Construction method of CD69 humanized mouse model and application thereof

Abstract

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.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal genetic engineering, and in particular to a method for constructing a CD69 humanized mouse model and applications thereof. BACKGROUND

[0002] The ultimate goal of biomedical research is to solve human diseases, however, the ethical limitations of human immune responses and in vivo disease research can lead to an insufficient understanding of the causes of diseases, resulting in errors in treatment. Therefore, people try to study through various experimental models, such as cells, tissues, and animal models. Animal models are undoubtedly the most suitable choice as substitutes for human biology. In animal models, mice are widely used due to their small size, ease of maintenance and handling, short reproductive cycle, shared genome and physiological characteristics with humans, and ease of genetic manipulation.

[0003] However, there are still limitations in using mouse models to study human biology. Although the genomes of mice and humans have a high degree of similarity, certain protein functional domains, such as receptor and donor binding sites, may still differ between mice and humans. In addition, research has found that certain human genes also lack homologous genes in mice. Therefore, in order to better reproduce human biological systems and more accurately reflect the pathogenesis of cancer or autoimmune diseases, people have further developed humanized mouse models. Currently, humanized mice have made significant progress in the study of human infectious diseases, cancer, regenerative medicine, graft-versus-host disease, allergy, and immunity. In the future, with the continuous development of humanized mice, this model will be applied to more preclinical experiments of diseases.

[0004] Cancer immunotherapy, also known as immuno-oncology, is a form of cancer treatment that uses the body's own immune system to prevent, control, and eliminate cancer. In recent decades, immune checkpoint blockade therapy has become the main force of cancer immunotherapy, such as the most representative anti-PD-1 / PD-L1 therapy, which mediates anti-tumor activity by disrupting co-inhibitory T cell signals; in addition, the immune response mediated by it can also effectively prevent the exhaustion of effector T cells in the tumor microenvironment. However, the mechanisms of inducing and preventing T cell exhaustion are still largely unknown. CD69 can regulate inflammation through T cell migration and retention in tissues, and it plays an important role in inducing tumor-infiltrating T cell exhaustion.

[0005] CD69 is a type 2 glycoprotein with a C-type lectin-like domain. As an early activation marker of leukocytes, CD69 is only expressed on activated immune cells, including T cells, B cells, NK cells, granulocytes, macrophages and dendritic cells. CD69 can be divided into three domains according to its structure and function: the extracellular domain can interact with ligand proteins, the transmembrane domain mediates the binding and internalization of sphingosine-1-phosphate receptor 1 (S1P1), and the intracellular domain rich in serine residues can mediate signal transduction. It is known that CD69 regulates the retention of lymphocytes in lymphoid organs by internalizing S1P1 during immune homeostasis and immune activation. In tumor immunity, CD69 is highly expressed on T cells in TME and TDLN. CD69-deficient mice exhibit enhanced anti-tumor immunity and a phenotype of alleviated immune exhaustion. Literature shows that Anti-CD69 monoclonal antibody therapy alleviates T cell exhaustion and tumor progression in tumor-bearing mice. These findings highlight the new role of CD69 in controlling tumor immune escape mediated by T cell exhaustion, suggesting that CD69 may be a new target for cancer immunotherapy. At the same time, CD69 also plays a negative regulatory mechanism in a variety of inflammatory diseases and autoimmune diseases. Studies have shown that Cd69 - / - Mice increase the incidence and severity of various T cell-dependent autoimmune and inflammatory diseases, such as collagen II-induced arthritis, allergic asthma, skin contact hypersensitivity, and autoimmune myocarditis. Therefore, CD69 is also considered a key target for treating immune-mediated inflammatory diseases.

[0006] The CD69 humanized mouse refers to a method of genetic modification, replacing the mouse-derived CD69 with a human-derived gene in a mouse with a healthy immune system, to construct a mouse model that can interact with anti-human CD69 monoclonal antibodies. Compared with ordinary mice, this model realizes the humanization modification of the key target molecule, while retaining the complete immune system, and can be used to screen and evaluate human CD69 neutralizing antibodies or small molecule drugs that inhibit human CD69 activity, and is an ideal preclinical drug test animal model. Therefore, the construction of a CD69 humanized mouse model has high application value in screening and evaluating CD69 target drugs. Existing research on the function of human CD69 in vivo can only be inferred from the function of animal homologous genes (such as mouse CD69) or simulated by in vitro experiments. At present, there is no literature reported on the construction method of an animal model expressing human CD69 and its application in target drug screening. SUMMARY

[0007] In view of the existing problems, the first aspect of the present application provides a construction method of a CD69 humanized mouse model. The mouse model obtained by the construction method expresses a human-mouse chimeric CD69 protein, and the mouse model is partially knocked out of the mouse-derived Cd69 protein.

[0008] Preferably, the extracellular region sequence encoded by the mouse model mouse-derived Cd69 gene obtained by the construction method is replaced by the extracellular region sequence encoded by the human-derived CD69 gene, while the intracellular region sequence encoded by the mouse-derived Cd69 gene is retained.

[0009] Preferably, the selected human-derived CD69 gene amino acid sequence in the mouse model obtained by the construction method is shown in SEQ ID No. 1, and the replaced mouse-derived CD69 gene amino acid sequence is shown in SEQ ID No. 2.

[0010] Preferably, the mouse model mouse-derived Cd69 protein is knocked out using CRISPR-Cas9 gene editing technology.

[0011] Preferably, the sgRNA gene sequence used in the CRISPR-Cas9 gene editing technology is (a) SEQ ID NO. 5 and SEQ ID NO. 6, or (b) SEQ ID NO. 7 and SEQ ID NO. 8.

[0012] More preferably, the sgRNA gene sequence used in the CRISPR-Cas9 gene editing technology is SEQ ID NO. 7 and SEQ ID NO. 8.

[0013] Preferably, the mouse model construction method comprises the following steps:

[0014] (1) Construct a targeting vector expressing a humanized CD69 gene for insertion of the humanized CD69 gene;

[0015] (2) Design sgRNA targeting the extracellular and transmembrane regions of the mouse Cd69 gene, and obtain the sgRNA using in vitro transcription technology;

[0016] (3) Co-inject or co-electrotransfer the targeting vector constructed in step (1), the sgRNA obtained in step (2), and the Cas9 protein into the cytoplasm or nucleus of mouse zygotes, and transplant the zygotes into pseudopregnant mice, genotype the pseudopregnant offspring mice, and screen positive F0 mice successfully inserted with the correct human fragment;

[0017] (4) Breed F0 mice with background mice to obtain F1 mice, genotype the F1 mice, and screen CD69 humanized mouse models.

[0018] Preferably, the step (1) comprises the following steps: according to the structure and function of human CD69, the extracellular region encoded by the mouse Cd69 gene is replaced by the extracellular region encoded by the human CD69 gene, the intracellular region sequence of the mouse is retained, the selected human CD69 gene amino acid sequence is shown as SEQ ID No. 1, and the replaced mouse CD69 gene amino acid sequence is shown as SEQ ID No. 2.

[0019] Preferably, the step (1) comprises the following steps: selecting 62-199 AA of the human CD69 gene, and replacing 62-199 AA of the mouse Cd69 gene by using the homologous recombination technology, and the selected human CD69 gene sequence is shown as SEQ ID No. 3.

[0020] Preferably, the successful targeting vector sequence constructed in the step (1) is shown as SEQ ID No. 4.

[0021] Preferably, the gene sequence of the sgRNA in the step (2) is (a) SEQ ID NO. 5 and SEQ ID NO. 6, or (b) SEQ ID NO. 7 and SEQ ID NO. 8.

[0022] More preferably, the gene sequence of the sgRNA in the step (2) is SEQ ID NO. 7 and SEQ ID NO. 8.

[0023] Preferably, the strain of the fertilized egg mouse and the pseudopregnant mouse provided in the step (3) is BALB / c.

[0024] Preferably, the 5' end identification primer used for the genotype identification of the F0 mouse in the step (3) is shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the 3' end identification primer is shown as SEQ ID NO. 11 and SEQ ID NO. 12.

[0025] Preferably, the PCR reaction system used for the genotype identification of the F0 mouse in the step (3) is as follows:

[0026]

[0027] Preferably, the PCR reaction conditions used for the genotype identification of the F0 mouse in the step (3) are as follows:

[0028]

[0029] The second aspect of the present application provides the application of the mouse obtained by the above construction method in the research of the function and mechanism of CD69 gene.

[0030] Preferably, the application is for non-diagnostic and non-therapeutic purposes.

[0031] The third aspect of the present application provides the use of the mouse obtained by the construction method described above in screening drugs for treating diseases related to the CD69 gene.

[0032] Preferably, the application is for non-diagnostic and non-therapeutic purposes.

[0033] Preferably, the disease related to the CD69 gene is asthma.

[0034] Advantages of the present application:

[0035] The animal model of the present application designs sgRNA for cutting mouse Cd69 gene, and designs Donor containing human CD69 gene at the same time. The sgRNA, Donor and Cas9 are mixed and injected into fertilized eggs of BALB / cJGpt background mice to perform homologous recombination, obtain positive F0, and mate F0 mice with BALB / c mice to obtain stable genetic positive F1 mouse model. The animal model of the present application solves the problems of studying the function of human gene coding protein in animals and studying the pathogenesis of human diseases, and provides a strong scientific basis and technology for the clinical safety and efficacy research of candidate anti-human CD69 antibody drugs. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is the preparation strategy of the humanized CD69 mouse model of the present application;

[0037] Figure 2 is the electropherogram of the 5' end and 3' end gene identification results of CD69-KI F0 mice;

[0038] Figure 3 is the electropherogram of the 5' end and 3' end gene identification results of CD69-KI F1 mice;

[0039] Figure 4 is the flow cytometry graph of the CD8 T cell and B cell subpopulation detection of the spleen of BALB / c mice and BALB / c-hCD69 homozygous mice; +

[0040] Figure 5 is the flow cytometry graph of the CD4 T cell and NK cell subpopulation detection of the spleen of BALB / c mice and BALB / c-hCD69 homozygous mice; +

[0041] Figure 6 is the flow cytometry graph of the neutrophil and monocyte subpopulation detection of the peripheral blood of BALB / c mice and BALB / c-hCD69 homozygous mice; ​​

[0042] Figure 7 is a flow cytometry chart of peripheral blood eosinophil and macrophage subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0043] Figure 8 is a flow cytometry chart of peripheral blood T / NK cell and B cell subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0044] Figure 9 is a flow cytometry chart of peripheral blood Tc / Th cell and DC cell subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0045] Figure 10 is a flow cytometry chart of spleen neutrophil and monocyte subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0046] Figure 11 is a flow cytometry chart of spleen eosinophil and macrophage subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0047] Figure 12 is a flow cytometry chart of spleen T / NK cell and B cell subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0048] Figure 13 is a flow cytometry chart of spleen Tc / Th cell and DC cell subpopulation detection of BALB / c mice and BALB / c-hCD69 homozygous mice;

[0049] Figure 14 is a food intake chart of each test group of anti-CD69 antibody asthma test;

[0050] Figure 15 is a classification count chart of lymphocytes, neutrophils and eosinophils in the bronchoalveolar lavage fluid of each test group of anti-CD69 antibody asthma test;

[0051] Figure 16 is a lung tissue airway wall thickness analysis chart of each test group of anti-CD69 antibody asthma test;

[0052] Figure 17 is a mRNA level analysis chart of inflammatory factors in the lung tissue of each test group of anti-CD69 antibody asthma test. DETAILED DESCRIPTION

[0053] The present application is further illustrated by way of the following examples, but the present application is not limited to the following examples only.

[0054] Establishment of CD69 humanized mouse model

[0055] The present application uses CRISPR / Cas9 technology to replace the mouse CD69 gene with the human CD69 gene in BALB / c background mice, thereby constructing a mouse model that can express human CD69, and the specific method is as follows:

[0056] 1. Determine the replacement region of the human fragment and the inserted human sequence

[0057] According to the structure and function of the CD69 gene, the gene expression element of the human CD69 protein extracellular region with a length of about 2.8 kb is operatively connected to the mouse transmembrane and intracellular expression element, so that the expression element cooperates with the remaining mouse transmembrane and intracellular expression element at this position. The expression effect obtained after insertion is that the partial amino acid sequence AA: 62-199 (as shown in SEQ ID No. 1) of the human CD69 gene is replaced with the partial amino acid sequence AA: 62-199 (as shown in SEQ ID No. 2) of the mouse CD69 gene.

[0058] SVGQYNCPGQYTFSMPSDSHVSSCSEDWVGYQRKCYFISTVKRSWTSAQNACSEHGATLAVIDSEKDMNFLKRYAGREEHWVGLKKEPGHPWKWSNGKEFNNWFNVTGSDKCVFLKNTEVSSMECEKNLYWICNKPYK (SEQ ID No. 1)

[0059] NVGKYNCPGLYEKLESSDHHVATCKNEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKNEANQTWKWANGKEFNSWFNLTGSGRCVSVNHKNVTAVDCEANFHWVCSKPSR (SEQ ID No. 2)

[0060] 2. Inject positive mice

[0061] 1) Determine the replacement region of the human fragment and the inserted human sequence

[0062] According to the human-mouse homology of the human CD69 protein and the comparison of the extracellular functional domain, the homology of the human-mouse CD69 protein is only 57.8%. When performing pharmacodynamic studies, the antibody targeting the human CD69 protein cannot well recognize the mouse protein domain. Therefore, the 62-199AA of the human CD69 gene is used to replace the 62-199AA of the mouse Cd69 gene, and the selected sequence of the human CD69 gene replacement is shown in SEQ ID No. 3.

[0063]

[0064] 2) Humanized targeting vector construction

[0065] The coding sequence of 62-199 AA of human-derived CD69 gene was constructed into a targeting vector, which was inserted into the corresponding position of murine-derived CD69 gene by homologous recombination technology. The sequence of the successfully constructed targeting vector is shown in SEQ ID No. 4 (KI fragment is indicated in italics).

[0066] 1 TTTTCA CTGTTA ATTATA ATCTTA TTCAAG GAGCCA TTTTGT ATTAAA GCTTCCATGAGC

[0067] 61 ATTAAA AAATTC ACACTG AAGAAT TTGGGA ACATAC AAGGGC ATTCTA GTGGGGGCATAC

[0068] 121 TGTTTT GAATCA TGAGGG TTCACC CTGTAA AAAACA AAATCC AAAATC TAAAGCCAAAAA

[0069] 181 TTGACT TTCCCT CTGCAG TCACCT ATTTAC ATGCTA GTACAG GCATAT TTCTATCTATCC

[0070] 241 AAATCC TGATAC AGAAAC ATTTAT TAGTTA AAGGTA ACTGTG ACTTTG TTCAACCTTTAT

[0071] 301 ACAGTA AGTTAA ATTGGG GGGGGG TCTGTT CAATAT GTTTTA TAGCAT TGTTACTTTTCA

[0072] 361 CTTATT ATTTTA ATCCAT CTTAGA AAATGT AATGTT TCACTG TTCATT CTTACATTATCA

[0073] 421 CTTTAA CTGTGC TATAAT TTATAT GCAATT AATGAA CACACC ATAGGA GTGCGGTGTAAG

[0074] 481 GACTCA ACAAAT GTATAG CCACAA TTCCCA TCAATA TTTGAA TATTCC CGTGACTCTAGG

[0075] 541 GCACTC TGTAGG TGCCAT TGTCAC AAGCAT AAATGC AAACAC TTTTCT TACATTTGACAC

[0076] 601 CACACC TTATTT TTGTCT AAAGTG CCAGTT ACATAG AAATAC ATAATA TGTAATCTTTAA

[0077] 661 TGAATA TACTGT TTTCAT TTAACA TTATTT TTTAAG ATTTAT CATATG GTATGCATCAGG

[0078] 721 GCTTTA TTTTAA TTGCTA GAATCA TTTTTT TGTGTG GAAATA CTAAAA AGTGTTTATTCA

[0079] 781 TTCTCA TGCTGA CAGGTA TTTGGA TTATTT TCAGGA CTTGGT CTTAGT GATGATAATTCT

[0080] 841 ATGAAT ATTGTT GTGCAA GGCATT TTAAAA ACATGG GGTTTT TTTAGA GGTGGTGGTGGT

[0081] 901 GGTGGT ATGTGT GTGTAG GACTGT CTGGAT CTTACA GTAGTA TATGTA TAACAATGTTTT

[0082] 961 TTATTT ATGTCT GTGGAA GTGTAG GTCATG TGTGTA CAGGTG TCTTCA AAAGCCAGAAGA

[0083] 1021 GGGTAC TGGAAC CTTCAG AGCTAT AGTGAC AGATGG TTGTGA GCCACC TGATGTGGGTTC

[0084] 1081 TGGGAA CCAAAC ACTGGT CCTCTA CAAAAA CAGTAC ATGCTC TCTAAC ACTGAGGCATCT

[0085] 1141 CTTCAG CCCCTG TGTGTT CATAAC AATGTA AAAAAG CGTATT TTTCCA GGTTAGCCTCTT

[0086] 1201 AGCATT AATATA TGAAAG TTTGCT GCACTG GTATTA CCAAGA AACAAA ATCAAGTTTGCA

[0087] 1261 ATTTTT TAAATT TTATCC TACTTT TGGATA TTTATG CATAAA TTCTCA TTGAAAACATTT

[0088] 1321 ATTCTG AGCATT CCATCT CCCATA AACAGT AGCTAA CTCCAA GGAGAC TAGGGAACTACA

[0089] 1381 GCCAGG ATACTC TTTTTA TATGTC TCCTAT AGCAAG TGGGTG CTTAGA AGTCTTAAATAA

[0090] 1441 TGATAT GCTTGA ATTTTT CATAAC TATGAA ATATAC CCATTA TGCCTA CTAAATACATAT

[0091] 1501 ATAAAT TATATA TTTATG CATATA TACATA CATACA TACATA CATACA TACATACATACA

[0092] 1561 TACATA GTGAAG ATACCT ATTAAG TCAGCA TTTTAT TTGCTT TGCCTT ATCTCTAGCCAA

[0093] 1621 CAAGAC GTTTTC CAAATG CACTCA GAATTT GGTAAA ATTGAC CATTAA TATTTTTTATTA

[0094] 1681 GTGGAA CAACAC ATTACT ATGCTT AATTTT ATATTC CTCAGG GAATAT CAACTGCTCCAT

[0095] 1741 CTTACA GCCTGA ATTCAC CCCACT TCTCTC CCTTTT CAGACC ATGGCA CCAGTATACATT

[0096] 1801 TTGAGA AGCATC ATGAAG GATCCA TTCAAG TTTCTA TCCCTT GGGCTG TGTTAATAGTGG

[0097] 1861 TCCTCA TCACGT CCTTAA TAATAG CTCTCA TTGCCT TA TCAG GTGGGT CTACAC TTCATC

[0098] 1921 AATTCT TTCAAA GCCTCC TAGCTG TAAGCA TTCATG CCCAGC TGAAAT CAATAT TAGTTT

[0099] 1981 AGTCTA GTTAAT CAGAAA CTAGCT AATTCA GAATCA TCCCAT GTACCA TATATG GCCAAT

[0100] 2041 TTCCTA ATCAAT ACTAAA CATAAT TCCACA ATCCTT GAATAA TTAATG TAATAC TTTTAT

[0101] 2101 AAGTAT TAAAAG TATCTA TGATTT ATATAT TACTGT AGAATA AAATTG CATTTG AAAAGA

[0102] 2161 TGTTTA CATCTA GAATGA AGGGAG CTGATT TGAATT CTGAAT AAATAC CTATTT AGCTAG

[0103] 2221 AAAAAA ATGTGA ACTTCA CATTCA TTGATT ATGGAA AAAAAT CGTATC TAAAGC CATAGA

[0104] 2281 TTAATC AGGAAG TGGCCA GTCTTT TGTGTT TTATCT CAAAGG ACACAG AATGGT AGTTCT

[0105] 2341 GAATAA TTAAGA TTATAT TTAAAA GTTCCC AGAGAG TCAGTA GTGATT TGGTAT TCAATA

[0106] 2401 CCTCCA GCTGTA CCAAGA TATATT TGTATC TTTTAC AAATGG GAATGT TTACCA TTGTTT

[0107] 2461 TGAGAT ATTCTG AGTCAT ATGTCT TTCTGT ATTTTG AATTGG TTCCCT AAAAGT TAGTTA

[0108] 2521 CTCTCT CCATCT TTCCAG CCTAGT GCTATA AATACT GCATCT TCAAAT TCTTCC GCCTCT

[0109] 2581 GCATTC AGATTC AAAACC TTATTT GAAATG TATAAT GCACTT TATCCT TTCTAG TCCAAA

[0110] 2641 GAAATA TCTAGT CCAAAT TTTCAG AATTGT TCTATA TTTCTG TCATTT TCTGAC ATTATG

[0111] 2701 ACCAAA AGTACA TCCCCT CATCCT CATATC TCCATC CAATGA TGATTA GGCACA ATGGAA

[0112] 2761 GATCAG ATATGG TCCTGA GAACTT TCTTTC TTCATA CCCTTT TCAGAA TTCTAC TTTTTA

[0113] 2821 GCTTTT ATCCTT TACTTT ATCACC ACCTAG CACAAC ATAGCA TCCTAA GGCTAG TTTTGC

[0114] 2881 ATGCTG TGGGTA TTTGGT TTCTTT TGTTAC TAAAAC AAAGCA TTTTTC TGTTCA CAGTGG

[0115] 2941 GCCAAT ACAATT GTCCAG GCCAAT ACACAT TCTCAA TGCCAT CAGACA GCCATG TTTCTT

[0116] 3001 CATGCT CTGAGG ACTGGG TTGGCT ACCAGA GGAAAT GCTACT TTATTT CTACTG TGAAGA

[0117] 3061 GGAGCT GGACTT CAGCCC AAAATG CTTGTT CTGAAC ATGGTG CTACTC TTGCTG TCATTG

[0118] 3121 ATTCTG AAAAGG ACATGG TAAGAA TAAAAA AAAGAT AAAATA CTACTA TTTTAA TTCATA

[0119] 3181 TGGCTA ATCAGT GCTTTC CTTCCG TTTCTA AGTGGA GAAAAC AAACAA ACAAAC AAACAT

[0120] 3241 TGTACC TATTTC AGATGT AGGAAA ATGTCA TCCCAG AGAACC ATATGA TCAGCC TGAGGT

[0121] 3301 CAAAAT GAGATT TAATTT AATCTT TTAAAT TTGATT TGATTG CTTAGC TTCAAA GACCAG

[0122] 3361 AAATCC AAATAT GCTGAT CCTATA TAGAAT GAGTAC ATGGTC AGATAA GCTGTC ATGCCT

[0123] 3421 CAAGTT GCAGCT TTTGAG AAACAG GATTTA CTATCT ACTTCC CCAGAA GGGTGT TTAATG

[0124] 3481 TAACAA ACTCCT TTGTGC TTTCTT TAAGAA CTTTCT AAAACG ATACGC AGGTAG AGAGGA

[0125] 3541 ACACTG GGTTGG ACTGAA AAAGGA ACCTGG TCACCC ATGGAA GTGGTC AAATGG CAAAGA

[0126] 3601 ATTTAA CAACTG GTAAGT CTCCAG AAGTCT CTTTAT ATTCCC CAGGGT GGCATC TCAGGA

[0127] 3661 AAATCA CTTTCC TTTTCT CCTTTC AAAGCA CTTTAA ATATAA AGGCTG AGCTCT TCAAGA

[0128] 3721 GCTCTA ACAATC TCTTCT GAATGC GTATAC TCTGGA ATTCTT TAATAA CAGGCT TTATTA

[0129] 3781 TTATTC TTTTTC TTTATC CACACT TATAAT ACATAA AATAAA TGCTTA TAGTAA TTTTTC

[0130] 3841 TCAGCA AACTCT AATGTG AGTAGT AGATCT GGTTAA TTTGGA AATATT TCAATA ATAATT

[0131] 3901 ATGATG ATGCAA CAAAAA CCCATT TTCAAT GTCACA TTTAGT CTTTAC TAATAA GACAGT

[0132] 3961 TGCTGG AAAATA AATCCT TCTTAC TATTTC TCAGTT TTCATC TCCACA GAATAA AGAATA

[0133] 4021 GTTTCA ATGCCT AATTAT GAGTTT CTATAC ACCTGA AAGAAA ATTTGC CCCAGA ATTAGA

[0134] 4081 TACTCT ATTTTC ATTACC TTTAGT TAAAAT ACATTT AATGTT TGAAGA TCATTT TATAAG

[0135] 4141 TTTGCT GTTGAT CATAAT ACTGAT AAAGTA TCGAGA TTAAAA TTCAGG TTTCTA GAAAAG

[0136] 4201 GGATTC CTAATA TTTGTC CTATGG CTCCAT ATCCAC AGATGG GCTTCA GGAGCC ATAAAC

[0137] 4261 CCCTTA AAGCTG TATGCA CTAGTA TGCATG GCTGTT GAATGC ATACAA TTTTGT GAAATT

[0138] 4321 ATTTAT TGAAAA ATCTAT GAACTC AGAAAT GACCAA GAATCA CTTATC TGGTGC CATGCC

[0139] 4381 AACCAA ATGAAT GGCGTG CTTTCC TCTTAC TTCCAT ATTTTT TCTCCA CATCCT ATTTTT

[0140] 4441 TTTTCA ATTCTT GTCCAT TGTCTC TACAGA TTAGTC TATGAT TATTTG ACTTGT GTATCC

[0141] 4501 CACTAA GAATTT AAAAGA AGCAGA GGTCAC CATCTC CGAGTT CATTCT CATGTA ACTGAA

[0142] 4561 TGAAAA CAATGA GCTGAA AATAAT TCTAAG TTTTTT TATTGT TTGACA GGTTCA ACGTTA

[0143] 4621 CAGGGT CTGACA AGTGTG TTTTTC TGAAAA ACACAG AGGTCA GCAGCA TGGAAT GTGAGA

[0144] 4681 AGAATT TATACT GGATAT GTAACA AACCTT ACAAAT AA CGAG GATACA TAGATGTATAAA

[0145] 4741 TGACTG TGCCAT AGCACC ACAGGA AAGTTG TGTAAC TGGATA CTGCTC TGCAGTTGAATT

[0146] 4801 TTCCACA AAAGAC TGCACA AACCAA CTTTAC ATCATC CTGGAT CAACTC CAGATAGAACTG

[0147] 4861 TGGGAC ATGAGG AAGAAG ATCCAG GAAGAT CCCTTG ACCAGG AGCTGA AACTGTCACCAA

[0148] 4921 CTGACT GCTAAT CACATC AAGGAT GAGAGG ATGGCT ATGAAG CCTCAT CAACGCACTTTA

[0149] 4981 TTTTTT TTTAAA CCTAGA ATGAAT AGAACT GACTAG CTCTAG AGTTAC TATTTATTGCCG

[0150] 5041 AATGAC TGCTAC AGCCAG TGCCTT TACGCA TTTGCA CTATTT GGAGGG GTTTCAGTGGTA

[0151] 5101 GGAAGA GCTGAA TGTAGG TACAGG AAGATT TGACTG ATTAAT GATTTT CTTAAATTCAAA

[0152] 5161 AAGCAT TCACAA ATAGAA CAATGC TTATGA AACCAA GCTTTG CAGTAA CTCCATCTATTT

[0153] 5221 ACAGAA ACTTTG CCTCAT TATTTT GTCAAT TGTTCT CCCCCA AAGAAT AAGAGTGGCCTT

[0154] 5281 TTCTTT AACTTC CTCCGT GTAGAC AGCTAA TTTTCA ATGGTA CATATT TCTTAACCTTTA

[0155] 5341 AAAACC TCTGTA GCGTAT TTCAAG GAAGAA AACAAA AGCACA GCATAT GAGTAATATTTG

[0156] 5401 TAGAGC AGATTT CAAAGT GCTGGA AAGAAA AGTGCA ATACGT GTAGTG GCAGATCTCTGT

[0157] 5461 CAGGAC ACACCC TGTGGT TTGACC TTGGAA TAACTC CAGTCT TGTCAC ATAACCTGTTTG

[0158] 5521 TGTCTT TCCCAG TCCTTT GTCCAA AATATT TCCCAT GTGCAA TAAAGT GTTTATATATTT

[0159] 5581 GTTTGT GTTTTA AAACCC ACTATA AAGTTT AAGCTG TTTCTA TGTATG TGAAAATAAAAG

[0160] 5641 AATAGA TAAATG GTTTCC AGCGGT TCTCAT TCAAAC TGCATT CAAAGT ACCGTGTTATAG

[0161] 5701 CATCAA ACTGCC TTCCAG GAAGGG GGGTCT CAAAAC CTTTAG ATTAGT GAAGTTCACAAA

[0162] 5761 CCTCAC CAGAGA AGAGTC TTTGTG CAGTGG CCAGTG GTCAAT GCAAGG TCTCCAAACTAG

[0163] 5821 TCAGAG CATGGA GAATTG GAGGCT GTGAAG TGCACA GCCACA AAATAA GAACTCTCCCCA

[0164] 5881 AAAAGG TGCAGG AAATAT CATGGA AGCCAG GGCAGC AAAACT GTAAGA TCCAGAGGTCAG

[0165] 5941 GGAGGA CTGGGT AAGAAA CATCTT CTGGGC TTGAGA GAACCA TTGCAC TCATGAATTTAT

[0166] 6001 AGCCAC AAGACC TGCACA GAATCA AGCCAG GCAACA TCCCCA CAGGAT GGAAGAGAGCA

[0167] 6061 AGGGCA TCCTCA CTCCTA CCTGAG AAGCTA TTGACA GTCAAT AGCTAC TAGGAGAGAAGC

[0168] 6121 AGACAG TTCTTA TTCCCA AGTATG TCCCCA GTAATT GACTGT GCTCCA GTGAATGACCCC

[0169] 6181 ATATCC ATGCAT TATGAA CAGCAT TAATTG GTGAAT TAATTG TGAATT AGCAGCATTAAT

[0170] 6241 TGGACT HELPGG GATTA TAAAAA GAAAAG HELPG AAGTTT GGAGGA GTTGGAGGACAA

[0171] 6301 GGTGGT GGTGAA TATGAT TGGAAC ACTTTG CATACA TGTATG AAATTC TCAAAGAATAGC

[0172] 6361 AGCAAG TTATAC ASSISTANCE GGAAAA AGCAAG ACAGTA GAACGC CTACTA AAAGTCTTCCAC

[0173] 6421 AATGCA AAACCC TAGTCT TATTAA CACACA TTCATG CCAAAA GGATAC ACTCATTTTTGC

[0174] 6481 ATATAT CAGATG CTCTGA ATCTCA ACATAT CCAAGG TTCTGA AAACAT TTTGTTCAACTC

[0175] 6541 TGTTCT GAAAAC ATCTTT CTCACT ATATAC TTTATT CACACA CACTAA TTCATGTAGTAT

[0176] (SEQ ID No.4).

[0177] 3) Construction of sgRNA

[0178] Using the PrimerStar or PrimerStar Max system, with sgRNA-F and sgRNA-R as primers, and the correctly sequenced puc57-sgRNA plasmid (1:30 dilution) as a template, PCR was performed. PCR conditions are shown in Tables 1 and 2. The PCR product was purified to prepare a template for sgRNA transcription. SgRNA transcription was performed using the T7-ShortScript in vitro transcription kit (AM1354).

[0179] Table 1. PCR amplification system for sgRNA

[0180]

[0181] Table 2 PCR reaction conditions for sgRNA

[0182]

[0183] 4) Screening of sgRNA from CD69 humanized mice

[0184] Two sets of sgRNAs (Cd69-5S1+Cd69-3S1 and Cd69-5S2+Cd69-3S2, with specific sequence information shown in Table 3) were designed and synthesized. The sgRNA recognition sites are located at both ends of the extracellular and transmembrane regions of the mouse Cd69 gene. Each pair of sgRNAs was then incubated with Cas9 protein and injected into 0.5-day-old fertilized eggs. After culturing to the blastocyst stage, the KO positivity rate of the mouse Cd69 gene was identified to verify the sgRNA cleavage activity.

[0185] sgRNA cleavage assay method: The collected blastocysts were subjected to PCR amplification. The PCR protocol is shown in Table 3-4. The amplified bands were subjected to next-generation sequencing. The results were compared with the WT bands, and the probability of mutation was calculated.

[0186] Table 3 sgRNA Information

[0187]

[0188] 5) Establishment of CD69 humanized mouse model

[0189] The selected sgRNAs (Cd69-5S1+Cd69-3S1 or Cd69-5S2+Cd69-3S2) were used to design and construct ssDNA donors carrying human sequences. The ssDNA donors and the Cas9 / sgRNA system were injected into 0.5-day-old mouse zygotes and transplanted into 0.5-day-old pseudopregnant female mice. After the mice were born, the target mice (F0) were selected by genetic identification.

[0190] 6) Genotyping of humanized F0 mice

[0191] The tail genomic DNA of the obtained F0 mice was subjected to PCR identification at both ends after target insertion using the two primer pairs shown in Table 4. The PCR reaction conditions and procedures are shown in Tables 5 and 6. Primers mCD69-5tF1 / hCD69-5tR1 were located outside the 5' homologous arm and inside the human fragment of the ssDNA donor, respectively. If the amplification of this primer pair produced PCR products, it indicated that the target donor had been effectively inserted at the 5' end of the mouse genome. hCD69-3tF1 / mCD69-3tR1 were located inside the human fragment of the ssDNA donor and outside the 3' homologous arm, respectively. If the amplification of this primer pair produced PCR primers, it indicated that the target donor had been effectively inserted at the 3' end of the mouse genome.

[0192] Table 4. F0 identification primers

[0193]

[0194] Table 5 PCR reaction system

[0195]

[0196] Table 6 PCR Reaction Conditions

[0197]

[0198] A total of 46 F0 mice were obtained in this experiment (21 mice (numbered 1-21) were obtained by injection with sgRNA containing a combination of Cd69-5S1 and Cd69-3S1, and 25 mice (numbered 22-46) were obtained by injection with sgRNA containing a combination of Cd69-5S2 and Cd69-3S2). Positive F0 mice were detected using the above identification protocol. Four positive F0 mice were obtained by targeting against a BALB / c mouse background. Genomic PCR identification of hCD69 F0 mouse tails is shown below. Figure 2As shown (WT control is BALB / c genomic DNA; N is blank control; P is positive control; M is DNA Marker: 8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp). The human CD69 gene 5' and 3' markers in mice #5, #28, #44, and #46 were all positive in identification and sequencing, indicating that these mice are F0 positive mice that underwent correct gene recombination.

[0199] After reaching sexual maturity, positive F0 generation mice were mated with wild-type background mice. The genomes of the resulting F1 generation were extracted by tail clipping and genetic identification was performed. Figure 3 (DNA Marker: 8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp). PCR identification results showed that the 5' and 3' bases of the human CD69 gene in mice 51-53# and 56-59# were positive (among which, the hCD69 gene in mice 51-53#, 57#, and 58# had base mutations, specifically intron mutations), indicating that these mice were positive mice that had undergone correct gene recombination. After large-scale propagation of the F1 generation, cross-pollination was performed to obtain homozygous CD69 humanized mice, abbreviated as BALB / c-hCD69.

[0200] Experiment 2: Detection of human CD69 protein expression and validation of the immune system in CD69 humanized mice.

[0201] 1. Test Methods

[0202] The presence of human CD69 protein in BALB / c-hCD69 homozygous mice was analyzed by flow cytometry. If the humanized mice expressed human CD69 protein but not mouse Cd69 protein, and did not cause significant abnormalities in the immune system, it indicates that the model was successfully constructed and can be used for drug efficacy experiments.

[0203] The spleen, a major expression tissue of CD69, was selected. One day before flow cytometry, CD3E antibody was injected intraperitoneally into mice for 24 h of stimulation. The spleen of the mice was then harvested, the required experimental weight was cut off, ground and digested, Fc block was added for blocking, and then incubated with human CD69 antibody before flow cytometry analysis.

[0204] For immune system validation, peripheral blood or the spleen, a major immune organ, was selected from mice. Blood was collected from the heart of CD69 humanized homozygous mice and corresponding background mice into EP tubes containing anticoagulant. After blood collection, the spleen was removed, and extracellular proteins of tissue cells were stained with mouse-derived immune cell surface antibodies. The number of immune cells was detected by flow cytometry.

[0205] 2. Test Results

[0206] The spleen, a major CD69-expressing tissue, was selected to detect CD69 protein expression. Compared with wild-type mice, BALB / c-hCD69 homozygous mice were able to express CD4+ protein. + / CD8 + Humanized CD69 protein was successfully expressed on the surface of T cells, B cells, and NK cells (see...). Figure 4 and Figure 5 Meanwhile, the increase in chimeric CD69 protein expression after stimulation was similar to the increase in mCD69 expression in wild-type mice, suggesting that chimeric CD69 protein can effectively and correctly transmit activation signals in the BALB / c-hCD69 mouse model.

[0207] Peripheral blood or spleen was collected from BALB / c-hCD69 homozygous mice and BALB / c background mice. Extracellular proteins in the tissue cells were stained with mouse-derived T, B, NK, DC, and macrophage surface antibodies. After washing the cells with PBS, flow cytometry was performed to detect T(CD4+) antibodies. + CD8 + The percentages of various immune cells, including T, B, NK, DC, and macrophages, were observed in the peripheral blood and spleen of BALB / c-hCD69 homozygous mice. The percentages of each immune cell type, such as T, B, and NK, in these cells were similar to those in BALB / c background mice (see [link to relevant documentation]). Figures 6 to 13 The above data indicate that human CD69 participates in the immune response process in mice. Humanized mice have normal immune systems and are no different from ordinary background mice. They can be used in drug efficacy evaluation systems that require the use of the immune system for drug evaluation.

[0208] Experiment 3: Evaluation of the efficacy of anti-CD69 antibody in asthma treatment using CD69 humanized mice.

[0209] 1. Test Methods

[0210] In vitro data have validated that CD69 humanized mice can normally express human CD69 protein, and the immune response to this protein in mice is normal. However, whether these mice can be used to reflect the actual situation of hCD69 in humanized mice remains to be verified. Therefore, an hCD69 mouse asthma model was induced by intratracheal instillation of OVA, and the efficacy of CD69 humanized antibody drugs against autoimmune diseases was evaluated.

[0211] Male SPF-grade BALB / c-hCD69 mice aged 10-13 weeks were randomly divided into four groups according to body weight: control group (G1), induction group (G2), hCD69 antibody drug 1 group (G3), and hCD69 antibody drug 2 group (G4). Groups G2-G4 underwent initial sensitization by intraperitoneal injection of 200 μl Solution A on days 0, 7, and 14; and induced sensitization by intratracheal infusion of 20 μl Solution B on days 21-26 (Solution A: 0.1 mg / ml OVA + alum adjuvant (0.1 mg / ml) + PBS; Solution B: 10 mg / ml OVA + PBS) to establish an asthma model. Groups G3 and G4 received intraperitoneal injection of 10 mg / kg anti-hCD69 monoclonal antibody on days 19, 22, and 25 before model establishment (dosing regimens are shown in Table 7 below, where hCD69 antibody drug 1 was a self-produced antibody, and anti-hCD69 2.8 mAb was a known antibody). 24-hour food intake was measured daily. On day 26, 2-3 hours after model establishment, mice were euthanized. Bronchoalveolar lavage fluid (BALF) was collected; 1 mL of BALF was injected into the lungs after tracheal intubation for alveolar lavage, repeated three times. The collected BALF was centrifuged at 400g for 7 minutes, and the cell pellet was resuspended for cell counting and flow cytometry analysis of eosinophils, T cells, and neutrophils. Lungs were harvested; the left lung was fixed and embedded, and then used for HE staining to detect airway wall thickening. The upper and middle lobes of the right lung were flash-frozen in liquid nitrogen and stored at -80℃. The mRNA expression of IL-4, IL-6, IL-33, and TNF-α was detected using qRT-PCR.

[0212] Table 7. Dosing regimens for OVA-induced BALB / c-hCD69 mouse asthma model

[0213]

[0214] 2. Test Results

[0215] The efficacy of anti-CD69 antibodies was evaluated by establishing an OVA-induced BALB / c-hCD69 mouse asthma model. Food intake was monitored daily in each group of mice. Mice in the G2 group showed decreased appetite and reduced food intake after continuous OVA challenge (see [link to OVA model]). Figure 14 Bronchoalveolar lavage fluid (BALF) was collected, and cell classification and counting were performed by flow cytometry. The results showed that compared with the G1 group, the number of lymphocytes (p<0.05), neutrophils (p<0.05), and eosinophils (p<0.05) in the BALF of mice in the G2 group was significantly increased. Treatment with hCD69 monoclonal antibody in the G3 and G4 groups reduced the number of lymphocytes, neutrophils, and eosinophils in the BALF, but the difference was not statistically significant (see [link to relevant documentation]).Figure 15 ).

[0216] Lungs were collected, and the left lung was fixed and embedded. After embedding, airway wall damage and thickness were observed and measured by HE staining. It was found that compared with the G1 group, the G2 group mice had bronchial stenosis, meaning the tracheal wall thickness was higher than the control group. Treatment with hCD69 antibody drugs in the G3 and G4 groups could alleviate airway wall thickening, but there were no statistically significant differences (see...). Figure 16 ).

[0217] The right lung was flash-frozen with liquid nitrogen, RNA was extracted, and the mRNA expression of inflammatory factors was detected. The results showed that, compared with the model control group G2, the mRNA levels of mIL-4, mIL-6, mIL-33, and mTNFα in lung tissue were reduced in groups G3 and G4, but the differences were not statistically significant (see [link to study].) Figure 17 ).

[0218] The efficacy study results indicate that three doses of the CD69 antibody drug effectively inhibited asthma development, specifically manifested in a reduction of inflammatory cells in BALF, decreased expression of inflammatory factors in lung tissue, and relief of airway wall thickening. Therefore, CD69 humanized mice are a powerful tool for evaluating the in vivo efficacy of CD69 inhibitors or CD69-targeting antibodies.

[0219] Although the implementation steps of this method have been described in detail, those skilled in the art can still modify some parameters and the overall scheme within the scope of this invention. Therefore, any changes, substitutions, adjustments, etc., made within the spirit and principles of this invention should be considered within the scope of this invention.

Claims

1. A method for constructing a CD69 humanized mouse model, characterized in that, The mouse model obtained by the construction method expresses human-mouse chimeric CD69 protein, and the mouse Cd69 protein in the mouse model is partially knocked out; the construction method selects 62-199 AA of the human CD69 gene to replace 62-199 AA of the mouse Cd69 gene.

2. The construction method according to claim 1, characterized in that, The amino acid sequence of the human CD69 gene selected in the mouse model obtained by the construction method is shown in SEQ ID No. 1, and the amino acid sequence of the replaced mouse CD69 gene is shown in SEQ ID No.

2.

3. The construction method according to claim 1, characterized in that, The construction method includes the following steps: (1) Construct a targeting vector expressing the humanized CD69 gene for insertion of the humanized CD69 gene; (2) Design sgRNAs targeting the extracellular and transmembrane regions of the mouse Cd69 gene and obtain the above sgRNAs using in vitro transcription technology; (3) The targeting vector constructed in step (1), the sgRNA obtained in step (2) and the Cas9 protein are co-injected or co-electrotransduced into the cytoplasm or nucleus of mouse zygote cells, and the zygote is transplanted into pseudopregnant mice. Genotyping of pseudopregnant mice is performed, and positive F0 mice with the correct human fragment inserted are screened. (4) F0 mice were bred with background mice to obtain F1 mice. Gene identification was performed on the tails of F1 mice to screen out CD69 humanized mouse models.

4. The construction method according to claim 3, characterized in that, Step (1) includes the following steps: based on the structure and function of human CD69, the extracellular region encoded by the mouse Cd69 gene is replaced by the extracellular region encoded by the human CD69 gene, while retaining the intracellular region sequence of the mouse. The selected human CD69 gene amino acid sequence is shown in SEQ ID No.1, and the replaced mouse CD69 gene amino acid sequence is shown in SEQ ID No.

2.

5. The construction method according to claim 3, characterized in that, Step (1) includes the following steps: selecting 62-199 AA of the human CD69 gene and replacing 62-199 AA of the mouse Cd69 gene with homologous recombination technology. The selected human CD69 gene sequence is shown in SEQ ID No.

3.

6. The construction method according to claim 3, characterized in that, The successful target vector sequence constructed in step (1) is shown in SEQ ID No.

4.

7. The construction method according to claim 3, characterized in that, The gene sequence of sgRNA in step (2) is (a) SEQ ID NO.5 and SEQ ID NO.6, or (b) SEQ ID NO.7 and SEQ ID NO.

8.

8. The application of the mice obtained by the construction method according to any one of claims 1-7 in the study of CD69 gene-related functions and mechanisms of action.

9. The use of mice obtained by the construction method according to any one of claims 1-7 in screening drugs for treating diseases related to the CD69 gene.

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