CN115011606A

CN115011606A - Construction method and application of CD37 gene humanized non-human animal

Info

Publication number: CN115011606A
Application number: CN202210655021.1A
Authority: CN
Inventors: 李志霞; 尚诚彰; 聂琰晖
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-06-11
Filing date: 2022-06-10
Publication date: 2022-09-06

Abstract

The invention provides a humanized CD37 protein, a humanized CD37 gene, a targeting vector of a CD37 gene, a humanized non-human animal of the CD37 gene, a construction method thereof and application thereof in the field of biomedicine, wherein a nucleotide sequence for coding the human CD37 protein is introduced into the genome of the non-human animal by utilizing a homologous recombination mode, the animal can normally express the human or humanized CD37 protein, and the humanized CD37 gene can be used as an animal model for researching human CD37 signal mechanism and screening tumor and autoimmune disease drugs, and has important application value for the research and development of new drugs of immune targets.

Description

Construction method and application of CD37 gene humanized non-human animal

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a CD37 gene modified non-human animal model and application thereof in the field of biomedicine.

Background

CD37, also known as GP52-40, tetraspanin-26 (tetraspanin-26)), belongs to one of the family members of the tetraspanin (TM4SF) protein family, is expressed almost exclusively in cells of the immune system, especially B cells (pre-B to peripheral mature B cell stage, not expressed on plasma cells), at lower levels in T cells, macrophages/monocytes, granulocytes and dendritic cells, involved in cell membrane organization and cooperative signaling. In mice, homozygous mutation of the CD37 gene resulted in decreased IgG1 immunoglobulin levels and impaired antibody responses to T cell-dependent antigens. CD37 knockout aged mice develop B cell lymphomas by losing CD 37-mediated negative feedback in the IL6/STAT3 (interleukin 6/signaling and transcriptional activator 3) pathway. CD37 has been found to be widely expressed on most B cell malignancies, including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphoma (NHL), and is a drug hot-target for B cell malignancies. There are currently several anti-CD 37 antibodies that have been introduced into human clinics worldwide, including Otletuzumab by Abbott, 177Lu-Lilotomab by Nordic Nanovector, Naratuximab Emtansine by ImmunoGen, BI-836826 by BI, DuoHexaBody-CD37 by Genmab, and the like. Genmab agreed a cooperative agreement of up to $ 40 billion with AbbVie on 3antibody drugs including DuoHexaBody-CD37 at 6 months 2020.

The experimental animal disease model is an indispensable research tool for researching etiology and pathogenesis of human diseases, developing prevention and treatment technologies and developing medicines. However, due to the differences between the physiological structures and metabolic systems of animals and humans, the traditional animal models cannot reflect the real conditions of human bodies well, and the establishment of disease models closer to the physiological characteristics of human bodies in animal bodies is an urgent need of the biomedical industry. With the continuous development and maturation of genetic engineering technology, the replacement or substitution of animal homologous genes with human genes has been realized, and the development of humanized experimental animal models in this way is the future development direction of animal models. The gene humanized animal model is one animal model with normal or mutant gene replaced with homologous gene in animal genome and similar physiological or disease characteristics. The gene humanized animal not only has important application value, for example, the humanized animal model of cell or tissue transplantation can be improved and promoted by gene humanization, but also more importantly, the human protein can be expressed or partially expressed in the animal body due to the insertion of the human gene segment, and the gene humanized animal can be used as a target of a drug which can only recognize the human protein sequence, thereby providing possibility for screening anti-human antibodies and other drugs at the animal level. However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, how to construct an "efficient" humanized animal model for new drug development remains the greatest challenge.

In view of the huge application potential of CD37 in the field of tumor therapy, there is an urgent need in the art to develop a non-human animal model of CD 37-related signaling pathway in order to further explore its relevant biological properties, improve the effectiveness of preclinical drug efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failures. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

In a first aspect of the invention, there is provided a humanized CD37 protein, wherein the humanized CD37 protein comprises all or part of a human CD37 protein.

Preferably, the humanized CD37 protein comprises all or part of the extracellular domain of human CD37 protein.

Preferably, the humanized CD37 protein further comprises all or part of the transmembrane and/or cytoplasmic domain of human CD37 protein.

Preferably, the humanized CD37 protein comprises a portion of the transmembrane region of human CD37 protein that is encoded by exon 2, exon 3, exon 4, exon 7, and/or exon 8 of the human CD37 gene.

Preferably, the humanized CD37 protein comprises a portion of the cytoplasmic region of human CD37 protein that is encoded by a portion of exon 3 and/or exon 8 of the human CD37 gene.

Preferably, the humanized CD37 protein comprises all or part of the extracellular, transmembrane and/or cytoplasmic region of human CD37 protein, further preferably, the humanized CD37 protein comprises all or part of the extracellular region of human CD37 protein, and the humanized CD37 protein comprises at least 20 amino acids of the extracellular region of human CD37 protein, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 130, 150, 151 amino acids of the extracellular region of human CD37 protein, further preferably, comprises SEQ ID NO:2, positions 39-59, 112-241; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized CD37 protein further comprises all or part of a transmembrane region of human CD37 protein, the humanized CD37 protein comprises a transmembrane region of human CD37 protein of at least 10 amino acids, such as at least 10, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 87 amino acids of the human CD37 protein, and further preferably comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with position 266 of 18-38 or 27-38, 60-74, 86-111, 242 and the like of SEQ ID NO:2 or comprises an amino acid sequence as shown at position 266 of 18-38 or 27-38, 60-74, 86-111, 242 and the like of SEQ ID NO: 2; the humanized CD37 protein further comprises all or part of a cytoplasmic region of the human CD37 protein, and the humanized CD37 protein comprises a cytoplasmic region of the human CD37 protein of at least 5 amino acids, such as a cytoplasmic region of the human CD37 protein of at least 5, 10, 20, 25, 26, 27, 28, 29, 30, 40, 43 amino acids, and further preferably comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 75-85, 267-281 and/or 1-17 of SEQ ID NO:2 or comprises an amino acid sequence as shown at positions 75-85, 267-281 and/or 1-17 of SEQ ID NO: 2.

Preferably, the humanized CD37 protein comprises all or part of an amino acid sequence encoded by exons 1 to 8 of the human CD37 gene. Further preferably, the humanized CD37 protein comprises all or part of an amino acid sequence encoded by a combination of two or more consecutive exons among exons 1 to 8 of human CD37 gene. Still more preferably, the humanized CD37 protein comprises the amino acid sequence encoded by part of exon 2 to part of exon 8, more preferably part of exon 2, all of exon 3 to 7 and part of exon 8 of human CD37 gene, wherein part of exon 2 comprises at least 20bp nucleotide sequence, such as at least 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73bp nucleotide sequence, and part of exon 8 comprises at least 50bp nucleotide sequence, such as at least 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 200, 300, 356bp nucleotide sequence.

Preferably, the portion of human CD37 protein comprises SEQ ID NO:2, amino acid sequence shown in positions 27-281; or, comprising a nucleotide sequence identical to SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical in amino acid sequence from position 27-281; or, comprising a nucleotide sequence identical to SEQ ID NO: amino acid sequence shown at positions 27-281 differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 27-281, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized CD37 protein further comprises a portion of a non-human animal CD37 protein. Further preferably at least comprises all or part of the transmembrane region and/or cytoplasmic region of the non-human animal CD37 protein, more preferably at least comprises part of the transmembrane region and part of the cytoplasmic region of the non-human animal CD37 protein.

Preferably, the portion of the non-human animal CD37 protein comprises SEQ ID NO:1, 1-26; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-26, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-26, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-26, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment, the humanized CD37 protein comprises at least the entirety of the extracellular region of the human CD37 protein, a portion of the transmembrane region of the human CD37 protein, a portion of the cytoplasmic region of the human CD37 protein, a portion of the transmembrane region of the non-human animal CD37 protein, and/or a portion of the cytoplasmic region of the non-human animal CD37 protein. The part of the human CD37 protein transmembrane region is a transmembrane region coded by parts of exon 1, exon 2, exon 3, exon 4, exon 7 and/or exon 8 of the human CD37 gene; the part of the cytoplasmic region of the human CD37 protein is the cytoplasmic region encoded by the part of exon 3 and/or exon 8 of the human CD37 gene; the part of the cytoplasmic region of the non-human animal CD37 protein is a cytoplasmic region partially encoded by the exon 1 of the non-human animal CD37 gene, and the part of the transmembrane region of the non-human animal CD37 protein is a transmembrane region partially encoded by the exon 1 of the non-human animal CD37 gene and the exon 2.

Preferably, the amino acid sequence of the humanized CD37 protein derived from human CD37 has the amino acid sequence shown in SEQ ID NO:2 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

Preferably, the amino acid sequence of the humanized CD37 protein derived from the non-human animal CD37 protein is identical to the amino acid sequence of SEQ ID NO:1 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

Preferably, the portion of the non-human animal CD37 protein comprises a sequence encoding SEQ ID NO:1, positions 1-26; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-26, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-26, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-26, comprising substitution, deletion and/or insertion of one or more amino acid residues. Is an extracellular, transmembrane and/or cytoplasmic region of a non-human animal CD37 protein, preferably comprising the amino acid sequence of SEQ ID NO:1, 1-17, 1-26, 1-38 or 242-281; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-17, 1-26 or 1-38 or 242-281, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-17, 1-26 or 1-38 or 242-281, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 1-17, 1-26 or 1-38 or 242-281, including substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the present invention, the partial amino acid sequence of the human CD37 protein contained in the humanized CD37 protein comprises one of the following groups:

A) is SEQ ID NO:2, all or part of the amino acid sequence from position 27 to 281;

B) and SEQ ID NO:2, at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% amino acid sequence identity;

C) and SEQ ID NO:2 from position 27 to 281 of the polypeptide sequence does not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by more than 1 amino acid; or

D) And SEQ ID NO:2, and (b) an amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues shown in positions 27 to 281 of the above-mentioned amino acid sequence.

In one embodiment of the present invention, the amino acid sequence of the humanized CD37 protein is selected from one of the following groups:

a) is SEQ ID NO: 9 all or part of an amino acid sequence;

b) and SEQ ID NO: 9 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% amino acid sequence identity;

c) and SEQ ID NO: 9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or

d) And SEQ ID NO: 9, comprising substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the non-human animal can be selected from any non-human animal such as rodent, zebrafish, pig, chicken, rabbit, monkey, etc. which can be genetically modified to make a gene humanized.

Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or a mouse.

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. Still 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) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a second aspect of the invention, there is provided a nucleic acid encoding the humanized CD37 protein.

In a third aspect of the invention, there is provided a humanized CD37 gene, wherein the humanized CD37 gene comprises a portion of the human CD37 gene.

Preferably, the humanized CD37 gene comprises all or part of the nucleotide sequence of an extracellular region encoding human CD37 protein, wherein the part of the extracellular region of human CD37 protein comprises at least 20 amino acids of the extracellular region of human CD37 protein, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 130, 150, 151 amino acids of the extracellular region of human CD37 protein, further preferably, the nucleotide sequence of the extracellular region comprising SEQ ID NO:2, positions 39-59, 112-241; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized CD37 gene further comprises a nucleotide sequence encoding all or part of the human CD37 protein transmembrane region, the part of the human CD37 protein transmembrane region comprises at least 10 amino acids of the human CD37 protein transmembrane region, such as at least 10, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 87 amino acids of the human CD37 protein transmembrane region, further preferably comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with positions 18-38 or 27-38, 60-74, 86-111, 242-minus one of SEQ ID NO:2 or comprises an amino acid sequence as shown at positions 18-38 or 27-38, 60-74, 86-111, 242-minus one of SEQ ID NO: 2; the humanized CD37 gene further comprises a nucleotide sequence encoding all or part of the cytoplasmic domain of the human CD37 protein, and the part of the cytoplasmic domain of the human CD37 protein comprises a cytoplasmic domain of the human CD37 protein of at least 5 amino acids, such as a cytoplasmic domain of the human CD37 protein of at least 5, 10, 20, 25, 26, 27, 28, 29, 30, 40, 43 amino acids, and further preferably comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with positions 75-85, 267-281 and/or 1-17 of SEQ ID NO:2 or comprises an amino acid sequence shown at positions 75-85, 267-281 and/or 1-17 of SEQ ID NO: 2.

Preferably, said portion of the human CD37 gene comprises a nucleotide sequence encoding SEQ ID NO:2 from position 27 to 281; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from 27 to 281 nucleotide sequence identities of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from positions 27-281 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, a polypeptide having the sequence encoding SEQ ID NO:2, positions 27-281, including nucleotide sequences in which one or more nucleotides are substituted, deleted and/or inserted.

Preferably, the humanized CD37 gene comprises all or part of the nucleotide sequence of exons 1 to 8 of human CD37 gene. Further preferably, the humanized CD37 gene comprises all or part of a nucleotide sequence of a combination of two or more consecutive exons among exons 1 to 8 of human CD37 gene. Still more preferably, the humanized CD37 gene comprises the partial nucleotide sequence of exon 2, the entire exon 3-7 and the partial nucleotide sequence of exon 8 of human CD37 gene. More preferably, the humanized CD37 gene further includes intron 2-3, intron 3-4, intron 4-5, intron 5-6, intron 6-7 and/or intron 7-8 of the human CD37 gene. Wherein, the part of exon 2 comprises at least 20bp nucleotide sequence, such as at least 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73bp nucleotide sequence, and the part of exon 8 comprises at least 50bp nucleotide sequence, such as at least 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 200, 300, 356bp nucleotide sequence.

Preferably, the part of the human CD37 gene comprises a nucleotide sequence of at least 100-4046bp starting from the inside of exon 2 of the human CD37 gene to the downstream.

In one embodiment, the part of human CD37 in the humanized CD37 gene refers to a nucleotide sequence of 4046bp in length from the inside of exon 2 of human CD37 gene until the last nucleotide of the coding region of exon 8.

In one embodiment of the present invention, the humanized CD37 gene comprises a partial nucleotide sequence of human CD37 gene comprising one of the following groups:

(A) is SEQ ID NO: 5, all or part of a nucleotide sequence set forth in seq id no;

(B) and SEQ ID NO: 5 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

(C) and SEQ ID NO: 5 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

(D) Has the sequence shown in SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the humanized CD37 gene further includes a portion of a non-human animal CD37 gene.

Preferably, said portion of the non-human animal CD37 gene preferably comprises a nucleotide sequence encoding SEQ ID NO:1, 1-26; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:1, positions 1-26, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:1, positions 1-26, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:1, positions 1-26, including nucleotide sequences with substitutions, deletions and/or insertions of one or more nucleotides.

Preferably, the humanized CD37 gene includes all or part of exon 1 of the non-human animal CD37 gene. Further preferably, the part of exon 1 of non-human animal CD37 gene at least comprises a non-coding region of exon 1 of non-human animal CD37 gene.

Preferably, the humanized CD37 gene includes all of exon 1, exon 2-encoded portion and non-coding region of exon 8 of the non-human animal CD37 gene.

Preferably, the nucleotide sequence of the humanized CD37 gene further comprises a nucleotide sequence identical to SEQ ID NO: 6 and/or SEQ ID NO: 7, or a nucleotide sequence having at least 60%, 70%, 80%, 90%, or at least 95% identity to SEQ ID NO: 6 and/or SEQ ID NO: 7.

In one embodiment of the present invention, the mRNA transcribed from the nucleotide sequence of the humanized CD37 gene is selected from one of the following groups:

(a) is SEQ ID NO: 8, or a portion or all of the nucleotide sequence set forth in seq id no;

(b) and SEQ ID NO: 8 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

(c) and SEQ ID NO: 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or

(d) And SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the humanized CD37 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible.

In one embodiment of the invention, the specific inducer is selected from the tetracycline System (Tet-Off System/Tet-On System) or Tamoxifen System (Tamoxifen System).

In one embodiment of the present invention, the humanized CD37 gene encodes the humanized CD37 protein described above.

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. Still further 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) mice,Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a fourth aspect of the invention, there is provided a targeting vector comprising a donor nucleotide sequence, said donor nucleotide sequence comprising one of the following group:

A) all or part of a nucleotide sequence encoding a human or humanized CD37 protein;

B) a nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic domain of human CD37 protein, wherein the portion of the extracellular domain of human CD37 protein comprises at least 20 amino acids of the extracellular domain of human CD37 protein, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 130, 150, 151 amino acids of the extracellular domain of human CD37 protein; the portion of the human CD37 protein transmembrane region comprises a human CD37 protein transmembrane region of at least 10 amino acids, such as a human CD37 protein transmembrane region of at least 10, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 87 amino acids; the portion of the cytoplasmic region of the human CD37 protein comprises a cytoplasmic region of the human CD37 protein of at least 5 amino acids, e.g., a cytoplasmic region of the human CD37 protein of at least 5, 10, 20, 25, 26, 27, 28, 29, 30, 40, 43 amino acids; preferably, the polypeptide comprises a sequence encoding SEQ ID NO:2 from position 27 to 281; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from 27 to 281 nucleotide sequence identities of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from positions 27-281 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, a polypeptide having the sequence encoding SEQ ID NO:2, nucleotide sequence from position 27 to 281, including nucleotide sequence with substitution, deletion and/or insertion of one or more nucleotides;

C) a human or humanized CD37 gene; or the like, or, alternatively,

D) all or part of exon 1 to exon 8 of the human CD37 gene, preferably all or part of exon 2 to exon 8, further preferably all or part of exon 2, exon 3 to exon 7 and part of exon 8, wherein part of exon 2 comprises at least a 20bp nucleotide sequence, for example at least a 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73bp nucleotide sequence, and part of exon 8 comprises at least a 50bp nucleotide sequence, for example at least a 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 200, 300, 356bp nucleotide sequence; further preferred comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or, comprises a polypeptide having the sequence of SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the donor nucleotide comprises all or part of the human CD37 gene.

Preferably, the portion of the human CD37 gene contained in the donor nucleotide corresponds to the portion of the human CD37 gene contained in the humanized CD37 gene described above.

Preferably, the targeting vector further comprises a first DNA segment, i.e.the 5 'arm, homologous to the 5' end of the transition region to be altered, selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD37 gene. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000073.7. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 3 or SEQ ID NO: 13 or as shown in SEQ ID NO: 3 or SEQ ID NO: 13, respectively.

Preferably, the targeting vector further comprises a second DNA fragment homologous to the 3 'end of the transition region to be altered, i.e.the 3' arm, selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the non-human animal CD37 gene. Further preferred, said 3' arm is a nucleotide having at least 90% homology with NCBI accession No. NC — 000073.7. Still more preferably, the 3' arm sequence is identical to SEQ ID NO: 4 or SEQ ID NO: 14 or as shown in SEQ ID NO: 4 or SEQ ID NO: as shown at 14.

Preferably, the transition region to be altered is located at the CD37 locus of the non-human animal. Further preferably, the transition region to be altered is located on exons 1 to 8 of the non-human animal CD37 gene, preferably exons 2 to 8 of the non-human animal CD37 gene.

Preferably, the targeting vector further comprises a non-human animal CD37 gene 5' UTR.

Preferably, the targeting vector further comprises a 3' UTR of the non-human animal CD37 gene.

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

Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Still more preferably, the gene encoding the negative selection marker is a gene encoding diphtheria toxin subunit a (DTA).

In one embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone selection. Further preferably, the resistance gene selected by the positive clone is neomycin phosphotransferase coding sequence Neo.

In one embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (a conventional LoxP recombination system can also be selected). The specific recombination system is provided with two Frt recombination sites which are respectively connected to two sides of the resistance gene.

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. Still further 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) mice, NOD/SCID mice or nude mice.

In a fifth aspect of the invention, there is provided a sgRNA molecule that targets a non-human animal CD37 gene, while the sequence of the sgRNA molecule is unique on the target sequence on the CD37 gene to be altered.

Preferably, the target site of the sgRNA molecule is located on exon 2 and/or exon 8 sequences of the CD37 gene.

Further preferably, the 5' target site sequence of the sgRNA molecule comprises the sequence set forth as SEQ ID NO: 15-20, the 3' end target site sequence comprises the sequence set forth in SEQ ID NO: 21-28.

In a specific embodiment of the invention, the sgRNA-targeted 5' target site sequence comprises the sequence set forth in SEQ ID NO: 19, and the 3' end target site sequence comprises a sequence shown as SEQ ID NO: 26, or a sequence as shown in fig.

In a sixth aspect of the invention, a DNA molecule encoding the sgRNA molecule is provided.

Preferably, the double strand of the DNA molecule is an upstream sequence and a downstream sequence of the sgRNA molecule, or a forward oligonucleotide sequence or a reverse oligonucleotide sequence after the addition of the enzyme cleavage site. Further preferably, TAGG is added to the 5' end of the sgRNA sequence, and AAAC is added to the complementary strand thereof.

In one embodiment of the invention, the DNA molecule may be SEQ ID NO: 29 and SEQ ID NO: 31, SEQ ID NO: 30 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 35, or, SEQ ID NO: 34 and SEQ ID NO: 36.

in a seventh aspect of the present invention, there is provided a sgRNA vector including the above sgRNA molecule or the above DNA molecule.

In an eighth aspect of the present invention, a preparation method of a sgRNA vector is provided, which includes:

(i) providing the sgRNA molecule, and preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence, wherein the sgRNA targets a CD37 gene, the sgRNA molecule is unique on a target sequence on a CD37 gene to be changed, and a target site of the sgRNA molecule is positioned on exons 1 to 8 of the CD37 gene to be changed, preferably on exons 2 to 8 of the CD37 gene to be changed;

(ii) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, carrying out enzyme digestion on the fragment DNA through EcoRI and BamHI to be connected to a skeleton vector, and carrying out sequencing verification to obtain a pT7-sgRNA vector;

(iii) (iii) denaturing and annealing the forward and reverse oligonucleotides obtained in step (i) to form a double strand that can be ligated to the pT7-sgRNA vector of step (ii);

(iv) and (5) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step (iii) with pT7-sgRNA vectors, and screening to obtain the sgRNA vectors.

Preferably, the T7 promoter and sgRNA scaffold fragment DNA in step (ii) are as shown in SEQ ID NO: shown at 35.

In a ninth aspect of the present invention, there is provided a cell comprising the targeting vector, the sgRNA molecule, the DNA molecule, or the sgRNA vector.

In a specific embodiment of the invention, the cell comprises the targeting vector and sgRNA molecule described above.

Preferably, the cells include cells that can develop into an animal subject and cells that cannot develop into an animal subject.

A tenth aspect of the present invention provides use of the targeting vector, the sgRNA molecule, the DNA molecule or the sgRNA vector, the sgRNA vector obtained by the preparation method, or a cell containing the targeting vector or the sgRNA molecule in modification of CD37 gene. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In an eleventh aspect of the invention, there is provided a method of constructing a non-human animal which expresses a human or humanized CD37 protein.

Preferably, the method of construction comprises introducing a donor nucleotide sequence into the non-human animal CD37 locus to form a human or humanized CD37 gene.

Preferably, the humanized CD37 gene is identical to the nucleic acid or the humanized CD37 gene described above.

Preferably, the humanized CD37 protein is identical to the humanized CD37 protein described above.

Preferably, the humanized CD37 gene is regulated in the non-human animal by endogenous or exogenous regulatory elements. Further preferably, the regulatory element is a promoter.

In one embodiment, a donor nucleotide sequence comprising a portion of the human CD37 gene described above is introduced at the non-human animal CD37 locus to form a humanized CD37 gene.

Preferably, the human CD37 gene is introduced into the corresponding region of the non-human animal CD37 locus.

Further preferably, the introduced donor nucleotide sequence is regulated by endogenous regulatory elements.

In one embodiment, the donor nucleotide sequence comprises one of the following groups:

B) a nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic domain of human CD37 protein, wherein the portion of the extracellular domain of human CD37 protein comprises at least 20 amino acids of the extracellular domain of human CD37 protein, such as at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 130, 150, 151 amino acids of the extracellular domain of human CD37 protein; the portion of the human CD37 protein transmembrane region comprises a human CD37 protein transmembrane region of at least 10 amino acids, such as a human CD37 protein transmembrane region of at least 10, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78, 79, 80, 87 amino acids; the portion of the cytoplasmic region of the human CD37 protein comprises at least a5 amino acid cytoplasmic region of the human CD37 protein, e.g., at least a5, 10, 20, 25, 26, 27, 28, 29, 30, 40, 43 amino acid cytoplasmic region of the human CD37 protein; preferably, the polypeptide comprises a sequence encoding SEQ ID NO:2 from position 27 to 281; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from 27 to 281 nucleotide sequence identities of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from positions 27-281 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO:2, nucleotide sequence from position 27 to 281, including nucleotide sequence with substitution, deletion and/or insertion of one or more nucleotides;

C) a human or humanized CD37 gene; or the like, or a combination thereof,

D) all or part of exon 1 to exon 8 of the human CD37 gene, preferably all or part of exon 2 to exon 8, further preferably all or part of exon 2, exon 3 to exon 7 and part of exon 8, wherein part of exon 2 comprises at least a 20bp nucleotide sequence, for example at least a 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 73bp nucleotide sequence, and part of exon 8 comprises at least a 50bp nucleotide sequence, for example at least a 50, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, 200, 300, 356bp nucleotide sequence; more preferably, all introns between exon No. 2 and exon No. 8 are also included; further preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, endogenous regulatory sequences (5' UTR) of the CD37 gene of the non-human animal are retained, ensuring normal expression of the CD37 gene.

Preferably, endogenous regulatory sequences (3' UTR) of the CD37 gene of the non-human animal are retained, ensuring normal expression of the CD37 gene.

Preferably, the construction method comprises introducing a nucleotide sequence comprising all or part of the nucleotide sequence encoding human CD37 protein into the non-human animal CD37 locus.

Preferably, the construction method comprises introducing a cDNA sequence encoding human CD37 protein into the non-human animal CD37 locus.

Preferably, the construction method comprises introducing a nucleotide sequence comprising the humanized CD37 gene into the non-human animal CD37 locus.

Preferably, the construction method comprises introducing a nucleotide sequence encoding the humanized CD37 protein into the non-human animal CD37 locus.

Preferably, the introduction described herein includes, but is not limited to, insertion, substitution or transgene, and the substitution is preferably in situ.

The introduction is replacement or insertion, specifically, the introduced non-human animal CD37 locus is replacement of a corresponding region of the non-human animal, preferably replacement of all or part of a nucleotide sequence encoding endogenous CD37 protein in the genome of the non-human animal. It is further preferred to replace the nucleotide sequence encoding positions 27-281 of SEQ ID NO. 1 in the genome of a non-human animal.

Preferably, exon 2 through exon 8 of the non-human animal CD37 gene are replaced in whole or in part, and further preferably, exon 2, exon 3 through exon 7 and exon 8 of the non-human animal CD37 gene are replaced in part.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD37 gene in the non-human animal and then performing the insertion operation. Or the insertion step can cause frame shift mutation to the endogenous CD37 gene and can realize the step of inserting the human sequence.

Preferably, the nucleotide sequence encoding the humanized CD37 protein is substituted at the corresponding position on the non-human animal CD37 locus, preferably, all or part of exon 1 to exon 8 of the non-human animal CD37 gene is substituted, and further preferably, all or part of intron between exon 2 and exon 8 of the non-human animal is substituted.

Preferably, the human or humanized CD37 gene in a humanized non-human animal of the CD37 gene is homozygous or heterozygous.

Preferably, the construction method comprises modifying the coding frame of the non-human animal CD37 gene, and inserting the coding frame containing the exogenous nucleotide sequence of claim 46 into the endogenous regulatory element of the non-human animal CD37 gene, wherein the coding frame of the modified non-human animal CD37 gene can adopt a functional region for knocking out the non-human animal CD37 gene or a sequence for inserting a protein with reduced expression or no function, so that the non-human animal CD37 protein is not expressed, and further preferably, the coding frame of the modified non-human animal CD37 gene can knock out all or part of the nucleotide sequence from exon 2 to exon 8 of the non-human animal CD37 gene.

The construction method comprises introducing the donor nucleotide sequence of the non-human animal CD37 gene into the endogenous regulatory element.

Preferably, the non-human animal is constructed using gene editing techniques including gene targeting using embryonic stem cells, CRISPR/Cas9, zinc finger nuclease, transcription activator-like effector nuclease, homing endonucleases, or other molecular biology techniques.

Preferably, the construction of the non-human animal is performed using the targeting vector and/or sgRNA described above.

In another embodiment of the present invention, the construction method comprises introducing the targeting vector into embryonic stem cells of a non-human animal, introducing the targeting vector into a blastocyst which is isolated in advance after a short culture, transplanting the obtained chimeric blastocyst into an oviduct of a recipient female mouse, allowing the chimeric blastocyst to develop, and identifying and screening the non-human animal humanized with the CD37 gene.

According to some embodiments of the invention, the constructing method further comprises: the CD37 gene-humanized non-human animal is mated with another genetically modified non-human animal, fertilized in vitro or directly subjected to gene editing, and then screened to obtain a polygene-modified non-human animal.

Preferably, the other gene is at least one gene selected from the group consisting of PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3 and CD 20.

Preferably, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3, and CD20 proteins.

According to some embodiments of the invention, the human or humanized CD37 gene and/or the other gene is homozygous or heterozygous for the endogenous modified locus.

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. Still further 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) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice. In a twelfth aspect of the invention, there is provided a humanized non-human animal, which isExpressing human or humanized CD37 protein in an object.

Preferably, the expression of endogenous CD37 protein in the non-human animal is reduced or absent.

Preferably, the humanized CD37 protein is selected from the humanized CD37 protein.

Preferably, the genome of the non-human animal comprises a portion of the human CD37 gene. Further preferably, the genome of the non-human animal contains the humanized CD37 gene described above.

Preferably, the non-human animal lacks all or part of exons 1-8 of the endogenous CD37 gene, preferably all or part of exons 2 to 8.

Preferably, the non-human animal lacks part of exon 2, all of exon 3, all of exon 4, all of exon 5, all of exon 6, all of exon 7, and the coding region of exon 8 of the endogenous CD37 gene.

Preferably, the non-human animal lacks all or part of an intron between exons 2-8 of the CD37 gene.

Preferably, the nucleotide sequence of the human or humanized CD37 gene is operably linked to a non-human animal endogenous regulatory element.

Preferably, the non-human animal further comprises additional genetic modifications, said additional genes being selected from at least one of PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3 and CD20, preferably, said CD37 gene and said additional genes are optionally homozygous or heterozygous.

Preferably, the non-human animal is constructed using gene editing techniques including gene targeting techniques using embryonic stem cells, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques.

In a specific embodiment, the non-human animal is constructed using the targeting vector and/or sgRNA.

In one embodiment, the non-human animal is constructed using the construction methods described above.

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. Still 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) mice, NOD/SCID mice or nude mice.

In a thirteenth aspect of the invention, there is provided a non-human animal deficient in the CD37 gene, said non-human animal being deficient in all or part of exons 2 to 8 of the endogenous CD37 gene.

Preferably, the CD37 gene deleted non-human animal is constructed by using the targeting vector.

In a fourteenth aspect of the present invention, a method for constructing a non-human animal with a deletion of CD37 gene is provided, the method comprising constructing a non-human animal using the targeting vector and/or the sgRNA. Wherein the sgRNA targets the non-human animal CD37 gene, while the sequence of the sgRNA is unique on the target sequence on the CD37 gene to be altered.

Preferably, the target site of the sgRNA is located on exon 1 to exon 8 sequences of the CD37 gene, preferably, on exon 2 and/or exon 8.

Further preferably, the sequence of the target site at the 5' end targeted by the sgRNA is shown in SEQ ID NO: 15-20, the 3' end target site sequence is shown in SEQ ID NO: 21-28.

In a fifteenth aspect of the present invention, there is provided a method for constructing a polygene-modified non-human animal, comprising the steps of:

I) providing the non-human animal or the non-human animal obtained by the construction method;

II) mating the non-human animal provided in step I) with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a polygenic modified non-human animal.

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized with genes PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3, or CD 20.

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

Preferably, each of the plurality of genes humanized in the genome of the polygenic modified non-human animal may be homozygous or heterozygous.

In a sixteenth aspect of the present invention, there is provided a non-human animal humanized with CD37 gene, a non-human animal with CD37 gene knockout, or a multi-gene modified non-human animal or progeny thereof obtained by the above construction method.

In a seventeenth aspect of the present invention, an animal model is provided, wherein the animal model 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 progeny. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

The eighteenth aspect of the present invention provides a method for producing an animal model, which comprises the step of constructing the above-mentioned non-human animal having a humanized CD37 gene, a CD37 knock-out non-human animal, or a multi-gene-modified non-human animal or progeny thereof. Preferably, the animal model is a tumor-bearing or inflammatory animal model, and more preferably, the method for preparing the tumor-bearing animal model further comprises the step of implanting tumor cells.

In a nineteenth aspect of the present invention, there is provided a use of the above-mentioned CD37 gene-humanized non-human animal, CD37 gene-knocked out non-human animal, multi-gene-modified non-human animal or progeny thereof, or the above-mentioned CD37 gene-humanized non-human animal, CD37 gene-knocked out non-human animal, multi-gene-modified non-human animal or progeny thereof obtained by the above-mentioned construction method for producing an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a twentieth aspect of the present invention, there is provided a cell or cell line or primary cell culture derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned animal model.

Preferably, the cell or cell line or primary cell culture comprises a cell or cell line or primary cell culture that can develop into an animal subject and that cannot develop into an animal subject.

In a twenty-first aspect of the present invention, there is provided a tissue or organ or culture thereof derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or progeny thereof, or the above-mentioned animal model.

Preferably, the tissue or organ or culture thereof includes a tissue or organ or culture thereof that can develop into an animal subject and cannot develop into an animal subject.

In a twenty-second aspect of the present invention, there is provided a tumor tissue after tumor bearing, wherein the tumor tissue is derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, or the above tumor bearing animal model.

In a twenty-third aspect of the invention, there is provided a cell humanised with the CD37 gene, said cell expressing a human or humanised CD37 protein.

Preferably, the humanized CD37 protein is the humanized CD37 protein of the first aspect of the invention.

Preferably, the expression of endogenous CD37 protein is reduced or absent in said cell.

Preferably, the genome of said cell comprises all or part of the human CD37 gene. Further preferably, the cell comprises the humanized CD37 gene described above.

In a twenty-fourth aspect of the present invention, there is provided a CD37 gene knock-out cell, wherein all or part of the nucleotide sequence of CD37 gene is deleted.

Preferably, the cell is deleted for all or part of exons 1 to 8 of the non-human animal CD37 gene. Further preferably, all or part of the nucleotide sequence of a combination of two or more consecutive exons among exons 2 to 8 of the non-human animal CD37 gene is deleted. Still more preferably, all or part of the nucleotide sequence of exons 2 to 8 of the non-human animal CD37 gene is deleted. In a specific embodiment, the deleted portion is a nucleotide sequence starting from the inside of exon 2 to the inside of exon 8 of the non-human animal CD37 gene.

In a twenty-fifth aspect of the invention, there is provided a construct expressing the humanized CD37 protein described above. Preferably, the construct comprises a humanized CD37 gene.

In a twenty-sixth aspect of the invention, there is provided a cell comprising the above construct.

In a twenty-seventh aspect of the invention, there is provided a tissue comprising the above-described cells.

Preferably, the tissue includes tissue that can develop into an animal subject and tissue that cannot develop into an animal subject.

In a twenty-eighth aspect of the present invention, there is provided a use of the humanized CD37 protein, the humanized CD37 gene, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, the above-mentioned tumor-bearing or inflammation model, the above-mentioned cell or cell line or primary cell culture, the above-mentioned tissue or organ or its culture, the above-mentioned tumor-bearing tissue, the above-mentioned cell, the above-mentioned construct, the above-mentioned cell or the above-mentioned tissue, the use comprising:

(A) use in the development of products involving CD 37-related immune processes in human cells;

(B) use as a model system in pharmacological, immunological, microbiological and medical research associated with CD 37;

(C) relates to the production and use of animal experimental disease models for the research of CD 37-related etiology and/or for the development of diagnostic strategies

And/or for use in developing therapeutic strategies;

(D) the application of the human CD37 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo;

alternatively, the first and second electrodes may be,

(E) the functions of the CD37 gene are researched, the medicine and the drug effect aiming at the target site of the human CD37 are researched, and the application in the aspects of the medicine for the immune-related diseases related to the CD37 and the anti-tumor medicine is researched. Preferably, the use includes therapeutic and diagnostic purposes for both disease and non-disease.

In a twenty-ninth aspect of the invention, there is provided a method of screening for a modulator specific for human CD37, said method comprising administering the modulator to an individual implanted with tumour cells and detecting tumour suppressivity; wherein the individual is selected from the group consisting of the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or a progeny thereof, or the above tumor-bearing or inflammatory model.

Preferably, the modulator is selected from CAR-T, a drug. Further preferably, the drug is an antibody.

Preferably, the modulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

Preferably, the detection comprises determining the size and/or proliferation rate of the tumor cells.

Preferably, the detection method comprises vernier caliper measurement, flow cytometry detection and/or animal in vivo imaging detection.

Preferably, the detecting comprises assessing the weight, fat mass, activation pathways, neuroprotective activity or metabolic changes in the individual, including changes in food consumption or water consumption.

Preferably, the tumor cell is derived from a human or non-human animal.

Preferably, the screening methods for modulators specific for human CD37 include therapeutic and non-therapeutic methods.

In one embodiment, the method is used to screen or evaluate drugs, test and compare the potency of candidate drugs to determine which candidate drugs can act as drugs and which cannot act as drugs, or to compare the potency sensitivity of different drugs, i.e., the therapeutic effect is not necessarily but only a possibility.

In a thirtieth aspect of the present invention, there is provided an evaluation method of an intervention program, the evaluation method comprising implanting tumor cells into an individual, applying the intervention program to the individual in which the tumor cells are implanted, and detecting and evaluating a tumor suppression effect of the individual after the application of the intervention program; wherein the individual is selected from the non-human animal, the non-human animal obtained by the construction method, the non-human animal or its offspring, or the animal model.

Preferably, the intervention regimen is selected from CAR-T, drug therapy. Further preferably, the drug is an antigen binding protein. The antibody binding protein is an antibody.

Preferably, the tumor cell is derived from a human or non-human animal.

Preferably, the methods of assessing the intervention program include both therapeutic and non-therapeutic methods.

In one embodiment, the assessment method detects and assesses the efficacy of an intervention program to determine whether the intervention program is therapeutically effective, i.e., the efficacy of the treatment is not necessarily, but is merely a possibility.

In a thirty-first aspect of the present invention, there is provided a use of the non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the above animal model in the preparation of a human CD 37-specific modulator.

In a thirty-second aspect of the present invention, there is provided a use of the non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, and the above-mentioned animal model in the preparation of a medicament for treating tumor, inflammation, or autoimmune disease.

Preferably, the non-human animal in all aspects above may be selected from rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc., which may be genetically engineered to become genetically humanized.

Preferably, the non-human animal in all aspects above is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or a mouse.

Preferably, the non-human animal in all aspects above is an immunodeficient non-human mammal. Further preferably, said immunodeficiencyThe non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

The "immune-related diseases" 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 diseases, diabetes, pain, or neurological disorders, etc.

The term "inflammation" as used herein includes acute inflammation as well as chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

"tumors" as referred to herein 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, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

The CD37 gene humanized non-human animal can normally express human or humanized CD37 protein in vivo, can be used for drug screening, drug effect evaluation, immune disease and tumor treatment aiming at a human CD37 target site, can accelerate the development process of a new drug, and can save time and cost. Provides effective guarantee for researching the functions of the CD37 protein and screening related disease drugs.

The invention relates to a whole or part, wherein the whole is a whole, and the part is a part of the whole or an individual forming the whole.

The humanized CD37 protein of the present invention includes part derived from human CD37 protein and part of non-human CD37 protein. Wherein, the "human CD37 protein" is identical to the whole human CD37 protein, namely the amino acid sequence of the "human CD37 protein" is identical to the full-length amino acid sequence of the human CD37 protein. The "part of human CD37 protein" is continuous or alternate 5-281 amino acid sequences identical to that of human CD37 protein. Preferably, the amino acid sequences are identical to the amino acid sequence of the human CD37 protein in a sequence or at intervals of 10-255, more preferably in a sequence of 5, 10, 20, 30, 40, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 255, 260, 270 and 281.

The "whole transmembrane region of human CD37 protein", "whole cytoplasmic region of human CD37 protein" or "whole extracellular region of human CD37 protein" according to the present invention means that the amino acid sequence thereof is identical to the full-length amino acid sequence of the transmembrane region, cytoplasmic region or extracellular region of human CD37 protein, respectively.

The "humanized CD37 gene" of the present invention comprises a part derived from human CD37 gene and a part derived from non-human CD37 gene. Wherein, the "human CD37 gene" is the same as the whole "human CD37 gene", namely, the nucleotide sequence of the "human CD37 gene" is consistent with the full-length nucleotide sequence of the human CD37 gene. The 'part of the human CD37 gene' is a continuous or spaced 20-5648bp (preferably 20-4606bp, 20-1259bp or 20-768bp) nucleotide sequence which is consistent with the nucleotide sequence of the human CD37 or has more than 70 percent of homology with the nucleotide sequence of the human CD 37.

The whole or part and the whole are integrated; a "portion" is a part of the whole, or an individual in the whole. For example, "all or part of exon No. 1 to exon No. 8" and "all of exon No. 1 to exon No. 8" are the whole, i.e., the whole nucleotide sequence of exon No. 1 to exon No. 8; "part of exon 1 to exon 8" is a whole individual, partially or wholly, i.e., one or more consecutive or spaced nucleotide sequences of exon 2 to exon 8.

The "exon" from xx to xxx or all of the "exons from xx to xxx" in the present invention include nucleotide sequences of exons and introns in between, for example, the "exons 1 to 8" include nucleotide sequences of exon 1, intron 1-2, exon 2, intron 2-3, exon 3, intron 3-4, exon 4, intron 4-5, exon 5, intron 5-6, exon 6, intron 6-7, exon 7, intron 7-8 and exon 8.

The "two or more continuous exons" as used herein refers to, for example, 2, 3, 4, 5, 6, 7, 8, 2, 3, 4, 3, 5, 4, 6, 5, 6, 7, 8, 2, 3, 4, 5, 6, 7, 5, 8, 5, 6, 5, 6, 4, 5, 7, 6, 5, 7, 8, 2, 3, 4, 5, 6, 5, 7, 8, 5, 6, 7, 4, 5, 6, 7, 8, 2, 3, 4, 5, 6, 7, 3, 4, 5, 6, 7, 5, 4, 5. 6, 7, 8 are consecutive, exons 2, 3, 4, 5, 6, 7, 8 are consecutive.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the portion of exon 2 of the human CD37 gene comprises contiguous or spaced nucleotide sequences of 5-73bp, preferably 10-64, identical to the nucleotide sequence of exon 2 of the human CD37 gene. In one embodiment of the present invention, the "part of exon 2" contained in the "human CD37 gene" at least comprises the nucleotide sequence from the first nucleotide sequence of exon 2 coding extracellular region to the last nucleotide sequence of exon 2. In a specific embodiment of the present invention, the "part of exon 8" contained in the "humanized CD37 gene" at least includes the nucleotide sequence from the first nucleotide sequence of exon 8 to the last amino acid encoded by the coding region.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "CD 37 locus" refers to a DNA fragment of any one of exons 1 to 8 of CD37 gene. In one embodiment of the invention, the replaced CD37 locus may be a DNA fragment of an optional stretch of exon 1 to 8 of the CD37 gene.

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

"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

"homology" in the context of the present invention refers to the fact that, in the context of using amino acid sequences or nucleotide sequences, a person skilled in the art can adjust the sequences to 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.

One skilled in the art can determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the crimyspascimyscimysciaenopsis (for example of the crimysciaeidae (for example of the hamsters, the new world rats and the new world rats, the rats and the rats, the. In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In a particular embodiment, the non-human animal is a rodent selected from the group consisting of BALB/C, A/He, A/J, A/WySN, 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 C57BL, C58 NOBr, A/Ca, PrCBA/34/CBA, PrCBA J, CBA/CBD, SCID-SCID strain ^scid IL-2rg ^null Background mice.

The practice of the present invention will employ, unless otherwise indicated, 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 documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mulliserial.u.s.pat.no. 4, 683, 195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds. inchief, Academic Press, Inc., New York), specific, Vols.154and 155(Wuetal. eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Caloseds, 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. week and c.c. blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: schematic comparison of mouse CD37 locus and human CD37 locus (not to scale);

FIG. 2: schematic representation of humanization of mouse CD37 gene (not to scale);

FIG. 3: CD37 gene targeting strategy and targeting vector V1 design schematic (not to scale);

FIG. 4: FRT recombination process schematic (not to scale) for F1 mouse;

FIG. 5 is a schematic view of: f1 mouse genotype identification results, wherein M is Marker, WT is wild control, and PC is positive control; h ₂ O is water control;

FIG. 6: CD37 gene targeting strategy and targeting vector V2 design schematic (not to scale);

FIG. 7: cas9/sgRNA activity detection results, wherein Con is a blank control, and PC is a positive control;

FIG. 8: f0 mouse genotype identification results, wherein M is Marker, WT is wild type control, H ₂ O is water control;

FIG. 9: genotype identification results of F1-generation mice, wherein M is Marker, WT is wild-type control, and H ₂ O is water control;

FIG. 10: southern blot assay results, where WT is wild type control;

FIG. 11: RT-PCR detection results of humanized mice (H/H) and wild type C57BL/6 mice (+/+) spleen tissues of CD37 gene, wherein H is ₂ O is water control;

FIG. 12: flow assay results for humanized mouse (H/H) and wild-type C57BL/6 mouse (+/+) spleen tissues of CD37 gene;

FIG. 13: flow detection results of humanized mouse (H/H) and wild type C57BL/6 mouse (+/+) lymph node tissues of the CD37 gene;

FIG. 14: flow detection results of peripheral blood of a humanized mouse (H/H) and a wild type C57BL/6 mouse (+/+) of the CD37 gene;

FIG. 15 is a schematic view of: PCR identification result of mouse tail of knockout mouse, wherein M is Marker, WT is wild type, H ₂ O is water control.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

In each of the following examples, the equipment and materials were obtained from several companies as indicated below:

BbsI, EcoRI, BamHI, AseI, EcoRV enzymes were purchased from NEB under the respective accession numbers R0539L, R0101M, R0136M, R0526M, R0195M;

c57BL/6 mice were purchased from the national rodent laboratory animal seed center of the Chinese food and drug testing institute;

ambion in vitro transcription kit purchased from Ambion, cat # AM 1354;

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

UCA kit comes from Baiosai chart company, Cat number BCG-DX-001;

Brilliant Violet 510 ^TM anti-mouse CD45Antibody, available from Biolegend under cat No. 103138;

Brilliant Violet 605 ^TM anti-mouse CD19Antibody, available from Biolegend under cat No. 115540;

PE anti-mouse CD37Antibody, available from Biolegend under cat number 146203;

FITC anti-human CD37Antibody was purchased from Biolegend, cat # 356303;

Zombie NIR ^TM fixable Viabilitkit was purchased from Biolegend, cat # 423106;

purified anti-mouse CD16/32Antibody, available from Biolegend under cat No. 101302;

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, available from Biolegend under cat # 108426;

Brilliant Violet 421 ^TM anti-mouse CD4Antibody, available from Biolegend under cat number 100438;

FITC anti-mouse F4/80Antibody from Biolegend, cat # 123108;

PE anti-mouse CD8a Antibody, available from Biolegend under cat No. 100708;

PE/Cy ^TM 7Mouse anti-Mouse NK1.1Antibody from BD Pharmingen, cat # 552878;

APC anti-mouse/rat Foxp3Antibody was purchased from eBioscience under cat # 17-5773-82;

FITC anti-Mouse CD19Antibody, available from Biolegend under cat # 115506;

PerCP/Cy5.5 anti-mouse TCR β chain from Biolegend, cat # 109228;

Brilliant Violet 605 ^TM anti-mouse CD11cAntibody, available from Biolegend under cat number 117334;

PE anti-mouse/human CD11bAntibody was purchased from Biolegend under cat # 101208.

Example 1 humanized mouse with CD37 Gene

A schematic comparison of the mouse CD37 Gene (NCBI Gene ID: 12493, Primary source: MGI: 88330, UniProt: Q61470, located at positions 44883056 to 44888701 of chromosome 7 NC-000073.7, based on transcript NM-007645.4 and its encoded protein NP-031671.1 (SEQ ID NO: 1)) and the human CD37 Gene (NCBI Gene ID: 951, Primary source: HGNC: 1666, UniProt ID: P11049, located at positions 49334961 to 49340608 of chromosome 19 NC-000019.10, based on transcript NM-001774.3 and its encoded protein NP-001765.1 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purpose of the present invention, a nucleotide sequence encoding a human CD37 protein may be introduced at the endogenous CD37 locus of a mouse, so that the mouse expresses a human or humanized CD37 protein. Specifically, the humanized CD37 locus schematic diagram is shown in FIG. 2, which is obtained by replacing the corresponding sequence of a mouse with a nucleotide sequence encoding human CD37 protein under the control of a mouse CD37 gene regulatory element by using a gene editing technology, so that the humanized transformation of the mouse CD37 gene is realized.

Further design the targeting strategy as shown in figure 3, which shows targeting vector V1 containing the homologous arm sequences upstream and downstream of the mouse CD37 gene, and an a fragment comprising the nucleotide sequence encoding human CD37 protein. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from position 44887988 to 44891655 of NCBI accession No. NC-000073.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is the same as the nucleotide sequence from position 44878915 to 44883324 of NCBI accession No. NC-000073.7; the nucleotide sequence of the human CD37 gene contained in fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence at positions 49335723 to 49340328 of NCBI accession No. NC-000019.10.

The targeting vector V1 also comprises a resistance gene for positive clone screening, namely a neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the connection between the 5' end of the Neo-box and the human CD37 gene is designed to be 5-

(SEQ ID NO: 6), wherein sequence "ACCCCThe last "C" in "is the last nucleotide, sequence, of a human

"G" in (1) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo cassette and the human CD37 gene is designed to be 5-

(SEQ ID NO: 7), wherein sequence "TGTACAThe last "A" in "is the last nucleotide of the Neo cassette, sequence

The first "G" in (a) is the first nucleotide of a human. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The protein sequence expressed by the reconstructed humanized mouse is shown as SEQ ID NO: shown at 9.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into embryonic stem cells of a C57BL/6 mouse by electroporation, the obtained cells are screened by using a positive clone screening marker gene, the integration condition of an exogenous gene is detected and confirmed by using PCR and Southern Blot technology, and correct positive clone cells are screened. The selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 4 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous mice for the CD37 gene. The genotype of somatic cells of progeny mice can be identified by PCR primer pairs WT-F/WT-R and WT-F/Mut-R (the primer sequences and the length of target fragments are shown in Table 1), and the identification results of exemplary F1 generation mice (with the Neo marker gene removed) are shown in FIG. 5, wherein 2 mice numbered F1-01 and F1-02 are all positive heterozygous mice. This shows that using this method, humanized mice of the CD37 gene can be constructed which can be stably passaged and have no random insertion.

TABLE 1 PCR detection of primer sequences and recombinant fragment sizes

In addition, a CRISPR/Cas system can be introduced for gene editing, and a targeting strategy as shown in FIG. 6 is designed, wherein a homologous arm sequence containing the upstream and downstream of a mouse CD37 gene and a human CD37 nucleotide sequence are shown on a targeting vector V2. Wherein, the sequence of the upstream homology arm (5 'homology arm, SEQ ID NO: 13) is the same as the nucleotide sequence from position 44887988 to 44889377 of NCBI accession NC-000073.7, the sequence of the downstream homology arm (3' homology arm, SEQ ID NO: 14) is the same as the nucleotide sequence from position 44881900 to 44883324 of NCBI accession NC-000073.7, and the nucleotide sequence of human CD37 is shown in SEQ ID NO: 5, respectively. The mRNA sequence of the humanized mouse CD37 after being transformed is shown as SEQ ID NO: 8, the amino acid sequence of the expressed protein is shown as SEQ ID NO: shown at 9.

Given that human CD37 and murine CD37 have multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, direct synthesis and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The correct targeting vector was verified by sequencing for subsequent experiments.

The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for constructing sgRNA expression vectors. sgRNA sequences recognizing the target site were designed and synthesized, and the target sequence of each sgRNA on CD37 gene was as follows:

sgRNA1 target site (SEQ ID NO: 15): 5'-GACCAGCTTCGTGTCCTTTGTGG-3'

sgRNA2 target site (SEQ ID NO: 16): 5'-GTGAGGGGGGCTGCGCAGTAGGG-3'

sgRNA3 target site (SEQ ID NO: 17): 5'-TCCTGATATCTCACTCGCCCTGG-3'

sgRNA4 target site (SEQ ID NO: 18): 5'-CCTGATATCTCACTCGCCCTGGG-3'

sgRNA5 target site (SEQ ID NO: 19): 5'-CTGATATCTCACTCGCCCTGGGG-3'

sgRNA6 target site (SEQ ID NO: 20): 5'-AGCTCTGACTGGCACCCCACTGG-3'

sgRNA7 target site (SEQ ID NO: 21): 5'-AGAGCGTCATGAAGCCGAGCTGG-3'

sgRNA8 target site (SEQ ID NO: 22): 5'-CCGGCTGGCCCGGTACCGCTAGG-3'

sgRNA9 target site (SEQ ID NO: 23): 5'-GGCTGGGGCCTAGCGGTACCGGG-3'

sgRNA10 target site (SEQ ID NO: 24): 5'-ATCTGGATCACGTCTATGACCGG-3'

sgRNA11 target site (SEQ ID NO: 25): 5'-GGATCACGTCTATGACCGGCTGG-3'

sgRNA12 target site (SEQ ID NO: 26): 5'-CCTAGCGGTACCGGGCCAGCCGG-3'

sgRNA13 target site (SEQ ID NO: 27): 5'-GGCACAGACGGAGGCTGTTGCGG-3'

sgRNA14 target site (SEQ ID NO: 28): 5'-GGGCTGGGGCCTAGCGGTACCGG-3'

The activity of multiple sgrnas is detected by using a UCA kit, and the sgrnas have different activities as shown in the results, and the detection results are shown in table 2 and fig. 7. From these, sgRNA5 and sgRNA12 were selected for subsequent experiments. The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide sequence and a reverse oligonucleotide sequence (see table 3), and after annealing, the annealed products are connected to pT7-sgRNA plasmid (the plasmid is firstly linearized by BbsI), so that expression vectors pT7-CD37-5 and pT7-CD37-12 are obtained.

Table 2 sgRNA activity assay results

Table 3 list of sgRNA5 and sgRNA12 sequences

pT7-sgRNA vector was synthesized by plasmid synthesis company as a fragment DNA (SEQ ID NO: 37) containing the T7 promoter and sgRNA scaffold, and ligated to a backbone vector (Takara, cat. No. 3299) by enzyme digestion (EcoRI and BamHI) in sequence, and sequencing by the professional sequencing company was verified, and the result indicated that the objective plasmid was obtained.

Taking mouse prokaryotic fertilized eggs, such as C57BL/6 mice, and injecting in-vitro transcription products of pT7-CD37-5 and pT7-CD37-12 plasmids (using an Ambion in-vitro transcription kit and performing transcription according to the method of the specification) and a targeting vector and Cas9mRNA into cytoplasm or nucleus of the mouse fertilized eggs after being premixed by using a microinjector. Microinjection of fertilized eggs was performed according to the method in the manual of experimental manipulation of mouse embryos (third edition), published by chemical industry, 2006, and the fertilized eggs after injection were transferred to a culture medium for short-term culture and then transplanted into the oviduct of a recipient mother mouse for development, and the obtained mice (generation F0) were subjected to hybridization and selfing to expand the population number and establish a stable humanized mouse strain of CD37 gene.

The somatic cell genotype of F0 generation mice can be identified by conventional detection methods (e.g., PCR) using 5 'and 3' primers (see Table 4 for PCR primers), and exemplary identification results for some F0 generation mice are shown in FIG. 8. The 5 'end primer detection result and the 3' end primer detection result are combined, and the mouse with the number of F0-01 in the figure 8 is further verified to be a positive mouse through sequencing.

TABLE 4PCR detection primer sequences and recombinant fragment sizes

Wherein the primer L-GT-F is located at the left side of the 5 'homology arm, R-GT-R is located at the right side of the 3' homology arm, and L-GT-R, R-GT-F is located on the human CD37 sequence.

Humanized mice with the CD37 gene identified as positive for F0 were mated with wild-type mice to give F1-generation mice. Genotype identification can be performed on F1 generation mice using the primers in Table 4, and exemplary test results are shown in FIG. 9, showing 2 mice numbered F1-01 and F1-02 as positive mice.

Southern blot assays were performed on mice identified as positive for F1 PCR to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting genome with AseI enzyme or EcoRV enzyme, transferring membrane, and hybridizing. The 5 'probe and the 3' probe are located outside the 5 'homology arm and on the 3' homology arm, respectively, and the lengths of the specific probes and the target fragment are shown in Table 5. The Southern blot results are shown in FIG. 10, and the results of combining the 3 'probe and the 5' probe indicate that 2 mice numbered F1-01 and F1-02 had no random inserts. This shows that the method can be used for constructing the humanized gene engineering mouse of the CD37 gene which can be stably passaged and has no random insertion.

TABLE 5 length of the particular probes and target fragments

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				AseI	5’Probe	6.0kb	4.3kb
EcoRV	3’Probe	10.0kb	12.8kb

The probe synthesis primers were as follows:

5’Probe-F(SEQ ID NO：42)：5’-CTGTCTGGGTGAAAAGGTCCGGG-3’，

5’Probe-R(SEQ ID NO：43)：5’-AAGGAATCAAAGCACTGGGGTCACA-3’；

3’Probe-F(SEQ ID NO：44)：5’-TCACAACCTGCCAGAACAGGCT-3’，

3’Probe-R(SEQ ID NO：45)：5’-AGGGAGTTCCTTTGATTTGGGGGT-3’；

heterozygous mice identified as positive in the F1 generation were mated with each other to obtain humanized homozygote mice of the CD37 gene in the F2 generation.

The expression of humanized CD37 mRNA protein in positive mice can be confirmed by conventional detection methods, for example, RT-PCR. 1 each of 10-week-old female wild-type C57BL/6 mice and the humanized homozygote CD37 mouse prepared in this example was selected, spleen tissues were collected after cervical dislocation, and RT-PCR was performed using the primer sequences shown in Table 6, and the results are shown in FIG. 11. As can be seen from the figure, only murine CD37 mRNA was detected in wild type C57BL/6 mice (+/+), and only humanized CD37 mRNA was detected in humanized homozygote CD37 (H/H) mice.

TABLE 6 RT-PCR detection of primer sequences and recombinant fragment sizes

Detection of mouse body by flow cytometryExpression of the internal humanized CD37 protein. Specifically, 1 mouse of 10-week-old wild-type C57BL/6 male mice and 1 mouse of humanized homozygote of CD37 gene prepared in this example were each selected, spleen tissues were harvested after cervical euthanization, spleen single cell suspensions were prepared, and the anti-mouse CD45antibody Brilliant Violet510 was used ^TM anti-mouse CD45Antibody (mCD45), anti-murine CD19Antibody Brilliant Violet 605 ^TM Flow detection is carried out after anti-mouse CD19Antibody (mCD19), anti-mouse CD37Antibody PE anti-mouse CD37Antibody (mCD37) or anti-human CD37Antibody FITC anti-human CD37Antibody (hCD37) are identified and stained, and detection results show that the proportion of mouse CD37 protein (characterized by mCD45+ mCD19+ mCD37+) is 95.3% and the proportion of humanized CD37 protein (characterized by mCD45+ mCD19+ hCD37+) is 0.36% in wild type C57BL/6 mouse spleen B cells (characterized by mCD45+ mCD19 +; the proportion of humanized CD37 protein (characterized by mCD45+ mCD19+ hCD37+) and the proportion of mouse CD37 protein (characterized by mCD45+ mCD19+ mCD37+) in the spleen B cells of a humanized homozygote mouse with the CD37 gene were 91.9% and 0.75%, respectively. The results show that the humanized mouse with the CD37 gene can successfully express the humanized CD37 protein.

Further adopting flow cytometry to detect the immunophenotyping condition of the leukocyte subtype and the T cell subtype in the mouse body. 3 female wild-type C57BL/6 mice of 7 weeks old and 3 humanized homozygote mice of CD37 gene prepared in this example were selected, and after cervical dislocation, spleen (spleens), lymph node (lymph node) and peripheral blood (blood) were collected and used as anti-mouse CD45antibody Brilliant Violet510 ^TM anti-mouse CD45Antibody (mCD45), anti-mouse Gr-1 Antibody PerCP anti-mouse Ly-6G/Ly-6Cantibody (mGr-1), anti-mouse CD4Antibody Brilliant Violet 421 ^TM anti-mouse CD4Antibody (mCD4), anti-mouse F4/80Antibody FITC anti-mouse F4/80Antibody (mF4/80), anti-mouse CD8 Antibody PE anti-mouse CD8a Antibody (mCD8), anti-mouse NK1.1Antibody PE/Cy ^TM 7Mouse anti-Mouse NK1.1Antibody (mNK1.1), anti-Mouse Foxp3Antibody APC anti-Mouse/rat Foxp3Antibody (mFoxp3), anti-Mouse CD19Antibody FITC anti-Mouse CD19Antibody (mCD19), anti-Mouse TCR beta Antibody PerCP/Cy5.5 anti-Mouse TCR beta chain Antibody (mTCR beta), anti-Mouse CD11c Antibody Brilliant Violet 605 ^TM anti-mouse CD11c Antibody (mCD11c), and anti-mouse/human CD11b Antibody PE anti-mouse/human CD11b Antibody (mCD11b) were identified and stained for flow assay. Among them, T cells are characterized by mCD45+ mTCR β +, B cells are characterized by mCD45+ mCD19+, NK cells are characterized by mCD45+ mTCR β -mnkg 1.1+, dendritic cells are characterized by mCD45+ mTCR β -mCD11c +, granulocytes are characterized by mCD45+ mGr-1+, monocytes and macrophages are characterized by mCD45+ mGr-1-mCD11B + mF4/80+, helper T cells (CD4+ T cells) are characterized by mCD45+ mCD4+ mfxp 3+, and killer T cells (CD8+ T cells) are characterized by mCD45+ mCD8 +.

The results of the detection of spleen, lymph node and peripheral blood are shown in FIGS. 12, 13 and 14, respectively. As can be seen from the figure, the percentage of leukocyte subtypes including T cells, B cells, NK cells, Dendritic cells (Dendritic cells), Granulocytes (Granulocytes), Monocytes (Monocytes) and Macrophages (Macrophages) in humanized homozygous mice (H/H) of the CD37 gene substantially coincided with that of wild-type C57BL/6 mice (+/+); the percentages of T cell subtypes, including CD4+ T cells, CD8+ T cells, regulatory T cells (tregs), were also substantially identical to wild-type C57BL/6 mice, indicating that humanization of the CD37 gene in wild-type mice did not affect the overall development, differentiation or distribution of leukocyte subtypes and T cell subtypes in the spleen, lymph nodes and peripheral blood of the mice in vivo.

In addition, since the cleavage of Cas9 causes double strand break of genomic DNA, insertion/deletion mutations are randomly generated by the repair mode of chromosome homologous recombination, and it is possible to obtain a knockout mouse with the function of CD37 protein being lost. For this purpose, a pair of primers was designed for detecting knockout mice, and the results are shown in FIG. 15, and the mice with the number KO-1 was further verified to be CD37 knockout mice by sequencing. The primers were located on the left side of the 5 'target site and on the right side of the 3' target site, respectively, and the primer sequences and recombinant fragment sizes are shown in Table 7.

TABLE 7 identification of PCR primer sequences and recombinant fragment sizes for CD37 Gene knock-out mouse genotypes

Example 2 preparation of double-humanized or multiple double-humanized mice

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared CD37 gene humanized mouse. For example, in example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3, and CD20, or can be used to obtain a two-gene or multi-gene modified mouse model of CD37 and other gene modifications based on humanized CD37 mice by using isolated mouse ES embryonic stem cells and gene recombination targeting technology. The homozygote or heterozygote of the CD37 mouse obtained by the method can be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of humanized CD37 and heterozygote of double gene or multiple gene modification of other gene modification can be obtained with a certain probability according to Mendel genetic rule, the heterozygote is mated with each other to obtain homozygote of double gene or multiple gene modification, and the in vivo efficacy verification of targeted human CD37 and other gene regulators can be carried out by utilizing the mouse of double gene or multiple gene modification.

Example 3 drug efficacy verification

The humanized mouse with CD37 gene or the mouse with multiple gene modifications prepared by the method can be used for evaluating the drug effect of the targeted human CD37 antibody. For example, a tumor animal model is constructed by taking a humanized homozygote mouse of the CD37 gene, the mouse is divided into a control group or a treatment group, the treatment group is injected with an antibody medicament targeting human CD37, and the control group is injected with an equal volume of physiological saline. Then the body weight, the tumor volume and the tumor related indexes of each group of mice are monitored, and the safety and the in vivo efficacy of the antibody drug in the humanized CD37 mice can be effectively evaluated.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Baiosai Diagram (Beijing) pharmaceutical science and technology Co., Ltd

Construction method and application of <120> CD37 gene humanized non-human animal

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<151> 2021-06-11

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<151> 2021-11-09

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35 40 45

Leu Ser Phe Val Pro Leu Gln Thr Trp Ser Lys Val Leu Ala Val Ser

50 55 60

Gly Val Leu Thr Met Ala Leu Ala Leu Leu Gly Cys Val Gly Ala Leu

65 70 75 80

Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr Phe Gly Met Leu Leu Leu

85 90 95

Leu Phe Ala Thr Gln Ile Thr Leu Gly Ile Leu Ile Ser Thr Gln Arg

100 105 110

Val Arg Leu Glu Arg Arg Val Gln Glu Leu Val Leu Arg Thr Ile Gln

115 120 125

Ser Tyr Arg Thr Asn Pro Asp Glu Thr Ala Ala Glu Glu Ser Trp Asp

130 135 140

Tyr Ala Gln Phe Gln Leu Arg Cys Cys Gly Trp Gln Ser Pro Arg Asp

145 150 155 160

Trp Asn Lys Ala Gln Met Leu Lys Ala Asn Glu Ser Glu Glu Pro Phe

165 170 175

Val Pro Cys Ser Cys Tyr Asn Ser Thr Ala Thr Asn Asp Ser Thr Val

180 185 190

Phe Asp Lys Leu Phe Phe Ser Gln Leu Ser Arg Leu Gly Pro Arg Ala

195 200 205

Lys Leu Arg Gln Thr Ala Asp Ile Cys Ala Leu Pro Ala Lys Ala His

210 215 220

Ile Tyr Arg Glu Gly Cys Ala Gln Ser Leu Gln Lys Trp Leu His Asn

225 230 235 240

Asn Ile Ile Ser Ile Val Gly Ile Cys Leu Gly Val Gly Leu Leu Glu

245 250 255

Leu Gly Phe Met Thr Leu Ser Ile Phe Leu Cys Arg Asn Leu Asp His

260 265 270

Val Tyr Asp Arg Leu Ala Arg Tyr Arg

275 280

<210> 2

<211> 281

<212> PRT

<213> human (human)

<400> 2

Met Ser Ala Gln Glu Ser Cys Leu Ser Leu Ile Lys Tyr Phe Leu Phe

1 5 10 15

Val Phe Asn Leu Phe Phe Phe Val Leu Gly Ser Leu Ile Phe Cys Phe

20 25 30

Gly Ile Trp Ile Leu Ile Asp Lys Thr Ser Phe Val Ser Phe Val Gly

35 40 45

Leu Ala Phe Val Pro Leu Gln Ile Trp Ser Lys Val Leu Ala Ile Ser

50 55 60

Gly Ile Phe Thr Met Gly Ile Ala Leu Leu Gly Cys Val Gly Ala Leu

65 70 75 80

Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr Phe Gly Met Leu Leu Leu

85 90 95

Leu Phe Ala Thr Gln Ile Thr Leu Gly Ile Leu Ile Ser Thr Gln Arg

100 105 110

Ala Gln Leu Glu Arg Ser Leu Arg Asp Val Val Glu Lys Thr Ile Gln

115 120 125

Lys Tyr Gly Thr Asn Pro Glu Glu Thr Ala Ala Glu Glu Ser Trp Asp

130 135 140

Tyr Val Gln Phe Gln Leu Arg Cys Cys Gly Trp His Tyr Pro Gln Asp

145 150 155 160

Trp Phe Gln Val Leu Ile Leu Arg Gly Asn Gly Ser Glu Ala His Arg

165 170 175

Val Pro Cys Ser Cys Tyr Asn Leu Ser Ala Thr Asn Asp Ser Thr Ile

180 185 190

Leu Asp Lys Val Ile Leu Pro Gln Leu Ser Arg Leu Gly His Leu Ala

195 200 205

Arg Ser Arg His Ser Ala Asp Ile Cys Ala Val Pro Ala Glu Ser His

210 215 220

Ile Tyr Arg Glu Gly Cys Ala Gln Gly Leu Gln Lys Trp Leu His Asn

225 230 235 240

Asn Leu Ile Ser Ile Val Gly Ile Cys Leu Gly Val Gly Leu Leu Glu

245 250 255

Leu Gly Phe Met Thr Leu Ser Ile Phe Leu Cys Arg Asn Leu Asp His

260 265 270

Val Tyr Asn Arg Leu Ala Arg Tyr Arg

275 280

<210> 3

<211> 3668

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctggggaat gtagctcgtt tggttgaatg cttgccctga atgcacaggg ccctaggttt 60

cacccccagt atctcattag aggagttttg atggcctgtg acagaaatca cagtccttgg 120

gaagtagaga tgagaggact tgaataaagt cctttgttat ccttgaccac acaacgtgtt 180

ctaggccagc ctgggctagt tgagaccctg tctcaaaagc taatcaagtt gaggccaaca 240

agatggctca gtgggtaaaa gcacttgctg ccagttctga tgacctacat tccatcctgt 300

tctccacatg aagacaagag agaactaact cccacaagtt gtcttctgac ccccacacat 360

gtcagggcac acgcaccaga gactcatatc taaacgatca gtcaatcact agaccgcaat 420

aacttttcaa agataggcat ggtgaggaag tagaggttta cctgcggcac acaaaacttc 480

acaactaggt attttgccca tatcaaaata aatgagtcat ctcttcaaat acaataagtc 540

aaaaatctca aactttgata gtgttgtccc tgactctgta gtccatcgga caatagtaac 600

aatttaataa caggcaagtt gtgtgaaagg agatcgattt gcctaactag aacttgttta 660

aaaacaagca gaagtgaaaa cagttttctg gacaatccgg tcaactggaa ctttcagttg 720

cagaactgag cgctggattt gattcctcgt ttaatgtatt catttctccg gtcagtactt 780

ggacttgggc cttgtccctg ctaggcaagt gatctctaac tgagctccag ccccagccac 840

tcctgaaccc tccgacccca ggactgagcc cgtggctgtc acaggtccct aggagcctcc 900

tcggaaacgc cccctgtgct cactttacct ccttccctgg aactatggcc tcagaaggga 960

aagcaagctg gtgtcctgag tcctccgaac aggaggtggc tgtccctagc gggcgggaaa 1020

tgaagccggg tcgggttgcc gcctcttagg cgccttccca atagagcagc cgtcaagagt 1080

tgcgggaggt tgactctcag gcctggcctc aggcaggcgc agccgaggag cgaggaggca 1140

gttttccggg ttctgacgga gagggggcac ggtgaccttg acctcgcgac tcccccctcc 1200

ctccctcctt cctggcgtcc tgtggagagc ctggggcagc acaatggccg ccccgtcggt 1260

ctcaccgccc gggcctcgag atccccgctg aactctccag cggctgtctg ggtgaaaagg 1320

tccgggaggc ctcggcatgg aagcttagcg acctccccag gccctccggc cccctggaag 1380

gcagttccca catcgtgaac ccacgaggcc ttccctgccc tttctgtgtc tctgcgctat 1440

tgctcccgtc tcgcctcctt cctctaccca cgcgaaggag tttgctgtgg cgcttccggt 1500

gcgcgtgcac atacacgtgc tcgcagcttc tgactgcgcg tgcgccgcag cgcctttgcc 1560

tctgggcgca gccttccacc cctgtgtgac cccagtgctt tgattccttg atgccacagg 1620

cagaaatggt gcaaagactt aatcttcaca attattccgt gttgatttca aaccactgtt 1680

aattactaaa cgcctccctt tcaggagcaa aataaacaca cacacacaca cacacacaca 1740

cacacacaca cacacacaca cacacaaagg ccaattaggg attttaagga cagcagataa 1800

gaattaaaaa ctggcaaatg agaggcccag atgtgccacc aaacggtgtt ccatcccaga 1860

ccccacagga ggaagggaag ataagctaag tgtctcaacc tatctgtccg attattggga 1920

caatgaagca caaagaaagg ggaatctccc cctaggaatc tcatgctggg aaagggaaaa 1980

gatgccaacc aatggttagc gtgtccctga ataggatttc acacggctct aagagcttgg 2040

gcgaccacag gttgaggttg aagcccagac gatggtgggt cagtggtggg taggaccgca 2100

tggtggactg catagacatg cataacatta gccttctatt tacacctcag tctcagtgag 2160

ggaccccaaa aatgaattga aggggatcac cagatcgctt tgtgcccttc taaatgtgac 2220

cacactgtgt gtaaaccttt ttctgtttcc tctagaaatt tggttctttt acttggctta 2280

ttggagatgg cagaacctgc cttggagcac agcctgtggc aggagaggcc cagtcccgaa 2340

aatttatcta tcttctgccc tctataagca ccatgtggcg cagacataca cacacacaca 2400

catcgttaaa atttttttaa aaaaagatga acagcttcca atgttgtcct ctggcctcca 2460

aaggcgcgcg cgcgcacaca cacacacaca cacacacaca cacacacaca cacactaaca 2520

catacaaata cacatacaga aagaaaatag atgagacatg tctcagaagg cagctgctgt 2580

cccagagaac taggctttgg gtagataggt gataggaact tcccacatta gaaatctgtg 2640

aggaactggg gacttgacaa gtgtcaggga cttctttctg ggcacatcat aggaaacttt 2700

gaagatgcta ggtatgctca agtgaccccc actccagtgc ctggcttttc tggggcctcc 2760

tcccctcagg tttctccgca gaggtaggaa gtgccatctg aggttcactt cctccaccgg 2820

atattcaggc actgcctcgc tgccctcctc actcctggga ctgtcacacc tcctctgcag 2880

ccctttcact tccccagggg tccaaccctg gcccccaagc acatcggagc acctgctcta 2940

gaagaaagag agcaaagaga gagacccagg gaaagagaca cacaggacag agaccgcaaa 3000

ggcccagaga gacagagaca gagaggcaga gacagcgcgg tggctcgagg ctaatgggtg 3060

ctaagcagaa gatcacaggg tgcagggggc ggagccagta gccagtgagg tcagagtgtc 3120

tgtgaggttg aactctgcag aaacaacctt gaagtcccag gacccacgtg agtggaagtc 3180

catcagaagc ccctgtgcgc cctgcctgag ctatggacac ctgcgaggaa cccattgtgt 3240

ccctggcacc cacatattcc aaggaccctc aggtactgct gtgcactcga tggaaccctg 3300

aagaagatag ggcccccgct gacagtcact aaatgaacag ggcggggtgg agctccaccc 3360

ccccggaact tcctctttct ctgccggttt cctttgtgcc tctcagtctc tgtctttctt 3420

tgcccactat tccccttccc ccctgcggcc aacacctctt ctgtctgggt ttctcgagtg 3480

ggtcattccc catctaggcg aagatgtccg cccaagagag ttgcctcagc ctcatcaagt 3540

acttcctctt cgttttcaac ctcttcttct ttgtgagtct actcatggct acccagccgg 3600

ggttccaggg ccacagtggc ccttgatccg ccctagcctc atttcttctc ttcccctagg 3660

tactaggc 3668

<210> 4

<211> 4410

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gccccagccc acccatcggc acccacgcac ctccatgcag tcaggaaatg tttccttcct 60

ggttggtcac aggcttggag cccatgcctc aggcctcatc ccctggggac ccagctgtct 120

gccctatcga cagccttcac tttccccaca tggccaggga cttttgtgcc cagcttcctt 180

cccttcctgg ctccctcctc cccacaccat ctgtctgcca cctcccacat gacacctcaa 240

ataaatcccc tttgggtttt tggctttttt tttttttttt tggtaaaaat agctttattc 300

tccttcaaac ataaaccatc actcttgggg aaaggaaggt ggcagggtgg tccaaggctc 360

acttaaatgg ggtgggggag attaagaagt cccaccccac tgcctagctg agataagatt 420

acatccctaa cactgtgtat aaatatctcc ttatattaaa attttttttc aggtcccact 480

tcaccctacc ccaagctggg aaaacattat cttcgggttg tgacgtcaca gttcccagag 540

aaagggtgtg gggggagagg cagatatgag acacgtggga agcaggaagg ttacaaggca 600

gtcagtaaga caccccacat agcccagctc cctttccaga agagcaagga ggggccctgt 660

agaacggagg cccgtgctgt acctgtgagc tcactcctgt ccccaatccc tgatgctcag 720

gagtggtgtg tcctgtcaaa tgttgtctct gaggtcctcg tgttggggat ctaaactcaa 780

acccagctca ctcagctcaa taaccaggct tccggttcct ttctaagagg agccagcccc 840

cgtggctcat gcttctgaga ttaggccttt ttaaaaatca tgggcccctc tccaaatctc 900

tcttacagag agagcaaaag ggcccttccc aggaaggggg tgttgtgaaa aacaccagcg 960

gcgggggctc catcagtccc tgaccaggcg gtagatgtgg tactgggctc ctcgttcact 1020

ggtggagact tcaaagacgt aggcagtaca cagcagcagt tcctgagtgt ccctgtttgt 1080

cacaacctgc cagaacaggc ttacattgta gggtggggat ggggtacagg acatggactg 1140

ccctgcccac cctagttatc aagaaagaga aacctgccat gctatgtacc ccaagaagaa 1200

tgtcctgcgg ctcaggttcc ttgggcaatg aggttacccc taagctgaga gatggtagga 1260

aggggcctcc ccatcatatc acattcccga gaaaagggtt ccccaccatg ccaccaggca 1320

gagagagagg gtcagcagat catattagca caacccccaa atcaaaggaa ctcccttctc 1380

tgaacttctc agagaagcca gatgcttaga agccgaggta tctcagctgc cctagggaag 1440

atgggctctc cacataggac agctcggtcc tgtcctcccg cccccacccc caccatgttc 1500

tggatgaagc aatctcatct ttggagtccc caggagctga gggtctgcct gccccagcgt 1560

atcacctgga ggatggtgaa gttctccagg acactgttca tcatgtagcg ttcaggcagc 1620

tgacggagct tgtgcaggaa attaaccagg tactcacaca tgggagagcg cagcagacgg 1680

tacacaaagc gcccgtcctc cagctgggcc cgttccgtct gggagtggga aacacagtag 1740

ctgagagaga tcacggccac cctaggaccg aagcttcacc cttagcctct gagaggccac 1800

tgtcaactcc cttggccaga gtccctcaag caacctcaac tttaatgacc agagacccta 1860

ggcctctgaa cctgcaaaca aggtaccccc cccccccttc tcctctgtca tggtctgaaa 1920

ccccagtgac ctccaaactc cctcaatagc atctccacgc tgagcttgag gcttcaagac 1980

ctcacaggcc ctgaagtata acatacctac tggctaatta cacagctcct accataccct 2040

taagagtctt tgtcatcctg cagaatccat ctctatacct ttcaaccctt gaaataagcc 2100

cgagttgtga cttttaatgg cttgtttatg tttagtggtt tgagacaggg tctcatgtag 2160

cccaggctag cctggaactc cttatgtagc taaggataga cttgaaactc tcgatgcttg 2220

gtttacatga gggtaagcac tccacaaagt gaactatatc ctcagccttt gaagtcttga 2280

gttaattcct agtgcatata ctaactgcca gagaagcggg gatagccctg cctcacacac 2340

acacacacac acacacacac acacacacac acacgcaatg ctgtgactgc ttcctcgaga 2400

tgcatgcagt accctagccc tgggtcttac ctccaccttc tctaccactt gcttgccaaa 2460

ggagcagacc ttggaggagc aggtgagtgt catgagctcc cggctctcat actggctgct 2520

cactccatag aagccaccgc cacctccgct gctccctgcc tcctcggcac tggggcccca 2580

gttcaggtcc gcctgagagg agatagaggg gcacgggtga gctggcggga acccagcaaa 2640

tgctgaccca gagcctggct ccgagctgca cacacttgcc cgtgaccaca ccctgactca 2700

gcttccaaag accccaggtc agcatgagct gtgctctata gaagacttac ctactgttcc 2760

aggaggacta gagaagccaa ggtccccaga atagtcctga ctctcattct ctaggagcca 2820

tacaatccaa ctgttttgcc tgaagcccct agctctctag agttcagctc tactcaggca 2880

tagcagtgca agcttataat gccggcactt aagaggccga tacagagccg ggcgtggtgg 2940

cgcacgcctt taatcccagc actcgggagg cagaggcagg cgaatttctg agttcgaggc 3000

cagcctggtc tacagagtga gttccaggac agccagggct ataaagagaa accctgtctc 3060

gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaccaa aaaaaaaaaa aaagaggccg 3120

atacaggggg gctgtgagtt tgagaccaac atggtcacat acagtgggag actgggtctc 3180

aaaatgagaa gaaaaaaaaa aggaaaaaaa aaacaaaaga gggaagatgg ggttggaagt 3240

tttaagaaaa tggagggggg ccgggcagtg gtggcgctct cctttaatcc tagcacttgg 3300

gaggcagagg caggcggatt tctgagttcg aggccaacct ggtctacaga gtgagttcca 3360

ggacagccag gactacacag agaaaccctg tttcgaaaaa gaaaaaaaga aagaaaaaaa 3420

gaaaggaaaa gaaaaggaaa ggaaatggag gaactagaga aagcacccaa ggaactaaag 3480

ggatcttcaa ccctatgggt ggaacaacaa tatgaactaa gcagtacccc ggagctcttg 3540

tctctagctg catatgtatc aaaagatggc ctagtcggcc atcactgcaa agagaggccc 3600

gttggacttg caaactttat atgccccagt acaggggaac gccagggcca aaaaggggga 3660

gtgggtgagt aggggggtgg gtgggtatgg gggacttttg gtatagcatt ggaaatgtaa 3720

atgagctaat aaaaaatgga aaaggaaagg aaaagaagat ggaggggaga caaatgaatg 3780

ttagctctgc caagggccga gttactagca ggcatgttac ccctagcaac tggttatggt 3840

ttcatatcct ccctcaaccc tccccagact ggccgtgaaa tcaagtggtg actacccatg 3900

agtagtaagg cctacctccc ccctagagca ctatctgtca atcaggaacg cactcttgct 3960

catccaaagg ccaggcgggc agagctgggc aagtcttata aagcctgtgg tcttgagagt 4020

ggagagtgcc tgttctctgt cctgacttta gctgcagctc tttggccagg actccaccta 4080

ccaccccagc tacaagcatt tcaatacaag gcaaatccct atgcctgtgg ccacagctca 4140

ccactggaac cccccagcac ttaggaggct gaagccagaa gaaaccagcc taggctacac 4200

tgtgaagggg aatctgcctg acaatggaag accacctggg ttggtgttca gccttaatcc 4260

cagcgcttgg aggcagagga aggcaagtct ctggagttca aggccagcct gggctacaga 4320

gctggttcga ggacaggtag gaaggccaca gcagctgctt ccaacagcct gtaacagccg 4380

tttctgagcc atctcaggcc tccacccaga 4410

<210> 5

<211> 4606

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcctgatct tctgcttcgg catctggatc ctcattgaca agaccagctt cgtgtccttt 60

gtgggtgagg ggggctgggg caggtgggag ggcctccccc aacccaagca acttcctggg 120

gtctcccttg tctcagggag acctacggtg ccctactctg caggcaggct acaggctccc 180

atccactgct caccggaggc ttcttggagc ctgagtcttc ttccggcaaa tggggttgtg 240

catacaaaca acaatgatca tgagagccat ttctcaagca cttcctatct gtcgggactt 300

gtggctggtc agatgccccc acaaaccttc ccctcgcttg aggttcagag aggggaagcc 360

ccttgatgga tgtcatacag caaggacgta aaagagccaa aataacatta ataataggaa 420

aataataatg aagtatgacc ctaaataata atgaaatata accctaaacg ctcatattta 480

agaagtcttt gccctgtgtt gaggactggg ttgaacacat gtcctcatgt gtctctcaaa 540

atgaccctag gggccgggcg cggtggctca cacctgtaat tccagcactt tgggaagccg 600

aggcaggtgg atcacttgag gtcaggagtt cgagaatagc ctggccaaca tggtgaaacc 660

ctgtctctac tcaaaaatac aaaacgttag ccaggcatgg tggcacatgc ctgtagtcgc 720

agctactagg gaggctgagg caggagaatc acttgaacct gggaggcaga ggttgcagtg 780

agccgagatc acatcactgc actccagcct gggggacaga gcgaggctcc aactcaaaaa 840

aaaaccctaa gaaatagata tcatctccta tgcctcaggg aggtttaagg agccaagcca 900

atgtcacaga acaagtgata gggctgaaac tactggggac gaggaggagc tgaaggagga 960

aggagagggt aagaggaaga gagacagaga tccagggacg ggaaacaggc ccagaaagaa 1020

gagaaagttt cagaaggaaa cagggactca gggtggggca gggacagagt cccagagaga 1080

ggggcaccaa gatactgctc cccacttggg tggctgccta ggtgggaaat ggggctgcct 1140

ggagctgtgc cacacagctc atcatcactc ccctcacctc tcccaggctt ggccttcgtg 1200

cctctgcaga tctggtccaa agtcctggcc atctcaggaa tcttcaccat gggcatcgcc 1260

ctcctgggtt gtgtgggggc cctcaaggag ctccgctgcc tcctgggcct ggtgagtgca 1320

ccatccctct ccgtccctag gaacactcct atccccaccc tcaaaaatgg cctgggccgg 1380

ggttgcaggg gtggtcagcc tgatctctcc actctgctcc ccagtatttt gggatgctgc 1440

tgctcctgtt tgccacacag atcaccctgg gaatcctcat ctccactcag cgggcccagg 1500

tgagcttcct gcagtggcca ccacccaccc ccagcaggga ggagagggaa gggagtggtg 1560

ggagagggtg gagagagacc gaaacaaggg cagacagggg ctaacctaga taaagagaaa 1620

taagaggctg ggcctggtgg ctcacgcctg taatcccata atcccagcac tttgggaggc 1680

tgaggtggga gtccaggagt ttgagaccag cctgggcaac atagtgagac cccatctata 1740

tttaaaaaaa aaatccgggt gtgatggtgt gcacctgggg tcccagctac ttgggaggtc 1800

gaggtgggag gatcacttga gccagggagt ttgaggctac agtgagccat gcactcagcc 1860

tgggtgacag agtgagaccc tgtctcaaaa aataaattaa tagaaagaca caccgacctg 1920

gaccaaggga gacaggcata aacagctcca aagggaaaca cacgggaaaa agagagaaca 1980

agagaaagaa atagacgccc tgaaagaaga gacagagact tatgagccgt agaaatagtg 2040

gaagaaacag aaagacagac ctgaagtaca cagagctagc cattcagtaa ggatttgaga 2100

ctggcccaga gatgcacagg gcccgcctga ggctggcaca gcctgagagg gggaggggtg 2160

gggcggggaa gataaggccc agcctcactg gtggcctctc agctggagcg aagcttgcgg 2220

gacgtcgtag agaaaaccat ccaaaagtac ggcaccaacc ccgaggagac cgcggccgag 2280

gagagctggg actatgtgca gttccaggta agcccccctc ctccagttcc ctcccggact 2340

gacccgcctc agccctgtgc ttggaggaga ctccacccca acgtgggccc gacccccagc 2400

tctacgatcc taacacaccc caatccctcc caggcccgac gctccccact ccccagatga 2460

cacaactgtc cccggcgtcg cctggtctcc cagtacccag accctggcgt ggcttcgcca 2520

tctacctcga gagactccgc ccccgccccc gccccgacca gaggttgtca ggcccctagt 2580

cccaagaccc tagcgagaca cggcttccta ccccgtagtg actctggctt ccaccggacc 2640

ccgcccccga tgagactcac atggtctcgc actcctattc acctgtcggg tatcagaggc 2700

tcaattgcac cacccaccca atctctagtc cacagcgcga ccctgcagct ccttccccag 2760

ccccaggtca ccccgtgact cgtctttccc agccccatgt tcccgtaatg tcccctggct 2820

ccggccccat cgtgacccca gcccactgtc ccccactcca cacccaccac ttagtcccct 2880

gctccccgac ctgacctcat acccatcacc ttgtcccctg atccccaaca tcatatgtct 2940

ccagtcccgg tccctctgac tcgtatccct ctcccagctg cgctgctgcg gctggcacta 3000

cccgcaggac tggttccaag tcctcatcct gagaggtaac gggtcggagg cgcaccgcgt 3060

gccctgctcc tgctacaact tgtcggcgac caacgactcc acaatcctag ataaggtgat 3120

cttgccccag ctcagcaggc ttggacacct ggcgcggtcc agacacagtg cagacatctg 3180

cgctgtccct gcagagagcc acatctaccg cgaggtgggc aggggttcgg agcataaacc 3240

tgtcgaatgg ggcggggcct gcgggagggg gagggctgtc agtgagtagc ggcctgagaa 3300

agggcggggt ctacgagaaa aggaaaggta cggcaggagg ggcggggccc tctgaagggg 3360

gcggggtctg caggaagggc agggcctaaa gaaaaggctg ggctggctct ggaatacaga 3420

tgtctaggga ggggccaggc ttggaaaagg tgaagcgagg gtgcactagt aaggagacta 3480

gagtgccctg gtgactaggg gagcgggtag atgcctgaag acggtgaggg ttggcctgaa 3540

aagaacgctg ggcctgggct tgagagtccc agaaagaatc cctttaactt ttccctacac 3600

cccccagggc tgcgcgcagg gcctccagaa gtggctgcac aacaacctta tttccatagt 3660

gggcatttgc ctgggcgtcg gcctactcga ggtgatctgg ccccgccccc acccgcgatc 3720

ggccctaaat ccctagatgg ccctgccctt catttcgcgt ccttcggttg cctgggaagg 3780

acgagctcag ggcggagcgc agcccacccc ggccctcccg ccgctccacc cagcaccgga 3840

gggtgggggc ggcccagctt cagggagccc tgattgggtg tacgcaggga aagcctcctg 3900

ctattggctg cgatctccct cccctttctc cgcagatgac tgtcatggtg ctgagcgtac 3960

agctacagcg cagggcactc cgccggaaat gcgagccgca cgtgccgggc gctggggatt 4020

cgagccccgg gcccagcctg atcgctgacg gcggcggcgg gcacagcggc agtctgtggg 4080

gtggctgggg catggcgggt gcctgcccca actggggaga caaggcaccg cagggcaagc 4140

tgcccatggc cctggggctc tggccgctgt gggttcaaga cgaggaccag cctgacactg 4200

gaagtgcggg cgcagaatta gaggaggcac aattagaggc tgaggcagag ggggaagaca 4260

gatgagcctc caaaataaag gaccctgggc ttgcttccga ccttactcct tctcagcctc 4320

tacccccact tgtagcagct attcccgcct catcagccag ccctgcggca gttcccgtcg 4380

agccccgccc ttttctacct atccccttct ccagcccctt cccgcccaat tcacggcccc 4440

acccctgacc tttctcgccc gggtgggcat cacccccgtc tcgccagcac cccttcgact 4500

tctctgacct catctccttt ctctatagct cgggttcatg acgctctcga tattcctgtg 4560

cagaaacctg gaccacgtct acaaccggct cgctcgatac cgttag 4606

<210> 6

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccaagaccc tagcgagaca cggcttccta ccccgttaac gaattccgaa gttcctattc 60

tctagaaagt ataggaactt 80

<210> 7

<211> 90

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tctctagaaa gtataggaac ttcatcagtc aggtacataa tggtggatcc tgtacagtag 60

tgactctggc ttccaccgga ccccgccccc 90

<210> 8

<211> 1226

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tttgtgcctc tcagtctctg tctttctttg cccactattc cccttccccc ctgcggccaa 60

cacctcttct gtctgggttt ctcgagtggg tcattcccca tctaggcgaa gatgtccgcc 120

caagagagtt gcctcagcct catcaagtac ttcctcttcg ttttcaacct cttcttcttt 180

gtactaggca gcctgatctt ctgcttcggc atctggatcc tcattgacaa gaccagcttc 240

gtgtcctttg tgggcttggc cttcgtgcct ctgcagatct ggtccaaagt cctggccatc 300

tcaggaatct tcaccatggg catcgccctc ctgggttgtg tgggggccct caaggagctc 360

cgctgcctcc tgggcctgta ttttgggatg ctgctgctcc tgtttgccac acagatcacc 420

ctgggaatcc tcatctccac tcagcgggcc cagctggagc gaagcttgcg ggacgtcgta 480

gagaaaacca tccaaaagta cggcaccaac cccgaggaga ccgcggccga ggagagctgg 540

gactatgtgc agttccagct gcgctgctgc ggctggcact acccgcagga ctggttccaa 600

gtcctcatcc tgagaggtaa cgggtcggag gcgcaccgcg tgccctgctc ctgctacaac 660

ttgtcggcga ccaacgactc cacaatccta gataaggtga tcttgcccca gctcagcagg 720

cttggacacc tggcgcggtc cagacacagt gcagacatct gcgctgtccc tgcagagagc 780

cacatctacc gcgagggctg cgcgcagggc ctccagaagt ggctgcacaa caaccttatt 840

tccatagtgg gcatttgcct gggcgtcggc ctactcgagc tcgggttcat gacgctctcg 900

atattcctgt gcagaaacct ggaccacgtc tacaaccggc tcgctcgata ccgttaggcc 960

ccagcccacc catcggcacc cacgcacctc catgcagtca ggaaatgttt ccttcctggt 1020

tggtcacagg cttggagccc atgcctcagg cctcatcccc tggggaccca gctgtctgcc 1080

ctatcgacag ccttcacttt ccccacatgg ccagggactt ttgtgcccag cttccttccc 1140

ttcctggctc cctcctcccc acaccatctg tctgccacct cccacatgac acctcaaata 1200

aatccccttt gggtttttgg cttttt 1226

<210> 9

<211> 281

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ser Ala Gln Glu Ser Cys Leu Ser Leu Ile Lys Tyr Phe Leu Phe

1 5 10 15

Val Phe Asn Leu Phe Phe Phe Val Leu Gly Ser Leu Ile Phe Cys Phe

20 25 30

Gly Ile Trp Ile Leu Ile Asp Lys Thr Ser Phe Val Ser Phe Val Gly

35 40 45

Leu Ala Phe Val Pro Leu Gln Ile Trp Ser Lys Val Leu Ala Ile Ser

50 55 60

Gly Ile Phe Thr Met Gly Ile Ala Leu Leu Gly Cys Val Gly Ala Leu

65 70 75 80

Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr Phe Gly Met Leu Leu Leu

85 90 95

Leu Phe Ala Thr Gln Ile Thr Leu Gly Ile Leu Ile Ser Thr Gln Arg

100 105 110

Ala Gln Leu Glu Arg Ser Leu Arg Asp Val Val Glu Lys Thr Ile Gln

115 120 125

Lys Tyr Gly Thr Asn Pro Glu Glu Thr Ala Ala Glu Glu Ser Trp Asp

130 135 140

Tyr Val Gln Phe Gln Leu Arg Cys Cys Gly Trp His Tyr Pro Gln Asp

145 150 155 160

Trp Phe Gln Val Leu Ile Leu Arg Gly Asn Gly Ser Glu Ala His Arg

165 170 175

Val Pro Cys Ser Cys Tyr Asn Leu Ser Ala Thr Asn Asp Ser Thr Ile

180 185 190

Leu Asp Lys Val Ile Leu Pro Gln Leu Ser Arg Leu Gly His Leu Ala

195 200 205

Arg Ser Arg His Ser Ala Asp Ile Cys Ala Val Pro Ala Glu Ser His

210 215 220

Ile Tyr Arg Glu Gly Cys Ala Gln Gly Leu Gln Lys Trp Leu His Asn

225 230 235 240

Asn Leu Ile Ser Ile Val Gly Ile Cys Leu Gly Val Gly Leu Leu Glu

245 250 255

Leu Gly Phe Met Thr Leu Ser Ile Phe Leu Cys Arg Asn Leu Asp His

260 265 270

Val Tyr Asn Arg Leu Ala Arg Tyr Arg

275 280

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtccgcccaa gagagttgcc tca 23

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctgggctcca acccactgtg tagt 24

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggaagtgctt gagaaatggc tctca 25

<210> 13

<211> 1390

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tattggagat ggcagaacct gccttggagc acagcctgtg gcaggagagg cccagtcccg 60

aaaatttatc tatcttctgc cctctataag caccatgtgg cgcagacata cacacacaca 120

cacatcgtta aaattttttt aaaaaaagat gaacagcttc caatgttgtc ctctggcctc 180

caaaggcgcg cgcgcgcaca cacacacaca cacacacaca cacacacaca cacacactaa 240

cacatacaaa tacacataca gaaagaaaat agatgagaca tgtctcagaa ggcagctgct 300

gtcccagaga actaggcttt gggtagatag gtgataggaa cttcccacat tagaaatctg 360

tgaggaactg gggacttgac aagtgtcagg gacttctttc tgggcacatc ataggaaact 420

ttgaagatgc taggtatgct caagtgaccc ccactccagt gcctggcttt tctggggcct 480

cctcccctca ggtttctccg cagaggtagg aagtgccatc tgaggttcac ttcctccacc 540

ggatattcag gcactgcctc gctgccctcc tcactcctgg gactgtcaca cctcctctgc 600

agccctttca cttccccagg ggtccaaccc tggcccccaa gcacatcgga gcacctgctc 660

tagaagaaag agagcaaaga gagagaccca gggaaagaga cacacaggac agagaccgca 720

aaggcccaga gagacagaga cagagaggca gagacagcgc ggtggctcga ggctaatggg 780

tgctaagcag aagatcacag ggtgcagggg gcggagccag tagccagtga ggtcagagtg 840

tctgtgaggt tgaactctgc agaaacaacc ttgaagtccc aggacccacg tgagtggaag 900

tccatcagaa gcccctgtgc gccctgcctg agctatggac acctgcgagg aacccattgt 960

gtccctggca cccacatatt ccaaggaccc tcaggtactg ctgtgcactc gatggaaccc 1020

tgaagaagat agggcccccg ctgacagtca ctaaatgaac agggcggggt ggagctccac 1080

ccccccggaa cttcctcttt ctctgccggt ttcctttgtg cctctcagtc tctgtctttc 1140

tttgcccact attccccttc ccccctgcgg ccaacacctc ttctgtctgg gtttctcgag 1200

tgggtcattc cccatctagg cgaagatgtc cgcccaagag agttgcctca gcctcatcaa 1260

gtacttcctc ttcgttttca acctcttctt ctttgtgagt ctactcatgg ctacccagcc 1320

ggggttccag ggccacagtg gcccttgatc cgccctagcc tcatttcttc tcttccccta 1380

ggtactaggc 1390

<210> 14

<211> 1425

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gccccagccc acccatcggc acccacgcac ctccatgcag tcaggaaatg tttccttcct 60

ggttggtcac aggcttggag cccatgcctc aggcctcatc ccctggggac ccagctgtct 120

gccctatcga cagccttcac tttccccaca tggccaggga cttttgtgcc cagcttcctt 180

cccttcctgg ctccctcctc cccacaccat ctgtctgcca cctcccacat gacacctcaa 240

ataaatcccc tttgggtttt tggctttttt tttttttttt tggtaaaaat agctttattc 300

tccttcaaac ataaaccatc actcttgggg aaaggaaggt ggcagggtgg tccaaggctc 360

acttaaatgg ggtgggggag attaagaagt cccaccccac tgcctagctg agataagatt 420

acatccctaa cactgtgtat aaatatctcc ttatattaaa attttttttc aggtcccact 480

tcaccctacc ccaagctggg aaaacattat cttcgggttg tgacgtcaca gttcccagag 540

aaagggtgtg gggggagagg cagatatgag acacgtggga agcaggaagg ttacaaggca 600

gtcagtaaga caccccacat agcccagctc cctttccaga agagcaagga ggggccctgt 660

agaacggagg cccgtgctgt acctgtgagc tcactcctgt ccccaatccc tgatgctcag 720

gagtggtgtg tcctgtcaaa tgttgtctct gaggtcctcg tgttggggat ctaaactcaa 780

acccagctca ctcagctcaa taaccaggct tccggttcct ttctaagagg agccagcccc 840

cgtggctcat gcttctgaga ttaggccttt ttaaaaatca tgggcccctc tccaaatctc 900

tcttacagag agagcaaaag ggcccttccc aggaaggggg tgttgtgaaa aacaccagcg 960

gcgggggctc catcagtccc tgaccaggcg gtagatgtgg tactgggctc ctcgttcact 1020

ggtggagact tcaaagacgt aggcagtaca cagcagcagt tcctgagtgt ccctgtttgt 1080

cacaacctgc cagaacaggc ttacattgta gggtggggat ggggtacagg acatggactg 1140

ccctgcccac cctagttatc aagaaagaga aacctgccat gctatgtacc ccaagaagaa 1200

tgtcctgcgg ctcaggttcc ttgggcaatg aggttacccc taagctgaga gatggtagga 1260

aggggcctcc ccatcatatc acattcccga gaaaagggtt ccccaccatg ccaccaggca 1320

gagagagagg gtcagcagat catattagca caacccccaa atcaaaggaa ctcccttctc 1380

tgaacttctc agagaagcca gatgcttaga agccgaggta tctca 1425

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaccagcttc gtgtcctttg tgg 23

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gtgagggggg ctgcgcagta ggg 23

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tcctgatatc tcactcgccc tgg 23

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cctgatatct cactcgccct ggg 23

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ctgatatctc actcgccctg ggg 23

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agctctgact ggcaccccac tgg 23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

agagcgtcat gaagccgagc tgg 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ccggctggcc cggtaccgct agg 23

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggctggggcc tagcggtacc ggg 23

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atctggatca cgtctatgac cgg 23

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ggatcacgtc tatgaccggc tgg 23

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cctagcggta ccgggccagc cgg 23

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ggcacagacg gaggctgttg cgg 23

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gggctggggc ctagcggtac cgg 23

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ctgatatctc actcgccctg 20

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

taggctgata tctcactcgc cctg 24

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

cagggcgagt gagatatcag 20

<210> 32

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aaaccagggc gagtgagata tcag 24

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cctagcggta ccgggccagc 20

<210> 34

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

taggcctagc ggtaccgggc cagc 24

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gctggcccgg taccgctagg 20

<210> 36

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

aaacgctggc ccggtaccgc tagg 24

<210> 37

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ggaccgcatg gtggactgca tagac 25

<210> 39

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cctctcagga tgaggacttg gaacc 25

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

cgggacgtcg tagagaaaac catcc 25

<210> 41

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

aatttcctgc acaagctccg tcagc 25

<210> 42

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ctgtctgggt gaaaaggtcc ggg 23

<210> 43

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

aaggaatcaa agcactgggg tcaca 25

<210> 44

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tcacaacctg ccagaacagg ct 22

<210> 45

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

agggagttcc tttgatttgg gggt 24

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

agagagttgc ctcagcctca tc 22

<210> 47

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tgtctgcact gtgtctggac c 21

<210> 48

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ctctaaagga gctgcgctgt 20

<210> 49

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cagccctcac ggtagatgtg 20

<210> 50

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

tcaccatctt ccaggagcga ga 22

<210> 51

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

gaaggccatg ccagtgagct t 21

<210> 52

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

tgagtctact catggctacc cagcc 25

<210> 53

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

tgaaggctgt cgatagggca gaca 24

Claims

1. A humanized CD37 protein, wherein the humanized CD37 protein comprises a portion of human CD37 protein.

2. The humanized CD37 protein according to claim 1, wherein the humanized CD37 protein comprises all or part of the extracellular, transmembrane and/or cytoplasmic region of human CD37 protein, preferably wherein said part of human CD37 protein comprises all or part of the extracellular region of human CD37 protein, further preferably comprises at least 20 amino acids of the extracellular region of human CD37 protein, more preferably comprises SEQ ID NO:2, positions 39-59, 112-241; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 39-59, 112-241, including substitution, deletion and/or insertion of one or more amino acid residues.

3. The humanized CD37 protein of claim 1 or 2, wherein the humanized CD37 protein further comprises all or part of the transmembrane region of the human CD37 protein, preferably wherein the humanized CD37 protein comprises the transmembrane region of the human CD37 protein of at least 10 amino acids, further preferably comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with position 27-38, 60-74, 86-111, 242-and 266 of SEQ ID No. 2 or comprises the amino acid sequence shown at position 27-38, 60-74, 86-111, 242-and 266 of SEQ ID No. 2; and/or, the humanized CD37 protein further comprises all or part of the cytoplasmic region of the human CD37 protein, preferably, the humanized CD37 protein comprises the cytoplasmic region of the human CD37 protein of at least 5 amino acids, and further preferably, comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with positions 75-85, 267-281 of SEQ ID NO:2 or comprises an amino acid sequence shown in positions 75-85, 267-281 of SEQ ID NO: 2.

4. The humanized CD37 protein of any one of claims 1-3, wherein the human CD37 protein comprises the amino acid sequence of SEQ ID NO:2, amino acid sequence shown in positions 27-281; or, comprising a nucleotide sequence identical to SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical in amino acid sequence from position 27-281; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from positions 27-281, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, position 27-281, including substitution, deletion, and/or insertion of one or more amino acid residues.

5. The humanized CD37 protein of any one of claims 1-4, wherein the humanized CD37 protein further comprises a portion of a non-human animal CD37 protein, preferably, the portion of the non-human animal CD37 protein comprises a portion of a transmembrane region and a cytoplasmic region of a non-human animal CD37 protein.

6. The humanized CD37 protein of any one of claims 1 to 5, wherein the amino acid sequence of the humanized CD37 protein comprises any one of the following group:

A) SEQ ID NO: 9, and (b) the amino acid sequence shown in the figure;

B) and SEQ ID NO: 9 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

7. A nucleic acid encoding the humanized CD37 protein of any one of claims 1-6.

8. A humanized CD37 gene, wherein the humanized CD37 gene comprises a portion of the human CD37 gene.

9. The humanized CD37 gene according to claim 8, wherein the portion of human CD37 gene comprises all or part of exon 1 to exon 8 of human CD37 gene, preferably comprises all or part of exon 2 to exon 8, further preferably comprises part of exon 2, all of exon 3 to exon 7 and part of exon 8, wherein part of exon 2 comprises at least 20bp of nucleotide sequence and part of exon 8 comprises at least 50bp of nucleotide sequence.

10. The humanized CD37 gene of claim 8 or 9, wherein the portion of the human CD37 gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

11. The humanized CD37 gene according to any of claims 8 to 10, wherein the humanized CD37 gene comprises all or part of exon 1 and/or exon 8 of the non-human animal CD37 gene, and further preferably comprises the nucleotide sequence of part of exon 2.

12. The humanized CD37 gene of any one of claims 8 to 11, wherein the nucleotide sequence of the humanized CD37 gene comprises any one of the following group:

A) the transcribed mRNA is SEQ ID NO: 8;

B) the transcribed mRNA hybridizes to SEQ ID NO: 8 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) the transcribed mRNA is identical to SEQ ID NO: 8 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide;

D) the transcribed mRNA has the sequence of SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted; or the like, or, alternatively,

E) SEQ ID NO: 6 and/or 7.

13. A targeting vector, wherein said targeting vector comprises a donor nucleotide sequence, preferably wherein said donor nucleotide sequence comprises one of the group consisting of:

B) a nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD37 protein, preferably a nucleotide sequence encoding at least 20 amino acids of the extracellular region of the human CD37 protein, more preferably a nucleotide sequence encoding at least 10 amino acids of the transmembrane region of the human CD37 protein, more preferably a nucleotide sequence encoding at least 5 amino acids of the cytoplasmic region of the human CD37 protein, even more preferably a nucleotide sequence encoding SEQ ID NO:2 from position 27 to 281;

C) a human or humanized CD37 gene; or the like, or, alternatively,

D) all or part of exon 1 to exon 8 of the human CD37 gene, preferably all or part of exon 2 to exon 8, further preferably all or part of exon 2, exon 3 to exon 7 and part of exon 8, wherein part of exon 2 comprises at least a 20bp nucleotide sequence and part of exon 8 comprises at least a 50bp nucleotide sequence, more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

14. The targeting vector according to claim 13, wherein said targeting vector further comprises a5 'arm and/or a 3' arm, preferably wherein said 5 'arm is a nucleotide having at least 90% homology to NCBI accession No. NC — 000073.7, and further preferably wherein said 5' arm sequence is as set forth in SEQ ID NO: 3 or 13, the 3' arm is selected from 100-10000 nucleotides in length of the non-human animal CD37 gene genome DNA; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000073.7; further preferably, the 3' arm sequence is as set forth in SEQ ID NO: 4 or 14.

15. A method of constructing a non-human animal, wherein said non-human animal expresses a human or humanized CD37 protein.

16. The method of claim 15, wherein the humanized CD37 protein is the humanized CD37 protein of any one of claims 1-6.

17. The method of construction according to claim 15 or 16 wherein the genome of the non-human animal comprises the nucleic acid of claim 7 or the humanized CD37 gene of any one of claims 8-12.

18. The construct of any of claims 15-17, comprising introducing a donor nucleotide sequence into the non-human animal CD37 locus, preferably wherein the donor nucleotide sequence comprises one of the following groups:

C) a human or humanized CD37 gene; or the like, or, alternatively,

19. The method of claim 18, wherein the donor nucleotide sequence is regulated in the non-human animal by endogenous regulatory elements.

20. The method of construction according to any one of claims 15-19, wherein said introducing is a substitution or insertion, optionally said introducing is into the non-human animal CD37 locus to replace a corresponding region of the non-human animal, preferably, exon 2 to exon 8 of the non-human animal CD37 gene is replaced in whole or in part, further preferably, part of exon 2, all of exon 3 to exon 7 and part of exon 8 of the non-human animal CD37 gene is replaced.

21. The method of construction according to any one of claims 15 to 20, wherein the targeting vector according to any one of claims 13 to 14 is used for construction of a non-human animal.

22. The method of any one of claims 15-21, further comprising mating the CD37 humanized non-human animal with another genetically modified non-human animal, in vitro fertilization, or directly performing gene editing, and screening to obtain a polygenetically modified non-human animal, preferably wherein said another gene is selected from at least one of PD-1, PD-L1, IL4, IL4R, IL6, IL6R, IL17, CD3, and CD 20.

23. A cell, tissue or organ which expresses a humanized CD37 protein according to any one of claims 1 to 6 or which comprises a nucleic acid according to claim 7 or a humanized CD37 gene according to any one of claims 8 to 12 or a non-human animal obtained by a method of construction according to any one of claims 15 to 22.

24. A tumor tissue after tumor loading, wherein said tumor tissue expresses the humanized CD37 protein of any one of claims 1-6, or said tumor tissue comprises the nucleic acid of claim 6 or the humanized CD37 gene of any one of claims 7-11, or a non-human animal obtained by the construction method of any one of claims 15-22.

25. Use of the humanized CD37 protein of any one of claims 1 to 6, the nucleic acid of claim 8, the humanized CD37 gene of any one of claims 8 to 12 or the construct of any one of claims 15 to 22 in a non-human animal, the use comprising:

A) use in the development of products involving CD 37-related immune processes in human cells;

B) use as a model system in pharmacological, immunological, microbiological and medical research associated with CD 37;

C) to the production and use of animal experimental disease models for the research of CD 37-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) the application of the human CD37 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo; alternatively, the first and second liquid crystal display panels may be,

E) the functions of the CD37 gene are researched, the medicine and the drug effect aiming at the target site of the human CD37 are researched, and the application in the aspects of the medicine for the immune-related diseases related to the CD37 and the anti-tumor medicine is researched.

26. The humanized CD37 protein according to any one of claims 5 to 6, the nucleic acid according to claim 7, the humanized CD37 gene according to any one of claims 11 to 12, the method of construction according to claims 15 to 22, the cell, tissue or organ according to claim 23, the neoplastic tissue according to claim 24, wherein the non-human animal is a non-human mammal, preferably wherein the non-human mammal is a rodent, more preferably wherein the rodent is a rat or a mouse.