Humanized non-human animals with TGFBR2 gene, and construction method and application thereof

Abstract

The present invention provides a non-human animal with humanized TGFBR2 gene and its construction method, a humanized TGFBR2 protein, a humanized TGFBR2 gene, a targeting vector for TGFBR2 gene, a cell with humanized TGFBR2 gene and its application in the biomedical field. The construction method includes introducing a nucleotide sequence encoding human TGFBR2 protein into the genome of a non-human animal by means of homologous recombination. The human or humanized TGFBR2 protein can be normally expressed in this animal, which can be used as an animal model for studying the signal mechanism of human TGFBR2, screening drugs for tumors and immune-related diseases, and has important application value for the research and development of new drugs targeting immune targets.

Description

Technical Field

[0001] The present invention belongs to the fields of animal genetic engineering and gene genetic modification. Specifically, it relates to a non-human animal with humanized TGFBR2 gene, a method for constructing the same, and its application in the biomedical field. Background Art

[0002] TGFBR2 (transforming growth factor beta receptor 2) is a single-channel type I membrane protein receptor that is widely present in adipose tissue, lungs, and 25 other tissues, and is expressed in monocytes, T cells, B cells, and NK cells, especially highly expressed in eosinophils and naive CD4 T cells, with low tissue and cell specificity. There are three types of TGFβ in mammals, including TGFβ1, TGFβ2, and TGFβ3. The TGFβ receptor superfamily is divided into two categories, namely TGFβ receptor type 1 and TGFβ receptor type 2. The receptors for the three types of TGFβ are TGFβ receptor 1 and TGFβ receptor 2. A receptor complex composed of two TGFBR1 and two TGFBR2 molecules symmetrically binds to the cytokine dimer, resulting in constitutive activation of TGFBR2 phosphorylation and activation of TGFRB1. Activated TGFBR1 phosphorylates SMAD2, and SMAD2 dissociates from the receptor and interacts with SMAD4. The SMAD2-SMAD4 complex then translocates to the nucleus, where it regulates the transcription of TGF-β-regulated genes, which constitutes the typical SMAD-dependent TGF-β signaling cascade. The transmembrane serine / threonine kinase formed by TGFBR2 and TGFBR1 transduces TGFB1, TGFB2, and TGFB3 signals from the cell surface to the cytoplasm, thereby regulating a large number of physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis. The TGFβ signal plays a complex role in the occurrence and maintenance of tumors, and its tumor-suppressing and tumor-promoting effects may depend on the stage of tumorigenesis and the tumor microenvironment. The TGFβ signal plays a tumor-suppressing role in precancerous lesions, while it plays a promoting role in cancer metastasis.

[0003] Among the current 9 preclinical research drugs, 5 are TGFBR2 expression inhibitors. 3 of them are in Phase I clinical trials. LY3022859 (IMC-TR1) developed by Eli Lilly is an anti-TGFβR2 IgG1 monoclonal antibody that can inhibit receptor-mediated signal activation; the Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting the extracellular domains of PD-L1 (CD274) and TGFBR2 receptors, in advanced solid tumors by Merck & Co., Inc. Jiangsu Hengrui Medicine Co., Ltd. follows closely, and its investigational drug SHR-1701 is also in Phase I clinical development. It is a bifunctional fusion protein composed of a monoclonal antibody targeting PD-L1 (CD274) fused with the extracellular domain of TGFBR2, and is used for the treatment of metastatic or locally advanced solid tumors, including recurrent or metastatic nasopharyngeal carcinoma.

[0004] With the continuous development and maturity of genetic engineering technology, it has been realized to replace or substitute the homologous genes of animals with human genes. Developing humanized experimental animal models in this way is the future development direction of animal models. Among them, gene humanized animal models, that is, using gene editing technology to replace the homologous genes in the animal genome with human normal or mutant genes, can establish normal or mutant gene animal models that are closer to human physiological or disease characteristics. Gene humanized animals not only have important application values themselves. For example, gene humanization can improve and enhance cell or tissue transplantation humanized animal models. More importantly, due to the insertion of human gene fragments, human proteins can be expressed or partially expressed in animals, which can be used as targets for drugs that can only recognize human protein sequences, providing the possibility for screening anti-human antibodies and other drugs at the animal level. However, due to the differences between animals and humans in physiology and pathology, coupled with the complexity of genes. For example, the identity of the TGFBR2 proteins between humans and mice is 92%, and the identity of the main functional domains is 87%. How to construct an "effective" humanized animal model for new drug research and development remains the biggest challenge.

[0005] In view of the complex mechanism of action of TGFBR2 and its great application value in the field of tumor treatment, in order to further explore its related biological characteristics, improve the effectiveness of preclinical efficacy trials, increase the success rate of research and development, make preclinical trials more effective and minimize research and development failures, there is an urgent need in this field to develop non-human animal models of the TGFBR2-related signaling pathway. In addition, the non-human animals obtained by this method can be mated with other gene humanized non-human animals to obtain multi-gene humanized animal models, which are used for screening and evaluating the efficacy of human drugs and combination drugs targeting this signaling pathway. The present invention has broad application prospects in academic and clinical research. Summary of the Invention

[0006] In a first aspect of the present invention, there is provided a method for constructing a non-human animal with humanized TGFBR2 gene, wherein the non-human animal expresses human or humanized TGFBR2 protein in vivo.

[0007] Preferably, the expression of endogenous TGFBR2 protein in the non-human animal is reduced or absent.

[0008] Preferably, the humanized TGFBR2 protein comprises all or part of the human TGFBR2 protein.

[0009] Preferably, the humanized TGFBR2 protein comprises all or part of the signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the human TGFBR2 protein.

[0010] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2, wherein the partial extracellular region of human TGFBR2 comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0011] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 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 including substitution, deletion, and / or insertion of one or more amino acid residues as shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44.

[0012] Preferably, the humanized TGFBR2 protein comprises all or part of the signal peptide of human TGFBR2.

[0013] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2 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 1-22 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0014] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2.

[0015] More preferably, it comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0016] In a specific embodiment of the present invention, it comprises the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44 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 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0017] Preferably, the humanized TGFBR2 protein further comprises part of the extracellular region, the entire transmembrane region, the entire signal peptide and the entire cytoplasmic region of the TGFBR2 protein of a non-human animal.

[0018] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 1-28 and 183-592 of SEQ ID NO: 43, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-28 and 183-592 of SEQ ID NO: 43; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-28 and 183-592 of SEQ ID NO: 43 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 that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 1-28 and 183-592 of SEQ ID NO: 43.

[0019] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2 and all or part of the signal peptide.

[0020] In a specific embodiment of the present invention, it comprises the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 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 that includes substitution, deletion and / or insertion of one or more amino acid residues as shown in positions 1-166 of SEQ ID NO: 2.

[0021] Preferably, the humanized TGFBR2 protein further comprises part of the transmembrane region of non-human animal TGFBR2 and all of the cytoplasmic region.

[0022] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1; or comprises an amino acid sequence that differs from the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1 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 that includes substitution, deletion and / or insertion of one or more amino acid residues as shown at positions 167-567 of SEQ ID NO: 1.

[0023] Preferably, the humanized TGFBR2 protein comprises all or part of the amino acid sequence encoded by the human TGFBR2 gene. More preferably, it comprises all or part of the amino acid sequence encoded by exons 1 to 8 of the human TGFBR2 gene. Even more preferably, it comprises the amino acid sequence encoded by one, two or more than two exons among exons 1 to 8 of the human TGFBR2 gene; even more preferably, it comprises all or part of the amino acid sequence encoded by exons 1 to 5 of the human TGFBR2 gene.

[0024] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by a part of exon 1, all of exons 2 to 4, and a part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, and the part of exon 5 comprises at least 5 bp of nucleotide sequence.

[0025] Preferably, the humanized TGFBR2 protein comprises all or part of the amino acid sequence encoded by exons 1 to 7 of the human TGFBR2 gene. More preferably, it comprises the amino acid sequence encoded by a combination of one, two or more than two exons among exons 1 to 7 of the human TGFBR2 gene.

[0026] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by a part of exon 1, all of exon 2, all of exon 3, and a part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least the nucleotide sequence of the coding region, and the part of exon 4 comprises at least the nucleotide sequence of the extracellular region.

[0027] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises any one of the following groups:

[0028] A) The amino acid sequence shown by positions 1 - 166 of SEQ ID NO: 2 or positions 29 - 182 of SEQ ID NO: 44;

[0029] B) Having an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% with the amino acid sequence shown by positions 1 - 166 of SEQ ID NO: 2 or positions 29 - 182 of SEQ ID NO: 44;

[0030] C) Differing from the amino acid sequence shown by positions 1 - 166 of SEQ ID NO: 2 or positions 29 - 182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;

[0031] Or,

[0032] D) An amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues, as shown by positions 1 - 166 of SEQ ID NO: 2 or positions 29 - 182 of SEQ ID NO: 44.

[0033] Preferably, the humanized TGFBR2 protein comprises a part of the human TGFBR2 protein and a part of the non - human animal TGFBR2 protein.

[0034] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises all of the extracellular region and all of the signal peptide of the human TGFBR2 protein, and a part of the transmembrane region and all of the cytoplasmic region of the non - human animal TGFBR2 protein.

[0035] In another embodiment of the present invention, the humanized TGFBR2 protein comprises all or part of the extracellular region of the human TGFBR2 protein, and all of the signal peptide, all of the cytoplasmic region, all or part of the transmembrane region and all or part of the extracellular region of the non - human animal TGFBR2 protein.

[0036] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0037] A) All or part of SEQ ID NO: 53;

[0038] B) Having an identity of at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% with the amino acid sequence of SEQ ID NO: 53;

[0039] C) differs from the amino acid sequence of SEQ ID NO: 53 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0040] D) an amino acid sequence that includes substitutions, deletions, and / or insertions of one or more amino acid residues as shown in SEQ ID NO: 53.

[0041] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0042] a) all or part of the amino acid sequence of SEQ ID NO: 13;

[0043] b) having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 13;

[0044] c) differs from the amino acid sequence shown in SEQ ID NO: 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0045] d) an amino acid sequence that includes substitutions, deletions, and / or insertions of one or more amino acid residues as shown in SEQ ID NO: 13.

[0046] Preferably, the genome of the non-human animal contains a human or humanized TGFBR2 gene, and the humanized TGFBR2 gene contains a portion of the human TGFBR2 gene.

[0047] Preferably, the genome of the non-human animal contains all or part of the nucleotide sequence encoding the human TGFBR2 protein, preferably contains all or part of the nucleotide sequence encoding the extracellular region, transmembrane region, signal peptide, and / or cytoplasmic region of the human TGFBR2 protein, and further preferably contains all or part of the nucleotide sequence encoding the extracellular region, wherein the portion of the extracellular region contains at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the human TGFBR2 extracellular region. More preferably, it contains the nucleotide sequence encoding the amino acids at positions 23 - 166 of SEQ ID NO: 2 or positions 29 - 182 of SEQ ID NO: 44.

[0048] Preferably, the genome of the non-human animal further comprises all or part of the nucleotide sequence encoding the signal peptide of human TGFBR2, preferably comprising the nucleotide sequence encoding the amino acids shown in positions 1-22 of SEQ ID NO: 2.

[0049] Preferably, the genome of the non-human animal comprises all or part of the nucleotide sequence encoding the extracellular region of human TGFBR2 and all or part of the transmembrane region, preferably comprising at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2, and more preferably comprising the nucleotide sequence encoding the amino acids in positions 29-182 of SEQ ID NO: 44.

[0050] Preferably, the genome of the non-human animal comprises the nucleotide sequence encoding the extracellular region and signal peptide of human TGFBR2, preferably comprising the nucleotide sequence encoding the amino acids in positions 1-166 of SEQ ID NO: 2.

[0051] Preferably, the genome of the non-human animal comprises all or part of the human TGFBR2 gene; preferably, it comprises all or part of exons 1 to 8 of the human TGFBR2 gene, more preferably it comprises one, two or more combinations of exons 1 to 8 of the human TGFBR2 gene, and even more preferably it comprises all or part of exons 1 to 5 of the human TGFBR2 gene.

[0052] Preferably, the genome of the non-human animal comprises a part of exon 1 of the human TGFBR2 gene, all of exons 2 to 4 and a part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence, and the part of exon 5 comprises at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. Preferably, it also comprises introns 1-2 and / or introns 4-5 of the human TGFBR2 gene.

[0053] Preferably, the humanized TGFBR2 gene further comprises a part of the non-human animal TGFBR2 gene, more preferably it comprises a part of exon 1 of the non-human animal TGFBR2 gene, a part of exon 5 and all of exons 6 to 8 of the non-human animal TGFBR2 gene.

[0054] Preferably, the genome of the non-human animal contains a part of exon 1, the whole of exon 2, the whole of exon 3, and a part of exon 4 of the human TGFBR2 gene. Among them, the part of exon 1 contains at least 50 bp of nucleotide sequence, such as at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence. The part of exon 1 contains the nucleotide sequence of the coding region. The part of exon 4 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. The part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0055] Preferably, the humanized TGFBR2 gene further contains a part of the non-human animal TGFBR2 gene, preferably contains all or part of exon 1, all or part of exon 2, a part of exon 4, all of exon 5, all of exon 6, and all of exon 7 of the non-human animal TGFBR2 gene.

[0056] In a specific embodiment of the present invention, the genome of the non-human animal contains the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, contains 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: 7 or SEQ ID NO: 47; or, contains a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47, including substitution, deletion and / or insertion of one or more nucleotides.

[0057] Preferably, the nucleotide sequence encoding the human or humanized TGFBR2 protein or the nucleotide sequence of the human or humanized TGFBR2 gene is operably linked to an endogenous regulatory element at the endogenous TGFBR2 locus in at least one chromosome.

[0058] Preferably, the expression of the endogenous TGFBR2 protein in the non-human animal is reduced or absent.

[0059] Preferably, the construction method includes introducing a donor nucleotide sequence into the TGFBR2 locus of a non-human animal.

[0060] Preferably, the donor nucleotide sequence comprises all or part of the human TGFBR2 gene.

[0061] In some specific embodiments, the donor nucleotide sequence comprises any one of the following groups:

[0062] A) a nucleotide sequence encoding a human or humanized TGFBR2 protein;

[0063] B) all or part of a nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the human TGFBR2 protein; preferably, it comprises all or part of the nucleotide sequence encoding the extracellular region; more preferably, it further comprises the nucleotide sequence encoding the signal peptide; even more preferably, it comprises the nucleotide sequence encoding amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44; still more preferably, it comprises the nucleotide sequence encoding SEQ ID NO: 13 or SEQ ID NO: 53;

[0064] C) the nucleotide sequence of the human or humanized TGFBR2 gene; or,

[0065] D) all or part of exons 1 to 8 of the human TGFBR2 gene, preferably, one, two, or more than two nucleotide sequences among exons 1 to 8 of the human TGFBR2 gene, more preferably, all or part of exons 1 to 5 of the human TGFBR2 gene; even more preferably, part of exon 1, all of exons 2 to 4, and part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence, and the part of exon 5 comprises at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. Preferably, it further comprises introns 1-2 and / or introns 4-5.

[0066] In a specific embodiment of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO: 47;

[0067] E) All or part of exons 1 to 7 of the human TGFBR2 gene, preferably one, two or more combinations of exons 1 to 7 of the human TGFBR2 gene, more preferably part of exon 1, all of exon 2, all of exon 3 and part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 contains at least 50 bp of nucleotide sequence, for example, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence, the part of exon 1 contains the nucleotide sequence of the coding region, the part of exon 4 contains at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence, and the part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0068] In a specific embodiment of the present invention, it contains the nucleotide sequence shown in SEQ ID NO: 7.

[0069] In some specific embodiments, the donor nucleotide sequence further contains a nucleotide sequence encoding P2A; preferably, the nucleotide sequence encoding P2A is located upstream of the human TGFBR2 gene part; preferably, the nucleotide sequence encoding P2A is as shown in SEQ ID NO: 4.

[0070] Preferably, the donor nucleotide sequence further contains a part of the non-human animal TGFBR2 gene. In some specific embodiments, the part of the non-human animal TGFBR2 gene is part of exons 1 to 8 of the non-human animal TGFBR2 gene. Preferably, it includes part of exon 1, part of exon 5, and all or part of exons 6 - 8 of the non-human animal TGFBR2 gene. More preferably, it further contains introns 5 - 6 of the non-human animal TGFBR2 gene.

[0071] Preferably, the donor nucleotide sequence further contains a part of the non-human animal TGFBR2 gene, and the part of the non-human animal TGFBR2 gene is all or part of exons 1, 2, 4, 5, 6 and / or 7 of the non-human animal TGFBR2 gene; preferably, it is part of exon 4, all of exon 5, all of exon 6 and / or all or part of exon 7 of the non-human animal TGFBR2 gene; optionally, it further contains introns.

[0072] In some specific embodiments, the donor nucleotide sequence contains a portion of the human TGFBR2 gene and a portion of the non-human animal TGFBR2 gene, and the arrangement order is: a portion of exon 1 of the human TGFBR2 gene, the entire exon 2, the entire exon 3, a portion of exon 4, and a portion of exon 4 of the non-human animal TGFBR2 gene, the entire exon 5, the entire exon 6, and all or part of exon 7.

[0073] Preferably, the donor nucleotide sequence further contains a nucleotide sequence encoding P2A.

[0074] Preferably, the donor nucleotide sequence further contains a coding gene for a selection marker. More preferably, the marker gene is a coding gene for a negative selection marker. Even more preferably, the coding gene for the negative selection marker is the coding gene for diphtheria toxin A subunit (DTA).

[0075] In a specific embodiment of the present invention, the donor nucleotide sequence further includes a resistance gene for positive clone screening. More preferably, the resistance gene for positive clone screening is the coding sequence Neo of neomycin phosphotransferase.

[0076] In a specific embodiment of the present invention, the donor nucleotide sequence further includes a specific recombination system. More preferably, the specific recombination system is the Frt recombination site (the conventional LoxP recombination system can also be selected). The specific recombination system has two Frt recombination sites, which are respectively connected to both sides of the resistance gene.

[0077] Preferably, the humanized TGFBR2 gene contains a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 8 and / or SEQ ID NO: 9, or contains the nucleotide sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0078] Preferably, the humanized TGFBR2 gene contains a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 10 and / or SEQ ID NO: 11, or contains the nucleotide sequence shown in SEQ ID NO: 10 and / or SEQ ID NO: 11.

[0079] Preferably, the humanized TGFBR2 gene comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 39 and / or SEQ ID NO: 40, or comprises the nucleotide sequence shown in SEQ ID NO: 39 and / or SEQ ID NO: 40.

[0080] Preferably, the humanized TGFBR2 gene comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 48, 49, 50 and / or 51, or comprises the nucleotide sequence shown in SEQ ID NO: 48, 49, 50 and / or 51.

[0081] In some specific embodiments, the humanized TGFBR2 gene comprises the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 36, SEQ ID NO: 47 or SEQ ID NO: 54; or, comprises a nucleotide sequence having an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% with the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 36, SEQ ID NO: 47 or SEQ ID NO: 54; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 36, SEQ ID NO: 47 or SEQ ID NO: 54 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 the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 36, SEQ ID NO: 47 or SEQ ID NO: 54, including substitution, deletion and / or insertion of one or more nucleotides.

[0082] Preferably, a part of the human TGFBR2 gene or the humanized TGFBR2 gene is regulated by a regulatory element in a non-human animal. More preferably, the regulatory element is endogenous or exogenous.

[0083] Preferably, the regulatory element includes an endogenous promoter.

[0084] The introduction is insertion or replacement. Among them, the replacement can be replacement at the corresponding position or replacement of a non-corresponding sequence of a certain segment of the gene.

[0085] Preferably, the introduction of the non-human animal TGFBR2 locus is to replace all or part of exons 1 to 8 of the non-human animal TGFBR2 gene.

[0086] More preferably, it is to replace the corresponding position of the non-human animal TGFBR2 gene, and even more preferably, it is all or part of exons 1 to 5.

[0087] More preferably, it is to replace exon 2 of the non-human animal TGFBR2 gene, and even more preferably, it is to replace the nucleotide sequence encoding amino acids 61-88 of SEQ ID NO: 1 in the non-human animal genome.

[0088] Preferably, the introduction of the non-human animal TGFBR2 locus is to insert all or part of exons 1 to 8 of the non-human animal TGFBR2 gene, preferably insert exon 1 or exon 2 of the non-human animal TGFBR2 gene, and more preferably insert it at the start codon of the non-human animal TGFBR2 gene.

[0089] In a specific embodiment of the present invention, the construction method includes replacing all or part of exons 1 to 8 of the non-human animal TGFBR2 gene with all or part of exons 1 to 8 of the human TGFBR2 gene.

[0090] In a specific embodiment of the present invention, the construction method includes replacing one, two or a combination of more than two exons of the non-human animal exons 1 to 8 with a combination of one, two or a combination of more than two exons of the human TGFBR2 gene exons 1 to 8.

[0091] In a specific embodiment of the present invention, the construction method includes replacing all or part of exons 1 to 5 of the non-human animal TGFBR2 gene with all or part of exons 1 to 5 of the human TGFBR2 gene.

[0092] In a specific embodiment of the present invention, the construction method includes using a part of exon 1 of the human TGFBR2 gene (preferably at least containing a nucleotide sequence of 5 bp), all of exons 2 to 4, and a part of exon 5 (preferably at least containing a nucleotide sequence of 5 bp), and preferably also contains introns 1-2 and / or introns 4-5 to correspondingly replace the part of exon 1, all of exons 2 to 4, and the part of exon 5 of the non-human animal TGFBR2 gene.

[0093] In a specific embodiment of the present invention, the construction method includes replacing all or part of the extracellular region encoding the non-human animal TGFBR2 with all or part of the extracellular region encoding the human TGFBR2.

[0094] In a specific embodiment of the present invention, the construction method includes replacing part of the extracellular region encoding non-human animal TGFBR2 with all or part of the extracellular region encoding human TGFBR2.

[0095] In a specific embodiment of the present invention, the construction method includes inserting or replacing a sequence of a non-human animal exon 2 with a nucleotide sequence encoding the extracellular region, signal peptide of human TGFBR2, and part of the transmembrane region and cytoplasmic region of non-human animal TGFBR2. Preferably, it is the nucleotide sequence encoding amino acids 61-88 of SEQ ID NO: 1 in non-human animal exon 2.

[0096] In a specific embodiment of the present invention, the construction method includes inserting or replacing a sequence of a non-human animal exon 2 with a human or humanized TGFBR2 gene. Preferably, it is the nucleotide sequence encoding amino acids 61-88 of SEQ ID NO: 1 in non-human animal exon 2.

[0097] In a specific embodiment of the present invention, the construction method includes inserting or replacing a sequence of a non-human animal exon 2 with a nucleotide sequence encoding a human or humanized TGFBR2 protein. Preferably, it is the nucleotide sequence encoding amino acids 61-88 of SEQ ID NO: 1 in non-human animal exon 2.

[0098] In a specific embodiment of the present invention, the construction method includes inserting a nucleotide sequence encoding the extracellular region, signal peptide of human TGFBR2, and all or part of the transmembrane region and cytoplasmic region of non-human animal TGFBR2 into exon 1 of the non-human animal TGFBR2 gene. Preferably, it is inserted at a position before the ATG and after the UTR of the non-human animal TGFBR2 gene.

[0099] In a specific embodiment of the present invention, the construction method includes inserting a human or humanized TGFBR2 gene into exon 1 of the non-human animal TGFBR2 gene. Preferably, it is inserted at a position before the ATG and after the UTR of the non-human animal TGFBR2 gene.

[0100] In a specific embodiment of the present invention, the construction method includes inserting a nucleotide sequence encoding a human or humanized TGFBR2 protein into exon 1 of the non-human animal TGFBR2 gene. Preferably, it is inserted at a position before the ATG and after the UTR of the non-human animal TGFBR2 gene.

[0101] Preferably, a non-human animal is constructed using gene editing technology, 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 technologies.

[0102] Preferably, the construction method includes constructing a non-human animal using a targeting vector and / or sgRNA.

[0103] Preferably, the targeting vector includes a donor nucleotide sequence, and the donor nucleotide sequence includes any one of the following groups:

[0104] A) A nucleotide sequence encoding a human or humanized TGFBR2 protein;

[0105] B) All or part of a nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of a human TGFBR2 protein; preferably, it includes all or part of a nucleotide sequence encoding the extracellular region; more preferably, it further includes a nucleotide sequence encoding the signal peptide; even more preferably, it includes a nucleotide sequence encoding the amino acids at positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44; still more preferably, it includes a nucleotide sequence encoding SEQ ID NO: 13 or SEQ ID NO: 53;

[0106] C) A nucleotide sequence of a human or humanized TGFBR2 gene; or,

[0107] D) All or part of exons 1 to 8 of the human TGFBR2 gene, preferably, one, two, or more than two nucleotide sequences among exons 1 to 8 of the human TGFBR2 gene, more preferably, all or part of exons 1 to 5 of the human TGFBR2 gene; even more preferably, part of exon 1, all of exons 2 to 4, and part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 includes at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence, and the part of exon 5 includes at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence; preferably, it further includes introns 1-2 and / or introns 4-5.

[0108] E) all or part of exons 1 to 7 of the human TGFBR2 gene, preferably, one, two or more combinations of exons 1 to 7 of the human TGFBR2 gene, more preferably, a part of exon 1, the whole of exon 2, the whole of exon 3 and a part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 contains at least 50 bp of nucleotide sequence, for example, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence, the part of exon 1 contains the nucleotide sequence of the coding region, the part of exon 4 contains at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence, and the part of exon 4 contains the nucleotide sequence of the extracellular region coding region.

[0109] In a specific embodiment of the present invention, the targeting vector contains a nucleotide sequence encoding amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44, or a nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence of amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44; or a nucleotide sequence encoding an amino acid sequence that differs from the amino acid sequence of amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or a nucleotide sequence encoding an amino acid sequence shown in amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0110] In another specific embodiment of the present invention, the targeting vector comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47, or comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47 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 the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47, including substitution, deletion and / or insertion of one or more nucleotides.

[0111] Preferably, the targeting vector further comprises a part of the non-human animal TGFBR2 gene, preferably comprises a nucleotide sequence encoding the cytoplasmic region and transmembrane region of non-human animal TGFBR2, more preferably comprises a nucleotide sequence encoding the amino acids at positions 167-567 of SEQ ID NO: 1, and even more preferably, comprises the nucleotide sequence shown in SEQ ID NO: 31.

[0112] Preferably, the targeting vector further comprises a part of the non-human animal TGFBR2 gene. In some specific embodiments, the part of the non-human animal TGFBR2 gene is a part of exons 1 to 8 of the non-human animal TGFBR2 gene. Preferably, it includes a part of exon 1 of the non-human animal TGFBR2 gene, a part of exon 5 of the non-human animal TGFBR2 gene, all or part of exons 6 to 8 of the non-human animal TGFBR2 gene.

[0113] In some specific embodiments, the targeting vector further comprises a part of the non-human animal TGFBR2 gene, and the part of the non-human animal TGFBR2 gene is all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the non-human animal TGFBR2 gene; preferably, it is all or part of exon 2, all or part of exon 3, exon 4, exon 5, exon 6 and / or exon 7 of the non-human animal TGFBR2 gene.

[0114] In a specific embodiment, the targeting vector sequentially comprises all or part of exon 1 of the human TGFBR2 gene, all of exon 2, all of exon 3 and all or part of exon 4 from the 5'-end to the 3'-end, and a part of exon 4, all of exon 5, all of exon 6 and all or part of exon 7 of the non-human animal TGFBR2 gene.

[0115] In a specific embodiment, the targeting vector sequentially comprises, from the 5'-end to the 3'-end, a nucleotide sequence encoding the signal peptide and extracellular region of human TGFBR2 and a nucleotide sequence encoding all or part of the cytoplasmic region and transmembrane region of non-human animal TGFBR2.

[0116] In some specific embodiments, the targeting vector further comprises a nucleotide sequence encoding P2A; preferably, the nucleotide sequence encoding P2A is located upstream of the human TGFBR2 gene portion; preferably, the nucleotide sequence encoding P2A is as shown in SEQ ID NO: 4.

[0117] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 8 and / or SEQ ID NO: 9, or comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0118] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 10 and / or SEQ ID NO: 11, or comprises the nucleotide sequence shown in SEQ ID NO: 10 and / or SEQ ID NO: 11.

[0119] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 39 and / or SEQ ID NO: 40, or comprises the nucleotide sequence shown in SEQ ID NO: 39 and / or SEQ ID NO: 40.

[0120] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 48, 49, 50 and / or 51, or comprises the nucleotide sequence shown in SEQ ID NO: 48, 49, 50 and / or 51.

[0121] In a specific embodiment of the present invention, the targeting vector comprises the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54, or comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54; or comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54 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 the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54, including substitution, deletion and / or insertion of one or more nucleotides.

[0122] Preferably, the targeting vector encodes the above-mentioned humanized TGFBR2 protein.

[0123] Preferably, the targeting vector further comprises a 5' arm and / or a 3' arm.

[0124] The 5' arm (or 5' homologous arm) is a DNA fragment homologous to the 5' end of the non-human animal TGFBR2 gene, and it is selected from nucleotides with a length of 100-10,000 of the genomic DNA of the non-human animal TGFBR2 gene. Further preferably, the 5' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000075.7. More preferably, the 5' arm sequence has 90% homology with SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 37 or SEQ ID NO: 45, or is as shown in SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 37 or SEQ ID NO: 45.

[0125] The 3' arm (or 3' homologous arm) is a DNA fragment homologous to the 3' end of the non-human animal TGFBR2 gene, and it is selected from nucleotides with a length of 100-10,000 of the DNA of the non-human animal TGFBR2 gene. Further preferably, the 3' arm is a nucleotide having at least 90% homology with NCBI accession number NC_000075.7. More preferably, the 3' arm sequence has 90% homology with SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 38 or SEQ ID NO: 46, or is as shown in SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 38 or SEQ ID NO: 46.

[0126] Preferably, the targeting vector further comprises a non-human animal 3'UTR.

[0127] Preferably, the targeting vector further comprises a non-human animal 5'UTR.

[0128] In a specific embodiment, sgRNA is used for the construction of non-human animals. Among them, the sgRNA targets the non-human animal TGFBR2 gene, and at the same time, the sequence of the sgRNA is unique on the target sequence of the TGFBR2 gene to be modified.

[0129] Preferably, the target site of the sgRNA is located on the exon 2 sequence of the TGFBR2 gene.

[0130] More preferably, the target sequence of the sgRNA on the TGFBR2 gene is shown in any one of SEQ ID NO: 20-21.

[0131] Preferably, the target site of the sgRNA is located on the exon 1 sequence of the TGFBR2 gene.

[0132] More preferably, the target sequence of the sgRNA on the TGFBR2 gene is shown in SEQ ID NO: 41.

[0133] Preferably, the replacement of the human or humanized TGFBR2 gene for the endogenous TGFBR2 gene in the humanized non-human animal of the TGFBR2 gene is homozygous or heterozygous.

[0134] Preferably, the humanized TGFBR2 gene and / or other genes are homozygous for the endogenous modified locus;

[0135] Preferably, the humanized TGFBR2 gene and / or other genes are heterozygous for the endogenous modified locus. Preferably, the genome of the non-human animal contains the humanized TGFBR2 gene on at least one chromosome.

[0136] Preferably, at least one cell in the non-human animal expresses human or humanized TGFBR2 protein.

[0137] Preferably, the construction method includes introducing the donor nucleotide sequence into non-human animal cells using a targeting vector, culturing the cells, and then transplanting the cultured cells into the oviduct of a female non-human animal to allow it to develop, and identifying and screening to obtain a non-human animal with a humanized TGFBR2 gene.

[0138] Preferably, the construction method includes introducing the donor nucleotide sequence into the endogenous TGFBR2 locus of the non-human animal using a guide RNA targeting the endogenous TGFBR2 gene of the non-human animal.

[0139] Preferably, the construction method further includes mating the non-human animal with humanized TGFBR2 gene with other genetically modified non-human animals, in vitro fertilization or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal.

[0140] Preferably, the other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3, TIGIT.

[0141] The human or humanized TGFBR2 gene and / or the other gene in the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0142] In a second aspect of the present invention, there is provided a non-human animal with humanized TGFBR2 gene, and the non-human animal expresses human or humanized TGFBR2 protein in vivo.

[0143] Preferably, the humanized TGFBR2 protein comprises all or part of the human TGFBR2 protein.

[0144] Preferably, the humanized TGFBR2 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human TGFBR2 protein.

[0145] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2, wherein the partial extracellular region of human TGFBR2 comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0146] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 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 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0147] Preferably, the humanized TGFBR2 protein comprises all or part of the human TGFBR2 signal peptide.

[0148] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2 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 1-22 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0149] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2.

[0150] More preferably, it comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0151] In a specific embodiment of the present invention, it comprises the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 29-182 of SEQ ID NO: 44 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 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0152] Preferably, the humanized TGFBR2 protein further comprises a part of the extracellular region, the entire transmembrane region, the entire signal peptide and the entire cytoplasmic region of the non-human animal TGFBR2 protein.

[0153] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequences shown in positions 1-28 and 183-592 of SEQ ID NO: 43, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequences shown in positions 1-28 and 183-592 of SEQ ID NO: 43; or comprises an amino acid sequence that differs from the amino acid sequences shown in positions 1-28 and 183-592 of SEQ ID NO: 43 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 1-28 and 183-592 of SEQ ID NO: 43, including substitution, deletion and / or insertion of one or more amino acid residues.

[0154] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region and all or part of the signal peptide of human TGFBR2.

[0155] In a specific embodiment of the present invention, it comprises the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2 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 at positions 1-166 of SEQ ID NO: 2, including an amino acid sequence with substitution, deletion and / or insertion of one or more amino acid residues.

[0156] Preferably, the humanized TGFBR2 protein further comprises a part of the transmembrane region and the whole cytoplasmic region of non-human animal TGFBR2.

[0157] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1; or comprises an amino acid sequence that differs from the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1 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 at positions 167-567 of SEQ ID NO: 1, including an amino acid sequence with substitution, deletion and / or insertion of one or more amino acid residues.

[0158] Preferably, the humanized TGFBR2 protein comprises all or part of the amino acid sequence encoded by the human TGFBR2 gene. More preferably, it comprises all or part of the amino acid sequence encoded by exons 1 to 8 of the human TGFBR2 gene. Even more preferably, it comprises the amino acid sequence encoded by one, two or more than two exons among exons 1 to 8 of the human TGFBR2 gene; even more preferably, it comprises all or part of the amino acid sequence encoded by exons 1 to 5 of the human TGFBR2 gene.

[0159] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by a part of exon 1, the whole of exons 2 to 4 and a part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, and the part of exon 5 comprises at least 5 bp of nucleotide sequence.

[0160] Preferably, the humanized TGFBR2 protein comprises all or part of the amino acid sequence encoded by exons 1 to 7 of the human TGFBR2 gene. More preferably, it comprises the amino acid sequence encoded by one, two or more than two exons selected from exons 1 to 7 of the human TGFBR2 gene.

[0161] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by a part of exon 1, the whole of exon 2, the whole of exon 3 and a part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least the nucleotide sequence of the coding region, and the part of exon 4 comprises at least the nucleotide sequence of the extracellular region.

[0162] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises any one of the following groups:

[0163] A) The amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44;

[0164] B) Having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44;

[0165] C) Differing from the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;

[0166] Or,

[0167] D) An amino acid sequence which includes substitution, deletion and / or insertion of one or more amino acid residues and is shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44.

[0168] Preferably, the humanized TGFBR2 protein comprises a part of the human TGFBR2 protein and a part of the non-human animal TGFBR2 protein.

[0169] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the extracellular region and signal peptide of the human TGFBR2 protein and all or part of the transmembrane region and cytoplasmic region of the non-human animal TGFBR2 protein.

[0170] In another embodiment of the present invention, the humanized TGFBR2 protein comprises all or part of the extracellular region of the human TGFBR2 protein, and all of the signal peptide, all of the cytoplasmic region, all or part of the transmembrane region, and all or part of the extracellular region of the non-human animal TGFBR2 protein.

[0171] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0172] A) all or part of SEQ ID NO: 53;

[0173] B) having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 53;

[0174] C) differing from the amino acid sequence of SEQ ID NO: 53 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0175] D) an amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 53.

[0176] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0177] a) all or part of the amino acid sequence of SEQ ID NO: 13;

[0178] b) having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of SEQ ID NO: 13;

[0179] c) differing from the amino acid sequence shown in SEQ ID NO: 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0180] d) an amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 13.

[0181] The genome of the non-human animal contains all or part of the nucleotide sequence encoding human TGFBR2 protein, preferably contains all or part of the nucleotide sequence encoding the extracellular region, transmembrane region, signal peptide and / or cytoplasmic region of human TGFBR2 protein, more preferably contains all or part of the nucleotide sequence encoding the extracellular region, wherein the part of the extracellular region contains at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2. Even more preferably, it contains the nucleotide sequence encoding the amino acids at positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44.

[0182] Preferably, the genome of the non-human animal further contains all or part of the nucleotide sequence encoding the signal peptide of human TGFBR2, preferably contains the nucleotide sequence encoding the amino acids shown at positions 1-22 of SEQ ID NO: 2.

[0183] Preferably, the genome of the non-human animal contains all or part of the extracellular region of human TGFBR2, preferably contains at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2. More preferably, it contains the nucleotide sequence encoding the amino acids at positions 29-182 of SEQ ID NO: 44.

[0184] Preferably, the genome of the non-human animal contains the nucleotide sequence encoding the extracellular region and signal peptide of human TGFBR2, preferably contains the nucleotide sequence encoding the amino acids at positions 1-166 of SEQ ID NO: 2.

[0185] Preferably, the genome of the non-human animal contains all or part of the human TGFBR2 gene; more preferably, it contains all or part of exons 1 to 8 of the human TGFBR2 gene, even more preferably contains one, two or a combination of three or more of exons 1 to 8 of the human TGFBR2 gene, and even more preferably contains all or part of exons 1 to 5 of the human TGFBR2 gene.

[0186] Preferably, the genome of the non-human animal contains a part of exon 1, all of exons 2 to 4, and a part of exon 5 of the human TGFBR2 gene. Among them, the part of exon 1 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence; the part of exon 5 contains at least 5 bp of nucleotide sequence, such as at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. Preferably, it also contains introns 1-2 and / or introns 4-5 of the human TGFBR2 gene.

[0187] Preferably, the humanized TGFBR2 gene further contains a part of the non-human animal TGFBR2 gene, and more preferably contains a part of exon 1, a part of exon 5, and all of exons 6 to 8 of the non-human animal TGFBR2 gene.

[0188] Preferably, the genome of the non-human animal contains a part of exon 1, all of exon 2, all of exon 3, and a part of exon 4 of the human TGFBR2 gene. Among them, the part of exon 1 contains at least 50 bp of nucleotide sequence, such as at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence; the part of exon 1 contains the nucleotide sequence of the coding region; the part of exon 4 contains at least 20 bp of nucleotide sequence, such as at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence, and the part of exon 4 contains the nucleotide sequence of the extracellular region coding region.

[0189] Preferably, the humanized TGFBR2 gene further contains a part of the non-human animal TGFBR2 gene, preferably contains all or part of exon 1, all or part of exon 2, a part of exon 4, all of exon 5, all of exon 6, and all of exon 7 of the non-human animal TGFBR2 gene.

[0190] In a specific embodiment of the present invention, the genome of the non-human animal contains the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, contains a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, contains a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, contains a nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47, including substitution, deletion and / or insertion of one or more nucleotides.

[0191] Preferably, the nucleotide sequence encoding a human or humanized TGFBR2 protein or the nucleotide sequence of a human or humanized TGFBR2 gene is operably linked to an endogenous regulatory element at the endogenous TGFBR2 locus in at least one chromosome.

[0192] Preferably, the expression of the endogenous TGFBR2 protein in the non-human animal is reduced or absent.

[0193] Preferably, the non-human animal is constructed by the construction method as described above in the present invention.

[0194] Preferably, the non-human animal further comprises other gene modifications, and the other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3, TIGIT.

[0195] Preferably, the TGFBR2 gene, the human or humanized TGFBR2 gene and the other genes in the non-human animal are homozygous for the endogenous modified locus;

[0196] Preferably, the TGFBR2 gene, the human or humanized TGFBR2 gene and the other genes in the non-human animal are heterozygous for the endogenous modified locus. Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare gene humanization.

[0197] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0198] Preferably, the non-human animal is an immunodeficient non-human mammal. More 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 NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mouse, NOD / SCID mouse or nude mouse.

[0199] In a third aspect of the present invention, there is provided a targeting vector, the targeting vector comprising a donor nucleotide sequence. Preferably, the donor nucleotide sequence comprises any one of the following groups:

[0200] A) a nucleotide sequence encoding a human or humanized TGFBR2 protein;

[0201] B) all or part of a nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of a human TGFBR2 protein; preferably comprising all or part of the nucleotide sequence encoding the extracellular region; more preferably further comprising the nucleotide sequence encoding the signal peptide; even more preferably, comprising the nucleotide sequence encoding amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44; still more preferably, comprising the nucleotide sequence encoding SEQ ID NO: 13 or SEQ ID NO: 53;

[0202] C) the nucleotide sequence of the human or humanized TGFBR2 gene; or,

[0203] D) All or part of exons 1 to 8 of the human TGFBR2 gene, preferably, one, two or more than two nucleotide sequences among exons 1 to 8 of the human TGFBR2 gene, more preferably, all or part of exons 1 to 5 of the human TGFBR2 gene; even more preferably, part of exon 1, all of exons 2 to 4 and part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence, and the part of exon 5 contains at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence; preferably, it also contains introns 1-2 and / or introns 4-5.

[0204] E) All or part of exons 1 to 7 of the human TGFBR2 gene, preferably, one, two or more than two combinations among exons 1 to 7 of the human TGFBR2 gene, more preferably, part of exon 1, all of exon 2, all of exon 3 and part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 contains at least 50 bp of nucleotide sequence, for example, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence, the part of exon 1 contains the nucleotide sequence of the coding region, and the part of exon 4 contains at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence, and the part of exon 4 contains the nucleotide sequence of the extracellular region.

[0205] In a specific embodiment of the present invention, the targeting vector comprises a nucleotide sequence encoding amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44, or a nucleotide sequence encoding an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44; or a nucleotide sequence encoding an amino acid sequence that differs from the amino acid sequence of amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or a nucleotide sequence encoding an amino acid sequence shown by amino acids 1-166 of SEQ ID NO: 2 or amino acids 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0206] In another specific embodiment of the present invention, the targeting vector comprises the nucleotide sequence shown by SEQ ID NO: 7 or SEQ ID NO: 47, or comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown by SEQ ID NO: 7 or SEQ ID NO: 47; or comprises a nucleotide sequence that differs from the nucleotide sequence shown by SEQ ID NO: 7 or SEQ ID NO: 47 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 the nucleotide sequence shown by SEQ ID NO: 7 or SEQ ID NO: 47, including substitution, deletion and / or insertion of one or more nucleotides.

[0207] Preferably, the targeting vector further comprises a part of the non-human animal TGFBR2 gene, preferably comprises a nucleotide sequence encoding the cytoplasmic region and transmembrane region of non-human animal TGFBR2, more preferably comprises a nucleotide sequence encoding amino acids 167-567 of SEQ ID NO: 1, and even more preferably, comprises the nucleotide sequence shown by SEQ ID NO: 31.

[0208] Preferably, the targeting vector further comprises a part of the non-human animal TGFBR2 gene. In some specific embodiments, the part of the non-human animal TGFBR2 gene is a part of exons 1 to 8 of the non-human animal TGFBR2 gene. Preferably, it includes a part of exon 1 of the non-human animal TGFBR2 gene, a part of exon 5 of the non-human animal TGFBR2 gene, all or part of exons 6 to 8 of the non-human animal TGFBR2 gene.

[0209] In some specific embodiments, the targeting vector further comprises a portion of the non-human animal TGFBR2 gene, and the portion of the non-human animal TGFBR2 gene is all or part of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and / or exon 7 of the non-human animal TGFBR2 gene; preferably, it is all or part of exon 2, all or part of exon 3, exon 4, exon 5, exon 6, and / or exon 7 of the non-human animal TGFBR2 gene.

[0210] In one specific embodiment, the targeting vector sequentially comprises, from the 5'-end to the 3'-end, all or part of exon 1 of the human TGFBR2 gene, all of exon 2, all of exon 3, and all or part of exon 4 of the human TGFBR2 gene, and part of exon 4, all of exon 5, all of exon 6, and all or part of exon 7 of the non-human animal TGFBR2 gene.

[0211] In one specific embodiment, the targeting vector sequentially comprises, from the 5'-end to the 3'-end, a nucleotide sequence encoding the signal peptide and extracellular region of human TGFBR2 and a nucleotide sequence encoding all or part of the cytoplasmic region and transmembrane region of non-human animal TGFBR2.

[0212] In some specific embodiments, the targeting vector further comprises a nucleotide sequence encoding P2A; preferably, the nucleotide sequence encoding P2A is located upstream of the human TGFBR2 gene portion; preferably, the nucleotide sequence encoding P2A is as shown in SEQ ID NO: 4.

[0213] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, or at least 95% identity with SEQ ID NO: 8 and / or SEQ ID NO: 9, or comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0214] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, or at least 95% identity with SEQ ID NO: 10 and / or SEQ ID NO: 11, or comprises the nucleotide sequence shown in SEQ ID NO: 10 and / or SEQ ID NO: 11.

[0215] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 39 and / or SEQ ID NO: 40, or comprises the nucleotide sequence shown in SEQ ID NO: 39 and / or SEQ ID NO: 40.

[0216] Preferably, the targeting vector comprises a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 48, 49, 50 and / or 51, or comprises the nucleotide sequence shown in SEQ ID NO: 48, 49, 50 and / or 51.

[0217] In a specific embodiment of the present invention, the targeting vector comprises the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54, or comprises a nucleotide sequence having an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% with the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54; or comprises a nucleotide sequence having 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: 3, SEQ ID NO: 36 or SEQ ID NO: 54; or comprises a nucleotide sequence having substitutions, deletions and / or insertions of one or more nucleotides as shown in the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36 or SEQ ID NO: 54.

[0218] Preferably, the targeting vector encodes the above-mentioned humanized TGFBR2 protein.

[0219] Preferably, the targeting vector further comprises a 5'-arm and / or a 3'-arm.

[0220] The 5'-arm (or 5'-homologous arm) is a DNA fragment homologous to the 5'-end of the non-human animal TGFBR2 gene, which is selected from nucleotides with a length of 100-10,000 of the genomic DNA of the non-human animal TGFBR2 gene. Further preferably, the 5'-arm has at least 90% homology with the NCBI accession number NC_000075.7. More preferably, the 5'-arm sequence has 90% homology with SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 37 or SEQ ID NO: 45, or is as shown in SEQ ID NO: 5, SEQ ID NO: 18, SEQ ID NO: 37 or SEQ ID NO: 45.

[0221] The described 3' arm (or 3' homologous arm) is a DNA fragment homologous to the 3' end of the non-human animal TGFBR2 gene, and it is selected from nucleotides with a length of 100 - 10,000 of the non-human animal TGFBR2 gene DNA. Further preferably, the 3' arm is a nucleotide having at least 90% homology with the NCBI accession number NC_000075.7. Even more preferably, the 3' arm sequence has 90% homology with SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 38 or SEQ ID NO: 46, or is as shown in SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 38 or SEQ ID NO: 46.

[0222] Preferably, the targeting vector further comprises the 3' UTR of the non-human animal.

[0223] Preferably, the targeting vector further comprises the 5' UTR of the non-human animal.

[0224] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be used for gene editing to prepare gene humanized animals.

[0225] 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.

[0226] 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 a rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mouse, NOD / SCID mouse or nude mouse.

[0227] Preferably, the targeting vector further comprises a coding gene for a selection marker. Further preferably, the marker gene is a coding gene for a negative selection marker. Even more preferably, the coding gene for the negative selection marker is the coding gene for diphtheria toxin A subunit (DTA).

[0228] In a specific embodiment of the present invention, the targeting vector further includes a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is the coding sequence Neo of neomycin phosphotransferase.

[0229] In a specific embodiment of the present invention, the targeting vector further includes a specific recombination system. Further preferably, the specific recombination system is the Frt recombination site (the conventional LoxP recombination system can also be selected). The specific recombination system has two Frt recombination sites, which are respectively connected to both sides of the resistance gene.

[0230] In the fourth aspect of the present invention, a sgRNA molecule is provided, wherein the sgRNA targets the TGFBR2 gene of a non-human animal, and at the same time, the sequence of the sgRNA is unique on the target sequence of the TGFBR2 gene to be modified.

[0231] Preferably, the target site of the sgRNA is located on the exon 2 sequence of the TGFBR2 gene.

[0232] Further preferably, the target sequence of the sgRNA on the TGFBR2 gene is as shown in any one of SEQ ID NO: 20-21.

[0233] Preferably, the target site of the sgRNA is located on the exon 1 sequence of the TGFBR2 gene.

[0234] Further preferably, the target sequence of the sgRNA on the TGFBR2 gene is as shown in SEQ ID NO: 41.

[0235] In the fifth aspect of the present invention, a cell containing the above-mentioned targeting vector and / or sgRNA is provided.

[0236] Preferably, the cell cannot develop into an animal individual.

[0237] In the sixth aspect of the present invention, the application of the above-mentioned targeting vector, the above-mentioned sgRNA, and the above-mentioned cell in the modification of the TGFBR2 gene is provided. Preferably, the application includes, but is not limited to, knockout, insertion, or replacement.

[0238] In the seventh aspect of the present invention, a humanized TGFBR2 protein is provided, and the humanized TGFBR2 protein contains all or part of the human TGFBR2 protein.

[0239] The humanized TGFBR2 protein contains the signal peptide, extracellular region, transmembrane region, and / or cytoplasmic region of the human TGFBR2 protein.

[0240] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2, wherein the part of the extracellular region of human TGFBR2 comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0241] In one specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 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 23-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0242] Preferably, the humanized TGFBR2 protein comprises all or part of the signal peptide of human TGFBR2.

[0243] In one specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown in positions 1-22 of SEQ ID NO: 2 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 1-22 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acid residues.

[0244] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region of human TGFBR2.

[0245] More preferably, it comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2.

[0246] In a specific embodiment of the present invention, it comprises the amino acid sequence shown in positions 29 - 182 of SEQ ID NO: 44, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 29 - 182 of SEQ ID NO: 44; or comprises an amino acid sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequence shown in positions 29 - 182 of SEQ ID NO: 44; or comprises an amino acid sequence shown in positions 29 - 182 of SEQ ID NO: 44, including substitution, deletion and / or insertion of one or more amino acid residues.

[0247] Preferably, the humanized TGFBR2 protein further comprises a part of the extracellular region, the entire transmembrane region, the entire signal peptide and the entire cytoplasmic region of the non - human animal TGFBR2 protein.

[0248] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequences shown in positions 1 - 28 and 183 - 592 of SEQ ID NO: 43, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequences shown in positions 1 - 28 and 183 - 592 of SEQ ID NO: 43; or comprises an amino acid sequence having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acid sequences shown in positions 1 - 28 and 183 - 592 of SEQ ID NO: 43; or comprises an amino acid sequence shown in positions 1 - 28 and 183 - 592 of SEQ ID NO: 43, including substitution, deletion and / or insertion of one or more amino acid residues.

[0249] Preferably, the humanized TGFBR2 protein comprises all or part of the extracellular region and all or part of the signal peptide of human TGFBR2.

[0250] In a specific embodiment of the present invention, it comprises the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2; or comprises an amino acid sequence that differs from the amino acid sequence shown at positions 1-166 of SEQ ID NO: 2 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 at positions 1-166 of SEQ ID NO: 2, including an amino acid sequence with substitution, deletion and / or insertion of one or more amino acid residues.

[0251] Preferably, the humanized TGFBR2 protein further comprises a part of the transmembrane region and the whole cytoplasmic region of non-human animal TGFBR2.

[0252] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1; or comprises an amino acid sequence that differs from the amino acid sequence shown at positions 167-567 of SEQ ID NO: 1 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 at positions 167-567 of SEQ ID NO: 1, including an amino acid sequence with substitution, deletion and / or insertion of one or more amino acid residues.

[0253] Preferably, the humanized TGFBR2 protein comprises the amino acid sequence encoded by all or part of the human TGFBR2 gene. More preferably, it comprises the amino acid sequence encoded by all or part of exons 1 to 8 of the human TGFBR2 gene. Even more preferably, it comprises the amino acid sequence encoded by one, two or more than two exons among exons 1 to 8 of the human TGFBR2 gene; even more preferably, it comprises the amino acid sequence encoded by all or part of exons 1 to 5 of the human TGFBR2 gene.

[0254] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by a part of exon 1, the whole of exons 2 to 4, and a part of exon 5 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least 5 bp of nucleotide sequence, and the part of exon 5 comprises at least 5 bp of nucleotide sequence.

[0255] Preferably, the humanized TGFBR2 protein comprises all or part of the amino acid sequence encoded by exons 1 to 7 of the human TGFBR2 gene. More preferably, it comprises the amino acid sequence encoded by one, two or more than two combinations of exons 1 to 7 of the human TGFBR2 gene.

[0256] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the amino acid sequence encoded by part of exon 1, all of exon 2, all of exon 3 and part of exon 4 of the human TGFBR2 gene, wherein the part of exon 1 comprises at least the nucleotide sequence of the coding region, and the part of exon 4 comprises at least the nucleotide sequence of the extracellular region.

[0257] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises any one of the following groups:

[0258] A) The amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44;

[0259] B) Having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44;

[0260] C) Differing from the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid;

[0261] Or,

[0262] D) An amino acid sequence which includes substitution, deletion and / or insertion of one or more amino acid residues as compared with the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44.

[0263] Preferably, the humanized TGFBR2 protein comprises part of the human TGFBR2 protein and part of the non-human animal TGFBR2 protein.

[0264] In a specific embodiment of the present invention, the humanized TGFBR2 protein comprises the extracellular region and signal peptide of the human TGFBR2 protein and all or part of the transmembrane region and cytoplasmic region of the non-human animal TGFBR2 protein.

[0265] In another embodiment of the present invention, the humanized TGFBR2 protein comprises all or part of the extracellular region of the human TGFBR2 protein, and all of the signal peptide, all of the cytoplasmic region, all or part of the transmembrane region, and all or part of the extracellular region of the non-human animal TGFBR2 protein.

[0266] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0267] A) All or part of SEQ ID NO: 53;

[0268] B) Having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 53;

[0269] C) Differing from the amino acid sequence of SEQ ID NO: 53 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0270] D) An amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 53.

[0271] In a specific embodiment of the present invention, the amino acid sequence of the humanized TGFBR2 protein comprises one of the following groups:

[0272] a) All or part of the amino acid sequence of SEQ ID NO: 13;

[0273] b) Having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 13;

[0274] c) Differing from the amino acid sequence shown in SEQ ID NO: 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0275] d) An amino acid sequence including substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 13.

[0276] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare gene humanization.

[0277] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0278] Preferably, the non-human animal is an immunodeficient non-human mammal. More 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 a rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mouse, NOD / SCID mouse or nude mouse.

[0279] In the eighth aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned humanized TGFBR2 protein.

[0280] In the ninth aspect of the present invention, there is provided a humanized TGFBR2 gene, and the humanized TGFBR2 gene contains all or part of the human TGFBR2 gene.

[0281] Preferably, the humanized TGFBR2 gene encodes the above-mentioned humanized TGFBR2 protein.

[0282] Preferably, the humanized TGFBR2 gene contains all or part of exons 1 to 8 of the human TGFBR2 gene. More preferably, it contains one, two or a combination of three or more of exons 1 to 8 of the human TGFBR2 gene. Even more preferably, it contains all or part of exons 1 to 5 of the human TGFBR2 gene.

[0283] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises a part of exon 1, all of exons 2 to 4, and a part of exon 5 of the human TGFBR2 gene. Among them, the part of exon 1 contains at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 476 bp of nucleotide sequence, and the part of exon 5 contains at least 5 bp of nucleotide sequence, for example, at least 5, 10, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. Preferably, it also contains introns 1-2 and / or introns 4-5 of the human TGFBR2 gene.

[0284] Preferably, the humanized TGFBR2 gene further comprises a part of the non-human animal TGFBR2 gene, and more preferably comprises a part of exon 1, a part of exon 5, and all of exons 6 to 8 of the non-human animal TGFBR2 gene.

[0285] Preferably, the humanized TGFBR2 gene further comprises a part of the non-human animal TGFBR2 gene, preferably comprises a part of exon 1, a part of exon 5, and all of exons 6 to 8 of the non-human animal TGFBR2 gene. More preferably, it also comprises introns 5-6 of the non-human animal TGFBR2.

[0286] Preferably, the humanized TGFBR2 gene further comprises a part of the non-human animal TGFBR2 gene, preferably comprises all or part of exon 1, all or part of exon 2, a part of exon 4, all of exon 5, all of exon 6, and all of exon 7 of the non-human animal TGFBR2 gene.

[0287] Preferably, the humanized TGFBR2 gene comprises all or part of exons 1 to 7 of the human TGFBR2 gene. More preferably, it comprises a combination of one, two or more than two exons among exons 1 to 7 of the human TGFBR2 gene.

[0288] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises a part of exon 1, the whole of exon 2, the whole of exon 3, and a part of exon 4 of the human TGFBR2 gene. Among them, the part of exon 1 contains at least 50 bp of nucleotide sequence, for example, at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 100, 150, 200, 300, 350, 377 bp of nucleotide sequence. The part of exon 1 contains the nucleotide sequence of the coding region. The part of exon 4 contains at least 20 bp of nucleotide sequence, for example, at least 20, 30, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 500, 600, 700, 800 bp of nucleotide sequence. The part of exon 4 contains the nucleotide sequence encoding the extracellular region.

[0289] In a specific embodiment of the present invention, it comprises all or part of exons 1 to 7 of the non-human animal TGFBR2 gene.

[0290] Preferably, the humanized TGFBR2 gene comprises the nucleotide sequence encoding the human TGFBR2 protein. More preferably, it comprises all or part of the extracellular region, transmembrane region, cytoplasmic region, and / or signal peptide of the human TGFBR2 protein.

[0291] Preferably, the humanized TGFBR2 gene comprises the nucleotide sequence encoding all or part of the extracellular region of the human TGFBR2 protein. More preferably, it comprises all or part of the signal peptide of the human TGFBR2 protein.

[0292] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises the entire nucleotide sequence encoding the extracellular region and signal peptide of the human TGFBR2 protein.

[0293] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises all or part of the extracellular region of the human TGFBR2.

[0294] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises the nucleotide sequence encoding at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of the human TGFBR2 protein.

[0295] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises a nucleotide sequence encoding at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144 consecutive amino acids of the extracellular region of the human TGFBR2 protein and a nucleotide sequence encoding at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22 consecutive amino acids of the signal peptide of human TGFBR2.

[0296] Preferably, the humanized TGFBR2 gene comprises a nucleotide sequence encoding a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with the nucleotide sequence of positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, or a nucleotide sequence encoding the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44; or, it comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, it comprises a nucleotide sequence having the nucleotide sequence encoding positions 1-166 of SEQ ID NO: 2 or positions 29-182 of SEQ ID NO: 44, including a nucleotide sequence with substitution, deletion and / or insertion of one or more nucleotides.

[0297] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises a nucleotide sequence encoding at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144 consecutive amino acids of the extracellular region of the human TGFBR2 protein, a nucleotide sequence encoding at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22 consecutive amino acids of the signal peptide of human TGFBR2, and a nucleotide sequence of the entire cytoplasmic region and a partial transmembrane region of the non-human animal TGFBR2 protein.

[0298] Preferably, the humanized TGFBR2 gene comprises a nucleotide sequence encoding a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequence of positions 167 - 567 of SEQ ID NO: 1, or a nucleotide sequence encoding the amino acid sequence shown in positions 167 - 567 of SEQ ID NO: 1; alternatively, it comprises a nucleotide sequence that differs from the nucleotide sequence encoding positions 167 - 567 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or it comprises a nucleotide sequence having the nucleotide sequence encoding positions 167 - 567 of SEQ ID NO: 1, including a nucleotide sequence with substitution, deletion and / or insertion of one or more nucleotides.

[0299] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises a nucleotide sequence encoding at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 138, 140, 144, 150, 151, 152, 153, 154, 160, 161 consecutive amino acids of the extracellular region of human TGFBR2, as well as the nucleotide sequences of the extracellular region of non - human animals, the entire transmembrane region, the cytoplasmic region and the signal peptide.

[0300] Preferably, the humanized TGFBR2 gene comprises a nucleotide sequence encoding a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequences of positions 1 - 28 and 183 - 592 of SEQ ID NO: 43, or a nucleotide sequence encoding the amino acid sequences shown in positions 1 - 28 and 183 - 592 of SEQ ID NO: 43; alternatively, it comprises a nucleotide sequence that differs from the nucleotide sequences encoding positions 1 - 28 and 183 - 592 of SEQ ID NO: 43 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or it comprises a nucleotide sequence having the nucleotide sequences encoding positions 1 - 28 and 183 - 592 of SEQ ID NO: 43, including a nucleotide sequence with substitution, deletion and / or insertion of one or more nucleotides.

[0301] In some specific embodiments, the humanized TGFBR2 gene further comprises a nucleotide sequence encoding P2A; preferably, the nucleotide sequence encoding P2A is located upstream of the human TGFBR2 gene portion; preferably, the nucleotide sequence encoding P2A is as shown in SEQ ID NO: 4.

[0302] In some specific embodiments, the humanized TGFBR2 gene comprises the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47 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 the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 47, including substitution, deletion and / or insertion of one or more nucleotides.

[0303] In some specific embodiments, the humanized TGFBR2 gene comprises the nucleotide sequence shown in SEQ ID NO: 31; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 31; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 31 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 the nucleotide sequence shown in SEQ ID NO: 31, including substitution, deletion and / or insertion of one or more nucleotides.

[0304] In one specific embodiment, the humanized TGFBR2 gene sequentially comprises, from the 5'-end to the 3'-end, a part of exon 1 of the non-human animal TGFBR2 gene, a part of exon 1 of the human TGFBR2 gene, all of exons 2 to 4, and a part of exon 5 of the human TGFBR2 gene, a part of exon 5 of the non-human animal TGFBR2 gene, and all of exons 6 to 8. Preferably, it also comprises introns 1-2 and 4-5 of human TGFBR2, and introns 5-6 of non-human animal TGFBR2.

[0305] In one specific embodiment, the humanized TGFBR2 gene sequentially comprises, from the 5'-end to the 3'-end, a part of exon 1 of the non-human animal TGFBR2 gene, all or part of exon 1, all of exon 2, all of exon 3 and a part of exon 4 of the human TGFBR2 gene, and a part of exon 4, all of exon 5, all of exon 6 and all of exon 7 of the non-human animal TGFBR2 gene.

[0306] In a specific embodiment, the humanized TGFBR2 gene sequentially includes a part of exons 1-2 of the non-human animal TGFBR2 gene, all or part of exon 1 of the human TGFBR2 gene, all or part of exon 2, all of exon 3, and all or part of exon 4, as well as all or part of exon 4 of the non-human animal TGFBR2 gene, all of exon 5, all of exon 6, and all or part of exon 7.

[0307] Preferably, the humanized TGFBR2 gene is regulated by regulatory elements in the non-human animal. More preferably, the regulatory elements are endogenous or exogenous.

[0308] Preferably, the regulatory elements include an endogenous promoter.

[0309] Preferably, the replacement of the endogenous TGFBR2 gene by the human or humanized TGFBR2 gene in the humanized TGFBR2 gene non-human animal is homozygous or heterozygous.

[0310] Preferably, the replacement of the TGFBR2 gene of the non-human animal at the endogenous TGFBR2 gene locus is homozygous or heterozygous.

[0311] Preferably, the humanized TGFBR2 gene is included on at least one chromosome in the genome of the non-human animal.

[0312] Preferably, at least one cell in the non-human animal expresses the human or humanized TGFBR2 protein.

[0313] Preferably, the humanized TGFBR2 gene includes the 5'UTR and / or 3'UTR of the non-human animal TGFBR2 gene.

[0314] Preferably, the nucleotide sequence of the humanized TGFBR2 gene includes a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 8 and / or SEQ ID NO: 9, or includes the nucleotide sequence shown in SEQ ID NO: 8 and / or SEQ ID NO: 9.

[0315] Preferably, the nucleotide sequence of the humanized TGFBR2 gene includes a nucleotide sequence having at least 60%, 70%, 80%, 90% or at least 95% identity with SEQ ID NO: 39 and / or SEQ ID NO: 40, or includes the nucleotide sequence shown in SEQ ID NO: 39 and / or SEQ ID NO: 40.

[0316] In a specific embodiment of the present invention, the humanized TGFBR2 gene comprises the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 36, and SEQ ID NO: 54; or, comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 36, and SEQ ID NO: 54; or, comprises a nucleotide sequence that differs from the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 36, and SEQ ID NO: 54 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 the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 36, and SEQ ID NO: 54, including substitution, deletion and / or insertion of one or more nucleotides.

[0317] In a specific embodiment of the present invention, the mRNA transcribed from the nucleotide sequence of the humanized TGFBR2 gene comprises one of the following groups:

[0318] (a) All or part of the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 42, or SEQ ID NO: 52;

[0319] (b) Having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 42, or SEQ ID NO: 52;

[0320] (c) Differing from the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 42, or SEQ ID NO: 52 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or,

[0321] (d) Having the nucleotide sequences shown in SEQ ID NO: 12, SEQ ID NO: 42, or SEQ ID NO: 52, including substitution, deletion and / or insertion of one or more nucleotides.

[0322] Preferably, the humanized TGFBR2 gene further comprises a specific inducer or repressor. More preferably, the specific inducer or repressor can be a substance that can be induced or repressed conventionally. 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).

[0323] Preferably, the non-human animal can be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc. that can be genetically edited to prepare gene humanization.

[0324] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0325] Preferably, the non-human animal is an immunodeficient non-human mammal. More 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 a rat. Most preferably, the immunodeficient mouse is NOD-Prkdc scid IL-2rγ null mice, NOD-Rag 1 - / - -IL2rg - / - (NRG) mice, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice or nude mice.

[0326] In the tenth aspect of the present invention, a gene-humanized cell is provided, and the cell expresses the above-mentioned human or humanized TGFBR2 protein.

[0327] Preferably, the cell contains the above-mentioned humanized TGFBR2 gene.

[0328] Preferably, the cell further comprises other gene modifications, and the other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3, TIGIT.

[0329] In the eleventh aspect of the present invention, a method for constructing a non-human animal with TGFBR2 gene knockout is provided. The construction method includes using the targeting vector and / or the sgRNA to construct a non-human animal. Among them, the sgRNA targets the TGFBR2 gene of the non-human animal, and at the same time, the sequence of the sgRNA is unique to the target sequence on the TNFSF9 gene to be modified. The sgRNA targets the TGFBR2 gene of the non-human animal, and the target sequence of the sgRNA on the TGFBR2 gene is as shown in any one of SEQ ID NO: 20-21 or 41.

[0330] In the twelfth aspect of the present invention, a non-human animal with TGFBR2 gene knockout is provided. The non-human animal lacks all or part of the TGFBR2 gene.

[0331] Preferably, it lacks all or part of exon 1 to exon 8 of the TGFBR2 gene. More preferably, it lacks one, two or more combinations of exons 1 to 8 of the TGFBR2 gene. Preferably, it lacks all or part of exon 2 or all or part of exons 1 to 5.

[0332] In some specific embodiments, the non-human animal with TGFBR2 gene knockout is obtained by the construction method described above.

[0333] In the thirteenth aspect of the present invention, a method for constructing a multi-gene modified non-human animal is provided, including the following steps:

[0334] I) Provide the above-mentioned non-human animal, or a non-human animal obtained by the above construction method;

[0335] II) Mate the non-human animal provided in step I) with other gene-modified non-human animals, perform in vitro fertilization or directly perform gene editing, and screen to obtain a multi-gene modified non-human animal.

[0336] Preferably, the other gene-modified non-human animals include non-human animals with humanized genes of PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3 and / or TIGIT.

[0337] Preferably, the multi-gene modified non-human animal is a dual-gene humanized non-human animal, a triple-gene humanized non-human animal, a quadruple-gene humanized non-human animal, a quintuple-gene humanized non-human animal, a sextuple-gene humanized non-human animal, a septuple-gene humanized non-human animal, an octuple-gene humanized non-human animal or a nonuple-gene humanized non-human animal.

[0338] Preferably, each of the multiple humanized genes in the genome of the multi-gene modified non-human animal can be homozygous or heterozygous for replacement of the endogenous locus.

[0339] In a fourteenth aspect of the present invention, there is provided a non-human animal with humanized TGFBR2 gene, a non-human animal with knocked-out TGFBR2 gene, or a multi-gene modified non-human animal or its offspring obtained by the above construction method.

[0340] In a fifteenth aspect of the present invention, there is provided an animal model, which is derived from the above non-human animal, the non-human animal obtained by the above construction method, or the above non-human animal or its offspring. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0341] In a sixteenth aspect of the present invention, there is provided a method for preparing a tumor-bearing or inflammatory animal model of an animal, the preparation method including the step of constructing the above non-human animal with humanized TGFBR2 gene, the non-human animal with knocked-out TGFBR2 gene, the above animal model or the multi-gene modified non-human animal or its offspring. Preferably, it further includes the step of implanting tumor cells.

[0342] In a seventeenth aspect of the present invention, there is provided the use of the above non-human animal with humanized TGFBR2 gene, the non-human animal with knocked-out TGFBR2 gene, the multi-gene modified non-human animal or its offspring, the above animal model, or the non-human animal with humanized TGFBR2 gene, the non-human animal with knocked-out TGFBR2 gene, the multi-gene modified non-human animal or its offspring obtained by the above construction method in the preparation of an animal model.

[0343] In an eighteenth aspect of the present invention, there is provided the above-mentioned cell, tissue or organ, wherein the cell, tissue or organ expresses the humanized TGFBR2 protein, or the genome of the cell, tissue or organ contains the above-mentioned human or humanized TGFBR2 gene, or the cell, tissue or organ is derived from the above-mentioned humanized non-human animal, or from the non-human animal obtained by the above construction method, or from the above animal model.

[0344] The cell, tissue or organ cannot develop into an individual animal.

[0345] In a nineteenth aspect of the present invention, there is provided a tumor tissue after tumor-bearing, which is derived from the above non-human animal or its offspring, the non-human animal obtained by the above construction method, or the above animal model.

[0346] In a twentieth aspect of the present invention, there is provided a genome of a non-human animal with humanized TGFBR2 gene.

[0347] Preferably, the genome contains all or part of the human or humanized TGFBR2 gene, and / or contains all or part of the nucleotide sequence encoding the human or humanized TGFBR2 protein.

[0348] Preferably, the humanized TGFBR2 gene is the above-mentioned humanized TGFBR2 gene.

[0349] Preferably, the humanized TGFBR2 protein is the above-mentioned humanized TGFBR2 protein.

[0350] Preferably, the genome contains, at the endogenous TGFBR2 locus of a non-human animal, a genomic fragment of the human TGFBR2 gene (preferably all or part of the sequence encoding the signal peptide and / or extracellular region of human TGFBR2, or all or part of the sequence encoding the extracellular region of human TGFBR2), and / or a genomic fragment of the TGFBR2 gene of the non-human animal (preferably all or part of the sequence encoding the transmembrane region and / or cytoplasmic region of the non-human animal TGFBR2), and the genomic fragment of the non-human animal TGFBR2 gene is introduced to form a modified TGFBR2 gene.

[0351] Preferably, the genome contains, at the endogenous TGFBR2 locus of a non-human animal, a humanized TGFBR2 gene introduced into the genomic fragment of the non-human animal TGFBR2 gene to form a modified TGFBR2 gene.

[0352] The modified TGFBR2 gene encodes a humanized TGFBR2 protein.

[0353] Preferably, the introduction is insertion or replacement.

[0354] Preferably, the insertion is at the start codon of exon 1 of the non-human animal TGFBR2 gene.

[0355] Preferably, the replacement is at exon 2 of the non-human animal TGFBR2 gene, or at part of exon 1, all of exons 2 to 3, and part of exon 5 of the non-human animal TGFBR2 gene.

[0356] Preferably, the expression of the modified TGFBR2 gene is controlled by the endogenous regulatory elements of the non-human animal.

[0357] Preferably, the non-human animal can be selected from any non-human animal such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc. that can be used for gene editing to prepare gene humanization.

[0358] Preferably, the non-human animal is a non-human mammal. More preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0359] In the twenty-first aspect of the present invention, there is provided a cell, tissue or organ comprising the genome of the above-mentioned humanized non-human animal of the TGFBR2 gene.

[0360] Preferably, any of the above-mentioned cells, tissues or organs or tumor tissues after tumor inoculation include cells, tissues or organs or tumor tissues after tumor inoculation that can develop into an animal individual or cannot develop into an animal individual.

[0361] In the twenty-second aspect of the present invention, there is provided the use of the above-mentioned humanized TGFBR2 protein, the above-mentioned nucleic acid, the above-mentioned humanized TGFBR2 gene, the above-mentioned non-human animal or its offspring, the above-mentioned cell of humanized TGFBR2 gene, the above-mentioned non-human animal obtained by the above-mentioned construction method, the above-mentioned animal model, the above-mentioned cell, tissue or organ, the above-mentioned tumor tissue after tumor inoculation, and the use comprises:

[0362] A) Use in the development of products related to the immune process associated with TGFBR2 in human cells;

[0363] B) Use as a model system related to TGFBR2 in pharmacological, immunological, microbiological and medical research;

[0364] C) Use related to the production and utilization of animal experimental disease models for etiological research related to TGFBR2 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0365] D) Use in the screening, efficacy detection, evaluation of efficacy, verification or evaluation of human TGFBR2 signaling pathway regulators in vivo; or,

[0366] E) Use in studying the function of the TGFBR2 gene, studying the drugs and drug effects targeting human TGFBR2 target sites, studying drugs for immune-related diseases related to TGFBR2 and anti-tumor drugs.

[0367] Preferably, the use includes methods for the treatment and / or diagnosis of diseases, or, methods for the treatment and / or diagnosis of non-diseases.

[0368] In the twenty-third aspect of the present invention, there is provided a method for screening a human TGFBR2-specific regulator, the screening method comprising applying a regulator to an individual implanted with tumor cells and detecting tumor inhibition; wherein, the individual is selected from the above-mentioned non-human animal or its offspring, the above-mentioned non-human animal obtained by the above-mentioned construction method, or the above-mentioned tumor-bearing or inflammation model.

[0369] Preferably, the regulator is selected from CAR-T and drugs. More preferably, the drug is an antibody.

[0370] Preferably, the regulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0371] Preferably, the detection includes measuring the size and / or proliferation rate of tumor cells.

[0372] Preferably, the detection methods include measurement with a vernier caliper, flow cytometry, and / or in vivo imaging of animals.

[0373] Preferably, the detection includes evaluating the body weight, fat mass, activation pathway, neuroprotective activity, or metabolic changes of an individual, and the metabolic changes include changes in food consumption or water consumption.

[0374] Preferably, the tumor cells are derived from humans or non-human animals.

[0375] Preferably, the screening method for the human TGFBR2-specific regulator includes therapeutic and non-therapeutic methods.

[0376] In a specific embodiment, this 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, or compare the sensitivity of the efficacy of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0377] In the twenty-fourth aspect of the present invention, a method for evaluating an intervention plan is provided. The evaluation method includes implanting tumor cells into an individual, applying an intervention plan to the individual implanted with tumor cells, and detecting and evaluating the tumor suppression effect of the individual after applying the intervention plan; wherein, the individual is selected from the above-mentioned non-human animals, non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animals or their offspring, or the above-mentioned tumor-bearing or inflammation models.

[0378] Preferably, the intervention plan is selected from CAR-T and drug treatment. More preferably, the drug is an antigen-binding protein. The antibody-binding protein is an antibody.

[0379] Preferably, the tumor cells are derived from humans or non-human animals.

[0380] Preferably, the evaluation method of the intervention plan includes therapeutic and non-therapeutic methods.

[0381] In a specific embodiment, this evaluation method detects and evaluates the effect of the intervention plan to determine whether the intervention plan has a therapeutic effect, that is, the therapeutic effect is not inevitable, but only a possibility.

[0382] In the twenty-fifth aspect of the present invention, there is provided the use of the non-human animal or its offspring as described above, the non-human animal obtained by the construction method as described above, or the tumor-bearing or inflammation model as described above in the preparation of a human TGFBR2-specific regulator.

[0383] In the twenty-sixth aspect of the present invention, there is provided the use of the non-human animal or its offspring as described above, the non-human animal obtained by the construction method as described above, or the tumor-bearing or inflammation model as described above in the preparation of a drug for treating tumors, inflammation or immune-related diseases.

[0384] The "immune-related diseases" as described in the present invention include, but are not limited to, allergy, 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 nerve disorders, etc.

[0385] The "inflammation" as described in the present invention includes acute inflammation and also chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotic inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

[0386] The "tumors" as described in the present invention include, but are not limited to, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, 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. Among them, the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the 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 Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0387] The non-human animal with humanized TGFBR2 gene according to the present invention can normally express human or humanized TGFBR2 protein in vivo, and can be used for drug screening, efficacy evaluation, treatment of immune diseases and tumors targeting human TGFBR2 target sites, which can accelerate the new drug R & D process, save time and cost. It provides an effective guarantee for studying the function of TGFBR2 protein and drug screening for related diseases.

[0388] The "whole or part" described in the present invention, "whole" refers to the whole, "part" refers to the local part in the whole, or the individual constituting the whole.

[0389] The "humanized TGFBR2 protein" described in the present invention comprises a part derived from human TGFBR2 protein and a part of non-human TGFBR2 protein. Among them, the "human TGFBR2 protein" is the same as the whole of human TGFBR2 protein, that is, its amino acid sequence is identical to the full-length amino acid sequence of human TGFBR2 protein. The "part of human TGFBR2 protein" is a continuous or spaced 5-592 amino acid sequence identical to the amino acid sequence of human TGFBR2 protein. Preferably, it is continuous or spaced 10-567, 10-166, 10-154, specifically 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 166, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 567, 592 amino acid sequences identical to the amino acid sequence of human TGFBR2 protein.

[0390] The "whole transmembrane region of human TGFBR2 protein", "whole cytoplasmic region of human TGFBR2 protein" or "whole extracellular region of human TGFBR2 protein" described in the present invention represents that their amino acid sequences are respectively identical to the full-length amino acid sequences of the transmembrane region, cytoplasmic region or extracellular region of human TGFBR2 protein.

[0391] The "part of the extracellular region of human TGFBR2 protein" described in the present invention is a continuous or spaced 5-144 amino acid sequence identical to the amino acid sequence of the extracellular region of human TGFBR2 protein, preferably 10-139, specifically 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 144 continuous amino acid sequences identical to the amino acid sequence of the extracellular region of human TGFBR2 protein.

[0392] The "portion of the non-human animal TGFBR2 protein" described in the present invention is a continuous or spaced 5 - 567 or 5 - 592 amino acid sequence that is identical to the amino acid sequence of the non-human animal TGFBR2 protein. Specifically, it is a continuous 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 450, 500, 550, 567, 592 amino acid sequence that is identical to the amino acid sequence of the non-human animal TGFBR2 protein.

[0393] The "humanized TGFBR2 gene" described in the present invention contains a portion derived from the human TGFBR2 gene and a portion of the non-human TGFBR2 gene. Among them, the "human TGFBR2 gene" is the entirety of the human TGFBR2 gene, that is, its nucleotide sequence is identical to the full-length nucleotide sequence of the human TGFBR2 gene.

[0394] In a specific embodiment, the "portion of the human TGFBR2 gene" is a continuous or spaced 20 - 87542 bp nucleotide sequence that is identical to the nucleotide sequence of the human TGFBR2 gene. Preferably, it is a 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 4000, 6000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 70000, 80000, 850000, 87542 bp nucleotide sequence that is identical to the nucleotide sequence of the human TGFBR2 gene.

[0395] In some specific embodiments, the "portion of the human TGFBR2 gene" is a continuous or spaced 20 - 498 bp nucleotide sequence that is identical to the nucleotide sequence of the human TGFBR2 gene. Preferably, it is a 20, 50, 100, 200, 250, 300, 350, 400, 450, 498 bp nucleotide sequence that is identical to the nucleotide sequence of the human TGFBR2 gene.

[0396] In a specific embodiment, the "portion of the human TGFBR2 gene" is a nucleotide sequence of 20 - 4842 bp, either continuous or spaced, that is identical to the nucleotide sequence of the human TGFBR2 gene. Preferably, it is a nucleotide sequence of 20, 50, 100, 200, 300, 400, 500, 600, 690, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3955, 4000 or 4842 bp that is identical to the nucleotide sequence of the human TGFBR2 gene.

[0397] The "portion of an exon" as described in the present invention means that several, dozens or hundreds of nucleotide sequences, either continuous or spaced, are identical to the entire exon nucleotide sequence. For example, the portion of exon 1 of the human TGFBR2 gene contains 5 - 476 or 5 - 377 bp, preferably 5 - 10 nucleotide sequences, either continuous or spaced, that are identical to the nucleotide sequence of exon 1 of the human TGFBR2 gene.

[0398] The "exon No. xx to xxx" or "the entire exon No. xx to xxx" as described in the present invention includes the nucleotide sequences of the exon and the introns in between. For example, "exon No. 2 to 4" includes the entire nucleotide sequences of exon 2, intron 2 - 3, exon 3, intron 3 - 4 and exon 4.

[0399] The "intron No. x - xx" as described in the present invention refers to the intron between exon x and exon xx. For example, "intron No. 1 - 2" refers to the intron between exon 1 and exon 2.

[0400] The "portion of the non-human animal TGFBR2 gene" described in the present invention is a nucleotide sequence that is continuous or spaced by 20 - 90,973 bp and is identical to the nucleotide sequence of the non-human animal TGFBR2 gene. Specifically, it is a nucleotide sequence of 20, 50, 100, 200, 300, 400, 500, 599, 690, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000, 300000, 40000, 50000, 60000, 70000, 80000, 85000, 90000, 90,973 nucleotides that is identical to the nucleotide sequence of the non-human animal TGFBR2 gene.

[0401] The "locus" described in the present invention generally represents the position occupied by a gene on a chromosome, and specifically represents a DNA fragment on a certain gene, that is, it can be a gene or a part of a gene. For example, the "TGFBR2 locus" refers to an arbitrary DNA fragment on exons 1 to 8 of the TGFBR2 gene. In a specific embodiment of the present invention, the TGFBR2 locus to be replaced can be an arbitrary DNA fragment on exons 1 to 8 of the TGFBR2 gene.

[0402] The "nucleotide sequence" described in the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Preferably, it is DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

[0403] The "more than three" described in the present invention includes more than three consecutive or spaced ones, specifically including but not limited to three, four, five, six, seven or more than eight, etc.

[0404] The "treatment" described in the present invention means slowing down, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, disease, or illness, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, illnesses, or disorders, and refers to a therapeutic intervention that improves the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0405] As used herein, "homology" refers to the aspect of using amino acid sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs on the premise of ensuring similar structures or functions to known sequences. Compared with the sequences obtained by the prior art, the sequences used 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.

[0406] Those skilled in the art can determine and compare sequence elements or degrees of identity to distinguish additional murine and human sequences.

[0407] 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 references. 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 (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. 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. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0408] In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as Dipodidae. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the Muridae family. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (such as mouse-like hamsters), Cricetidae (such as hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (mountain mice, rock mice, tailed rats, Madagascar rats and mice), Platacanthomyidae (such as the spiny dormouse), and Spalacidae (such as mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice, and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0409] In a particular embodiment, the non-human animal is a rodent that is a mouse of the C57BL, C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, CBA / H strains selected from BALB / c, A, A / He, A / J, A / WySN, AKR, AKR / A, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola, and NOD, NOD / SCID, NOD-Prkdc scidIL-2rg null Background mice.

[0410] The above only summarizes some aspects of the present invention and should not be considered, nor should it be construed as, a limitation of the present invention in any way.

[0411] All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the spirit or scope of the present invention.

[0412] The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0413] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0414] Figure 1 : Schematic diagram of the comparison between the mouse TGFBR2 locus and the human TGFBR2 locus (not to scale);

[0415] Figure 2 : Schematic diagram of the humanization of the mouse TGFBR2 gene (not to scale);

[0416] Figure 3 : Schematic diagram of the TGFBR2 gene targeting strategy and the design of the targeting vector (not to scale);

[0417] Figure 4 : Schematic diagram of the FRT recombination process of the TGFBR2 gene humanized mouse (not to scale);

[0418] Figure 5 : Schematic diagram of the TGFBR2 gene targeting strategy and the design of the targeting vector (not to scale);

[0419] Figure 6 : Schematic diagram of the humanization of the mouse TGFBR2 gene (not to scale);

[0420] Figure 7 : Schematic diagram of the TGFBR2 gene targeting strategy and the design of the targeting vector (not to scale);

[0421] Figure 8 : Schematic diagram of the comparison between the mouse TGFBR2 locus and the human TGFBR2 locus (not to scale);

[0422] Figure 9 : Schematic diagram of the humanization of the mouse TGFBR2 gene (not to scale);

[0423] Figure 10: Schematic diagram of the TGFBR2 gene targeting strategy and targeting vector design (not to scale);

[0424] Figure 11 : PCR identification results of the tails of F1 generation mice with humanized TGFBR2 gene. Among them, WT is wild type, H2O is water control, and M is Marker;

[0425] Figure 12 : RT-PCR identification of mRNA expression in mice homozygous for humanized TGFBR2 gene. Among them, + / + is wild-type C57BL / 6 mice, H / H is mice homozygous for humanized TGFBR2 gene, and H2O is water control;

[0426] Figure 13 : Flow cytometry detection results of the proportion of leukocyte subsets (13A) and T cell subsets in the spleen (13B). Among them, + / + is wild-type C57BL / 6 mice, and H / H is mice homozygous for humanized TGFBR2 gene;

[0427] Figure 14 : Flow cytometry detection results of the proportion of leukocyte subsets (14A) and T cell subsets in the lymph nodes (14B). Among them, + / + is wild-type C57BL / 6 mice, and H / H is mice homozygous for humanized TGFBR2 gene;

[0428] Figure 15 : Flow cytometry detection results of the proportion of leukocyte subsets (15A) and T cell subsets in the blood (15B). Among them, + / + is wild-type C57BL / 6 mice, and H / H is mice homozygous for humanized TGFBR2 gene. Detailed implementation mode

[0429] 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 progresses. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0430] In each of the following embodiments, the equipment and materials are obtained from the following several companies:

[0431] StuI enzyme and NcoI enzyme are purchased from NEB, and the product numbers are R0187S and R3193S respectively;

[0432] C57BL / 6 mice and Flp tool mice are purchased from the National Rodent Laboratory Animal Seed Center of the National Institutes for Food and Drug Control;

[0433] Brilliant Violet 510 TM anti-mouse CD45 Antibody was purchased from Biolegend, catalog number 103138;

[0434] Brilliant Violet 605 TM anti-mouse CD19 Antibody was purchased from Biolegend, catalog number 115540;

[0435] Purified anti-mouse CD16 / 32 Antibody was purchased from Biolegend, catalog number 101302;

[0436] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number 423106;

[0437] Brilliant Violet 711 TM anti-mouse TCRβ chain Antibody was purchased from Biolegend, catalog number 109243;

[0438] Mouse TGF-beta RII PE-conjugated Antibody was purchased from R&D, catalog number FAB532P;

[0439] PE anti-human TGF-β Receptor II Antibody was purchased from Biolegend, catalog number 399703;

[0440] FITC anti-Mouse CD19 Antibody was purchased from Biolegend, catalog number 115506;

[0441] PerCP / Cyanine5.5 anti-mouse TCRβ chain Antibody was purchased from Biolegend, catalog number: 109228;

[0442] APC anti-human TGF-β Receptor II Antibody was purchased from Biolegend, catalog number: 399705;

[0443] Goat IgG PE-conjugated Antibody was purchased from R&D, catalog number: IC108P

[0444] The APC Rat IgG2b,κ Isotype Ctrl Antibody was purchased from Biolegend, catalog number: 400612;

[0445] Zombie NIR TM The Fixable Viability Kit was purchased from Biolegend, catalog number: 423106;

[0446] The PerCP anti-mouse Ly-6G / Ly-6C(Gr-1) Antibody was purchased from Biolegend, catalog number: 108426;

[0447] Brilliant Violet 421 TM The anti-mouse CD4 Antibody was purchased from Biolegend, catalog number: 100438;

[0448] The FITC anti-mouse F4 / 80 Antibody was purchased from Biolegend, catalog number: 123108;

[0449] The PE anti-mouse CD8a Antibody was purchased from Biolegend, catalog number: 100708;

[0450] PE / Cy TM The Mouse anti-mouse NK1.1 was purchased from BD Pharmingen, catalog number: 552878;

[0451] The APC anti-mouse / rat Foxp3 Antibody was purchased from eBioscience, catalog number: 17-5773-82;

[0452] The APC Hamster Anti-Mouse TCRβ Chain was purchased from BD Pharmingen TM ,catalog number: 553174;

[0453] Brilliant Violet 605 TM The anti-mouse CD11c Antibody was purchased from Biolegend, catalog number: 117334;

[0454] The PE anti-mouse / human CD11b Antibody was purchased from Biolegend, catalog number: 101208.

[0455] Example 1: Humanized Mouse of TGFBR2 Gene (I)

[0456] Schematic comparison of mouse TGFBR2 gene (NCBI Gene ID: 21813, Primary source: MGI:98729, UniProt: Q3UG22, located at positions 115916763 to 116004431 of NC_000075.7 on chromosome 9, based on transcript NM_029575.3 and its encoded protein NP_083851.3 (SEQ ID NO: 1)) and human TGFBR2 gene (NCBI Gene ID: 7048, Primary source: HGNC:11773, UniProt ID: P37173-1, located at positions 30606472 to 30694142 of NC_000003.12 on chromosome 3, based on transcript NM_003242.6 and its encoded protein NP_003233.4 (SEQ ID NO: 2)) is as Figure 1 shown.

[0457] To achieve the object of the present invention, a nucleotide sequence encoding human TGFBR2 protein can be introduced into the mouse endogenous TGFBR2 locus, so that the mouse expresses human or humanized TGFBR2 protein. Specifically, using gene editing technology, under the control of the mouse TGFBR2 gene regulatory element, the human TGFBR2 genomic coding region is used to replace the mouse TGFBR2 genomic coding region, or a chimeric gene sequence containing the human TGFBR2 gene and the mouse TGRBR2 gene is inserted or replaced into the mouse endogenous TGFBR2 locus, and the schematic diagram of the humanized TGFBR2 locus is as Figure 2 shown, realizing the humanization transformation of the mouse TGFBR2 gene.

[0458] Design as Figure 3The targeting strategy shown in the figure shows that the targeting vector contains homologous arm sequences upstream and downstream of the mouse TGFBR2 gene, as well as an A1 fragment (SEQ ID NO: 3) containing a P2A sequence, human TGFBR2, mouse TGFBR2, and a STOP sequence. Among them, the P2A sequence is shown in SEQ ID NO: 4; the upstream homologous arm sequence (5' homologous arm, SEQ ID NO: 5) is identical to the nucleotide sequence from positions 115960696 to 115964481 of NCBI accession number NC_000075.7, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 6) is identical to the nucleotide sequence from positions 115956141 to 115960230 of NCBI accession number NC_000075.7. The nucleotide sequence of human TGFBR2 on the A1 fragment (SEQ ID NO: 7) is identical to the nucleotide sequence from positions 284 to 781 of NCBI accession number NM_003242.6; the nucleotide sequence of mouse TGFBR2 on the A1 fragment is SEQ ID NO: 31; the connection design of the upstream of the A1 fragment sequence to the mouse is:

[0459] Among them, the sequence in which the "T" is the last nucleotide of the mouse, and the first "G" in the sequence GGAA is the first nucleotide of the A1 fragment. The connection design of the downstream of the A1 fragment sequence to the mouse is:

[0460] Among them, the sequence in which the last "C" is the last nucleotide of the A1 fragment, and the first "A" in the sequence AGAA is the first nucleotide of the mouse sequence.

[0461] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. Among them, the connection design of the 5' end of the Neo cassette to the STOP sequence is: Among them, the sequence in which the last "A" is the last nucleotide of the STOP sequence, and the first "G" of the sequence GTCG is the first nucleotide of the Neo cassette; the connection design of the 3' end of the Neo cassette to the mouse gene is:

[0462] Among them, the sequence The last C in the sequence is the last nucleotide of the Neo cassette. AGAAT The first A in the sequence is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (the coding gene for diphtheria toxin subunit A (DTA)) was constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the engineered humanized mouse TGFBR2 is shown in SEQ ID NO: 12, and the expressed protein sequence is shown in SEQ ID NO: 13.

[0463] Given that human TGFBR2 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

[0464] The targeting vector can be constructed using conventional methods such as digestion and ligation. After preliminary verification of the constructed targeting vector by digestion, it is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into embryonic stem cells of C57BL / 6 mice by electroporation. The obtained cells are screened using a positive clone selection marker gene, and the integration of the foreign gene is detected and confirmed using PCR and Southern Blot techniques. The correct positive clone cells are screened out. Clones identified as positive by PCR are further subjected to Southern Blot detection. Clones that are positive in Southern detection and further verified as positive by sequencing and without random insertion are used for the next experiment.

[0465] Among them, the PCR assay includes the following primers:

[0466] WT-F: 5'-AGTTAACAGTGATGTCATGGCCAGCG-3' (SEQ ID NO: 14)

[0467] WT-R: 5'-GTGGATGCTAAGAGGTGAAACGA-3' (SEQ ID NO: 15);

[0468] The correctly screened positive clone cells (black mice) are introduced into the isolated blastocysts (white mice) according to techniques known in the art. The obtained chimeric blastocysts are transferred to a culture medium for short-term culture and then transplanted into the oviduct of a recipient female mouse (white mouse) to 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 then the F1 generation heterozygous mice are 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 selection marker gene (a schematic diagram of this process is shown in Figure 4) After that, homozygous TGFBR2 gene humanized mice can be obtained by crossbreeding. The genotypes of the somatic cells of the offspring mice can be identified by PCR (the primer sequences are shown in SEQ ID NO: 14-17). Using this method, TGFBR2 gene humanized mice (V1) that can be stably passed on and have no random insertion can be constructed.

[0469] Among them, the PCR primers are:

[0470] WT-F: 5’-AGTTAACAGTGATGTCATGGCCAGCG-3’ (SEQ ID NO: 14)

[0471] Mut-R: 5’-GGAGAAGCAGCATCTTCCAGAATAAAGT-3’ (SEQ ID NO: 16)

[0472] Mut-F: 5’-CACACCTCCCCCTGAACCTGAAAC-3’ (SEQ ID NO: 17)

[0473] WT-R: 5’-GTGGATGCTAAGAGGTGAAACGA-3’ (SEQ ID NO: 15)

[0474] In addition, the CRISPR / Cas system can also be introduced for gene editing. Design the targeting strategy as Figure 5 shown. The figure shows that the targeting vector contains homologous arm sequences upstream and downstream of the mouse TGFBR2 gene, and the A2 fragment (SEQ ID NO: 54) containing human TNFBR2. Among them, the upstream homologous arm sequence (5’ homologous arm, SEQ ID NO: 18) is the same as the nucleotide sequence at positions 115960696 to 115962174 of NCBI accession number NC_000075.7, and the downstream homologous arm sequence (3’ homologous arm, SEQ ID NO: 19) is the same as the nucleotide sequence at positions 115958894 to 115960321 of NCBI accession number NC_000075.7. The nucleotide sequence of human TGFBR2 is the same as the nucleotide sequence at positions 284 to 781 of NCBI accession number NM_003242.6 (SEQ ID NO: 7).

[0475] The construction of the targeting vector can be carried out by conventional methods, such as restriction enzyme ligation, direct synthesis, etc. After the constructed targeting vector is preliminarily verified by restriction enzyme digestion, it is then sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is used for subsequent experiments.

[0476] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing the sgRNA expression vector. The sgRNA sequences recognizing the target sites were designed and synthesized, and the target sequences of each sgRNA on the TGFBR2 gene are as follows:

[0477] Target site of sgRNA1 (SEQ ID NO: 20): 5’-ATGAAGTCTGCGTGGCCGTGTGG-3’;

[0478] Target site of sgRNA2 (SEQ ID NO: 21): 5’-TCATGTGTGCTCGTGGCAAGAGG-3’;

[0479] After detecting the activity of the sgRNA using the UCA kit and determining that it can mediate high cleavage efficiency, restriction enzyme sites were added to the 5’ end and its complementary strand to obtain the forward and reverse oligonucleotide sequences (see Table 1). After annealing, the annealed product was ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain the expression vectors pT7-TGFBR2-1 and pT7-TGFBR2-2.

[0480] Table 1 List of sgRNA1 and sgRNA2 sequences

[0481]

[0482] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment of DNA (SEQ ID NO: 28) with a T7 promoter and an sgRNA scaffold, and was successively ligated to the backbone vector (source: Takara, catalog number 3299) through restriction enzyme digestion (EcoRI and BamHI). It was verified by sequencing by a professional sequencing company, and the results showed that the target plasmid was obtained.

[0483] Take prophase fertilized eggs of mice, such as C57BL / 6 mice. Using a microinjector, the in vitro transcription products of the pT7-TGFBR2-1 and pT7-TGFBR2-2 plasmids (transcribed using the Ambion in vitro transcription kit according to the instructions), the targeting vector, and Cas9 mRNA were premixed and then injected into the cytoplasm or nucleus of the mouse fertilized eggs. The microinjection of the fertilized eggs was carried out according to the method in the "Mouse Embryo Manipulation Experiment Manual (Third Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient female mouse for development. The obtained mice (F0 generation) were hybridized and self-crossed to expand the population size and establish a stable humanized mouse strain of the TGFBR2 gene.

[0484] The genotype of somatic cells of F0 generation mice can be identified by conventional detection methods (such as PCR analysis). Combining the detection results of 5'-end primers and the PCR detection results of 3'-end primers, and further verified as positive mice by sequencing. The TGFBR2 gene humanized mice identified as positive in F0 were mated with wild-type mice to obtain F1 generation mice. The same PCR method (the primer sequences are shown in Table 2) can be used to identify the genotype of F1 generation mice. The identification results of exemplary F1 generation mice are shown in Figure 11 , among which, the mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08, F1-09, F1-10, F1-11, F1-12, F1-13 and F1-14 are positive heterozygous mice.

[0485] Table 2 Primer sequences for PCR detection of F1 generation genotype and the size of recombinant fragments

[0486]

[0487] Southern blot detection was performed on the mice identified as positive by F1 generation PCR to confirm whether there was random insertion. The mouse tail was cut to extract genomic DNA, and the genomic DNA was digested with StuI enzyme or NcoI enzyme, transferred to the membrane, and hybridized. The 5' probe and 3' probe were located on the 5' homologous arm and outside the 3' homologous arm respectively. The specific probes and the lengths of the target fragments are shown in Table 3. Genetically engineered mice with humanized TGFBR2 gene that can be stably passaged and have no random insertion were obtained.

[0488] Table 3 Specific probes and the lengths of the target fragments

[0489] Restriction endonuclease Probe Wild-type fragment size Recombinant sequence fragment size StuI 5’Probe 4.7kb 7.0kb NcoI 3’Probe — 4.3kb

[0490] The primers for probe synthesis are as follows:

[0491] 5’Probe-F (SEQ ID NO: 32): 5’-TTTAGAGACCCAGGAAGCAGGTGTT-3’,

[0492] 5’Probe-R (SEQ ID NO: 33): 5’-CAGTAAGACAACCCTGCCATGCACTC-3’;

[0493] 3’Probe-F (SEQ ID NO: 34): 5’-AACTGATGAATGGGAGCAGTGGTGG-3’,

[0494] 3’Probe-R (SEQ ID NO: 35): 5’-GCAGACACTCTATGCCTGTGTGGAG-3’;

[0495] Heterozygous mice identified as positive in the F1 generation were mated with each other to obtain F2 generation homozygous humanized TGFBR2 gene mice (V2).

[0496] The expression of humanized TGFBR2 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. Specifically, one 8-week-old male C57BL / 6 wild-type mouse and one 8-week-old male TGFBR2 gene heterozygous humanized mouse were taken. After cervical dislocation for euthanasia, spleen tissues were taken and stained with anti-mouse TGFBR2 antibody Mouse TGF-beta RII PE-conjugated Antibody (mTGFBR2), anti-human TGFBR2 antibody PE anti-human TGF-β Receptor II Antibody (hTGFBR2), anti-mouse TCRβ antibody Brilliant Violet 711 TM anti-mouse TCRβ (mTCRβ), mouse leukocyte recognition antibody Brilliant Violet 510 TM anti-mouse CD45 (mCD45), anti-mouse CD19 antibody Brilliant Violet 605 TM anti-mouse CD19 (mCD19), anti-mouse CD16 / 32 antibody Purified anti-mouse CD16 / 32, etc., and then flow cytometry was performed to detect the expression of human or humanized TGFBR2 protein.

[0497] The results showed that in the spleen of C57BL / 6 mice, 0.97% of B cells (characterized by mCD45 + mCD19 +) were hTGFBR2 positive cells (characterized by mCD45 + mCD19 + hTGFBR2 +), and 23.7% were mTGFBR2 positive cells (characterized by mCD45 + mCD19 + mTGFBR2 +). In the spleen of TGFBR2 heterozygous mice, 12.2% of B cells (characterized by mCD45 + mCD19 +) were hTGFBR2 positive cells (characterized by mCD45 + mCD19 + hTGFBR2 +), and 10.8% were mTGFBR2 positive cells (characterized by mCD45 + mCD19 + mTGFBR2 +).

[0498] In the spleens of C57BL / 6 mice, 0.60% of T cells (characterized by mCD45+mTCRβ+) were positive for hTGFBR2 (characterized by mCD45+mTCRβ+hTGFBR2+), and 23.1% were positive for mTGFBR2 (characterized by mCD45+mTCRβ+mTGFBR2+). In the spleens of TGFBR2 heterozygous mice, 15.8% of T cells (characterized by mCD45+mTCRβ+) were positive for hTGFBR2 (characterized by mCD45+mTCRβ+hTGFBR2+), and 11.0% were positive for mTGFBR2 (characterized by mCD45+mTCRβ+mTGFBR2+).

[0499] In summary, the expression of mouse TGFBR2 protein could be detected in C57BL / 6 and TGFBR2 humanized heterozygous mice, but the expression of humanized TGFBR2 protein could only be detected in TGFBR2 humanized heterozygous mice. This indicates that the humanized TGFBR2 protein can be normally expressed in TGFBR2 humanized heterozygous mice.

[0500] Example 2 TGFBR2 Humanized Mice (II)

[0501] The human-mouse chimeric sequence in Example 1 was also inserted after the 5'UTR and before the ATG, and TGFBR2 humanized mice were successfully obtained. The schematic diagram of the modified mouse locus is as Figure 6 shown, achieving the humanization of mouse TGFBR2.

[0502] A targeting strategy was designed as Figure 7 shown. The figure shows that the targeting vector contains homologous arm sequences upstream and downstream of the mouse TGFBR2 gene, as well as the human TGFBR2 sequence A3 fragment (SEQ ID NO: 36). Among them, the upstream homologous arm sequence (5' homologous arm, SEQ ID NO: 37) is identical to the nucleotide sequence from position 116004106 to 116006016 of NCBI accession number NC_000075.7, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 38) is at least 99% identical or the same as the nucleotide sequence from position 116003093 to 116004102 of NCBI accession number NC_000075.7. The nucleotide sequence of human TGFBR2 on the A3 fragment (SEQ ID NO: 7) is identical to the nucleotide sequence from position 284 to 781 of NCBI accession number NM_003242.6; the nucleotide sequence of mouse TGFBR2 on the A3 fragment is SEQ ID NO: 31; the connection design between the upstream of the A3 fragment sequence and the mouse is as follows:

[0503] Among them, the sequence CTGCC the last C in it is the last nucleotide of the mouse, and the sequence ATGGG the A in it is the first nucleotide of the A3 fragment. The connection downstream of the A3 fragment sequence to the mouse is designed as:

[0504] Among them, the sequence the last C in it is the last nucleotide of the A3 fragment, and the sequence GGTC the first G in it is the first nucleotide of the mouse sequence.

[0505] The construction of the targeting vector is carried out in a manner known in the art. The target sequences of the sgRNA on the TGFBR2 gene are as follows:

[0506] sgRNA target site (SEQ ID NO: 41): 5’-GAGTGGCCAGGGCGCGACCCTGG-3’;

[0507] After detecting the activity of the sgRNA using the UCA kit and determining that it can mediate high cleavage efficiency, restriction enzyme sites are added to the 5’ end and the complementary strand respectively to obtain the forward oligonucleotide and reverse oligonucleotide sequences. After annealing, the annealed product is ligated to the pT7-sgRNA plasmid (the plasmid is first linearized with BbsI) to obtain the expression vector pT7-TGFBR2. The microinjection method and the method for detecting the mouse genotype can refer to Example 1, and a genetically engineered mouse (V3) with stable inheritance and no random insertion of the humanized TGFBR2 gene can be obtained. The mRNA sequence of the humanized mouse TGFBR2 after modification is shown in SEQ ID NO: 42, and the expressed protein sequence is shown in SEQ ID NO: 13.

[0508] The genotype of the F2 generation homozygous mice (V3) can be identified by RT-PCR method. One 6-week-old female C57BL / 6 wild-type mouse and one TGFBR2 gene humanized homozygous mouse are selected. After decapitation and euthanasia, spleen tissues are taken, and RT-PCR detection is carried out using the primers shown in Table 4. The identification results are shown in Figure 12 , mouse TGFBR2 mRNA can be detected in the spleen cells of C57BL / 6 wild-type mice, and human TGFBR2 mRNA can only be detected in TGFBR2 gene humanized homozygous mice.

[0509] Table 4 Primer sequences and recombinant fragment sizes for RT-PCR detection of F2 generation genotypes

[0510]

[0511]

[0512] The protein expression in C57BL / 6 wild-type mice and homozygous humanized TGFBR2 gene mice was detected by flow cytometry. Specifically, one 7-week-old female C57BL / 6 wild-type mouse and one homozygous humanized TGFBR2 gene mouse were taken respectively. After cervical dislocation euthanasia, spleen tissues were taken and stained with mouse leukocyte recognition antibody Brilliant Violet510 TM anti-mouse CD45 (mCD45), FITC anti-Mouse CD19 (anti-mouse CD19 antibody), PerCP / Cy5.5 anti-mouse TCRβ chain (anti-mouse TCRβ antibody), anti-human TGF-β APC receptor (anti-human TGF-β Receptor II Antibody), Mouse TGF-beta RII PE-conjugated Antibody (mTGFBR2, anti-mouse TGFBR2 antibody), Goat IgG PE-conjugated Antibody, APC Rat IgG2b,κ Isotype Ctrl Antibody, ZombieNIR TM Fixable Viability Kit, and Purified anti-mouse CD16 / 32 (anti-mouse CD16 / 32 antibody). After recognition staining, flow cytometry was performed to detect the expression of human or humanized TGFBR2 protein.

[0513] The results showed that in the spleens of C57BL / 6 mice, 2.75% of B cells were hTGFBR2-positive cells (characterized by mCD45 + mCD19 + hTGFBR2+), and 27.7% were mTGFBR2-positive cells (characterized by mCD45 + mCD19 + mTGFBR2+). In the spleens of TGFBR2 homozygous mice, 14.4% of B cells were hTGFBR2-positive cells (characterized by mCD45 + mCD19 + hTGFBR2+), and 5.53% were mTGFBR2-positive cells (characterized by mCD45 + mCD19 + mTGFBR2+).

[0514] In the spleen of C57BL / 6 mice, 3.02% of T cells are positive for hTGFBR2 (characterized by mCD45+mTCRβ+hTGFBR2+), and 16.9% of T cells are positive for mTGFBR2 (characterized by mCD45+mTCRβ+mTGFBR2+). In the spleen of TGFBR2 homozygous mice, 18.9% of T cells are positive for hTGFBR2 (characterized by mCD45+mTCRβ+hTGFBR2+), and 10.4% of T cells are positive for mTGFBR2 (characterized by mCD45+mTCRβ+mTGFBR2+).

[0515] In the spleen of C57BL / 6 mice, 2.42% of NK cells are positive for hTGFBR2 (characterized by mCD45+mTCRβ-mNK1.1+hTGFBR2+), and 28.6% of NK cells are positive for mTGFBR2 (characterized by mCD45+mTCRβ-mNK1.1+mTGFBR2+). In the spleen of TGFBR2 homozygous mice, 16.4% of NK cells are positive for hTGFBR2 (characterized by mCD45+mTCRβ-mNK1.1+hTGFBR2+), and 27.8% of NK cells are positive for mTGFBR2 (characterized by mCD45+mTCRβ-mNK1.1+mTGFBR2+).

[0516] It can be seen that only the expression of mouse TGFBR2 protein can be detected in C57BL / 6 mice. Since the Mouse TGF-beta RIIPE-conjugated Antibody has cross-reactivity with human TGFBR2, combined with the RT-PCR detection results, it can be confirmed that only human TGFBR2 protein can be detected in TGFBR2 gene humanized homozygous mice.

[0517] Furthermore, flow cytometry was used to perform immunophenotyping detection on the spleen, lymph nodes and blood tissues of C57BL / 6 wild-type mice and TGFBR2 gene humanized homozygous mice (H / H). Specifically, three 9-week-old female C57BL / 6 wild-type mice and three TGFBR2 gene humanized homozygous mice were sacrificed by cervical dislocation, and then the spleen, lymph nodes and blood tissues were taken and used with Purified anti-mouse CD16 / 32Antibody, Zombie NIR TM Fixable Viability Kit, Brilliant Violet 510 TM anti-mouse CD45 Antibody, PerCP anti-mouse Ly-6G / Ly-6C(Gr-1)Antibody, Brilliant Violet 421TM anti-mouse CD4 Antibody, FITC anti-mouse F4 / 80 Antibody, PE anti-mouse CD8a Antibody, PE / Cy TM 7 Mouse anti-mouse NK1.1, APC anti-mouse / rat Foxp3 Antibody, FITC anti-Mouse CD19 Antibody, PerCP / Cy5.5 anti-mouse TCRβ chain and other antibodies were used for immunophenotyping detection. The detection results of leukocyte subtypes and T cell subtypes in the spleen and blood are shown respectively as Figure 13 and Figure 15 shown. It can be seen from the figures that the leukocyte subtypes such as B Cells, T cells, NK cells, CD4+ T cells, CD8+ T cells, Granulocytes, Dendritic cells, Macrophages and Monocytes in the spleen and blood of TGFBR2 gene humanized homozygous mice are basically the same as those of C57BL / 6 wild-type mice ( Figure 13 (A) and Figure 15 (A)), and the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells are basically the same as those of C57BL / 6 wild-type mice ( Figure 13 (B) and Figure 15 (B)).

[0518] The detection results of leukocyte subtypes and T cell subtypes in the lymph nodes are shown respectively as Figure 14 (A) and Figure 14 (B). It can be seen from the figures that the leukocyte subtypes such as B cells, T cells, NK cells in the lymph nodes of TGFBR2 gene humanized homozygous mice are basically the same as those of C57BL / 6 wild-type mice, and the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells are basically the same as those of C57BL / 6 wild-type mice. It indicates that the humanization of the TGFBR2 gene has no effect on the differentiation, development and distribution of leukocytes and T cells in the spleen, lymph nodes and blood of mice.

[0519] Given that human TGFBR2 has multiple subtypes or transcripts, the methods described in this article can be applied to other subtypes or transcripts.

[0520] Schematic comparison of mouse TGFBR2 gene (NCBI Gene ID: 21813, Primary source: MGI:98729, UniProt: Q62312-1, located at positions 115916763 to 116004431 of NC_000075.7 on chromosome 9, based on transcript NM_009371.3 and its encoded protein NP_033397.3 (SEQ ID NO: 43)) and human TGFBR2 gene (NCBI Gene ID: 7048, Primary source: HGNC:11773, UniProt ID: P37173-2, located at positions 30606472 to 30694142 of NC_000003.12 on chromosome 3, based on transcript NM_001024847.2 and its encoded protein NP_001020018.1 (SEQ ID NO: 44)) is as shown in Figure 8 shown.

[0521] To achieve the object of the present invention, a nucleotide sequence encoding human TGFBR2 protein can be introduced into the endogenous TGFBR2 gene locus of a mouse, such that the mouse expresses human or humanized TGFBR2 protein. Specifically, using gene editing technology, under the control of the mouse TGFBR2 gene regulatory element, the genomic coding region of human TGFBR2 is used to replace the genomic coding region of mouse TGFBR2, or a sequence of approximately 64 kb from the partial sequence of exon 1 to the partial sequence of exon 5 of the human TGFBR2 gene is used to replace the sequence of approximately 64 kb from the partial sequence of exon 1 to the partial sequence of exon 5 of the mouse, and the schematic diagram of the humanized TGFBR2 gene locus is as shown in Figure 9 shown, realizing the humanization transformation of the mouse TGFBR2 gene.

[0522] Design a targeting strategy as shown in Figure 10 shown. The figure shows that the targeting vector contains homologous arm sequences upstream and downstream of the mouse TGFBR2 gene, and fragment A4 containing the human TGFBR2 sequence. Among them, the upstream homologous arm sequence (5' homologous arm, SEQ ID NO: 45) is the same as the nucleotide sequence at positions 116004022 to 116007886 of NCBI accession number NC_000075.7, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 46) is the same as the nucleotide sequence at positions 115933903 to 115939354 of NCBI accession number NC_000075.7. The nucleotide sequence of human TGFBR2 on fragment A4 (SEQ ID NO: 47) is the same as the nucleotide sequence at positions 30606968 to 30671654 of NCBI accession number NC_000003.12; the connection design between the upstream of the human TGFBR2 sequence and the mouse is: Among them, the last "T" in the sequence " CGTT " is the last nucleotide of the mouse, and the "C" in the sequence is the first nucleotide of the human. The connection between the downstream of the human TGFBR2 sequence and the mouse is designed as Among them, the "T" in the sequence " CAAT " is the last nucleotide of the human, and the first "C" in the sequence is the first nucleotide of the mouse sequence.

[0523] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human gene is designed as:

[0524] Among them, the "A" in the sequence "GGGA" is the last nucleotide of the human, and the first "G" in the sequence "GGAT" is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo cassette and the human gene is designed as Among them, the sequence the last "C" in is the last nucleotide of the Neo cassette, and the first "A" in the sequence " AAGA " is the first nucleotide of the human. In addition, a coding gene with a negative selection marker (the coding gene of diphtheria toxin A subunit (DTA)) was constructed downstream of the 3' homologous arm of the targeting vector. The mRNA sequence of the humanized mouse TGFBR2 after modification is shown in SEQ ID NO: 52, and the expressed protein sequence is shown in SEQ ID NO: 53. The subsequent steps can refer to the method for preparing TGFBR2 humanized mice from embryonic stem cells in Example 1 to obtain genetically engineered mice (V4) with stable inheritance and no random insertion of the TGFBR2 gene.

[0525] Example 3 Pharmacodynamic Verification

[0526] The TGFBR2 humanized mice prepared by this method can be used to evaluate the pharmacodynamics of antibody drugs targeting human TGFBR2. For example, take TGFBR2 humanized homozygous mice and subcutaneously inoculate colon cancer cells MC38. Wait until the tumor volume grows to about 100 mm 3Subsequently, they were divided into a control group or a treatment group according to the tumor volume. In the treatment group, an antibody drug targeting human TGFBR2 was injected, while in the control group, an equal volume of normal saline was injected. The tumor volume was measured regularly and the body weight of the mice was weighed. By comparing the changes in the body weight of the mice and the tumor volume, the safety and in vivo efficacy of the antibody drug in humanized TGFBR2 mice could be effectively evaluated.

[0527] Example 4 Preparation of Double Humanized or Multiple Double Humanized Mice

[0528] The TGFBR2 mice prepared by this method can also be used to prepare double humanized or multi-humanized mouse models. For example, in the aforementioned Example 1 or 2, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3, TIGIT, etc. Alternatively, on the basis of humanized TGFBR2 mice, by separating mouse ES embryonic stem cells and using gene recombination and targeting technology, a double-gene or multi-gene modified mouse model with TGFBR2 and other gene modifications can be obtained. The homozygous or heterozygous TGFBR2 mice obtained by this method can also be mated with homozygous or heterozygous mice with other gene modifications, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining heterozygous mice with humanized TGFBR2 and other gene modifications. Then, the heterozygotes can be mated with each other to obtain homozygotes with double-gene or multi-gene modifications. These double-gene or multi-gene modified mice can be used to verify the in vivo efficacy of targeted human TGFBR2 and other gene regulators, etc.

[0529] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0530] In addition, it should be noted that, in the case of no conflict, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0531] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing a non-human animal with humanized TGFBR2 gene, characterized in that, The humanized TGFBR2 protein is expressed in the non-human animal, the genome of the non-human animal contains the humanized TGFBR2 gene, the humanized TGFBR2 protein contains a part of the human TGFBR2 protein, the part of the human TGFBR2 protein is the entire signal peptide and extracellular region of the human TGFBR2 protein, the part of the human TGFBR2 protein is the amino acid sequence shown in positions 1-166 of SEQ ID NO: 2, the humanized TGFBR2 gene contains a part of the human TGFBR2 gene, and the part of the human TGFBR2 gene is the nucleotide sequence encoding the part of the human TGFBR2 protein. The part of the human TGFBR2 gene or the humanized TGFBR2 gene is regulated by endogenous regulatory elements in the non-human animal, and the amino acid sequence of the humanized TGFBR2 protein is SEQ ID NO:

13. The expression of the endogenous TGFBR2 protein in the non-human animal is reduced or absent, and the non-human animal is a mouse.

2. The construction method according to claim 1, characterized in that, The humanized TGFBR2 gene contains the nucleotide sequence shown in SEQ ID NO:

31.

3. The construction method according to claim 1, characterized in that The genome of the non-human animal contains the nucleotide sequence shown in SEQ ID NO:

7.

4. The construction method according to claim 1, wherein The construction method includes introducing a donor nucleotide sequence into the TGFBR2 gene locus of the non-human animal.

5. The construction method according to claim 4, wherein The donor nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO:

7.

6. The construction method according to claim 4, wherein The donor nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO:

31.

7. The construction method according to claim 4, characterized in that The donor nucleotide sequence sequentially contains a P2A sequence, a part of the human TGFBR2 gene, a part of the non-human animal TGFBR2 gene, and a STOP sequence from the 5' end to the 3' end.

8. The construction method according to claim 7, wherein The P2A sequence is the nucleotide sequence shown in SEQ ID NO: 4, the part of the human TGFBR2 is the nucleotide sequence shown in SEQ ID NO: 7, and the part of the non-human animal TGFBR2 is the nucleotide sequence shown in SEQ ID NO:

31.

9. The construction method according to claim 4, characterized in that, The donor nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO: 3, 54, or 36.

10. The construction method according to any one of claims 4-9, characterized in that, The introduction is by replacement or insertion.

11. The construction method according to claim 10, characterized in that, The introduction into the non-human animal TGFBR2 gene locus is an insertion into the non-human animal TGFBR2 gene locus.

12. The construction method according to claim 11, wherein, The introduction into the non-human animal TGFBR2 gene locus is an insertion into exon 2 of the non-human animal TGFBR2 gene.

13. The construction method according to claim 10, characterized in that, The introduction into the non-human animal TGFBR2 gene locus is an insertion after the 5' UTR and before the ATG of the non-human animal TGFBR2 gene.

14. The construction method according to any one of claims 4-9 and 11-13, characterized in that, A targeting vector is used for the construction of the non-human animal; the targeting vector includes the donor nucleotide sequence.

15. The construction method according to claim 14, characterized in that, The targeting vector further contains a 5' arm and a 3' arm; the 5' arm sequence is as shown in SEQ ID NO: 5, SEQ ID NO: 18, or SEQ ID NO: 37; The 3' arm sequence is as shown in SEQ ID NO: 6, SEQ ID NO: 19, or SEQ ID NO:

38.

16. The construction method according to any one of claims 1-9, 11-13, and 15, characterized in that, The described construction method further includes mating a non-human animal with humanized TGFBR2 gene with other genetically modified non-human animals, in vitro fertilization, or directly performing gene editing, and screening to obtain a multi-gene modified non-human animal.

17. The construction method according to claim 16, wherein, The described other genes are selected from at least one of PD-1, PD-L1, CD27, CD40, CD73, CD226, OX40, 4-1BB, LAG3, and TIGIT.

18. The construction method according to claim 17, wherein The humanized TGFBR2 gene and / or other genes are homozygous for the endogenous modified locus.

19. The construction method according to claim 17, characterized in that, The humanized TGFBR2 gene and / or other genes are heterozygous for the endogenous modified locus.

20. A targeting vector, characterized in that, The targeting vector contains a nucleotide sequence encoding a part of the human TGFBR2 protein. The targeting vector is used to introduce the humanized TGFBR2 protein expression at the TGFBR2 locus of the non-human animal. The part of the human TGFBR2 protein is the entire signal peptide and extracellular region of the human TGFBR2 protein. The part of the human TGFBR2 protein is the amino acid sequence shown at positions 1-166 of SEQ ID NO:

2. The amino acid sequence of the humanized TGFBR2 protein is SEQ ID NO:

13. The non-human animal is a mouse.

21. The targeting vector according to claim 20, wherein The targeting vector contains the nucleotide sequence shown in SEQ ID NO:

7.

22. The targeting vector according to claim 20, wherein, The targeting vector contains the nucleotide sequence shown in SEQ ID NO:

31.

23. The targeting vector according to claim 20, characterized in that, The targeting vector sequentially contains a P2A sequence, a part of the human TGFBR2 gene, a part of the non-human animal TGFBR2 gene, and a STOP sequence from the 5'-end to the 3'-end.

24. The targeting vector according to claim 23, characterized in that, The P2A sequence is the nucleotide sequence shown in SEQ ID NO:

4. The part of the human TGFBR2 is the nucleotide sequence shown in SEQ ID NO:

7. The part of the non-human animal TGFBR2 is the nucleotide sequence shown in SEQ ID NO:

31.

25. The targeting vector according to claim 20, wherein, The targeting vector contains the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 36, or SEQ ID NO:

54.

26. The targeting vector according to any one of claims 20-25, characterized in that, The targeting vector further contains a 5'-arm and a 3'-arm; The 5'-arm sequence is as shown in SEQ ID NO: 5, SEQ ID NO: 18, or SEQ ID NO: 37; The 3'-arm sequence is as shown in SEQ ID NO: 6, SEQ ID NO: 19, or SEQ ID NO:

38.

27. A humanized TGFBR2 protein, characterized in that, The humanized TGFBR2 protein contains a part of the human TGFBR2 protein. The part of the human TGFBR2 protein is the entire signal peptide and extracellular region of the human TGFBR2 protein. The part of the human TGFBR2 protein is the amino acid sequence shown at positions 1-166 of SEQ ID NO:

2. The amino acid sequence of the humanized TGFBR2 protein is SEQ ID NO:

13.

28. A humanized TGFBR2 gene, characterized in that, The humanized TGFBR2 gene described above contains a part of the human TGFBR2 gene, and the humanized TGFBR2 gene encodes the humanized TGFBR2 protein described in claim 27.

29. The humanized TGFBR2 gene according to claim 28, wherein The humanized TGFBR2 gene described above contains the nucleotide sequence shown in SEQ ID NO:

31.

30. The humanized TGFBR2 gene according to claim 28, wherein The humanized TGFBR2 gene described above contains the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 36 or SEQ ID NO:

54.

31. The humanized TGFBR2 gene according to any one of claims 28-30, characterized in that, The mRNA transcribed from the nucleotide sequence of the humanized TGFBR2 gene described above is the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO:

42.

32. A cell, tissue or organ, characterized in that, The cell, tissue or organ described above expresses the humanized TGFBR2 protein described in claim 27, or the genome of the cell, tissue or organ contains the humanized TGFBR2 gene described in any one of claims 28-31, or the cell, tissue or organ is derived from a non-human animal obtained by the construction method described in any one of claims 1-19, and the cell, tissue or organ cannot develop into an animal individual.

33. The cell, tissue or organ according to claim 32, wherein The tissue described above is a tumor tissue after tumor implantation.

34. Use of the humanized TGFBR2 protein described in claim 27 and the humanized TGFBR2 gene described in any one of claims 28-31 in the preparation of an animal model.

35. Use of a non-human animal obtained by the construction method described in any one of claims 1-19 in drug screening.

36. Use of the cell, tissue or organ described in any one of claims 32-33 as an animal model.

Images