Construction method and application of ERBB gene humanized non-human animal

Abstract

The invention provides an ERBB gene humanized non-human animal and a construction method thereof, a humanized ERBB protein, a humanized ERBB gene, a targeting vector of the ERBB gene and application of the targeting vector in the field of biological medicine, and a nucleotide sequence for coding the human ERBB protein is introduced into a non-human animal genome in a homologous recombination mode; the human or humanized ERBB protein can be normally expressed in the animal body, and the human or humanized ERBB protein can be used as an animal model for human ERBB signal mechanism research and inflammation, tumor or immune related disease drug screening, and has important application value for new drug research and development of immune targets.

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic engineering and gene genetic modification, and specifically relates to a method for constructing an ERBB gene-modified non-human animal model and its application in the field of biomedicine. Background Art

[0002] ERBB belongs to the ErbB tyrosine kinase receptor family and is a type I transmembrane glycoprotein widely distributed on the cell surfaces of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc. Activation of ERBB leads to autophosphorylation of receptor tyrosine kinase, thereby initiating a series of downstream signaling pathways, which play an important role in physiological processes such as cell proliferation, differentiation and survival. Its abnormal activation, such as receptor overexpression, mutation, ligand-dependent receptor dimerization, and ligand-independent activation, controls tumor growth through signal transduction and is closely related to angiogenesis, tumor invasion and metastasis. For example, ERBB is abnormally expressed in cancers such as glioma, intestinal cancer, head and neck cancer, breast cancer, rectal cancer, endometrial cancer, medulloblastoma, pancreatic cancer and prostate cancer, especially in ovarian cancer and non-small cell lung cancer. It is directly related to the poor cure of patients and is an important target for tumor treatment.

[0003] As a tumor target, ERBB is a hot spot for the research and development of antibody drugs. It can also be used in combination with other targets as a target for bispecific antibodies. For example, an antibody targeting the tumor ERBB target and an antibody targeting the stem cell target LGR5 are prepared into a bispecific antibody. ERBB as a tumor target can improve the targeting of LGR5 antibodies to tumor stem cells and reduce toxicity, preventing tumor cells from differentiating and transforming back into tumor stem cells; ERBB antibodies can also play a role in clearing tumor cells.

[0004] In the development of ERBB target-related drugs, the use of experimental animals for the discovery and screening of human drugs, especially antibody drugs, is a very important research tool; at the same time, the use of experimental animals for preclinical studies such as pharmacology and efficacy is an indispensable development step. However, due to the differences in physiological structure and metabolic system between animals and humans, traditional animal models cannot well reflect the actual condition of drugs in the human body. Establishing a humanized animal model that is closer to human physiological characteristics is an urgent need in the biopharmaceutical industry.

[0005] In view of the huge application potential of ERBB in the treatment of tumor diseases, in order to further explore the relevant biological characteristics, improve the effectiveness of preclinical trials of ERBB target-related drugs, and reduce the risks of clinical research, this field urgently needs to develop non-human animal models of ERBB-related signaling pathways. Summary of the invention

[0006] In a first aspect, the present invention provides a method for constructing a non-human animal with humanized ERBB gene, wherein the non-human animal expresses human or humanized ERBB protein in vivo.

[0007] Preferably, the expression of endogenous ERBB protein in the non-human animal is reduced or absent.

[0008] Preferably, the humanized ERBB protein comprises all or part of the human ERBB protein.

[0009] Preferably, the humanized ERBB protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human ERBB protein.

[0010] Further preferably, the humanized ERBB protein comprises a human or humanized extracellular region.

[0011] In a specific embodiment of the present invention, the humanized ERBB protein comprises an endogenous signal peptide, a human or humanized extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region.

[0012] In a specific embodiment of the present invention, the humanized ERBB protein comprises a human extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. Preferably, the humanized ERBB protein further comprises an endogenous ERBB signal peptide.

[0013] Preferably, the humanized extracellular region comprises the amino acid sequence shown at positions 37-643 of SEQ ID NO: 2. Preferably, it also comprises the amino acid sequence shown at positions 25-36 and / or 646-647 of SEQ ID NO: 1.

[0014] Preferably, the human extracellular region comprises the amino acid sequence shown at positions 25-645 of SEQ ID NO:2.

[0015] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by all or part of exons 1 to 28 of the human ERBB gene. Further preferably, it comprises the amino acid sequence encoded by all or part of any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 28. More preferably, it comprises the amino acid sequence encoded by all or part of exons 1 to 17. More preferably, the amino acid sequence encoded by the portion of exon 2 to the portion of exon 17, or the portion of exon 1 to the portion of exon 17, wherein the portion of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the portion of exon 1 contains the nucleotide sequence encoding the extracellular region in exon 1, and the portion of exon 2 contains at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135 4, 135, 140, 150, 152 bp nucleotide sequence; preferably, the portion of exon 2 includes the nucleotide encoding the 6th-15th (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N-terminus of the extracellular region to the last nucleotide of exon 2, and the portion of exon 17 includes at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 includes the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region without the nucleotide sequence.

[0016] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that differs from the amino acid sequence encoded by SEQ ID NO: 5 or 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in the amino acid sequence encoded by SEQ ID NO: 5 or 8, including substitution, deletion and / or insertion of one or more amino acids.

[0017] In a specific embodiment of the present invention, the humanized ERBB protein comprises all or part of the extracellular region of the human ERBB protein, preferably, comprises at least 200 consecutive amino acids in the extracellular region of the human ERBB protein, for example, comprises at least 200, 300, 400, 500, 600, 605, 606, 607, 608, 609, 610, 620, 621 consecutive amino acids; the extracellular region of the humanized ERBB protein comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645; or, comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID The amino acid sequence as set forth in NO:2 at positions 25-645 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical; or, comprises an amino acid sequence that differs from the amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645, including a substitution, deletion and / or insertion of one or more amino acid residues.

[0018] Preferably, the humanized ERBB protein extracellular region comprises the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises NO:2 The amino acid sequence shown at positions 37-639, 37-645 or 25-639, including substitution, deletion and / or insertion of one or more amino acid residues.

[0019] Preferably, the humanized ERBB protein also comprises a portion of a non-human animal ERBB protein, further preferably comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a non-human animal ERBB protein, more preferably comprises all of the signal peptide, transmembrane region and cytoplasmic region of a non-human animal ERBB protein, and further preferably also comprises a portion of the extracellular region.

[0020] In a specific embodiment of the present invention, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID The amino acid sequence shown in NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in SEQID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0021] Preferably, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The amino acid sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as shown in SEQ ID NO: 1 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0022] In a specific embodiment of the present invention, the amino acid sequence of the humanized ERBB protein comprises any one of the following groups:

[0023] A) the amino acid sequence shown in SEQ ID NO: 9;

[0024] B) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9;

[0025] C) differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0026] D) an amino acid sequence as shown in SEQ ID NO: 9, including substitution, deletion and / or insertion of one or more amino acid residues.

[0027] Preferably, the non-human animal genome comprises a human or humanized ERBB gene, and further preferably, the humanized ERBB gene comprises a portion of a human ERBB gene.

[0028] Preferably, the human or humanized ERBB gene encodes the human or humanized ERBB protein.

[0029] Preferably, the humanized ERBB gene comprises all or part of exons 1 to 28 of the human ERBB gene. Further preferably, the humanized ERBB gene comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons of exons 1 to 28 of the human ERBB gene. More preferably, it comprises all or part of exons 1 to 17. Even further preferably, the humanized ERBB gene comprises part of exon 2, all of exons 3 to 16, and part of exon 17 of the human ERBB gene, or part of exon 1, all of exons 2 to 16, and part of exon 17, preferably further comprising introns 1-2, introns 2-3, and / or introns 16-17, more preferably comprising any intron between exons 1-17 or 2-17, and even more preferably Preferably, the humanized ERBB gene comprises a portion of exon 2 to a portion of exon 17 of the human ERBB gene, or a portion of exon 1 to a portion of exon 17; wherein the portion of exon 1 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the portion of exon 1 comprises a portion of exon 1 to a portion of exon 17; The nucleotide sequence encoding the extracellular region in the exon, the portion of exon 2 comprises at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 140, 150, 152 bp of nucleotide sequence; preferably, the portion of exon 2 comprises the 6th to 15th (for example, 6, 7, 8, 9, 10, 11, 12, 13, 14) nucleotide sequence from the N-terminal of the coding extracellular region. , 15) amino acids to the last nucleotide of exon 2, the portion of exon 17 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 comprises the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region removed.

[0030] In a specific embodiment of the present invention, the portion of the human ERBB gene comprises the nucleotide sequence shown in SEQ ID NO:5; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:5; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO:5; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0031] Preferably, the humanized ERBB gene comprises all or part of the nucleotide sequence encoding the human ERBB protein. It is further preferred that the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human ERBB protein, wherein the humanized ERBB gene comprises the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that differs from the amino acid sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645. The nucleotide sequence of positions 37-643 of NO: 2 or positions 25-645 of SEQ ID NO: 2 includes a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0032] Preferably, the humanized ERBB gene comprises a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that differs from an amino acid sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639. NO: 2 The nucleotide sequence at positions 37-639, 37-645 or 25-639, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0033] Preferably, the humanized ERBB gene further comprises all or part of the non-human animal ERBB gene. It is further preferred to comprise all or part of exons 1 to 28 of the non-human animal ERBB gene, and more preferably comprises all of exon 1, part of exon 2, part of exon 17 to all of exon 28 of the non-human animal ERBB gene, or part of exon 1, part of exon 17 to all of exon 28.

[0034] In a specific embodiment of the present invention, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID The amino acid sequence of NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence encoding SEQ ID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0035] Preferably, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210; or, comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210. NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence shown in positions 1-24, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence comprises a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence shown in positions 1-36, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210.

[0036] In a specific embodiment of the present invention, the nucleotide sequence of the humanized ERBB gene comprises any one of the following groups:

[0037] A) the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8;

[0038] B) the transcribed mRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8;

[0039] C) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO:8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or,

[0040] D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0041] Preferably, the human or humanized ERBB gene is homozygous or heterozygous for the endogenous replaced locus.

[0042] Preferably, the humanized ERBB gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor can be a conventional substance that can be induced or repressed. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0043] Preferably, the construction method comprises introducing a donor nucleotide sequence into the ERBB locus of a non-human animal.

[0044] Preferably, the donor nucleotide sequence may be genomic DNA, CDS or cDNA.

[0045] Preferably, the donor nucleotide sequence comprises one of the following groups:

[0046] A) Nucleotide sequence encoding human or humanized ERBB protein;

[0047] B) all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a human ERBB protein, preferably, all or part of a nucleotide sequence encoding an extracellular region of a human ERBB protein, further preferably, comprising a nucleotide sequence encoding at least 200 consecutive amino acids of an extracellular region of a human ERBB protein, more preferably, comprising a nucleotide sequence encoding positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2;

[0048] C) the nucleotide sequence of a human or humanized ERBB gene; or,

[0049] D) All or part of exons 1 to 28 of the human ERBB gene. Preferably, it comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 28. Further preferably, it comprises all or part of exons 1 to 17 of the human ERBB gene, and further preferably, it comprises part of exon 2 to part of exon 17, or part of exon 1 to part of exon 17, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the part of exon 1 comprises the nucleotide sequence encoding the extracellular region in exon 1, and the part of exon 2 comprises at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133 , 134, 135, 140, 150, 152 bp nucleotide sequence; preferably, the portion of exon 2 includes the nucleotide encoding the 6th-15th (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N-terminus of the extracellular region to the last nucleotide of exon 2, and the portion of exon 17 includes at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 includes the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region removed; further preferably, the targeting vector includes SEQ ID NO:5; or, comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:5; or, comprising a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence as shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0050] Preferably, the introduction described in the present application includes but is not limited to insertion, replacement or transgenesis.

[0051] Preferably, the human or humanized ERBB gene is operably linked to endogenous regulatory elements of an endogenous ERBB gene on at least one chromosome.

[0052] Preferably, the human or humanized ERBB gene is regulated in a non-human animal by a regulatory element. Further preferably, the regulatory element may be endogenous or exogenous. The exogenous regulatory element is derived from the human ERBB gene.

[0053] Preferably, the regulatory element includes but is not limited to an endogenous promoter.

[0054] In a specific embodiment of the present invention, the endogenous regulatory element is derived from a non-human animal ERBB gene.

[0055] Preferably, the introduced position is located after the endogenous regulatory element of the ERBB gene.

[0056] Preferably, the introduction is replacement or insertion, preferably, the introduction into the non-human animal ERBB locus is replacement of the corresponding region of the non-human animal, further preferably, the replacement comprises replacement of all or part of exons 1 to 17 of the non-human animal ERBB gene, more preferably, the replacement comprises replacement of part of exon 2 to part of exon 17 of the non-human animal, or, replacement of part of exon 1 to part of exon 17.

[0057] Preferably, the nucleotide sequence encoding the amino acids shown at positions 37 to 645 of SEQ ID NO: 1 or positions 25 to 647 of SEQ ID NO: 1 is replaced.

[0058] In a specific embodiment of the present invention, the construction method comprises replacing the endogenous nucleotide sequence encoding the ERBB protein of a non-human animal with a nucleotide sequence encoding the human or humanized ERBB protein.

[0059] In a specific embodiment of the present invention, the construction method comprises replacing all or part of the nucleotide sequence encoding the extracellular region of ERBB in a non-human animal endogenous manner with all or part of the nucleotide sequence encoding the extracellular region of ERBB in a non-human animal endogenous manner.

[0060] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 endogenously in a non-human animal with a nucleotide sequence encoding a human or humanized ERBB protein.

[0061] In a specific embodiment of the present invention, the construction method comprises replacing the endogenous nucleotide sequence encoding SEQ ID NO: 1 in a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or 9.

[0062] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence endogenously encoding SEQ ID NO: 1 at positions 37-645 or SEQ ID NO: 1 at positions 25-647 in a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-643 or SEQ ID NO: 2 at positions 25-645.

[0063] In a specific embodiment of the present invention, the construction method comprises replacing the corresponding endogenous region of a non-human animal with a nucleotide sequence encoding positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2.

[0064] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence endogenously encoding SEQ ID NO: 1 in a non-human animal with a nucleotide sequence comprising SEQ ID NO: 8.

[0065] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence of the endogenous ERBB gene of the non-human animal with a nucleotide sequence comprising the human ERBB gene.

[0066] In a specific embodiment of the present invention, the construction method comprises replacing a portion of exon 2 to exon 17 of an endogenous ERBB gene of a non-human animal with a portion of exon 2 to exon 17 of a human ERBB gene.

[0067] In a specific embodiment of the present invention, the construction method comprises replacing a portion of exon 1 to exon 17 of an endogenous ERBB gene of a non-human animal with a portion of exon 1 to exon 17 of a human ERBB gene.

[0068] In a specific embodiment of the present invention, the construction method comprises replacing the corresponding endogenous region of a non-human animal with a nucleotide sequence shown in SEQ ID NO:5.

[0069] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence at positions 37-645 of SEQ ID NO: 1 endogenously encoded by a non-human animal with the nucleotide sequence shown in SEQ ID NO: 5.

[0070] Preferably, gene editing technology is used to construct non-human animals with humanized ERBB genes, and the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

[0071] In a specific embodiment of the present invention, the construction method includes modifying the coding frame of the non-human animal ERBB gene, and inserting the nucleotide sequence encoding the human or humanized ERBB protein or the nucleotide sequence of the humanized ERBB gene after the endogenous regulatory element of the non-human animal ERBB gene. Among them, the coding frame of the modified non-human animal ERBB gene can be used to knock out the functional region of the non-human animal ERBB gene or to insert a sequence so that the non-human animal ERBB protein is not expressed or the expression is reduced or the expressed protein is non-functional. Further preferably, the coding frame of the modified non-human animal ERBB gene can be to knock out all or part of exon 1 to exon 17 of the non-human animal ERBB gene, preferably knocking out the part containing exon 2 to the part of exon 17, or, the part of exon 1 to the part of exon 17.

[0072] Preferably, a targeting vector is used for constructing a non-human animal, wherein the targeting vector comprises the donor nucleotide sequence.

[0073] Preferably, the targeting vector further comprises a 5' arm (5' homology arm) and / or a 3' arm (3' homology arm).

[0074] The 5' arm is a DNA fragment homologous to the 5' end of the switch region to be changed, which is selected from 100-10000 nucleotides of the genomic DNA of the ERBB gene of non-human animals. Preferably, the 5' arm has at least 90% homology with the nucleotides of NCBI accession number NC_000077.7. Further preferably, the 5' arm sequence is shown in SEQ ID NO: 3.

[0075] The 3' arm is a second DNA fragment homologous to the 3' end of the conversion region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the ERBB gene of non-human animals; preferably, the 3' arm has at least 90% homology with nucleotides with NCBI accession number NC_000077.7; further preferably, the 3' arm sequence is as shown in SEQ ID NO: 4.

[0076] Preferably, in order to improve the recombination efficiency, a sgRNA targeting the ERBB gene can also be used together with a targeting vector to construct a non-human animal, wherein the sgRNA targets the non-human animal ERBB gene, and the sequence of the sgRNA is on the target sequence on the ERBB gene to be changed.

[0077] Preferably, the sgRNA target site is located on the sequence from exon 2 to exon 17 of the ERBB gene.

[0078] Preferably, the target site of the sgRNA is located on the exon 2 and / or exon 17 sequence of the ERBB gene.

[0079] Preferably, the target sequence of the sgRNA on the ERBB gene is shown in SEQ ID NOs: 10 and 11.

[0080] In a specific embodiment of the present invention, the construction method includes introducing the above-mentioned targeting vector, sgRNA targeting the ERBB gene and Cas9 into non-human animal cells, culturing the cells (preferably fertilized eggs), and then transplanting the cultured cells into the fallopian tubes of female non-human animals, allowing them to develop, and identifying and screening to obtain non-human animals with humanized ERBB genes.

[0081] In another specific embodiment of the present invention, the construction method includes introducing the above-mentioned targeting vector into embryonic stem cells of non-human animals, and after a short culture, introducing it into a previously isolated blastocyst, and transplanting the obtained chimeric blastocyst into the oviduct of a recipient mother mouse, allowing it to develop, and identifying and screening to obtain non-human animals with humanized ERBB genes.

[0082] According to some embodiments of the present invention, the construction method further comprises: mating, in vitro fertilization or direct gene editing of non-human animals with humanized ERBB genes with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0083] Preferably, the other genes are at least one of NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R and HER2.

[0084] Preferably, the non-human animal also expresses at least one of human or humanized NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R and HER2 proteins.

[0085] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal is homozygous for the endogenous replaced locus.

[0086] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal is heterozygous for the endogenous replaced locus.

[0087] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0088] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0089] The non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0090] The second aspect of the present invention provides a non-human animal with humanized ERBB gene, wherein the non-human animal expresses human or humanized ERBB protein in vivo.

[0091] Preferably, the expression of endogenous ERBB protein in the non-human animal is reduced or absent.

[0092] Preferably, the humanized ERBB protein comprises all or part of the human ERBB protein.

[0093] Preferably, the humanized ERBB protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human ERBB protein.

[0094] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by all or part of exons 1 to 28 of the human ERBB gene. Further preferably, it comprises the amino acid sequence encoded by all or part of any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 28. More preferably, it comprises the amino acid sequence encoded by all or part of exons 1 to 17. More preferably, the amino acid sequence encoded by the portion of exon 2 to the portion of exon 17, or the portion of exon 1 to the portion of exon 17, wherein the portion of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the portion of exon 1 contains the nucleotide sequence encoding the extracellular region in exon 1, and the portion of exon 2 contains at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135 4, 135, 140, 150, 152 bp nucleotide sequence; preferably, the portion of exon 2 includes the nucleotide encoding the 6th-15th (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N-terminus of the extracellular region to the last nucleotide of exon 2, and the portion of exon 17 includes at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 includes the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region without the nucleotide sequence.

[0095] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that differs from the amino acid sequence encoded by SEQ ID NO: 5 or 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in the amino acid sequence encoded by SEQ ID NO: 5 or 8, including substitution, deletion and / or insertion of one or more amino acids.

[0096] In a specific embodiment of the present invention, the humanized ERBB protein comprises all or part of the extracellular region of the human ERBB protein, preferably, comprises at least 200 consecutive amino acids in the extracellular region of the human ERBB protein, for example, comprises at least 200, 300, 400, 500, 600, 605, 606, 607, 608, 609, 610, 620, 621 consecutive amino acids; preferably, the extracellular region of the humanized ERBB protein comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645; or, comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID The amino acid sequence as set forth in NO:2 at positions 25-645 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical; or, comprises an amino acid sequence that differs from the amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645, including a substitution, deletion and / or insertion of one or more amino acid residues.

[0097] Preferably, the humanized ERBB protein extracellular region comprises the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises NO:2 The amino acid sequence shown at positions 37-639, 37-645 or 25-639, including substitution, deletion and / or insertion of one or more amino acid residues.

[0098] Preferably, the humanized ERBB protein also comprises a portion of a non-human animal ERBB protein, further preferably comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a non-human animal ERBB protein, more preferably comprises all of the signal peptide, transmembrane region and cytoplasmic region of a non-human animal ERBB protein, and further preferably also comprises a portion of the extracellular region.

[0099] In a specific embodiment of the present invention, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID The amino acid sequence shown in NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in SEQID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0100] Preferably, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The amino acid sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as shown in SEQ ID NO: 1 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0101] In a specific embodiment of the present invention, the amino acid sequence of the humanized ERBB protein comprises any one of the following groups:

[0102] A) the amino acid sequence shown in SEQ ID NO: 9;

[0103] B) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9;

[0104] C) differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0105] D) an amino acid sequence as shown in SEQ ID NO: 9, including substitution, deletion and / or insertion of one or more amino acid residues.

[0106] Preferably, the non-human animal genome comprises a human or humanized ERBB gene, and further preferably, the humanized ERBB gene comprises a portion of a human ERBB gene.

[0107] Preferably, the humanized ERBB gene comprises all or part of exons 1 to 28 of the human ERBB gene. Further preferably, the humanized ERBB gene comprises all or part of a combination of any one, two, three or more, or two or more consecutive exons of exons 1 to 28 of the human ERBB gene. More preferably, it comprises all or part of exons 1 to 17. Even further preferably, the humanized ERBB gene comprises part of exon 2, all of exons 3 to 16, and part of exon 17 of the human ERBB gene, or part of exon 1, all of exons 2 to 16, and part of exon 17. More preferably, the humanized ERBB gene comprises part of exon 2 to part of exon 17 of the human ERBB gene, or part of exon 1 to part of exon 17. Preferably, the portion of exon 1 further comprises introns 1-2, 2-3 and / or 16-17, and more preferably comprises any intron between exons 1-17 or 2-17, wherein the portion of exon 1 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the portion of exon 1 comprises exon 1 The nucleotide sequence encoding the extracellular region in the exon, the portion of exon 2 comprises at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 140, 150, 152 bp of nucleotide sequence; preferably, the portion of exon 2 comprises the 6th to 15th (for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 152 bp) of nucleotide sequence from the N-terminal of the coding extracellular region. , 15) amino acids to the last nucleotide of exon 2, the portion of exon 17 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 comprises the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region removed.

[0108] In a specific embodiment of the present invention, the portion of the human ERBB gene comprises the nucleotide sequence shown in SEQ ID NO:5; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:5; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO:5; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0109] Preferably, the humanized ERBB gene comprises all or part of the nucleotide sequence encoding the human ERBB protein. It is further preferred that the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human ERBB protein, wherein the humanized ERBB gene comprises the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that differs from the amino acid sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645. The nucleotide sequence of positions 37-643 of NO: 2 or positions 25-645 of SEQ ID NO: 2 includes a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0110] Preferably, the humanized ERBB gene comprises a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that differs from an amino acid sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639. NO: 2 The nucleotide sequence at positions 37-639, 37-645 or 25-639, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0111] Preferably, the humanized ERBB gene further comprises all or part of the non-human animal ERBB gene. It is further preferred to comprise all or part of exons 1 to 28 of the non-human animal ERBB gene, and more preferably comprises all of exon 1, part of exon 2, part of exon 17 to all of exon 28 of the non-human animal ERBB gene, or part of exon 1, part of exon 17 to all of exon 28.

[0112] In a specific embodiment of the present invention, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID The amino acid sequence of NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence encoding SEQ ID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0113] Preferably, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210; or, comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210. NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence shown in positions 1-24, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence comprises a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence shown in positions 1-36, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210.

[0114] In a specific embodiment of the present invention, the nucleotide sequence of the humanized ERBB gene comprises any one of the following groups:

[0115] A) the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8;

[0116] B) the transcribed mRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8;

[0117] C) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO:8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or,

[0118] D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0119] Preferably, the humanized ERBB gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor can be a conventional substance that can be induced or repressed. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0120] Preferably, the nucleotide sequence of the human or humanized ERBB gene is operably linked to an endogenous regulatory element of an endogenous ERBB gene in at least one chromosome.

[0121] Preferably, the introduction described in the present application includes but is not limited to insertion, replacement or transgenesis.

[0122] Preferably, the non-human animal is constructed by the construction method of the present invention as described above.

[0123] According to some embodiments of the present invention, the non-human animal further comprises other gene modifications, and the other genes are selected from at least one of NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R and HER2.

[0124] According to some embodiments of the invention, the human or humanized ERBB gene and / or the other gene is homozygous for the endogenous replaced locus.

[0125] According to some embodiments of the invention, the human or humanized ERBB gene and / or the other gene is heterozygous for the endogenous replaced locus.

[0126] Preferably, the humanized ERBB gene further includes a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a conventional substance that can be induced or repressed.

[0127] In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0128] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0129] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0130] The non-human animal may be an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0131] The third aspect of the present invention provides a targeting vector, wherein the targeting vector comprises a donor nucleotide sequence.

[0132] Preferably, the donor nucleotide sequence comprises one of the following groups:

[0133] A) Nucleotide sequence encoding human or humanized ERBB protein;

[0134] B) all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a human ERBB protein, preferably, all or part of a nucleotide sequence encoding an extracellular region of a human ERBB protein, further preferably, comprising a nucleotide sequence encoding at least 200 consecutive amino acids of an extracellular region of a human ERBB protein, more preferably, comprising a nucleotide sequence encoding positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2;

[0135] C) the nucleotide sequence of a human or humanized ERBB gene; or,

[0136] D) All or part of exons 1 to 28 of the human ERBB gene. Preferably, it comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 28. Further preferably, it comprises all or part of exons 1 to 17 of the human ERBB gene, and further preferably, it comprises part of exon 2 to part of exon 17, or part of exon 1 to part of exon 17, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the part of exon 1 comprises the nucleotide sequence encoding the extracellular region in exon 1, and the part of exon 2 comprises at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133 , 134, 135, 140, 150, 152 bp nucleotide sequence; preferably, the portion of exon 2 includes the nucleotide encoding the 6th-15th (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N-terminus of the extracellular region to the last nucleotide of exon 2, and the portion of exon 17 includes at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 includes the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region removed; further preferably, the targeting vector includes SEQ ID NO:5; or, comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:5; or, comprising a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprising a nucleotide sequence as shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0137] Preferably, the targeting vector further comprises a 5' arm (5' homology arm) and / or a 3' arm (3' homology arm).

[0138] The 5' arm is a DNA fragment homologous to the 5' end of the switch region to be changed, which is selected from 100-10000 nucleotides of the genomic DNA of the ERBB gene of non-human animals. Preferably, the 5' arm has at least 90% homology with the nucleotides of NCBI accession number NC_000077.7. Further preferably, the 5' arm sequence is shown in SEQ ID NO: 3.

[0139] The 3' arm is a second DNA fragment homologous to the 3' end of the conversion region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the ERBB gene of non-human animals; preferably, the 3' arm has at least 90% homology with nucleotides with NCBI accession number NC_000077.7; further preferably, the 3' arm sequence is as shown in SEQ ID NO: 4.

[0140] Preferably, the switch region to be changed is located on exons 2 to 17 of the ERBB gene of a non-human animal.

[0141] Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Even further preferably, the gene encoding the negative selection marker is a gene encoding a diphtheria toxin A subunit (DTA).

[0142] In a specific embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo.

[0143] In a specific embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is an Frt recombination site (a conventional LoxP recombination system may also be selected). There are two specific recombination systems, preferably installed in the same direction on both sides of the resistance gene.

[0144] Preferably, the targeting vector further comprises SEQ ID NO: 6 and / or 7.

[0145] In a fourth aspect of the present invention, a sgRNA is provided, wherein the sgRNA targets the ERBB gene of a non-human animal, and the target site is located on the sequence from exon 2 to exon 17 of the ERBB gene.

[0146] Preferably, the target site of the sgRNA is located on the exon 2 and / or exon 17 sequence of the ERBB gene.

[0147] Preferably, the target sequence of the sgRNA on the ERBB gene is shown in SEQ ID NOs: 10 and 11.

[0148] In a fifth aspect of the present invention, a DNA molecule encoding the above sgRNA is provided. Preferably, the double strand of the DNA molecule is the upstream and downstream sequence of the sgRNA, or the forward oligonucleotide sequence or the reverse oligonucleotide sequence after adding a restriction site.

[0149] In a sixth aspect, the present invention provides a vector comprising the above-mentioned sgRNA.

[0150] In a seventh aspect, the present invention provides a cell comprising the above-mentioned targeting vector, the above-mentioned sgRNA, the above-mentioned DNA molecule encoding the sgRNA and / or the above-mentioned vector.

[0151] In an eighth aspect of the present invention, a use of the above-mentioned targeting vector, the above-mentioned sgRNA, the above-mentioned DNA molecule encoding sgRNA, the above-mentioned vector and / or the above-mentioned cell in ERBB gene editing is provided. Preferably, the application includes but is not limited to knockout, insertion or replacement.

[0152] The ninth aspect of the present invention provides a humanized ERBB protein, wherein the humanized ERBB protein comprises all or part of a human ERBB protein.

[0153] Preferably, the humanized ERBB protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human ERBB protein.

[0154] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by all or part of exons 1 to 28 of the human ERBB gene. Further preferably, it comprises the amino acid sequence encoded by all or part of any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 28. More preferably, it comprises the amino acid sequence encoded by all or part of exons 1 to 17. More preferably, the amino acid sequence encoded by the portion of exon 2 to the portion of exon 17, or the portion of exon 1 to the portion of exon 17, wherein the portion of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the portion of exon 1 contains the nucleotide sequence encoding the extracellular region in exon 1, and the portion of exon 2 contains at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135 4, 135, 140, 150, 152 bp nucleotide sequence; preferably, the portion of exon 2 includes the nucleotide encoding the 6th-15th (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N-terminus of the extracellular region to the last nucleotide of exon 2, and the portion of exon 17 includes at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, 142 bp of nucleotide sequence; preferably, the portion of exon 17 includes the nucleotide sequence encoding 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids at the C-terminus of the extracellular region without the nucleotide sequence.

[0155] Preferably, the humanized ERBB protein comprises the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence encoded by SEQ ID NO: 5 or 8; or, comprises an amino acid sequence that differs from the amino acid sequence encoded by SEQ ID NO: 5 or 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in the amino acid sequence encoded by SEQ ID NO: 5 or 8, including substitution, deletion and / or insertion of one or more amino acids.

[0156] In a specific embodiment of the present invention, the humanized ERBB protein comprises all or part of the extracellular region of the human ERBB protein, preferably, comprises at least 200 consecutive amino acids in the extracellular region of the human ERBB protein, for example, comprises at least 200, 300, 400, 500, 600, 605, 606, 607, 608, 609, 610, 620, 621 consecutive amino acids; the extracellular region of the humanized ERBB protein comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645; or, comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID The amino acid sequence as set forth in NO:2 at positions 25-645 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical; or, comprises an amino acid sequence that differs from the amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as set forth in SEQ ID NO:2 at positions 37-643 or SEQ ID NO:2 at positions 25-645, including a substitution, deletion and / or insertion of one or more amino acid residues.

[0157] Preferably, the humanized ERBB protein extracellular region comprises the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference with the amino acid sequence shown in SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises NO:2 The amino acid sequence shown at positions 37-639, 37-645 or 25-639, including substitution, deletion and / or insertion of one or more amino acid residues.

[0158] Preferably, the humanized ERBB protein also comprises a portion of a non-human animal ERBB protein, further preferably comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a non-human animal ERBB protein, more preferably comprises all of the signal peptide, transmembrane region and cytoplasmic region of a non-human animal ERBB protein, and further preferably also comprises a portion of the extracellular region.

[0159] In a specific embodiment of the present invention, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID NO: 1, positions 1-24, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 646-1210 or SEQ ID The amino acid sequence shown in NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence shown in SEQID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0160] Preferably, the humanized ERBB protein further comprises the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1, positions 1-24, 642-1210 or SEQ ID NO: 1, positions 1-36, 648-1210; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1, positions 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The amino acid sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as shown in SEQ ID NO: 1 1-36, 642-1210 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210, including substitution, deletion and / or insertion of one or more amino acid residues.

[0161] In a specific embodiment of the present invention, the amino acid sequence of the humanized ERBB protein comprises any one of the following groups:

[0162] A) the amino acid sequence shown in SEQ ID NO: 9;

[0163] B) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9;

[0164] C) differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0165] D) an amino acid sequence as shown in SEQ ID NO: 9, including substitution, deletion and / or insertion of one or more amino acid residues.

[0166] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0167] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0168] Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0169] The tenth aspect of the present invention provides a humanized ERBB gene.

[0170] Preferably, the humanized ERBB gene encodes the above-mentioned humanized ERBB protein.

[0171] Preferably, the humanized ERBB gene comprises a portion of the human ERBB gene.

[0172] Preferably, the humanized ERBB gene comprises all or part of exons 1 to 28 of the human ERBB gene. Further preferably, the humanized ERBB gene comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons of exons 1 to 28 of the human ERBB gene. More preferably, it comprises all or part of exons 1 to 17. Further preferably, the humanized ERBB gene comprises part of exon 2, all of exons 3 to 16, and part of exon 17 of the human ERBB gene, or part of exon 1, all of exons 2 to 16, and part of exon 17, preferably also comprising introns 1-2, introns 2-3, and / or introns 16-17. Further preferably, the humanized ERBB gene comprises introns 2 to 16 of the human ERBB gene. The part of exon 1 to the part of exon 17, or the part of exon 1 to the part of exon 17, more preferably including any intron between exons 1-17 or 2-17, wherein the part of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 6, 7, 8, 9, 10, 15, 16, 20, 50, 100, 200, 300, 349 bp of nucleotide sequence; preferably, the part of exon 1 contains 1 The portion of exon 2 comprises at least 50 bp of nucleotide sequence, for example, at least 50, 70, 90, 100, 110, 120, 130, 131, 132, 133, 134, 135, 140, 150, 152 bp of nucleotide sequence; preferably, the portion of exon 2 comprises the nucleotide sequence encoding the extracellular region from the 6th to the 15th (for example, 6, 7, 8, 9, 10, 11, 12, 13, 140, 151, 152 bp) N-terminal end of the exon 2; The present invention relates to a method for preparing an oligonucleotide encoding a polypeptide having at least one nucleotide sequence, wherein the oligonucleotide comprises at least one nucleotide sequence of at least 5 bp, such as at least 5, 6, 7, 8, 9, 10, 15, 16, 30, 50, 70, 100, 120, 140, or 142 bp of a polypeptide. The method comprises the following steps: first, the oligonucleotide comprises a nucleotide sequence encoding ...0, 1, 2, 3, 4, or 5 amino acids at the C-terminus of the extracellular region.

[0173] In a specific embodiment of the present invention, the portion of the human ERBB gene comprises the nucleotide sequence shown in SEQ ID NO:5; or, comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:5; or, comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference from the nucleotide sequence shown in SEQ ID NO:5; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

[0174] Preferably, the humanized ERBB gene comprises all or part of the nucleotide sequence encoding the human ERBB protein. It is further preferred that the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human ERBB protein, wherein the humanized ERBB gene comprises the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645; or, comprises a nucleotide sequence that differs from the amino acid sequence encoding SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 37-643 or SEQ ID NO: 2 25-645. The nucleotide sequence of positions 37-643 of NO: 2 or positions 25-645 of SEQ ID NO: 2 includes a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0175] Preferably, the humanized ERBB gene comprises a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639; or, comprises a nucleotide sequence that differs from an amino acid sequence encoding SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises a nucleotide sequence that encodes SEQ ID NO: 2 at positions 37-639, 37-645 or 25-639. NO: 2 The nucleotide sequence at positions 37-639, 37-645 or 25-639, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0176] Preferably, the humanized ERBB gene further comprises all or part of the non-human animal ERBB gene. It is further preferred to comprise all or part of exons 1 to 28 of the non-human animal ERBB gene, and more preferably comprises all of exon 1, part of exon 2, part of exon 17 to all of exon 28 of the non-human animal ERBB gene, or part of exon 1, part of exon 17 to all of exon 28.

[0177] In a specific embodiment of the present invention, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID NO: 1 positions 1-24, 648-1210; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 646-1210 or SEQ ID The amino acid sequence of NO:1 at positions 1-24, 648-1210 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence encoding SEQ ID NO:1 at positions 1-36, 646-1210 or SEQ ID NO:1 at positions 1-24, 648-1210, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0178] Preferably, the humanized ERBB gene further comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210; or, comprises a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210 having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a nucleotide sequence encoding SEQ ID NO: 1 positions 1-36, 642-1210 or SEQ ID NO: 1 positions 1-24, 642-1210 or SEQ ID NO: 1 positions 1-36, 648-1210. NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence shown in positions 1-24, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-36, 648-1210 The nucleotide sequence comprises a substitution, deletion and / or insertion of one or more nucleotides from the nucleotide sequence shown in positions 1-36, 642-1210 of SEQ ID NO: 1 or SEQ ID NO: 1 1-24, 642-1210 or SEQ ID NO: 1 1-36, 648-1210.

[0179] In a specific embodiment of the present invention, the nucleotide sequence of the humanized ERBB gene comprises any one of the following groups:

[0180] A) the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8;

[0181] B) the transcribed mRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8;

[0182] C) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO:8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or,

[0183] D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0184] Preferably, the humanized ERBB gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor can be a conventional substance that can be induced or repressed. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0185] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0186] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0187] Preferably, the non-human animal is an immunodeficient non-human mammal. Preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. More preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0188] In the eleventh aspect of the present invention, a non-human animal lacking an ERBB gene is provided, wherein the non-human animal lacks all or part of exon 1 to exon 17 of the endogenous ERBB gene, preferably lacks part of exon 2 to part of exon 17 of the endogenous ERBB gene, or part of exon 1 to part of exon 17.

[0189] The twelfth aspect of the present invention provides a method for constructing a non-human animal with ERBB gene deletion, the construction method comprising preparing the non-human animal using the above-mentioned targeting vector or the above-mentioned sgRNA.

[0190] The thirteenth aspect of the present invention provides a cell lacking the ERBB gene, wherein the cell lacks all or part of exon 1 to exon 17 of the ERBB gene, preferably lacks part of exon 2 to part of exon 17 of the endogenous ERBB gene, or part of exon 1 to part of exon 17.

[0191] In a fourteenth aspect of the present invention, a method for constructing cells lacking the ERBB gene is provided, comprising using the above-mentioned targeting vector and / or the above-mentioned sgRNA to construct cells lacking the ERBB gene.

[0192] The fifteenth aspect of the present invention provides a method for constructing a multi-gene modified non-human animal, comprising the following steps:

[0193] I) providing the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method;

[0194] II) mating the non-human animal provided in step I) with other genetically modified non-human animals, fertilizing them in vitro or directly performing gene editing, and screening to obtain multi-gene modified non-human animals.

[0195] Preferably, the other genetically modified non-human animals include non-human animals modified with at least one of the genes NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R and HER2.

[0196] Preferably, the multi-gene modified non-human animal is a two-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal, a five-gene humanized non-human animal, a six-gene humanized non-human animal, a seven-gene humanized non-human animal, an eight-gene humanized non-human animal or a nine-gene humanized non-human animal.

[0197] Preferably, each of the multiple humanized genes in the genome of the multi-gene modified non-human animal can be homozygous or heterozygous for the replacement of the endogenous locus.

[0198] In the sixteenth aspect of the present invention, a cell, tissue or organ is provided, wherein the cell, tissue or organ expresses the humanized ERBB protein, and the genome of the cell, tissue or organ contains the humanized ERBB gene. Alternatively, the cell, tissue or organ is derived from the non-human animal, or the non-human animal obtained by the construction method.

[0199] Preferably, the tissue comprises tumor tissue after tumor bearing.

[0200] In the seventeenth aspect of the present invention, a tumor tissue after tumor bearing is provided, wherein the tumor tissue expresses the above-mentioned humanized ERBB protein. Alternatively, the genome of the tumor tissue contains the above-mentioned humanized ERBB gene. Alternatively, the tumor tissue after tumor bearing is derived from the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method.

[0201] The eighteenth aspect of the present invention provides an animal model, wherein the animal model is derived from the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method. Preferably, the animal model is a tumor-bearing or inflammation animal model.

[0202] In a nineteenth aspect of the present invention, a method for constructing an animal model is provided, wherein the method is performed using the above-mentioned constructed non-human animal, non-human animal or its offspring, or gene-deficient non-human animal. Preferably, the animal model is a tumor-bearing or inflammatory animal model. Preferably, the animal model further comprises the step of implanting tumor cells.

[0203] The twentieth aspect of the present invention provides a use of the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method in constructing an animal model. Preferably, the animal model is a tumor-bearing or inflammation animal model.

[0204] The twenty-first aspect of the present invention provides a use of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model in the preparation of drugs for treating tumors, inflammation or immune-related diseases.

[0205] The twenty-second aspect of the present invention provides a cell with humanized ERBB gene, wherein the cell expresses human or humanized ERBB protein.

[0206] Preferably, the cells express the above-mentioned humanized ERBB protein.

[0207] Preferably, the genome of the cell comprises a portion of the human ERBB gene. More preferably, the cell comprises the humanized ERBB gene.

[0208] The twenty-third aspect of the present invention provides a construct comprising the humanized ERBB gene or a construct expressing the humanized ERBB protein. Preferably, the construct may be a plasmid.

[0209] The twenty-fourth aspect of the present invention provides a cell comprising the above construct.

[0210] The twenty-fifth aspect of the present invention provides a tissue comprising the above-mentioned cells.

[0211] The twenty-sixth aspect of the present invention provides a genome of a non-human animal with a humanized ERBB gene.

[0212] Preferably, the genome comprises all or part of a human or humanized ERBB gene, and / or comprises all or part of a nucleotide sequence encoding a human or humanized ERBB protein.

[0213] Preferably, the humanized ERBB gene is the above-mentioned humanized ERBB gene.

[0214] Preferably, the humanized ERBB protein is the above-mentioned humanized ERBB protein.

[0215] Preferably, the genome comprises a genomic fragment of a human ERBB gene (preferably encoding all or part of the sequence of the extracellular region of human ERBB) at the endogenous ERBB locus of a non-human animal, and / or a genomic fragment of a non-human animal ERBB gene is introduced into the genomic fragment of a non-human animal ERBB gene to form a modified ERBB gene.

[0216] Preferably, the genome comprises a genomic fragment of a non-human animal ERBB gene introduced into the non-human animal endogenous ERBB gene locus with a humanized ERBB gene to form a modified ERBB gene.

[0217] The modified ERBB gene encodes humanized ERBB protein.

[0218] Preferably, the introduction is insertion or replacement.

[0219] Preferably, the introduction of the non-human animal ERBB locus is to replace the corresponding region of the non-human animal. Further preferably, all or part of exons 1 to 17 of the non-human animal ERBB gene are replaced. More preferably, the replacement includes part of exon 2 to part of exon 17 of the non-human animal ERBB gene, or part of exon 1 to part of exon 17 is replaced.

[0220] Preferably, the expression of the modified ERBB gene is controlled by endogenous regulatory elements of the non-human animal.

[0221] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0222] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0223] The twenty-seventh aspect of the present invention provides cells, tissues or organs comprising the genome of the above-mentioned non-human animal with humanized ERBB gene.

[0224] Preferably, any of the above-mentioned cells, tissues or organs or tumor tissues after tumor development includes cells, tissues or organs or tumor tissues after tumor development that can develop into animal individuals or cannot develop into animal individuals.

[0225] In a twenty-eighth aspect of the present invention, there is provided an application of the above-mentioned humanized ERBB protein, the above-mentioned humanized ERBB gene, the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, any of the above-mentioned cells, tissues or organs, or tumor tissues, or the above-mentioned animal model, wherein the application comprises:

[0226] A) Application in product development for ERBB-related immune processes involving human cells;

[0227] B) Application as a model system related to ERBB for pharmacology, immunology, microbiology and medical research;

[0228] C) Applications involving the production and use of animal experimental disease models for the study of ERBB-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0229] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human ERBB signaling pathway modulators; or,

[0230] E) Study the function of ERBB gene, study the drugs and efficacy targeting human ERBB target sites, and study the application of drugs for immune-related diseases and anti-tumor drugs related to ERBB.

[0231] Preferably, the application includes a method for treating and / or diagnosing a disease, or a method for treating and / or diagnosing a non-disease.

[0232] The twenty-ninth aspect of the present invention provides a non-human animal derived from the above-mentioned non-human animal, a non-human animal obtained by the above-mentioned construction method, or the above-mentioned tumor-bearing or inflammatory animal model for screening of human ERBB-specific regulators.

[0233] The thirtieth aspect of the present invention provides a method for screening human ERBB-specific regulators, the screening method comprising applying a regulator to an individual implanted with tumor cells and detecting tumor inhibitory activity; wherein the individual is selected from the above-mentioned non-human animals or the non-human animals constructed using the above-mentioned method or the above-mentioned tumor-bearing animal model.

[0234] Preferably, the regulator is selected from CAR-T drugs. Further preferably, the drug is an antibody, specifically, the drug can be an anti-ERBB antibody.

[0235] Preferably, the regulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0236] Preferably, said detecting comprises measuring the size and / or proliferation rate of tumor cells.

[0237] Preferably, the detection method includes vernier caliper measurement, flow cytometry detection and / or in vivo animal imaging detection.

[0238] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0239] Preferably, the tumor cells are derived from humans or non-human animals.

[0240] Preferably, the screening method includes therapeutic and non-therapeutic methods.

[0241] In one embodiment, the screening method detects and evaluates the effect of a modulator to determine whether the modulator has a therapeutic effect, i.e., a therapeutic effect is not a certainty but only a possibility.

[0242] In the thirty-first aspect of the present invention, a method for screening or evaluating human drugs is provided, wherein the method comprises constructing a disease animal model individual, administering a candidate drug to the disease animal model individual, and performing drug efficacy detection and / or comparison on the individual administered the candidate drug. Wherein, the individual is selected from the non-human animal obtained by the above construction method, the above non-human animal or its offspring, or the above tumor-bearing or inflammatory animal model.

[0243] Preferably, the method for drug screening or evaluation includes therapeutic and non-therapeutic methods.

[0244] In one embodiment, the method is used to screen or evaluate drugs, detect and compare the efficacy of candidate drugs to determine which candidate drugs can be used as drugs and which cannot be used as drugs, or compare the sensitivity of the efficacy of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0245] Preferably, the candidate drug includes a targeted drug. Further preferably, the targeted drug is an antigen binding protein. In a specific embodiment of the present invention, the antigen binding protein is an antibody.

[0246] Preferably, the candidate drug is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0247] Preferably, the detection includes determining the size and / or proliferation rate of tumor cells; preferably, the detection method includes vernier caliper measurement, flow cytometry and / or in vivo animal imaging detection.

[0248] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0249] Preferably, any of the above non-human animals is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0250] Preferably, any of the above-mentioned non-human animals can also be selected from any non-human animals that can be gene-edited to prepare humanized genes, such as pigs, rabbits, monkeys, etc.

[0251] The "immune-related diseases" described in the present invention include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, etc.

[0252] The "tumor" described in the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, described leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myeloid leukemia; described lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, T cell lymphoma and Waldenstrom macroglobulinemia; described sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In a specific embodiment of the present invention, described tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

[0253] The "inflammation" described in the present invention can be inflammation occurring in any tissue, and the tissues include but are not limited to the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (such as renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicle, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and leukocytes. Further preferably, the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies (e.g., allergic rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity reactions, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis or interstitial cystitis.

[0254] The term "treating" (or "treat" or "treatment") as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treating" and the like refers to therapeutic interventions that improve the signs, symptoms, and the like of a disease or pathological state after the disease has begun to develop.

[0255] The "locus" described in the present invention broadly represents the position of a gene on a chromosome, and narrowly represents a DNA fragment on a gene, that is, it can be a gene or a part of a gene. For example, the "ERBB locus" described in the present invention represents an arbitrary DNA fragment on exons 1 to 28 of the ERBB gene. In a specific embodiment of the present invention, the replaced ERBB locus can be an arbitrary DNA fragment on exons 1 to 28 of the ERBB gene.

[0256] The "nucleotide sequence" of the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences, preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA.

[0257] The “all or part” mentioned in the present invention, “all” refers to the whole, and “part” refers to a part of the whole, or an individual that makes up the whole.

[0258] The "humanized ERBB protein" of the present invention includes a portion derived from a human ERBB protein. The "human ERBB protein" is the same as the "entire human ERBB protein", that is, its amino acid sequence is consistent with the full-length amino acid sequence of the human ERBB protein. The "portion of the human ERBB protein" is a continuous or intermittent 5-1210 (preferably 10-607) amino acid sequence that is consistent with the amino acid sequence of the human ERBB protein or has more than 70% homology with the amino acid sequence of the human ERBB protein.

[0259] The "humanized ERBB gene" described in the present invention includes a portion derived from the human ERBB gene and a portion derived from the non-human ERBB gene. The "human ERBB gene" is the same as the "entire human ERBB gene", that is, its nucleotide sequence is consistent with the full-length nucleotide sequence of the human ERBB gene. The "portion of the human ERBB gene" is a continuous or intermittent 20-2097266bp (preferably 20-30687bp, 20-9905bp or 20-1821bp) nucleotide sequence that is consistent with the human ERBB nucleotide sequence or has more than 70% homology with the human ERBB nucleotide sequence.

[0260] The "exons xx to xxx" or "all of exons xx to xxx" mentioned in the present invention include the nucleotide sequences of exons and introns therebetween, for example, the "exons 1 to 2" mentioned above include the entire nucleotide sequences of exon 1, introns 1-2, and exon 2.

[0261] The "intron x-xx" of the present invention refers to the intron between exon x and exon xx. For example, "intron 1-2" refers to the intron between exon 1 and exon 2.

[0262] The "part of an exon" of the present invention means that a continuous or intermittent nucleotide sequence of several, dozens or hundreds of nucleotides is consistent with the entire exon nucleotide sequence. For example, a part of exon 2 of the human ERBB gene includes a continuous or intermittent nucleotide sequence of 5-152 bp, preferably 10-132 bp, which is consistent with the nucleotide sequence of exon 2 of the human ERBB gene.

[0263] The "part of exon x to part of exon xx" or "part of exon x - all of exon xx" described in the present invention includes all or part of an exon and introns therebetween. For example, "part of exon 2 to all of exon 4" includes part of exon 2, all of intron 2, all of exon 3, all of intron 3, and all of exon 4.

[0264] The "cells" described in the present invention may be fertilized egg cells or other somatic cells, preferably including but not limited to platelets, monocytes, microglia and endothelial cells, neutrophils, activated macrophages, B cell precursors, dendritic cells, natural killer cells, late B cells or plasma cells, etc. Therefore, depending on the different sources of cells, some of the cells described in this application can develop into animal individuals, while some cannot.

[0265] The “ERBB protein” described in the present invention, such as “human ERBB protein”, “non-human animal ERBB protein” or “humanized ERBB protein”, all comprise a signal peptide, an extracellular region, an intracellular region and / or a transmembrane region.

[0266] The term "comprising" or "including" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. However, when used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0267] The "homology" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39 %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

[0268] One skilled in the art is able to determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

[0269] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0270] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0271] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature. For example: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DNGlovered., 1985); Oligonucleotide Synthesis (MJGaited., 1984); Mullisetal. US Pat. No. 4, 683, 195; Nucleic Acid Hybridization (BDHames&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RIFreshney, AlanR.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols.154and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (DMWeir and CCBlackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0272] Genetically humanized animal models, that is, using gene editing technology to replace homologous genes in the animal genome with normal or mutant human genes, can establish normal or mutant gene animal models that are closer to human physiological or disease characteristics. Genetically humanized animals not only have important application value in themselves, such as improving and enhancing cell or tissue transplantation humanized animal models through gene humanization, but more importantly, due to the insertion of human gene fragments, the animal body can express or partially express human proteins, which can be used as targets for drugs that can only recognize human protein sequences, making it possible to screen anti-human antibodies and other drugs at the animal level.

[0273] In addition, the non-human animals obtained by this method can also be mated with other gene humanized non-human animals to obtain multi-gene humanized animal models, which are used to screen and evaluate the efficacy of human drugs and combined drugs targeting the signaling pathway. The present invention has broad application prospects in academic and clinical research.

[0274] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0275] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention.

[0276] The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0277] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0278] Figure 1 : Schematic diagram comparing the mouse ERBB locus and the human ERBB locus (not to scale);

[0279] Figure 2 : Schematic diagram of the humanization of the mouse ERBB gene (not to scale);

[0280] Figure 3 : Schematic diagram of ERBB gene targeting strategy and targeting vector design (not to scale);

[0281] Figure 4 :Southern blot detection results, where WT is the wild-type control;

[0282] Figure 5 : Schematic diagram of the FRT recombination process of ERBB gene humanized mice (not to scale);

[0283] Figure 6 : F1 generation genotype identification results, where WT is the wild type control, PC is the positive control, and H2O is the water control;

[0284] Figure 7 : RT-PCR test results, where + / + is wild-type C57BL / 6 mice, H / H is ERBB gene humanized homozygous mice, H2O is water control, GAPDH is glyceraldehyde-3-phosphate dehydrogenase internal reference;

[0285] Figure 8 : IHC staining results of different tissues of C57BL / 6 mice and ERBB gene humanized homozygous mice, where + / + is wild-type C57BL / 6 mice, H / H is ERBB gene humanized homozygous mice, the positive control of C57BL / 6 wild mice is mouse liver tissue, the positive control of ERBB gene humanized homozygous mice is PDX tumor tissue, and the negative controls of both are canine liver tissue. DETAILED DESCRIPTION

[0286] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0287] In each of the following examples, equipment and materials were obtained from the following companies:

[0288] BbsI, EcoRI, BamHI, BgIII, ScaI, SpeI, and NdeI enzymes were purchased from NEB with catalog numbers R0539L, R0101M, R0136M, R0144L, R3122L, R3133L, and R0111L, respectively;

[0289] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0290] The Ambion in vitro transcription kit was purchased from Ambion, catalog number AM1354;

[0291] Cas9mRNA source: SIGMA, catalog number: CAS9MRNA-1EA;

[0292] UCA kit was obtained from Biocytogen, catalog number BCG-DX-001;

[0293] BAC genes were purchased from BACPAC, human BAC catalog number CH17-159M13, mouse BAC catalog number RP23-291E21.

[0294] Example 1 ERBB gene humanized mice

[0295] Mouse ERBB gene (NCBI Gene ID: 13649, Primary source: MGI: 95294, UniProt ID:

[0296] Q01279, located at positions 16700153 to 16868158 on chromosome 11 NC_000077.7, based on transcript NM_207655.2 and its encoded protein NP_997538.1 (SEQ ID NO: 1)) and human ERBB gene (NCBI Gene ID: 1956, Primary source: HGNC: 3236, UniProt ID: P00533, located at positions 55019017 to 552116282 on chromosome 7 NC_000007.14, based on transcript NM_005228.5 and its encoded protein NP_005219.2 (SEQ ID NO:

[0297] 2)) Comparison diagram as shown Figure 1 shown.

[0298] In order to achieve the purpose of the present invention, a nucleotide sequence encoding human ERBB protein can be introduced into the mouse endogenous ERBB locus, so that the mouse expresses human or humanized ERBB protein. Specifically, using gene editing technology, under the control of the mouse ERBB gene regulatory element, a partial nucleotide sequence encoding human ERBB protein (derived from human BAC: CH17-159M13) is used to replace the corresponding mouse sequence, and a humanized ERBB locus is obtained as shown in the schematic diagram Figure 2 As shown, the humanized transformation of the mouse ERBB gene was achieved.

[0299] Further design such as Figure 3Schematic diagram of the targeting strategy shown, showing the upstream and downstream homology arm sequences of the mouse ERBB gene on the targeting vector, and the A fragment containing the nucleotide sequence encoding the human ERBB protein. Among them, the upstream homology arm sequence (5' homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence of positions 16804993-16808915 of NCBI Accession No. NC_000077.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) has 99.9% identity with the nucleotide sequence of positions 16839923-16844278 of NCBI Accession No. NC_000077.7, and A is mutated to C at position 16839964, and G is mutated to C at position 16839967; the human ERBB sequence (SEQ ID NO: 5) contained in the A fragment is identical to the nucleotide sequence of positions 55142306-55172992 of NCBI Accession No. NC_000007.14.

[0300] The targeting vector also includes a resistance gene for positive clone screening, namely, the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette.

[0301] The connection between the 5' end of the Neo box and the human gene was designed to be 5'-TAACCCCTTTCAAGCTCGTTCAGAGAGTATTTCAC ACA ATGATAT CGTTCCTTATCATCTGGCGAATCGGACCCACAAGA-3' (SEQ ID NO: 6), wherein the sequence " ACAAT "T" is the last nucleotide in the human sequence. GATAT "G" in the "Neo box" is the first nucleotide; the connection between the 3' end of the Neo box and the human gene is designed to be 5'-TACATAGATTACCACAACTCCGAGCCCTTCCACCG GATCCCCAT G TG CTCATCTTAAAAGCCAAGGACCCAGAGGAGTCT-3' (SEQ ID NO: 7), wherein the sequence " GATCC The last "C" in the sequence is the last nucleotide of the Neo box. CCATG The first "C" in " is the first nucleotide of human. In addition, a coding gene with a negative selection marker (coding gene of diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence transcribed from the modified humanized mouse ERBB gene is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.

[0302] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0303] The CRISPR / Cas9 system can be introduced for gene editing. The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. The selection and design of efficient and specific target sequences are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize the 5' and 3' target sites, and screen out sgRNAs with better activity and higher sequence specificity for subsequent experiments. The exemplary target sequence of sgRNA A on the ERBB gene is shown below:

[0304] sgRNA1 target site (SEQ ID NO: 10): 5'-AGTTGTTGTACATCCTCTGCAGG-3'

[0305] sgRNA2 target site (SEQ ID NO: 11): 5'-TGGCCTCCTCTTCATAGTGGTGG-3'

[0306] Restriction sites were added to the 5' end and complementary strand of sgRNA to obtain forward and reverse oligonucleotide sequences. After annealing, the annealed products were connected to the pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-ERBB-1 and pT7-ERBB-2. The pT7-sgRNA vector was synthesized by a plasmid synthesis company with a fragment DNA (SEQ ID NO: 12) containing T7 promoter and sgRNA scaffold and was sequentially connected to the backbone vector (source Takar a, item number 3299) through restriction digestion (EcoRI and BamHI). The sequencing was verified by a professional sequencing company, and the results showed that the target plasmid was obtained.

[0307] The targeting vector and sgRNA plasmid verified by sequencing were electroporated into embryonic stem cells of C57BL / 6 mice, and the obtained cells were screened using the positive clone screening marker gene, and the integration of the exogenous gene was confirmed by PCR and Southern Blot methods to screen the correct positive clone cells. The clones identified as positive by PCR were then subjected to Southern Blot (cell DNA was digested with Bg III, ScaI, SpeI or NdeI restriction endonucleases and hybridized with 5'Probe, 3'Probe, Neo probe, A1 Probe and A2 Probe probes, and the specific probes and target fragment lengths are shown in Table 1). The Southern Blot results are shown in Table 1. Figure 4 The test results showed that among the clones identified as positive by PCR, the 7 cells numbered 1-A08, 1-F05, 1-H09, 2-C04, 2-D01, 2-F03 and 2-H01 were further verified by sequencing and were all positive clones without random insertions.

[0308] Table 1: Specific probes and target fragment lengths

[0309] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BgIII 5'-Probe 12.6Kb 17.7Kb ScaI 3'-Probe 13.2Kb 19.7Kb SpeI Neo Probe -- 20.3Kb N i A1 Probe -- 21.8Kb ScaI A2 Probe -- 19.7Kb

[0310] The PCR assay included the following primers:

[0311] PCR-F1: 5'-TGCCCTGGATTTGCAAGATGGAAGT-3' (SEQ ID NO: 13),

[0312] PCR-R1: 5'-TCCTATGGGGTCATTAGCGCCTATC-3' (SEQ ID NO: 14);

[0313] Neo-F: 5'-GCTCGACTAGAGCTTGCGGA-3' (SEQ ID NO: 15);

[0314] PCR-R2: 5'-AAGGTCTGCAAAAGCAGATCTCCAG-3' (SEQ ID NO: 16);

[0315] Southern Blot assay includes the following probe primers:

[0316] 5'Probe-F: 5'-TGAAACTGTGAGATTCGGTGGCAG-3' (SEQ ID NO: 17);

[0317] 5'Probe-R: 5'-GGGTTGGAGTACTTCTGCTGAGCG-3' (SEQ ID NO: 18);

[0318] 3'Probe-F: 5'-TATCCCTGTGAGGCAGATGGCTAGG-3' (SEQ ID NO: 19);

[0319] 3'Probe-R: 5'-GAAAAGCCAGAGGAGAGCTCGCAAT-3' (SEQ ID NO: 20);

[0320] Neo probe-F: 5'-GGATCGGCATTGAACAAGAT-3' (SEQ ID NO: 21);

[0321] Neo probe-R: 5'-CAGAAGAACTCGTCAAGAAGGC-3' (SEQ ID NO: 22);

[0322] A1 Probe-F: 5'-GGCCCTCTAACTGGTCAGGCATC-3' (SEQ ID NO: 23);

[0323] A1 Probe-R: 5'-CGGTGTATTAAATACCTGCTTGGCAC-3' (SEQ ID NO: 24);

[0324] A2 Probe-F: 5'-TATTGCATTCAGCAGGCAGGGAGAG-3' (SEQ ID NO: 25);

[0325] A2 Probe-R: 5'-GTGGGACACGCTGCCATCATTACTT-3' (SEQ ID NO: 26);

[0326] The correct positive clone cells (black mice) screened out are introduced into the separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for a short culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), which can produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (see the schematic diagram of this process). Figure 5 ) and then ERBB gene humanized homozygous mice can be obtained by mating with each other.

[0327] The genotype of mouse somatic cells can be identified by conventional detection methods (such as PCR). The results of the identification of F1 generation mice are shown in Figure 6 The results showed that the mouse numbered F1-01 was a positive mouse. PCR primers are shown in Table 2.

[0328] Table 2 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0329]

[0330] The primer WT-F is located at the 5' homology arm, WT-R is located at the second intron of mouse endogenous ERBB, and Mut-R is located on the A fragment sequence.

[0331] The results showed that this method can be used to construct ERBB gene humanized mice that can be stably propagated.

[0332] The expression of mRNA transcribed from the humanized ERBB gene or humanized ERB B protein in the humanized ERBB gene mice can be detected by conventional methods. Specifically, 1 6-week-old female wild-type C57BL / 6 mouse (+ / +) and 10-week-old female ERBB gene humanized homozygous (H / H) prepared in this embodiment were selected, and liver tissue was obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 3. The test results are shown in FIG. Figure 7 As shown in the figure, only mouse ERBB mRNA was detected in wild-type C57BL / 6 mice, and no human or humanized ERBB mRNA was detected; only humanized ERBB mRNA was detected in ERBB gene homozygous mice, and no mouse ERBB mRNA was detected.

[0333] Table 3 RT-PCR primer sequences and target fragment sizes

[0334]

[0335]

[0336] Further, IHC staining was used to detect the expression of human ERBB protein in mice. Specifically, one 8-week-old female C57BL / 6 wild-type mouse (+ / +) and one ERBB gene humanized homozygous mouse (H / H) prepared in this embodiment were selected and euthanized. The esophagus and back skin of the mice were fixed in 10% neutral formalin solution. All tissues were paraffin-embedded, sliced ​​and IHC-stained, and the tissues were analyzed under an optical microscope. Exemplary IHC scoring results are shown in Table 4. Scoring items include staining intensity and percentage of positive cells. The scoring criteria for staining intensity were: colorless for 0 points, light yellow for 1 point, brown for 2 points, and brown for 3 points; the scoring criteria for the percentage of positive cells were: negative for 0 points, ≤10% for 1 point, 11%-50% for 2 points, 51%-75% for 3 points, and >75% for 4 points; the total score was the product of the score of staining intensity and the score of positive cell percentage, with 0 points for no positive staining, 1-3 points for questionable positive, 4-6 points for weak positive, 7-9 points for moderate positive, and 10-12 points for strong positive.

[0337] Table 4: Total IHC scores for each mouse tissue

[0338]

[0339] Example tissue expression diagram of mouse Figure 8 As shown, the positive control for C57BL / 6 wild mice was mouse liver tissue, and the positive control for ERBB gene humanized homozygous mice was PDX tumor tissue; the negative controls for both were canine liver tissue.

[0340] IHC results showed that the expression of ERBB protein in C57BL / 6 mice was basically consistent with that in ERBB gene humanized homozygous mice, indicating that there was no significant difference in the expression of ERBB protein in tissues such as esophagus and skin of ERBB gene humanized homozygous mice and C57BL / 6 wild mice.

[0341] In addition, 8 female wild-type C57BL / 6 mice (+ / +) and ERBB humanized homozygous mice (H / H) aged 6-8 weeks were selected, and peripheral blood was collected for routine blood tests and blood biochemistry tests. The results showed that the humanized modification of the ERBB gene did not affect the composition and morphology of blood cells in mice, and the liver function status of the modified mice was basically consistent with that of wild-type mice.

[0342] Example 2 Pharmacodynamic Model

[0343] The ERBB gene humanized mice disclosed in the present invention can be used to induce the preparation of tumor disease models, which can be used to test the in vivo efficacy and safety evaluation of human-specific ERBB therapeutic drugs. For example, ERBB gene humanized mice can be used to evaluate the efficacy, pharmacokinetics of drugs (such as nucleic acid drugs, antibodies) of human-specific ERBB signaling pathways, and the in vivo efficacy and safety evaluation in various disease models known in the art.

[0344] For example, ERBB gene humanized mice homozygous for MC38 cells were subcutaneously inoculated and the tumor volume grew to about 100 mm. 3 The mice were then divided into a control group or a treatment group according to the tumor volume. The treatment group randomly selected a drug targeting human ERBB, and the control group was injected with an equal volume of normal saline. The tumor volume was measured regularly and the mice were weighed. By comparing the weight changes of the mice and the tumor size, the in vivo safety and in vivo efficacy of the drug targeting human ERBB can be effectively evaluated.

[0345] For another example, homozygous humanized mice with ERBB gene were taken and divided into a control group and multiple dosing groups. Multiple dosing groups randomly selected different drugs targeting human ERBB and / or different dosing regimens, and the control group was injected with an equal volume of normal saline. The weight of mice was measured regularly, blood was collected regularly for blood biochemistry and blood routine examinations, and the liver, skin, oral mucosa and other parts were collected for pathological evaluation. By comparing the changes in mouse weight, blood biochemistry and blood routine results, and pathological evaluation of different parts, the in vivo safety of drugs and / or dosing regimens targeting human ERBB can be effectively evaluated.

[0346] It is shown that the ERBB gene humanized mouse disclosed in the present invention can be used for the in vivo efficacy evaluation and in vivo safety evaluation of drugs targeting ERBB.

[0347] Example 3 Preparation of double humanized or multiply humanized mice

[0348] The ERBB gene humanized mouse prepared by the present method can also be used to prepare a double humanized or multi-humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice modified with other genes such as NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R, HER2, etc. Alternatively, on the basis of humanized ERBB mice, mouse ES embryonic stem cells can be separated and gene recombination targeting technology can be used to obtain a double or multi-gene modified mouse model of ERBB and other genes. The homozygous or heterozygous ERBB mice obtained by this method can also be mated with other genetically modified homozygous or heterozygous mice, and their offspring can be screened. According to Mendel's law of inheritance, there is a certain probability of obtaining humanized ERBB and other genetically modified double-gene or multi-gene modified heterozygous mice, and then the heterozygotes can be mated with each other to obtain double-gene or multi-gene modified homozygotes. These double-gene or multi-gene modified mice can be used to perform in vivo efficacy verification of targeted human ERBB and other gene regulators.

[0349] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0350] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0351] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a non-human animal with humanized ERBB gene, characterized in that: The non-human animal expresses human or humanized ERBB protein in vivo, and / or the genome of the non-human animal contains human or humanized ERBB gene.

2. The construction method according to claim 1, characterized in that: The humanized ERBB protein comprises a portion of a human ERBB protein. Preferably, the humanized ERBB protein comprises all or part of a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a human ERBB protein. Further preferably, the humanized ERBB protein comprises all or part of the extracellular region of a human ERBB protein. More preferably, the humanized ERBB protein comprises at least 200 consecutive amino acids of the extracellular region of a human ERBB protein. Even more preferably, the humanized ERBB protein comprises the amino acid sequence shown in positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence shown in positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence shown in positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2; NO:2, positions 37-643 or SEQ ID NO:2, positions 25-645 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising an amino acid sequence as shown in SEQ ID NO:2, positions 37-643 or SEQ ID NO:2, positions 25-645, including a substitution, deletion and / or insertion of one or more amino acid residues, Preferably, the humanized ERBB protein further comprises a portion of a non-human animal ERBB protein, preferably all or part of a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a non-human animal ERBB protein, and further preferably comprises an amino acid sequence as shown in positions 1-36 and positions 646-1210 of SEQ ID NO: 1 or positions 1-24 and positions 648-1210 of SEQ ID NO: 1, Preferably, the amino acid sequence of the humanized ERBB protein comprises any one of the following groups: A) the amino acid sequence shown in SEQ ID NO: 9; B) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9; C) differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or D) an amino acid sequence as shown in SEQ ID NO: 9, including substitution, deletion and / or insertion of one or more amino acid residues.

3. The construction method according to any one of claims 1-2, characterized in that: The humanized ERBB gene comprises a portion of the human ERBB gene. Preferably, the humanized ERBB gene comprises all or part of exons 1 to 28 of the human ERBB gene. Further preferably, the humanized ERBB gene comprises all or part of exons 1 to 17 of the human ERBB gene. More preferably, the humanized ERBB gene comprises a portion of exon 2 to a portion of exon 17 of the human ERBB gene, or a portion of exon 1 to a portion of exon 17, wherein the portion of exon 1 comprises at least 5 bp of nucleotide sequence, the portion of exon 2 comprises at least 50 bp of nucleotide sequence, and the portion of exon 17 comprises at least 5 bp of nucleotide sequence. Further preferably, the portion of the human ERBB gene comprises the nucleotide sequence shown in SEQ ID NO: 5; or comprises the same nucleotide sequence as SEQ ID NO:

5. The nucleotide sequence of SEQ ID NO:5 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical; or, comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having a substitution, deletion and / or insertion of one or more nucleotides as shown in the nucleotide sequence of SEQ ID NO:5, Preferably, the humanized ERBB gene further comprises a portion of a non-human animal ERBB gene. Preferably, the humanized ERBB gene comprises all of exon 1, part of exon 2, part of exon 17 to all of exon 28 of the non-human animal ERBB gene, or part of exon 1, part of exon 17 to all of exon 28. Preferably, the nucleotide sequence of the humanized ERBB gene comprises any one of the following groups: A) the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8; B) the transcribed mRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8; C) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO:8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or, D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

4. The construction method according to any one of claims 1 to 3, characterized in that: The construction method comprises introducing a donor nucleotide sequence into the ERBB locus of a non-human animal. Preferably, the donor nucleotide sequence comprises one of the following groups: A) Nucleotide sequence encoding human or humanized ERBB protein; B) all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a human ERBB protein, preferably, all or part of a nucleotide sequence encoding an extracellular region of a human ERBB protein, further preferably, comprising a nucleotide sequence encoding at least 200 consecutive amino acids of an extracellular region of a human ERBB protein, more preferably, comprising a nucleotide sequence encoding positions 37-643 of SEQ ID NO: 2 or positions 25-645 of SEQ ID NO: 2; C) the nucleotide sequence of a human or humanized ERBB gene; or, D) all or part of exons 1 to 28 of the human ERBB gene, preferably, all or part of exons 1 to 17 of the human ERBB gene, further preferably, part of exon 2 to part of exon 17 of the human ERBB gene, or part of exon 1 to part of exon 17, wherein the part of exon 1 contains at least 5 bp of nucleotide sequence, the part of exon 2 contains at least 50 bp of nucleotide sequence, and the part of exon 17 contains at least 5 bp of nucleotide sequence, more preferably, contains the nucleotide sequence shown in SEQ ID NO: 5; or contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 5; or contains The nucleotide sequence shown in NO:5 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:5, including substitution, deletion and / or insertion of one or more nucleotides.

5. The construction method according to any one of claims 1 to 4, characterized in that: The human or humanized ERBB gene is operably linked to an endogenous regulatory element of an endogenous ERBB gene on at least one chromosome; Preferably, the expression of endogenous ERBB protein in the non-human animal is reduced or absent.

6. The construction method according to any one of claims 1 to 5, characterized in that: The introduction is replacement or insertion. Preferably, the introduction into the non-human animal ERBB locus is replacement of the corresponding region of the non-human animal. Preferably, the replacement comprises replacement of all or part of exons 1 to 17 of the non-human animal ERBB gene. Further preferably, the replacement comprises replacement of part of exon 2 to part of exon 17 of the non-human animal, or, replacement of part of exon 1 to part of exon 17.

7. The construction method according to any one of claims 1 to 6, characterized in that: The construction method further comprises mating, in vitro fertilization or direct gene editing of non-human animals with humanized ERBB genes with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals, preferably, the other genes are selected from at least one of NKP46, HER3, TROP2, MET, HGF, PD-1, PD-L1, LAG3, 4-1BB, CD40, CTLA4, CD3, CD16, CD64, IGF1R and HER2, Preferably, the human or humanized ERBB gene and / or the other gene is homozygous or heterozygous for the endogenous replaced locus.

8. A humanized ERBB protein, characterized in that The humanized ERBB protein comprises a portion of the human ERBB protein.

9. The humanized ERBB protein according to claim 8, characterized in that The humanized ERBB protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human ERBB protein. Preferably, the humanized ERBB protein comprises all or part of the extracellular region of the human ERBB protein. Further preferably, it comprises at least 200 consecutive amino acids in the extracellular region of the human ERBB protein. More preferably, the humanized ERBB protein comprises the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2, positions 37-643 or SEQ ID NO: 2, positions 25-645. The amino acid sequence shown in positions 25-645 of SEQ ID NO:2 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises an amino acid sequence shown in positions 37-643 of SEQ ID NO:2 or positions 25-645 of SEQ ID NO:2, including a substitution, deletion and / or insertion of one or more amino acid residues, Preferably, the humanized ERBB protein further comprises a portion of a non-human animal ERBB protein, preferably all or part of a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region of a non-human animal ERBB protein, and further preferably comprises an amino acid sequence as shown in positions 1-36, 646-1210 of SEQ ID NO: 1 or positions 1-24, 648-1210 of SEQ ID NO: 1, Preferably, the amino acid sequence of the humanized ERBB protein comprises any one of the following groups: A) the amino acid sequence shown in SEQ ID NO: 9; B) is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9; C) differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or D) an amino acid sequence as shown in SEQ ID NO: 9, including substitution, deletion and / or insertion of one or more amino acid residues.

10. A humanized ERBB gene, characterized in that: The humanized ERBB gene encodes the humanized ERBB protein according to any one of claims 8-9.

11. The humanized ERBB gene according to claim 10, characterized in that: The humanized ERBB gene comprises all or part of exons 1 to 28 of the human ERBB gene. Preferably, the humanized ERBB gene comprises all or part of exons 1 to 17 of the human ERBB gene. Further preferably, the humanized ERBB gene comprises part of exon 2 to part of exon 17 of the human ERBB gene, or part of exon 1 to part of exon 17, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, the part of exon 2 comprises at least 50 bp of nucleotide sequence, and the part of exon 17 comprises at least 5 bp of nucleotide sequence. More preferably, the part of the human ERBB gene comprises the nucleotide sequence shown in SEQ ID NO: 5; or comprises the nucleotide sequence shown in SEQ ID NO:

6. The nucleotide sequence of SEQ ID NO:5 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical; or, comprises a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO:5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence having a substitution, deletion and / or insertion of one or more nucleotides as shown in the nucleotide sequence of SEQ ID NO:5, Preferably, the humanized ERBB gene further comprises a portion of a non-human animal ERBB gene. Further preferably, the humanized ERBB gene comprises all of exon 1, part of exon 2, and a portion of exon 17 to all of exon 28 of a non-human animal ERBB gene, or a portion of exon 1, a portion of exon 17 to all of exon 28. Preferably, the nucleotide sequence of the humanized ERBB gene comprises any one of the following groups: A) the transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8; B) the transcribed mRNA is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8; C) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO:8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or, D) The transcribed mRNA has the nucleotide sequence shown in SEQ ID NO: 8, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

12. A cell, tissue or organ, characterized in that: The genome of the cell, tissue or organ contains the humanized ERBB gene described in any one of claims 10-11, or the cell, tissue or organ expresses the humanized ERBB protein described in any one of claims 8-9, or the cell, tissue or organ is derived from a non-human animal obtained by the construction method described in any one of claims 1-7. Preferably, the tissue includes tumor tissue after tumor bearing.

13. A use of the humanized ERBB protein according to any one of claims 8-9, the humanized ERBB gene according to any one of claims 10-11, the non-human animal obtained by the construction method according to any one of claims 1-7, or the cell, tissue or organ according to claim 12, characterized in that: The applications described include: A) Application in product development for ERBB-related immune processes involving human cells; B) Application as a model system related to ERBB for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of ERBB-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human ERBB signaling pathway modulators; or, E) Study the function of ERBB gene, study the drugs and efficacy targeting human ERBB target sites, and study the application of drugs for immune-related diseases and anti-tumor drugs related to ERBB.

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