttcaga acagcattta tacagtgaag gaagagaaca 780

gccagaatga gcagagggag gacatgcctg catctgagag ggccttggtg ctagacagga 840

tagctaacaa tgtagctaag cgtaagagct ctatgccaca gaggtttgtg ggagagaatc 900

gtctgtcaga gctatctttc gagagtggct caggtgagct gatgcagccc catgtgattg 960

atcaggccat caacagtgca attagctatc tgggtgcaga gtccttgcgg cctctggttc 1020

agacctctcc tgggtccgcc gacatggtgg tcagccctct atacaacctg cacaagtcac 1080

aaacagctga aggcaatggc gtttctgcta aagacagcgc cgcagagcac cttctcttac 1140

tctctaagtc caaatccgcc tctgttgaca aagacggttc ccccagtccc agcgggcagg 1200

attccactga cactgagagc aacaatgagg agcgttcagc cggggtaagc ggaacagcag 1260

ccacaggtgg tctcatctac ctgaccaacc acatggctcc aggtatgaga aatggaggcc 1320

tgccaggggt gaaggaagaa caacagcggc attttgaggc tttgcgagca gcaggaatgg 1380

atttgagtat agcgtcatca gaaggattta aggtgctgag tggagatgga gaagaactga 1440

gggcgtaccg ctgtatccac tgcagagttt tgttcctgga tcatgtcatg tacaccatcc 1500

acatgggctg tcatggcttc cgagacccct ttgagtgcaa cctatgcggg taccgcagtc 1560

aggaccgtta tgagttctca tcgcacatca cacgtggaga gcaccgcatc tga 1613

<210> 4

<211> 1939

<212> DNA

<213> Danio rerio

<400> 4

atggcgaggc ggcacagaca cagtgtttac agtagcgacg aagatgatga tgatgtggag 60

atttatgatc atgactatga tggtcctcat gccaagacag gaaaacgcca cctcggcaag 120

acacgctgga cccgtgagga ggatgaaaag ttaaagaggc tggtagagca tcatggttct 180

gaagactgga aagtcatcgc cagctttcta ccgaatcgaa cagatgttca atgccagcat 240

cgttggcaga aagtcctcaa ccctgaactc attaaaggac catggactaa agaagaagac 300

caacgggtga ttgagttggt gcagaagtac ggccccaaac gttggtcagt aatcgcaaag 360

catttaaagg ggcgaatcgg aaagcagtgt agagagcgct ggcacaacca tctaaaccct 420

gaggtgaaga agacgtcctg gactgaggaa gaagatcaaa tcatttacca ggcgcacgag 480

aaacttggaa accgatgggc cgagattgcc aagttacttc cgggaagaac cgataatgcc 540

atcaagaatc actggaactc cactatgcgg cggaaagtgg agcaagaagg ttatttgcag 600

cacgctgcta aagtcagccc aactccgcta aacaacagct attctaaacc tcacctcctg 660

aactacaatc acacaccaag caacacatcc atgcctgcct cctccatgag caatcagtat 720

ccatactaca ctgaatcatc acgggtgcca tttccacttg ctctccagtt aaacatcttg 780

aactttccac aacatggcac tgctgctatc cagagacact acagcgacga ggaccccgaa 840

aaagaaaaga gggtgaaaga aatcgagatg ctgttgatgt caacagaaaa cgagctgaag 900

ggacaacagg cactaccatt cccccaccca gatctccatg aacggctacg gtggctggaa 960

cagaggctct ttggcggaca gttcggttgg cattgttgtt tcagtcccgg ctccatccct 1020

agagcaagga tgccttcctg aagaaagcgc acatggcaac acaaacagcc caaataacga 1080

ctccagctcc actttaccag agtttatcga tgctattgat tcggccccat cagtctgggc 1140

caacaaacac cctaaaacgg agtgccaaac cggtcagggt tcgcctgcaa acaacggcaa 1200

cagaagtgcc cccacttttc tttgctcaac gcccaaatat tcatccgtca gacacctgcc 1260

tttttctccc actcagttct ttaatgcttc agtgagtccc gattcagaca gtcaaaatat 1320

accagtcacg ccatcgccgg tctgctccca gaaagcactg cagcaagacc ttgctcttcg 1380

acctcagaag gaaaatgagc tgttccgaac tcccaacctg aggcgttcga tcatggagtg 1440

ttctcctcgc acacccactc cattcaaatc cacactgaca atgcaggaca gcaaatacgg 1500

accactgaag agggtccaca gtccttcgct ggactctgga gtggtcggca cgattaaaca 1560

agagccccag gagtgtgaga tcagtgttgg aggggtgcat ctagaccagc cccctctgaa 1620

aaagatcaaa caggaggtgg agtcagtgtg tcttcagtgg gaggggcaag atctccacac 1680

tcagctgttc ccttctaatg gccccgccca cgacatgcct gatctgctga ctagctctgt 1740

gctgatgctt cccaacacag agaagactga ggacggacac aaagctgccc agttgccccg 1800

cagacccata ggaagtcctt tgcagcagtt gaacacatgg gaacaggtgc tttgtgggaa 1860

gacggaagag cagacgattc cctctgaagc cacgcacaaa tacctcagca attattcttc 1920

gcgagctctg gtcatatga 1939

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

cactatggca tatggtgtag 20

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence

<400> 6

gcaaaagctg aagatgcgag t 21

<210> 7

<211> 57

<212> DNA

<213> Artificial sequence

<400> 7

taatacgact cactatagga tgctaatgaa ggcggaggtt ttagagctag aaatagc 57

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

agcaccgact cggtgccact 20

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence

<400> 9

cactgctgct atccagagac actac 25

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

agccgttcat ggagattggt ag 22

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<400> 11

catggagatc tgggtggggg 20

<210> 12

<211> 537

<212> PRT

<213> Danio rerio

<400> 12

Met Glu Thr Glu Glu Ala Gln Glu Met Ser Gln Ile Thr Gly Arg Asp

1 5 10 15

Ser Pro Met Asn Ala Asn Glu Gly Gly Glu Asp Gln Asp Glu Ala Met

20 25 30

Pro Val Pro Glu Asp Leu Ser Ala Ser Thr Gly Leu Gln His Asn Asn

35 40 45

Arg Thr Asp Lys Pro Leu Ala Cys Asn Ile Lys Val Glu Ala Arg Ser

50 55 60

Asp Glu Glu Asn Gly Leu Ser Cys Glu Met Asn Gly Glu Ala Glu Glu

65 70 75 80

Cys Ala Ala Glu Asp Leu Arg Ile Leu Asp Gly Ser Gly Ala Lys Val

85 90 95

Asn Gly Ser His Ala Gly Pro Asp Ser Lys Pro Ala Ala Tyr Pro Thr

100 105 110

Ala Gly Gly Ile Arg Leu Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys

115 120 125

Gly Ile Val Cys Ile Gly Pro Asn Val Leu Met Val His Lys Arg Ser

130 135 140

His Thr Glu Glu Arg Lys Ser Val Leu Glu Gln Gln Lys Gly Glu Arg

145 150 155 160

Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn

165 170 175

Leu Leu Arg His Ile Lys Leu His Ser Gly Glu Lys Pro Phe Lys Cys

180 185 190

His Leu Cys Asn Tyr Ala Cys Arg Arg Arg Asp Ala Leu Thr Gly His

195 200 205

Leu Arg Thr His Ser Val Gly Lys Pro His Lys Cys Ala Tyr Cys Gly

210 215 220

Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys

225 230 235 240

His Asn Tyr Leu Gln Cys Met Gly Leu Gln Asn Ser Ile Tyr Thr Val

245 250 255

Lys Glu Glu Asn Ser Gln Asn Glu Gln Arg Glu Asp Met Pro Ala Ser

260 265 270

Glu Arg Ala Leu Val Leu Asp Arg Ile Ala Asn Asn Val Ala Lys Arg

275 280 285

Lys Ser Ser Met Pro Gln Arg Phe Val Gly Glu Asn Arg Leu Ser Glu

290 295 300

Leu Ser Phe Glu Ser Gly Ser Gly Glu Leu Met Gln Pro His Val Ile

305 310 315 320

Asp Gln Ala Ile Asn Ser Ala Ile Ser Tyr Leu Gly Ala Glu Ser Leu

325 330 335

Arg Pro Leu Val Gln Thr Ser Pro Gly Ser Ala Asp Met Val Val Ser

340 345 350

Pro Leu Tyr Asn Leu His Lys Ser Gln Thr Ala Glu Gly Asn Gly Val

355 360 365

Ser Ala Lys Asp Ser Ala Ala Glu His Leu Leu Leu Leu Ser Lys Ser

370 375 380

Lys Ser Ala Ser Val Asp Lys Asp Gly Ser Pro Ser Pro Ser Gly Gln

385 390 395 400

Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Arg Ser Ala Gly Val

405 410 415

Ser Gly Thr Ala Ala Thr Gly Gly Leu Ile Tyr Leu Thr Asn His Met

420 425 430

Ala Pro Gly Met Arg Asn Gly Gly Leu Pro Gly Val Lys Glu Glu Gln

435 440 445

Gln Arg His Phe Glu Ala Leu Arg Ala Ala Gly Met Asp Leu Ser Ile

450 455 460

Ala Ser Ser Glu Gly Phe Lys Val Leu Ser Gly Asp Gly Glu Glu Leu

465 470 475 480

Arg Ala Tyr Arg Cys Ile His Cys Arg Val Leu Phe Leu Asp His Val

485 490 495

Met Tyr Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu

500 505 510

Cys Asn Leu Cys Gly Tyr Arg Ser Gln Asp Arg Tyr Glu Phe Ser Ser

515 520 525

His Ile Thr Arg Gly Glu His Arg Ile

530 535

<210> 13

<211> 641

<212> PRT

<213> Danio rerio

<400> 13

Met Ala Arg Arg His Arg His Ser Val Tyr Ser Ser Asp Glu Asp Asp

1 5 10 15

Asp Asp Val Glu Ile Tyr Asp His Asp Tyr Asp Gly Pro His Ala Lys

20 25 30

Thr Gly Lys Arg His Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp

35 40 45

Glu Lys Leu Lys Arg Leu Val Glu His His Gly Ser Glu Asp Trp Lys

50 55 60

Val Ile Ala Ser Phe Leu Pro Asn Arg Thr Asp Val Gln Cys Gln His

65 70 75 80

Arg Trp Gln Lys Val Leu Asn Pro Glu Leu Ile Lys Gly Pro Trp Thr

85 90 95

Lys Glu Glu Asp Gln Arg Val Ile Glu Leu Val Gln Lys Tyr Gly Pro

100 105 110

Lys Arg Trp Ser Val Ile Ala Lys His Leu Lys Gly Arg Ile Gly Lys

115 120 125

Gln Cys Arg Glu Arg Trp His Asn His Leu Asn Pro Glu Val Lys Lys

130 135 140

Thr Ser Trp Thr Glu Glu Glu Asp Gln Ile Ile Tyr Gln Ala His Glu

145 150 155 160

Lys Leu Gly Asn Arg Trp Ala Glu Ile Ala Lys Leu Leu Pro Gly Arg

165 170 175

Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys

180 185 190

Val Glu Gln Glu Gly Tyr Leu Gln His Ala Ala Lys Val Ser Pro Thr

195 200 205

Pro Leu Asn Asn Ser Tyr Ser Lys Pro His Leu Leu Asn Tyr Asn His

210 215 220

Thr Pro Ser Asn Thr Ser Met Pro Ala Ser Ser Met Ser Asn Gln Tyr

225 230 235 240

Pro Tyr Tyr Thr Glu Ser Ser Arg Val Pro Phe Pro Leu Ala Leu Gln

245 250 255

Leu Asn Ile Leu Asn Phe Pro Gln His Gly Thr Ala Ala Ile Gln Arg

260 265 270

His Tyr Ser Asp Glu Asp Pro Glu Lys Glu Lys Arg Val Lys Glu Ile

275 280 285

Glu Met Leu Leu Met Ser Thr Glu Asn Glu Leu Lys Gly Gln Gln Ala

290 295 300

Leu Pro Ile Ser Met Asn Gly Tyr Gly Gly Trp Asn Arg Gly Ser Leu

305 310 315 320

Ala Asp Ser Ser Val Gly Ile Val Val Ser Val Pro Ala Pro Ser Leu

325 330 335

Glu Gln Gly Cys Leu Pro Glu Glu Ser Ala His Gly Asn Thr Asn Ser

340 345 350

Pro Asn Asn Asp Ser Ser Ser Thr Leu Pro Glu Phe Ile Asp Ala Ile

355 360 365

Asp Ser Ala Pro Ser Val Trp Ala Asn Lys His Pro Lys Thr Glu Cys

370 375 380

Gln Thr Gly Gln Gly Ser Pro Ala Asn Asn Gly Asn Arg Ser Ala Pro

385 390 395 400

Thr Phe Leu Cys Ser Thr Pro Lys Tyr Ser Ser Val Arg His Leu Pro

405 410 415

Phe Ser Pro Thr Gln Phe Phe Asn Ala Ser Val Ser Pro Asp Ser Asp

420 425 430

Ser Gln Asn Ile Pro Val Thr Pro Ser Pro Val Cys Ser Gln Lys Ala

435 440 445

Leu Gln Gln Asp Leu Ala Leu Arg Pro Gln Lys Glu Asn Glu Leu Phe

450 455 460

Arg Thr Pro Asn Leu Arg Arg Ser Ile Met Glu Cys Ser Pro Arg Thr

465 470 475 480

Pro Thr Pro Phe Lys Ser Thr Leu Thr Met Gln Asp Ser Lys Tyr Gly

485 490 495

Pro Leu Lys Arg Val His Ser Pro Ser Leu Asp Ser Gly Val Val Gly

500 505 510

Thr Ile Lys Gln Glu Pro Gln Glu Cys Glu Ile Ser Val Gly Gly Val

515 520 525

His Leu Asp Gln Pro Pro Leu Lys Lys Ile Lys Gln Glu Val Glu Ser

530 535 540

Val Cys Leu Gln Trp Glu Gly Gln Asp Leu His Thr Gln Leu Phe Pro

545 550 555 560

Ser Asn Gly Pro Ala His Asp Met Pro Asp Leu Leu Thr Ser Ser Val

565 570 575

Leu Met Leu Pro Asn Thr Glu Lys Thr Glu Asp Gly His Lys Ala Ala

580 585 590

Gln Leu Pro Arg Arg Pro Ile Gly Ser Pro Leu Gln Gln Leu Asn Thr

595 600 605

Trp Glu Gln Val Leu Cys Gly Lys Thr Glu Glu Gln Thr Ile Pro Ser

610 615 620

Glu Ala Thr His Lys Tyr Leu Ser Asn Tyr Ser Ser Arg Ala Leu Val

625 630 635 640

Ile

<210> 14

<211> 57

<212> PRT

<213> Danio rerio

<400> 14

Met Glu Thr Glu Glu Ala Gln Glu Met Ser Gln Ile Thr Gly Arg Asp

1 5 10 15

Ser Pro Met Asn Ala Asn Glu Gly Gly Glu Asp Gly Met Arg Pro Cys

20 25 30

Leu Phe Leu Lys Thr Cys Gln Gln Ala Leu Ala Ser Asn Thr Thr Ile

35 40 45

Ala Gln Ile Asn His Trp Pro Val Ile

50 55

<210> 15

<211> 346

<212> PRT

<213> Danio rerio

<400> 15

Met Ala Arg Arg His Arg His Ser Val Tyr Ser Ser Asp Glu Asp Asp

1 5 10 15

Asp Asp Val Glu Ile Tyr Asp His Asp Tyr Asp Gly Pro His Ala Lys

20 25 30

Thr Gly Lys Arg His Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp

35 40 45

Glu Lys Leu Lys Arg Leu Val Glu His His Gly Ser Glu Asp Trp Lys

50 55 60

Val Ile Ala Ser Phe Leu Pro Asn Arg Thr Asp Val Gln Cys Gln His

65 70 75 80

Arg Trp Gln Lys Val Leu Asn Pro Glu Leu Ile Lys Gly Pro Trp Thr

85 90 95

Lys Glu Glu Asp Gln Arg Val Ile Glu Leu Val Gln Lys Tyr Gly Pro

100 105 110

Lys Arg Trp Ser Val Ile Ala Lys His Leu Lys Gly Arg Ile Gly Lys

115 120 125

Gln Cys Arg Glu Arg Trp His Asn His Leu Asn Pro Glu Val Lys Lys

130 135 140

Thr Ser Trp Thr Glu Glu Glu Asp Gln Ile Ile Tyr Gln Ala His Glu

145 150 155 160

Lys Leu Gly Asn Arg Trp Ala Glu Ile Ala Lys Leu Leu Pro Gly Arg

165 170 175

Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys

180 185 190

Val Glu Gln Glu Gly Tyr Leu Gln His Ala Ala Lys Val Ser Pro Thr

195 200 205

Pro Leu Asn Asn Ser Tyr Ser Lys Pro His Leu Leu Asn Tyr Asn His

210 215 220

Thr Pro Ser Asn Thr Ser Met Pro Ala Ser Ser Met Ser Asn Gln Tyr

225 230 235 240

Pro Tyr Tyr Thr Glu Ser Ser Arg Val Pro Phe Pro Leu Ala Leu Gln

245 250 255

Leu Asn Ile Leu Asn Phe Pro Gln His Gly Thr Ala Ala Ile Gln Arg

260 265 270

His Tyr Ser Asp Glu Asp Pro Glu Lys Glu Lys Arg Val Lys Glu Ile

275 280 285

Glu Met Leu Leu Met Ser Thr Glu Asn Glu Leu Lys Gly Gln Gln Ala

290 295 300

Leu Pro Phe Pro His Pro Asp Leu His Glu Arg Leu Arg Trp Leu Glu

305 310 315 320

Gln Arg Leu Phe Gly Gly Gln Phe Gly Trp His Cys Cys Phe Ser Pro

325 330 335

Gly Ser Ile Pro Arg Ala Arg Met Pro Ser

340 345

Claims (8)

1. A method for constructing an animal model with agranulocytosis, which is characterized by comprising the following steps in sequence:

s1: obtaining F0 zebra fish; the F0 generation zebra fish comprises a heterozygote ikzf1 with single gene mutation^+/-Strains and monogenic mutant hybridsZygote cmyb^+/-Strain;

s2: single gene mutated ikzf1^+/-Lines and single gene mutant cmyb^+/-Mating the strains to obtain F1-generation zebra fish; the F1 generation zebra fish comprises a heterozygote ikzf1 with double gene mutation^+/-cmyb^+/-Strain;

S3：ikzf1^+/-cmyb^+/-selfing the strain to obtain F2-generation zebra fish; the F2 generation zebra fish comprises ikzf1^+/-cmyb^-/-Strain, ikzf1^-/-cmyb^+/-Lines and ikzf1^-/-cmyb^-/-Strain;

wherein, + represents a wild-type allele, -represents a mutant allele; the mutant allele is obtained by gene knockout of the wild type allele.

2. The method for constructing the agranulocytosis animal model according to claim 1, wherein the zebrafish of the F3 generation is ikzf1^-/-cmyb^-/-And (5) strain.

3. The method for constructing an animal model with granulocytopenia as claimed in claim 2, wherein the cDNA sequence of the wild-type allele of ikzf1 gene is shown in SEQ ID No.1, and the cDNA sequence of the wild-type allele of cmyb gene is shown in SEQ ID No. 2.

4. The method for constructing an animal model with granulocytopenia as claimed in claim 3, wherein the cDNA sequence of mutant allele of ikzf1 gene is shown in SEQ ID NO.3, and the cDNA sequence of mutant allele of cmyb gene is shown in SEQ ID NO. 4.

5. The zebrafish model obtained by the method for constructing the animal model with agranulocytosis according to any one of claims 1 to 4.

Application of ikzf1 gene and cmyb gene in constructing model of granulocytopenia zebra fish.

7. The use of the ikzf1 gene and cmyb gene according to claim 6 in constructing a agranulocytosis zebrafish model, wherein one or both alleles of the ikzf1 gene are knocked out and both alleles of the cmyb gene are knocked out; or one or both alleles of the cmyb gene are knocked out and both alleles of the ikzf1 gene are knocked out.

8. The use of the ikzf1 gene and the cmyb gene in constructing a agranulocytosis zebrafish model according to claim 7, wherein all alleles of the ikzf1 gene and all alleles of the cmyb gene are knocked out.

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