CN115260303A

CN115260303A - Construction method and application of CD70 gene humanized non-human animal

Info

Publication number: CN115260303A
Application number: CN202210609446.9A
Authority: CN
Inventors: 吕锐利; 李冲; 尚诚彰
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-05-31
Filing date: 2022-05-31
Publication date: 2022-11-01
Also published as: WO2022253219A1

Abstract

The invention provides a construction method of a CD70 gene humanized non-human animal, which is characterized in that a nucleotide sequence for coding a human CD70 protein is introduced into the genome of the non-human animal in a homologous recombination mode, the animal body can normally express the human or humanized CD70 protein, can be used as an animal model for researching the human CD70 signal mechanism and screening drugs for tumors and immune related diseases, and has important application value for the research and development of new drugs for immune targets. The invention also provides a humanized CD70 protein, a humanized CD70 gene, a targeting vector of the CD70 gene and application of the constructed non-human animal in the field of biomedicine.

Description

Construction method and application of CD70 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 CD70 gene modified non-human animal model and application thereof in the field of biomedicine.

Background

CD70 is one of the Tumor Necrosis Factor (TNF) superfamily members, is a type II transmembrane protein, and is mainly expressed in activated T cells, B cells, natural Killer (NK) cells and dendritic cells. The interaction between CD70 and its receptor, CD27, promotes the activation, proliferation and differentiation of T cells, B cells and NK cells, which are essential conditions for the generation of immune responses. In cellular immunity, the main biological effect of CD70/CD27 is to promote the activation of T cells and the proliferation of T cells, so that the T cells become effector T cells and memory T cells; in humoral immunity, the primary role of CD70/CD27 is to promote B cell differentiation and proliferation of plasma cells; for NK cells, the direct action of CD27 and CD70 on the surface of NK cells can increase the killing activity of NK cells, promote the differentiation of NK cells and release IFN-gamma.

Under physiological conditions, CD70 is highly precisely regulated by antibodies, antigens, cytokines, etc., and thus is expressed on the surface of activated T cells and B cells for a short period of time. In addition to expression on normal lymphocytes, CD70 is also highly expressed on the surface of a variety of tumor cells, especially blood-borne tumors such as hodgkin's lymphoma, non-hodgkin's lymphoma, leukemia, and fahrenheit macroglobulinemia (Waldenstroms macroglobulinemia), and is a potential target for partial tumor targeting and immunotherapy; among non-blood-derived tumors, the high expression of CD70 is found in undifferentiated nasopharyngeal carcinoma and germ cell cancer at the earliest, and subsequent studies find that CD70 is also highly expressed in tumors such as thymoma, nephroblastoma, glioblastoma, astrocytoma and ovarian cancer, particularly kidney cancer, which is currently considered as a novel specific tumor marker for kidney cancer. In addition, the research finds that the costimulatory signal provided by the CD27/CD70 interaction plays a promoting role in the occurrence and development of various autoimmune diseases, and selective intervention of the costimulatory pathway can alleviate or reduce the development of certain autoimmune diseases. Such as Systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), etc., may be a specific therapeutic target for autoimmune diseases.

At present, monoclonal antibodies targeting CD70 have entered human clinics and show better safety and therapeutic effects. For example, humanized monoclonal antibody Cusatuzumab obtained by Janssen early-paid in 3 hundred million dollars and invested in 2 hundred million dollars shows good safety and tolerance, also shows a certain anti-cancer effect and shows the potential for treating the disease in a clinical test of stage I/II of CD70 positive advanced cutaneous T cell lymphoma; in another phase I/II clinical trial of acute myelogenous leukemia, cusatuzumab in combination with Azacitidine had a higher overall remission rate than the historical data for Azacitidine alone.

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. Therefore, because of the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, it remains the greatest challenge to construct "efficient" humanized animal models for new drug development.

In view of the huge application potential of the CD27/CD70 signal pathway in the treatment field of tumors, autoimmune diseases and the like, in order to further explore the relevant biological characteristics, improve the effectiveness of the preclinical pharmacodynamic test, improve the success rate of research and development, make the preclinical test more effective and minimize the research and development failure, the development of a non-human animal model of the CD27/CD70 signal pathway is urgently needed in the field. 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

The application uses human normal or mutant genes to replace homologous genes of animal genomes, and establishes a normal or mutant gene animal model which is closer to the human physiological or disease characteristics. The humanized animal model for cell or tissue transplantation is improved, and more importantly, the humanized animal model can express or partially express human protein in an animal body due to the insertion of human gene segments, can be used as a target of a drug only capable of recognizing human protein sequences, and provides possibility for screening anti-human antibodies and other drugs at the animal level.

In a first aspect of the invention, a humanized CD70 protein is provided, wherein the humanized CD70 protein comprises all or part of a human CD70 protein.

Preferably, the humanized CD70 protein comprises an extracellular domain, a transmembrane domain and/or a cytoplasmic domain of the human CD70 protein.

Preferably, the humanized CD70 protein comprises all or part of the extracellular domain of a human CD70 protein. Further preferred are those comprising at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD 70. Further preferably, the extracellular domain comprises an extracellular domain of human CD70 in which 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids are deleted from the C-terminus and/or N-terminus of the extracellular domain.

Preferably, the humanized CD70 protein further comprises a portion of a non-human animal CD70 protein, and further preferably, the portion of the non-human animal CD70 protein comprises a transmembrane region and/or a cytoplasmic region of the non-human animal CD70 protein; still more preferably, the polypeptide further comprises a part of extracellular domain of the non-human animal CD70 protein, and further preferably comprises 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) consecutive amino acid sequences of C-terminal and/or N-terminal of the extracellular domain of the non-human animal.

Preferably, the portion of the human CD70 protein and the portion of the non-human animal CD70 protein are directly linked or indirectly linked (e.g., a peptide linker).

In one embodiment of the present invention, the humanized CD70 protein comprises an extracellular domain of human CD70 protein, a transmembrane domain and a cytoplasmic domain of a non-human animal, and preferably further comprises a partial extracellular domain of a non-human animal.

Preferably, the portion of human CD70 protein comprises SEQ ID NO:2, amino acid sequence as shown in positions 48-193 or 39-193; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 48-193 or 39-193, is at least 60%,70%, 75%, 80%, 85%, 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 48-193 or 39-193, 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 48-193 or 39-193, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the portion of the non-human animal CD70 protein comprises SEQ ID NO:1, positions 1-44 or 1-49; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99% identical to the amino acid sequence shown in positions 1-44 or 1-49; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-44 or 1-49, 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, 1-44 or 1-49, including substitution, deletion and/or insertion of one or more amino acid residues.

In one embodiment of the present invention, the humanized CD70 protein comprises SEQ ID NO:2, amino acid sequence shown in positions 48-193 or 39-193 and SEQ ID NO:1, 1-44 or 1-49.

Preferably, said part of the human CD70 protein comprises all or part of the amino acid sequence encoded by the human CD70 gene.

Preferably, the part of the human CD70 protein comprises all or part of an amino acid sequence encoded by exon 1, exon 2 and/or exon 3 of the human CD70 gene; preferably, the humanized CD70 protein comprises all or part of an amino acid sequence encoded by exons 1 to 3 of the human CD70 gene. Further preferably, the humanized CD70 protein comprises all or part of an amino acid sequence encoded by one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of the human CD70 gene. Still more preferably, the humanized CD70 protein comprises part of exon 1 and the amino acid sequence encoded by exons 2 to 3 of human CD70 gene, wherein the part of exon 1 is preferably a nucleotide sequence encoding a transmembrane region and/or an extracellular region.

Preferably, the portion of the non-human animal CD70 protein comprises a partial amino acid sequence encoded by the non-human animal CD70 gene. Preferably a partial amino acid sequence encoded by exon 1 of the non-human animal CD70 gene.

In a specific embodiment of the present invention, the humanized CD70 protein comprises a part of the amino acid sequence encoded by exon 1 of the non-human animal CD70 gene, and all of exons 2 to 3 of the human CD70 gene, and preferably further comprises a part of the amino acid sequence encoded by exon 1 of the human CD70 gene.

Preferably, the non-human animal can be selected from any non-human animal such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc., which can be genetically engineered to become genetically 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 further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mouse, rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

Preferably, the amino acid sequence of the humanized CD70 protein derived from human CD70 protein is identical to the amino acid sequence of 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 CD70 protein derived from the non-human animal CD70 protein has a sequence similar to that 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 humanized CD70 protein comprises at least 50 amino acid sequences identical to the corresponding amino acid sequence of human CD70, and further, the 50 amino acid sequences are the amino acid sequences from the first amino acid of the C-terminus.

In one embodiment of the present invention, the amino acid sequence of the humanized CD70 protein comprises one of the following groups:

a) Is SEQ ID NO:11, all or part of an amino acid sequence;

b) And SEQ ID NO:11 is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical in amino acid sequence;

c) And SEQ ID NO:11 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:11, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a second aspect of the invention, there is provided a nucleic acid encoding the humanized CD70 protein.

In a third aspect of the invention, a humanized CD70 gene is provided, said humanized CD70 gene comprising a portion of a human CD70 gene.

Preferably, the humanized CD70 gene encodes the humanized CD70 protein.

Preferably, the humanized CD70 gene comprises all or part of exons 1 to 3 of human CD70 gene, further preferably comprises any one, two or three or two consecutive exons of exons 1 to 3 of human CD70 gene, further preferably comprises part of exon 1, all of exon 2 and part of exon 3 of human CD70 gene, wherein part of exon 1 comprises at least 10bp to at least 310bp of exon 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp contiguous nucleotide sequence, and part of exon 3 comprises at least 100bp to at least 567bp of exon 3, such as 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 385, 389, 390, 400, 500, 550, 7bp contiguous nucleotide sequence, preferably also comprises introns 1-2 bp and/or 2bp contiguous nucleotide sequence.

Preferably, the humanized CD70 gene comprises the genomic DNA sequence, cDNA sequence or CDs sequence of all or part of the human CD70 gene.

In one embodiment of the present invention, the humanized CD70 gene comprises a partial nucleotide sequence of the human CD70 gene comprising one of the following groups:

(A) 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 CD70 gene further comprises a portion of a non-human animal CD70 gene, and further preferably comprises all or part of exon 1 and/or all or part of exon 3 of a non-human animal CD70 gene.

In one embodiment of the present invention, the humanized CD70 gene comprises a portion of exon 1 of non-human animal CD70, a portion of exon 1 of human CD70 gene, all of exon 2, a portion of exon 3, and a portion of exon 3 of non-human animal CD70 gene. Preferably, the gene further comprises intron 1-2 and/or intron 2-3 of the human CD70 gene.

Preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the transmembrane, cytoplasmic, and/or extracellular regions of the human CD70 protein. Further preferably, the humanized CD70 gene comprises all or part of the nucleotide sequence encoding the extracellular domain of human CD70 protein.

Preferably, the humanized CD70 gene comprises a nucleotide sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193, is at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 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, the nucleotide sequence at the 39 th to 193 th positions or the 48 th to 193 th positions comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions.

Preferably, the humanized CD70 gene further comprises a partial nucleotide sequence encoding a non-human animal CD70 protein. Further preferably comprises all or part of the nucleotide sequence encoding the cytoplasmic region and/or the transmembrane region of the non-human animal CD70 protein, and still further preferably comprises part of the nucleotide sequence encoding the extracellular region of the non-human animal CD70 protein.

In one embodiment of the invention, the humanized CD70 gene comprises a nucleotide sequence identical to a nucleotide sequence encoding SEQ ID NO:1, amino acid sequence 1-44 or 1-49, or a nucleotide sequence that has at least 90% identity to a nucleotide sequence encoding SEQ ID NO:1, 1-44 or 1-49 amino acid sequence.

Preferably, the humanized CD70 gene further comprises 5'UTR and/or 3' UTR of the non-human animal CD70 gene.

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 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^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mice, rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence identical to SEQ ID NO:6 and/or SEQ ID NO:7 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:6 and/or SEQ ID NO: 7.

Preferably, the nucleotide sequence of the humanized CD70 gene comprises a nucleotide sequence identical to SEQ ID NO:8 and/or SEQ ID NO:9 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:8 and/or SEQ ID NO:9, or a nucleotide sequence shown in the specification.

In one embodiment of the present invention, the mRNA transcribed from the humanized CD70 gene comprises any one of the following groups:

(a) The amino acid sequence of SEQ ID NO:10, or a portion or all of a nucleotide sequence set forth in seq id no;

(b) And SEQ ID NO:10 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:10 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:10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the humanized CD70 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 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 CD70 protein;

b) A nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD70 protein, preferably encoding all or part of the extracellular region of the human CD70 protein, further preferably a nucleotide sequence encoding at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154, consecutive amino acids of the extracellular region of human CD70, further preferably a nucleotide sequence encoding an extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and/or the N-terminus of the extracellular region, more preferably a nucleotide sequence encoding an extracellular region comprising SEQ ID NO:2 from position 39-193 or from position 48-193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 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, the nucleotide sequence from 39 th to 193 th or from 48 th to 193 th, including nucleotide sequences with one or more nucleotide substitutions, deletions and/or insertions;

c) All or part of a human or humanized CD70 gene; or,

d) All or part of exons 1 to 3 of the human CD70 gene, preferably any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein part of exon 1 comprises at least 10bp to at least 310bp of exon 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp of a consecutive nucleotide sequence, and part of exon 3 comprises at least 100bp to at least 7bp of exon 3, such as 100, 200, 300, 350, 380, 400, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp of a consecutive nucleotide sequence, preferably also comprising exons 1-2 and/or 3-introns; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to 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%; 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, the targeting vector further comprises a 5 'arm and/or a 3' arm.

The 5' arm (or 5' homologous arm) and the transformation region 5' end homologous DNA fragment, which is selected from the non-human animal CD70 gene genome DNA 100-10000 nucleotides in length. Preferably, the 5' arm has at least 90% homology with NCBI accession No. NC — 000083.7. Further preferably, the 5' arm sequence is identical to SEQ ID NO:3 or as shown in SEQ ID NO:3, respectively.

The 3' arm (or 3' homologous arm) and the 3' end of the switching region to be changed are homologous DNA fragments selected from 100-10000 nucleotides in length of non-human animal CD70 gene genome DNA. Preferably, the 3' arm has at least 90% homology with NCBI accession No. NC — 000083.7. Further preferably, the 3' arm sequence is identical to SEQ ID NO:4 or as shown in SEQ ID NO:4, respectively.

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

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

Preferably, the transition region to be altered is located at the non-human animal CD70 locus. Further preferably, the transition region to be altered is located on exons 1 to 3 of the non-human animal CD70 gene. Further preferably, the gene is located in exon 1 and/or exon 3 of the non-human animal CD70 gene.

Preferably, the targeting vector comprises a sequence identical to SEQ ID NO:6 and/or SEQ ID NO:7 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:6 and/or SEQ ID NO: 7.

Preferably, the targeting vector comprises a sequence identical to SEQ ID NO:8 and/or SEQ ID NO:9 or a nucleotide sequence having at least 90% or at least 95% identity thereto or comprising SEQ ID NO:8 and/or SEQ ID NO:9, or a nucleotide sequence shown in the specification.

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.

In a fifth aspect of the invention, there is provided a cell comprising the targeting vector described above.

In a sixth aspect of the invention, the targeting vector and the cell containing the targeting vector are provided for use in CD70 gene modification. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In the seventh aspect of the invention, a method for constructing a non-human animal humanized with a CD70 gene is provided, wherein the non-human animal expresses a human or humanized CD70 protein, and/or the genome of the non-human animal comprises the human or humanized CD70 gene.

Preferably, the humanized CD70 protein is the humanized CD70 protein described above.

Preferably, the humanized CD70 gene is the humanized CD70 gene described above.

Preferably, the non-human animal has reduced or absent expression of endogenous CD70 protein.

Preferably, the genome of the non-human animal comprises a part of the human CD70 gene, further preferably comprises all or part of exons 1 to 3 of the human CD70 gene, further preferably comprises any one, two or three or two consecutive exons of exons 1 to 3 of the human CD70 gene, further preferably comprises part of exon 1, all of exon 2 and part of exon 3 of the human CD70 gene, wherein the part of exon 1 at least comprises at least 10bp to at least 310bp of exon 1, e.g., 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp contiguous nucleotide sequence, a portion of exon 3 comprising at least 100bp to at least 567bp of exon 3, e.g., 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp contiguous nucleotide sequence, preferably further comprising intron 1-2 and/or intron 2-3; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to 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%; 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, the genome of the non-human animal comprises all or part of a nucleotide sequence encoding a human CD70 protein, further preferably comprises all or part of a nucleotide sequence encoding an extracellular region, a cytoplasmic region and/or a transmembrane region of a human CD70 protein, further preferably comprises all or part of a nucleotide sequence encoding an extracellular region of a human CD70 protein, wherein the part of the extracellular region of a human CD70 protein comprises at least 50 to at least 154, such as 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154 consecutive amino acids of the extracellular region of human CD 70. Further preferably, the extracellular domain comprises an extracellular domain of human CD70 in which 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids are deleted from the C-terminus and/or N-terminus of the extracellular domain.

In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39-193 or from position 48-193 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, the nucleotide sequence at the 39 th to 193 th positions or the 48 th to 193 th positions comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions.

Preferably, the construction method comprises introducing any one of the following nucleotide sequences into the CD70 locus of the non-human animal:

b) A nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD70 protein, preferably encoding all or part of the extracellular region of the human CD70 protein, further preferably a nucleotide sequence encoding at least 50 to at least 154, e.g. 50, 80, 100, 120, 130, 140, 145, 146, 147, 150 or 154, consecutive amino acids of the extracellular region of human CD70, further preferably a nucleotide sequence encoding an extracellular region of human CD70 with 0-20 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids removed from the C-terminus and/or the N-terminus of the extracellular region, more preferably a nucleotide sequence encoding an extracellular region comprising SEQ ID NO:2 from position 39-193 or from position 48-193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 60%,70%, 75%, 80%, 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% identical; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from position 39 to 193 or from position 48 to 193 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, the nucleotide sequence from 39 th to 193 th or from 48 th to 193 th, and comprises a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions;

c) All or part of a human or humanized CD70 gene; or,

d) All or part of exons No. 1 to 3 of human CD70 gene, preferably any one, two or three or two consecutive exons of exons No. 1 to 3 of human CD70 gene, further preferably part of exons No. 1, all of exons No. 2 and part of exons No. 3 of human CD70 gene, wherein part of exons No. 1 comprises at least 10bp to at least 310bp of exons No. 1, such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 50, 100, 150, 200, 250, 300, 310bp of consecutive nucleotide sequence, and part of exons No. 3 comprises at least 100bp to at least 7bp of exons No. 3, such as 100, 200, 300, 350, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 500, 550, 567bp of consecutive nucleotide sequence, preferably also comprises exons No. 1-2 and/or 2-3; even more preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to 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%; 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 by 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, the human or humanized CD70 gene, nucleotide sequence encoding the human or humanized CD70 protein is regulated in the non-human animal by regulatory elements. The regulatory element is endogenous or exogenous.

Preferably, the regulatory element comprises an endogenous promoter.

Optionally, the endogenous regulatory element is a non-human animal CD70 gene regulatory element, and the exogenous regulatory element is a human CD70 gene regulatory element.

Preferably, the introduction is insertion or substitution.

Preferably, the CD70 locus is introduced into the non-human animal to replace a corresponding region of the non-human animal, preferably to replace all or part of a nucleotide sequence encoding a human CD70 protein in the genome of the non-human animal, more preferably to replace a nucleotide sequence encoding part of an extracellular domain of an endogenous CD70 protein in the genome of the non-human animal, and even more preferably to replace all or part of exons 1 to 3 of the CD70 gene in the genome of the non-human animal.

In a particular embodiment of the invention, the CD70 locus introduced into the non-human animal is a replacement for the nucleotide sequence encoding SEQ ID NO:1 from amino acid 50 to amino acid 195.

In a specific embodiment of the present invention, part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene are replaced; it is further preferred that the intron 1-2 and/or the intron 2-3 of the non-human animal are also replaced.

Preferably, the construction method comprises inserting or replacing at the non-human animal CD70 locus all or part of the nucleotide sequence encoding the non-human animal CD70 protein with a cDNA sequence comprising the protein encoding human CD70 or all or part of the nucleotide sequence encoding the human or humanized CD70 protein.

Preferably, the constructing method comprises inserting or replacing at the non-human animal CD70 locus all or part of the nucleotide sequence encoding the transmembrane region, cytoplasmic region and/or extracellular region of the non-human animal CD70 protein with all or part of the nucleotide sequence comprising the transmembrane region, cytoplasmic region and/or extracellular region encoding the human CD70 protein. Further preferably, all or part of the nucleotide sequence encoding the extracellular domain of the non-human animal CD70 protein at the CD70 locus is inserted or substituted with all or part of the nucleotide sequence comprising the extracellular domain encoding the human CD70 protein. In one embodiment, the polypeptide is encoded by a polypeptide comprising the sequence encoding SEQ ID NO:2, amino acids 48-193, or a substitution to a nucleotide sequence encoding SEQ ID NO:1, amino acids 50-195. In one embodiment, the polypeptide is encoded by a polypeptide comprising the sequence encoding SEQ ID NO:2, amino acids 39-193, or a substitution to a nucleotide sequence encoding SEQ ID NO:1, amino acids 45-195.

Preferably, the construction method comprises inserting or replacing a nucleotide sequence comprising the humanized CD70 gene at the non-human animal CD70 locus. Preferably, the construction method comprises inserting or replacing a portion of the endogenous CD70 gene at the CD70 locus of the non-human animal with a portion comprising the human CD70 gene. Preferably, the construction method comprises inserting or replacing all or part of exons 1 to 3 of the non-human animal CD70 gene with a recombinant vector comprising all or part of exons 1 to 3 of the human CD70 gene. Preferably, the construction method comprises inserting or replacing part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene with a gene comprising part of exon 1, all of exon 2, and part of exon 3 of the human CD70 gene, preferably intron 1-2 and/or intron 2-3 are replaced.

Preferably, the construction method comprises modifying the coding frame of the non-human animal CD70 gene, and inserting any nucleotide sequence comprising the above into the endogenous regulatory element of the non-human animal CD70 gene, wherein the coding frame of the modified non-human animal CD70 gene can adopt a functional region for knocking out the non-human animal CD70 gene or adopt a sequence for inserting a segment so that the non-human animal CD70 protein is not expressed or the protein with reduced expression or expressed is not functional, and further preferably, the coding frame of the modified non-human animal CD70 gene can be all or part of the nucleotide sequence from exon 1 to exon 3 of the non-human animal CD70 gene. Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD70 gene in the non-human animal and then performing the insertion operation. Or the insertion step can cause frame shift mutation to the endogenous CD70 gene and realize the step of inserting the human sequence.

Preferably, the genome of the non-human animal comprises a humanized CD70 gene on at least one chromosome.

Preferably, at least one cell in the non-human animal expresses a human or humanized CD70 protein.

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

Preferably, the targeting vector of the present invention is used for construction of a non-human animal.

Preferably, the construction method comprises introducing the targeting vector into non-human animal cells, culturing the cells, transplanting the cultured cells into oviduct of female non-human animals, allowing the female non-human animals to develop, and identifying and screening the non-human animals with humanized CD70 genes.

Preferably, the construction method further comprises mating the non-human animal humanized with the CD70 gene with another genetically modified non-human animal, performing in vitro fertilization or directly performing gene editing, and screening to obtain the polygenetically modified non-human animal.

Preferably, the other gene is at least one selected from the group consisting of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, and LAG 3. Preferably, the human or humanized CD70 gene or other gene is homozygous for the endogenously modified locus; preferably, the human or humanized CD70 gene or other gene is heterozygous for the endogenous modified locus.

In an eighth aspect of the invention, there is provided a humanized non-human animal, wherein the non-human animal expresses a human or humanized CD70 protein, and/or wherein the genome of the non-human animal comprises a human or humanized CD70 gene.

Preferably, the humanized CD70 protein is the humanized CD70 protein.

Preferably, the humanized CD70 gene is the humanized CD70 gene described above.

Preferably, the nucleotide sequence encoding the human or humanized CD70 protein or the nucleotide sequence of the human or humanized CD70 gene is operably linked to endogenous regulatory elements at the endogenous CD70 locus in at least one chromosome.

Preferably, the non-human animal further comprises other genetic modifications, the other genes selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, LAG 3.

Preferably, the human or humanized CD70 gene and/or said further gene is homozygous for the endogenous modified locus.

Preferably, the human or humanized CD70 gene and/or said other gene is heterozygous for the endogenous modified locus.

In a ninth aspect of the invention, a CD70 gene-deleted non-human animal is provided.

Preferably, the non-human animal lacks all or part of the nucleotide sequence of the CD70 gene.

Preferably, the non-human animal lacks all or part of exons 1 to 3 of the CD70 gene. Further preferably, all or part of the nucleotide sequence of one, two, three or a combination of two consecutive exons from exons 1 to 3 is deleted. Still more preferably, part of the nucleotide sequence of exon 1, all of exon 2 and part of exon 3 are deleted. Most preferably, the nucleotide sequence of part of exon 1, intron 1-2, all of exon 2, intron 2-3 and part of exon 3 are deleted.

Preferably, the non-human animal has a deleted portion of exon 1 of the CD70 gene that does not include at least a non-coding region, and more preferably, does not include a portion encoding a cytoplasmic region and/or a transmembrane region. Still more preferably, the non-human animal further lacks a nucleotide sequence encoding a portion of the extracellular domain of exon 1 of the CD70 gene.

In one embodiment of the present invention, the nucleotide sequence of the exon 3 of the endogenous CD70 gene deleted in the non-human animal does not include at least a non-coding region.

Preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the cytoplasmic, transmembrane and/or extracellular domain. Further preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular domain and/or the transmembrane domain. In one embodiment of the invention, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular region.

The tenth aspect of the invention provides a method for constructing the non-human animal with the CD70 gene deleted, wherein the method for constructing the non-human animal comprises the step of constructing the non-human animal by using the targeting vector.

In the eleventh 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 the step I) with other genetically modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-genetically modified non-human animal.

Preferably, the other genetically modified non-human animal comprises a non-human animal modified with the genes PD-1, PD-L1, CD27, CD40, OX40, 4-1BB and/or LAG 3.

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 modified non-human animal.

Preferably, each of the plurality of genes modified in the genome of the polygenic modified non-human animal may be homozygous for the endogenous modified locus.

Preferably, each of the plurality of genes modified in the genome of the polygenetically modified non-human animal may be heterozygous for the endogenous modified locus.

In a twelfth aspect of the present invention, there is provided a non-human animal or its progeny obtained by the above construction method.

In a thirteenth 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.

In a fourteenth aspect of the present invention, there is provided a method for producing an animal model, which comprises using the above-constructed humanized non-human animal of CD70 gene, a non-human animal lacking CD70 gene, or a multi-gene-modified non-human animal or its progeny. Preferably, the method further comprises the step of implanting the tumor cells.

In a fifteenth aspect of the present invention, there is provided a use of the above-mentioned humanized non-human animal of CD70 gene, a non-human animal deficient in CD70 gene, a multi-gene-modified non-human animal or progeny thereof, or a humanized non-human animal of CD70 gene, a non-human animal deficient in CD70 gene, a multi-gene-modified non-human animal or progeny thereof obtained by the above-mentioned construction method, for preparing an animal model.

In a sixteenth aspect, the invention provides the cell, the tissue or the organ, wherein the cell, the tissue or the organ expresses the humanized CD70 protein, or the genome of the cell, the tissue or the organ comprises the humanized CD70 gene, or the cell, the tissue or the organ is derived from the non-human animal or the non-human animal obtained by the construction method.

In a seventeenth aspect of the present invention, there is provided a tumor tissue after tumor loading, wherein the tumor tissue expresses the humanized CD70 protein, or the genome of the tumor tissue contains the humanized CD70 gene, or the tumor tissue is derived from the above non-human animal or its progeny, the non-human animal obtained by the above construction method, or the above animal model.

In the eighteenth aspect of the invention, a CD70 gene humanized cell is provided, wherein the cell expresses human or humanized CD70 protein, or the genome of the cell contains humanized CD70 gene.

Preferably, the humanized CD70 protein is the humanized CD70 protein according to the first aspect of the present invention.

Preferably, the expression of endogenous CD70 protein in said cell is reduced or absent.

Preferably, the genome of said cell comprises all or part of the human CD70 gene. Further preferably, the cell comprises the humanized CD70 gene described above.

In a nineteenth aspect, the present invention provides an application derived from the above-mentioned humanized CD70 protein, the above-mentioned humanized CD70 gene, the above-mentioned non-human animal or its progeny, the non-human animal obtained by the above-mentioned construction method, the above-mentioned animal model, the above-mentioned cell, tissue or organ, or the above-mentioned tumor-bearing tissue, the application comprising:

(A) Use in the development of products involving CD 70-related immune processes in human cells;

(B) Use as a model system in association with CD70 for pharmacological, immunological, microbiological and medical research;

(C) To the production and use of animal experimental disease models for the study of CD 70-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

(D) The application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human CD70 signal channel regulator is studied in vivo; or,

(E) The functions of the CD70 gene are researched, the medicine and the drug effect aiming at the target site of the human CD70 are researched, and the application in the aspects of CD 70-related immune-related disease medicines and anti-tumor medicines is researched;

(F) The application of the CD27/CD70 signal channel in the treatment fields of tumors, immune-related diseases and the like is researched.

Preferably, the use may be for therapeutic purposes.

Preferably, the use may be for non-therapeutic purposes.

In a twentieth aspect of the present invention, there is provided a method of screening for a human CD 70-specific modulator, said screening method comprising administering the modulator to an individual implanted with tumor cells, and detecting tumor suppression; wherein the individual is selected from the non-human animal or its offspring, the non-human animal obtained by the construction method, or the animal 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 subject's body weight, fat mass, activation pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

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

Preferably, the method of screening for a human CD 70-specific modulator may be for therapeutic purposes.

Preferably, the method of screening for a human CD 70-specific modulator may be for non-therapeutic purposes. The screening method is used for screening or evaluating drugs, and detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary but is only a possibility.

According to a twenty-first 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 applying the intervention program; 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, and the above animal model.

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

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

Preferably, the method of assessing the intervention regimen may be for therapeutic purposes.

Preferably, the method of assessing the intervention regimen may be for non-therapeutic purposes. The evaluation method detects and evaluates the effect of the intervention program to determine whether the intervention program has a therapeutic effect, i.e. the therapeutic effect is not necessarily but only a possibility.

In a twenty-second aspect of the present invention, there is provided a use of the non-human animal obtained by the above construction method or a progeny thereof, or the above animal model, in the preparation of a human CD 70-specific modulator.

In a twenty-third aspect of the present invention, there is provided a use of the non-human animal obtained by the above-mentioned construction method, or the above-mentioned animal model, in the preparation of a medicament for treating tumor, inflammation or immune-related diseases, derived from the above-mentioned non-human animal or its progeny.

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 CD70 gene humanized non-human animal can normally express human or humanized CD70 protein in vivo, can be used for drug screening, drug effect evaluation, immune disease and tumor treatment aiming at human CD70 target sites, can accelerate the development process of new drugs, and can save time and cost. Provides effective guarantee for researching CD70 protein function and screening related disease drugs.

The "cell" of the present invention may be a fertilized egg cell, an embryonic stem cell, an immune cell (e.g., a T cell, a B cell, a Natural Killer (NK) cell, and a dendritic cell), a tumor cell, or the like. The tumor cell may be blood-borne tumor such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia and Waldenstrom's macroglobulinemia, and may also be thymus tumor, nephroblastoma, glioblastoma, astrocytoma, ovarian cancer, etc. Thus, depending on the source of the cells, a portion of the cells described herein may develop into individual animals and a portion may not.

The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that include the stated specified components or steps, as well as any other specified components or steps, which are not materially affected. However, when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described herein.

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

The "humanized CD70 protein" of the present invention comprises a part derived from a human CD70 protein and a part of a non-human CD70 protein. Wherein, the "human CD70 protein" is identical to the whole human CD70 protein, namely the amino acid sequence of the "human CD70 protein" is identical to the full-length amino acid sequence of the human CD70 protein. The "part of human CD70 protein" is a continuous or alternate 5-193 amino acid sequence consistent with the amino acid sequence of human CD70 protein. Preferably, the amino acid sequence of the human CD70 protein is identical to the amino acid sequence of the human CD70 protein in a sequence or at intervals of 10-146, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 160, 170, 180, 190, 193.

The "whole transmembrane region of human CD70 protein", "whole cytoplasmic region of human CD70 protein" or "whole extracellular region of human CD70 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 CD70 protein, respectively.

The "part of the extracellular region of the human CD70 protein" of the present invention is a sequence of 5 to 155 (preferably 10 to 146) consecutive or spaced amino acids identical to the amino acid sequence of the extracellular region of the human CD70 protein, for example, a sequence of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 146, 150, 155 consecutive amino acids identical to the amino acid sequence of the extracellular region of the human CD70 protein.

The "part of the non-human animal CD70 protein" according to the present invention refers to a sequence of contiguous or separated 5 to 195 (preferably 10 to 50) amino acids identical to the amino acid sequence of the non-human animal CD70 protein, for example, contiguous 5, 10, 20, 30, 40, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195 amino acids identical to the amino acid sequence of the non-human animal CD70 protein.

The "humanized CD70 gene" of the present invention includes a portion derived from a human CD70 gene and a portion derived from a non-human CD70 gene. Wherein, the "human CD70 gene" is identical to the whole human CD70 gene, namely the nucleotide sequence is consistent with the full-length nucleotide sequence of the human CD70 gene. The "part of the human CD70 gene" is a nucleotide sequence of 20-9503bp (preferably 20-4842bp or 20-911 or 20-441) which is continuous or spaced and is consistent with the nucleotide sequence of the human CD70 gene, for example, 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 800, 900, 911, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 4000, 4842, 5000, 6000, 7000, 8000, 9000 or 9503bp is consistent with the nucleotide sequence of the human CD70 gene.

"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 1 of the human CD70 gene, which comprises consecutive or spaced nucleotide sequences of 5-310bp (preferably 10-21 bp) identical to the exon 1 nucleotide sequence of the human CD70 gene. For example, the portion of exon 3 of the human CD70 gene, which comprises consecutive or spaced nucleotide sequences of 5-567bp (preferably 10-386 bp) identical to the nucleotide sequence of exon 3 of the human CD70 gene. In a specific embodiment of the present invention, the "part of exon 1" contained in the "human CD70 gene" at least includes the nucleotide sequence from the 48 th amino acid of the coding human extracellular region to the last nucleotide sequence of exon 1. In a specific embodiment of the present invention, the "part of exon 3" contained in the "humanized CD70 gene" at least includes a nucleotide sequence from the first nucleotide sequence of exon 3 to a stop codon.

The "xx to xxx exon" or "all of the xx to xxx exons" of the present invention comprise nucleotide sequences of exons and introns therebetween, for example, the "1 to 3 exons" comprise all nucleotide sequences of exon 1, intron 1-2, exon 2, intron 2-3 and exon 3.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 1-2" means an intron between exon 1 and exon 2.

The "part of the non-human animal CD70 gene" of the present invention is a nucleotide sequence of 20-3781bp (20-764 bp or 20-441 bp) continuously or intermittently, which is identical to the nucleotide sequence of the non-human animal CD70 gene, for example, a nucleotide sequence of 20, 50, 100, 200, 300, 400, 441, 500, 600, 700, 764, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3781 nucleotides is identical to the nucleotide sequence of the non-human animal CD70 gene.

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 "CD70 locus" refers to a DNA fragment of an optional stretch of exons 1 to 3 of the CD70 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, one 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.

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); mullisetal.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.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1986).

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 of the method of the present invention, the genetically modified animal is from a member selected from the group consisting of the family Pomaceae (e.g., hamster-like hamster), the family Kangalidae (e.g., hamster, new world rat and mouse, hamster), the superfamily muridae (true mouse and rat, gerbil, spiny rat, crowned rat) the families of marmotacaceae (climbing mice, rock mice, tailed rats, madagascar rats and mice), acanthomyidae (e.g. thorny sleep rats) and spacidomyidae (e.g. mole rats, bamboo rats and zokors). 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 specific embodiment, the non-human animal is a rodent that is a C57BL, C58, CBA/Br, CBA/Ca, A/J, CBA/H/st, CBA/H, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola strain C57 BL/BL, C58, CBA/Br, CBA/Ca, A/J, CBA/ST, CBA/H, and CBA/H strainsMouse and NOD, NOD/SCID, NOD-Prkdc^scid IL-2rg^nullBackground mice.

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 CD70 gene and human CD70 locus (not to scale);

FIG. 2: schematic representation of humanization of mouse CD70 gene (not to scale);

FIG. 3: CD70 gene targeting strategies and targeting vector design schematic (not to scale);

FIG. 4: southern Blot assay results, where WT is wild type control;

FIG. 5: schematic representation (not to scale) of FRT recombination process for humanized mouse of CD70 gene;

FIG. 6: f1 generation mouse genotype identification result, wherein M is Marker, WT is wild type control, PC is positive control, H₂O is water control;

FIG. 7: the mouse thymus tissue RT-PCR detection result is shown, wherein M is Marker, +/-H + is wild type C57BL/6 mouse, H/H is CD70 gene humanized homozygote mouse, H₂O is water control.

Detailed Description

The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more 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:

BglII, draIII, aseI enzymes were purchased from NEB under the respective accession numbers R0144M, R0510L, R0526M;

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

Brilliant Violet 510^TManti-mouse CD45 Antibody was purchased from Biolegend, cat No. 103138;

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

Brilliant Violet 605^TManti-mouse CD11c Antibody, available from Biolegend under cat # 117334;

APC anti-mouse CD70 Antibody, available from Biolegend under cat No. 2204066;

PE anti-human CD70 Antibody from Biolegend, cat # 355103;

Zombie NIR^TMfixable visualization Kit available from Biolegend, cat # 423106;

purified anti-mouse CD16/32 was purchased from Biolegend, cat # 101302.

Example 1 preparation of CD70 Gene-humanized mouse

A comparison scheme of mouse CD70 genes (NCBI Gene ID:21948, primary source.

To achieve the object of the present invention, a nucleotide sequence encoding a human CD70 protein may be introduced at the endogenous CD70 locus of a mouse, so that the mouse expresses the human or humanized CD70 protein. Specifically, the gene editing technology is used to replace the corresponding sequence of the mouse with the nucleotide sequence coding the human CD70 protein under the control of the regulatory element of the mouse CD70 gene, and the schematic diagram of the humanized CD70 locus is shown in FIG. 2, so as to realize the humanized transformation of the mouse CD70 gene.

Further design as shown in figure 3 of the targeting strategy diagram, which shows the targeting vector containing the mouse CD70 gene upstream and downstream homology arm sequences, and contains encoding human CD70 protein nucleotide sequence of A fragment. Wherein, the sequence of the upstream homology arm (5 'homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from 57456455 to 57460839 of NCBI accession No. NC-000083.7, and the sequence of the downstream homology arm (3' homology arm, SEQ ID NO: 4) is the same as the nucleotide sequence from 57448179 to 57452246 of NCBI accession No. NC-000083.7; the human CD70 gene sequence (SEQ ID NO: 5) contained in fragment A is identical to the nucleotide sequence 6586020 to 6590861 of NCBI accession No. NC-000019.10; the connection between the upstream of the human CD70 sequence in the A fragment and the mouse is designed as

Wherein the sequence "aacag"g" in "is the last nucleotide, sequence of the mouse

The first "c" in (a) is the first nucleotide in humans; the connection of the downstream of the human CD70 sequence and the mouse is designed as

Wherein the sequence "cctga"A" in "is the last nucleotide, sequence, of human

The first "c" in (a) is the first nucleotide in the mouse.

The targeting vector also comprisesThe resistance gene selected by positive clone, namely 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 mouse gene is designed as

Wherein the sequence "cgcagThe last "g" of "is the last nucleotide, sequence, of the mouse

The first "C" in (a) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo box and the mouse gene is designed as

Wherein the sequence "GCCGCThe last "C" in "is the last nucleotide of the Neo cassette, sequence

The first "T" in (a) is the first nucleotide in the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin A subunit coding gene (DTA)) is constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CD70 after being transformed is shown as SEQ ID NO:10, and the expressed protein sequence is shown as SEQ ID NO: shown at 11.

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 method comprises the steps of transfecting a targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse by electroporation, screening the obtained cells by using a positive clone screening marker gene, detecting by using PCR and Southern Blot technology to confirm the integration condition of an exogenous gene, screening correct positive clone cells, detecting clones identified as positive by PCR by using Southern Blot (cell DNA is digested by BglII or DraIII or AseI respectively and hybridized by using 3 probes, the lengths of the probes and target fragments are shown in Table 1), detecting exemplary results are shown in FIG. 4, and detecting by further verification through sequencing, wherein 4 clones with numbers of 1-A11, 1-G02, 1-G12 and 3-E06 are positive clones without random insertion.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				BglII	5’Probe	8.4kb	5.8kb
DraIII	3’Probe	20.7kb	12.1kb
				AseI	Neo Probe	-	10.4kb

Wherein the PCR assay comprises the following primers:

PCR-F1：5’-CACTGCCACGGAATTCTTGAGGTGA-3’(SEQ ID NO：12)，

PCR-R1：5’-TCATTCTGTCTTTTCGGTCACGCG-3’(SEQ ID NO：13)；

PCR-F2：5’-GCTCGACTAGAGCTTGCGGA-3’(SEQ ID NO：14)，

PCR-R2：5’-ACAGTCTGGACCTCCGAGGCA-3’(SEQ ID NO：15)；

the Southern Blot detection comprises the following probe primers:

5’Probe：

5’Probe-F：5’-AGAAGCAACTTGCAGAAGGGACAGG-3’(SEQ ID NO：16)，

5’Probe-R：5’-TTTAGAGGCCCTCGGAGATTGGTCT-3’(SEQ ID NO：17)；

3’Probe：

3’Probe-F：5’-TCTCCTCTCTCTTTGTTGGCTCTGG-3’(SEQ ID NO：18)，

3’Probe-R：5’-AAGGCCTTCTAGCACAGGATCTCCA-3’(SEQ ID NO：19)；

Neo Probe：

Neo Probe-F：5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO：20)，

Neo Probe-R：5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO：21)。

the selected correctly positive clone 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 F0 generation chimeric mice and the wild mice are backcrossed to obtain F1 generation mice, and the F1 generation heterozygous mice are mutually mated to obtain F2 generation homozygous mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 5 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous CD70 gene mice. The somatic genotypes of the progeny mice can be identified by PCR (primers shown in Table 2), and the identification results of exemplary F1 mice (from which the Neo marker gene has been removed) are shown in FIG. 6, wherein 8 mice numbered F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, and F1-08 are all positive heterozygous mice. This indicates that using this method, humanized mice of the CD70 gene can be constructed that can be stably passaged without random insertions.

Table 2: primer name and specific sequence

The expression of humanized CD70mRNA in positive mice can be confirmed by conventional detection methods, such as RT-PCR and the like. Specifically, 1 mouse of 9-week-old wild-type C57BL/6 mouse and 10-week-old humanized homozygote CD70 mouse prepared by the method were selected, and after cervical dislocation, thymus tissue was taken, and the primer sequences shown in Table 3 were designed to detect the mRNA expression of the thymus tissue of the C57BL/6 mouse and the humanized homozygote CD70 mouse. The results showed that only murine CD70mRNA expression was detected in C57BL/6 mouse thymus tissue (FIG. 7A); only expression of humanized CD70mRNA was detected in the thymus tissue of humanized homozygote mouse of CD70 gene (FIG. 7B), and no expression of murine CD70mRNA was detected (FIG. 7A).

Table 3: RT-PCR primer sequences

The expression of humanized CD70 protein in positive mice can be confirmed by flow cytometry. Specifically, 1 mouse of 8-week-old wild-type C57BL/6 female mice and 1 mouse of 9-week-old humanized homozygote of CD70 gene prepared in this example were selected, bone marrow cells were harvested after cervical-denuded euthanasia, and after incubation for 24 hours with 1ug/ml Lipopolysaccharide (LPS), anti-mouse CD45 antibody Brilliant Violet 510 was used^TManti-mouse CD45 Antibody (mCD 45), murine specific T cell surface Antibody PerCP/Cy5.5 anti-mouse TCR beta chain (mTCR beta), anti-mouse CD11c Antibody Brilliant Violet 605^TMFlow-through CD70 Antibody (hCD 70) recognition staining was performed after anti-mouse CD11c Antibody (mCD 11 c), anti-mouse CD70 Antibody APC anti-mouse CD70 Antibody (mCD 70) or anti-human CD70 Antibody PE anti-human CD70 Antibody (hCD 70)The detection result shows that the proportion of mCD70 marker cells (characterized by mCD45+ mTCR beta-mCD 11C + mCD70 +) in Dendritic Cells (DC) (characterized by mCD45+ mTCR beta-mCD 11C +) in wild type C57BL/6 mouse bone marrow is 14.1 percent, and the proportion of hCD70 (characterized by mCD45+ mTCR beta-mCD 11C + hCD70 +) marker cells is 0.95 percent; in contrast, the percentage of hCD 70-labeled cells in CD 70-gene-humanized mouse bone marrow DC cells was 20.1%, and the percentage of mCD 70-labeled cells was 0.94%. The results show that the humanized CD70 protein can be successfully expressed in the bone marrow cells of the humanized mouse with the CD70 gene.

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

The method or the prepared CD70 gene humanized mouse can also be used for preparing a double-humanized or multi-humanized mouse model. For example, in example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other genetic modifications such as PD-1, PD-L1, CD27, CD40, OX40, 4-1BB, LAG3, etc., or can be humanized CD70 mice and then used to obtain a mouse model with CD70 and other genetic modifications by using isolated mouse ES embryonic stem cells and gene recombination targeting technology. The homozygote or heterozygote of the CD70 mouse obtained by the method can also 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 CD70 and double-gene or multi-gene modified heterozygote of other gene modification can be obtained with a certain probability according to Mendel genetic rules, then the heterozygote is mated with each other to obtain double-gene or multi-gene modified homozygote, and the in vivo efficacy verification of targeted human CD70 and other gene regulators can be carried out by utilizing the double-gene or multi-gene modified mice.

Example 3 drug efficacy verification

The humanized mouse with CD70 gene or the mouse modified by multiple genes prepared by the method can be used for evaluating the drug effect of the targeted human CD70 antibody. For example, a tumor animal model is constructed by taking a humanized homozygote mouse of the CD70 gene, the mouse is divided into a control group or a treatment group, the treatment group is injected with an antibody drug targeting human CD70, 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 drug effect of the antibody drug in the humanized CD70 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 the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition. 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

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ggaagacttg gggggatgag ggggacagga caggaccaat tcacccctga ccacaccagc 180

atccctcccc cagagaaatc taagcaggga ctccacccct gagccacgcc cccaaggcca 240

gtggatgagt gtggatgaat gggcaacact tgccaactct atactgctct agcttccatc 300

ctcaccactg caaataataa taataataat aataataata aacatgatgt aaatcattgg 360

tacatggatt ctttataaag ttcaaagtcc tccagcagtt aatttcctag tgtactgcat 420

agttttatgt caatacaaat agggttggac aaggcatgac tgcatttcct tgattaatgt 480

tcaatgtagg agggcccagc acacaacatt tccatggaga tggtcctaag ccctacaaga 540

atgtaggctg agcaagtcag taagccagag atccccctag gctcctgctt cagtcctgcc 600

ttctctcgat gatgaggaac tataaagagg aacaaatcct tccctctcca aggtcacggt 660

gttcatcaac aataaaacct ctagcatgaa ccacatacat tagatgcctc aaagccccca 720

aaatggagct gaagaaatga gttagttatg agcaccggct gggactctcg ctgactctgg 780

agggctccat ttcagctagg ctgatcgatg agctgcggca atcctcctgt ctctgcttct 840

cagcaggcta ccattacaga caggcactga tgaaccccac tttcaggtat caaactcaga 900

gttcccattc ttgcttgatg gggcctgtac cagtaacttt aatttttttt cagaccttat 960

tttcaatgat gattcttaaa cactttaacc ttccttatag cccaccaccc accagaggta 1020

gtgggaaaga aaggatacag gagaagtgga cctgtttaga aaggttcttt ggagcaactc 1080

ctgcctgtgt tgtcaggaaa gaagttcaca ggttagcagc cgcagcttga tccactcaca 1140

aacacttcac ggacacacca gcagtccagt ttggtagagt caagatagca aacaggaatg 1200

aatccgaagt agtggctcta cctagcacta ggagaagctc tctctcagct gtgcctcttt 1260

cagggaagtt aagaacaatt catcagaccc acaggcgttg tacagctagc tccttaagcc 1320

tagctccgcc tctgtcactg tccatcaagc actttttttt tttttcattt ttcgaaacag 1380

ggtttctctg tgtagccctg gctgtcctgg aactcatttt atagaccagg ctggcctcga 1440

actcagaaat ccgcctgcct ctgcctccca agtgctgggt gtaaaggcat gggccaccac 1500

gactggctca tcaagtcctt tttataccct ccaaacctca catgttctcc aagggtcttg 1560

cctcaacact ccgccaatca gcatgagtcc gaggaagctg caagaaactg cagcacacca 1620

ccagaagact tttggtgctc tttttctctc tatagagtcc ctgtgagacc ccgacttaga 1680

gcgtttctcc ctgggaggta aagcccctga atgttccccc aagcctgttt tccctgatct 1740

ctaaaaatac agccagaaag aagctctttg tcctctacag ccagcaaaaa gcctttttgt 1800

ttctatacct tataaccaga gatacgataa ttgtaatttt accaaggaca cctggcagag 1860

aagagctgta gccaactctc ctcccaactc ctcccaactc ctcccaattc ctcagctagc 1920

tgttaaaagc cccctacggt taccactcag ggtggaactc ccctgccctg cgcagcgaag 1980

aggagttcgt cctcagctag ctgataataa aacctcttgc agtttgcatc aggtgtggtt 2040

ttctcaagtc attggggtgc tggccagctc atcccgggac ttgagcggag gcccagcttc 2100

aggggtctta catcccgaca aaggcagcat gttgttagca aagatccttc atcacgtgtc 2160

ctttcacgtg cttgctttag tagaacatgc cctctgctgt gtctgcttca gctaaacgtt 2220

ccttcaggag tctgccttag tctatcacct gtgtccactt caagaaaaca ctccttcact 2280

tgtctgccct agcaaaacac caccaacaca actgactttc caaagaaccc ctaagtttcc 2340

acttcagggc ctgtatccac tgagccatat ccccagccta cttcatattt tttaaagggt 2400

ttgttttgtt ttaatgtatg tgtggggggg cgatatgtgc acaggtgccc atggaaacca 2460

gagttgccaa atactcctag aactggaatt tcagatcgtt ttaaaccatt tgacatgggt 2520

gctgggaagt gagccctctc tctgggatag acacactcaa cctctgagat gtctctccaa 2580

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acccatttca gaggcaacct ctccctggta ccctctcctg tagtaggcat ctcaacttcg 2760

gaaaaacttg agcttgtaga tgtgttccaa catttcatca aacagtctgg atcctgggta 2820

aaatagactt ccgtgaagaa cacacagcac tcttggggtg cccagagaag atccagaaga 2880

gaggacagca gagacaggag agatacaagc cccaggcctg ccaggctggg gcagctgctg 2940

cccagctcgg cacctcctgc ctcaagtgtt ttctcgcctc tgccagttct aagtaaactc 3000

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cacctgaaag caccaagcct ggttgtctgc ctgttcattc tttattgtct gacagcttcg 3120

tatatttgca aaaatgaatt gtagtcatga ccaccccctt tcccctctcc agtccactcc 3180

cctccttgtc ctgctgaaca ttttcttcca agcaagttct gctccccacc cctgtaatcg 3240

ccactgcctt acaggctttc gagtctcttt ctgtgtgggc tgcactgaat catgggcagg 3300

aggctggttt tctggggcaa tggcaactta ctggtggcca caacactgaa gaaagtgacc 3360

ttcctttccc agtggccaga actgcccaga aagcctcagg gaggggcaga atctcatggc 3420

ccagccccta tcaaggccaa accattcggg gacatcttgt gccaattttc acagctgagt 3480

taagtgtagg agtgcaatgg ctgtgtccac tttcaggtag gatcattgtc tcagtctccg 3540

ggtgacatgg atggggatgg gggtggggat cagcagtagc taatcaatat gctagctatc 3600

atattgagac caagccaagt gtggtggctc acaccagcaa tctagtagtc ctgcagtcag 3660

aaggatgtga gaggccagcc tgggctacat gacaagtttg acacctgcct gagctacttg 3720

aggagctgaa ggtcaccatg gacaccgtgg caaatactaa gccagcctgg gctctacggt 3780

gagttccaat gtgagctgtt tataatggcc ccccatggaa gagcctacaa tcgtgtccaa 3840

atattttggg aagcacaagg gattcctgga tgcggatgct tcctgatttc ctagcattca 3900

gcccaatact gaacctctaa tagatgctca gtgaatggtt tgtcgaatga atgaatgaat 3960

gaatgggcac cgagcagcag gagcaggggc tgaatgccca atcggaagct taaatattct 4020

gaagcaggga caggcctgct tcagtttgtc tgtgggatgg cagaaggtgc caaaagctcc 4080

aggggatttc cctgccctcc gagaagaggc ccagttcttc ccctgcatcg gacatccccg 4140

aggttctaag ggcaggtcaa ggcaggcaga agcttcaaaa gctcggctga ggaggctaca 4200

gcttcccgct gccttcaggc cgctgcttcc gtgcagggat gccggaggaa ggtcgccctt 4260

gcccctgggt tcgctggagc gggaccgcgt tccagcgcca atggccatgg ctgctgctgg 4320

tggtgtttat tactgtgttt tgctgttggt ttcattgtag cggactactc agtaagcagc 4380

aacag 4385

<210> 4

<211> 4068

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tactatggag gaggcctgtg cagaagctat aggcctctag gtgggaagtg agcactctaa 60

gcttgacctt caaagctccc tgtgataggt cacagctagg ccccacctcc aaagggtcca 120

catggaccct agattcaaac ctgaacattc caatcctggt cccaataggc ccatgggcat 180

ctcacagtac aaatgtattt aattcaaatt caacaccctc caccccttaa caccttcaat 240

gtctttcaaa agactgagtt cagaggcagc agaaacagct cagcagttag agcaccagct 300

gctcttgcag aggacctggg tttggttcca gcacctatag ggcagctcac agccagctct 360

aactccagct ccagggatcc cacactccgt tctggcctct gtgggcacag tgcacacatg 420

ttgcacagac agacatgaag gcaaaacacc catgcacaga aaaaatttta aaaacagaga 480

ggcctgtcta acagtctaaa ccaactaatc cttaactcgg gggggggggg ggagggcact 540

atgaaactag aatccatcaa ggttttcccg tggtagaaaa taaatcatcc aactgtcaag 600

tggggaggat ggaataggga ggcagatgag gccgaggcaa ggtgaaaccc cagcattgca 660

agttttgctc cctctactcc aaacctggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 720

tgtgtgtgtg tgtttaaatg tcaaagggtt aaattgacag acaaaagttc attaaacaga 780

cagctaacct agcagacatc ctacctccct gacagcttct ttttgtgctc cagagaacag 840

aacacagcac ttacagtttg cacgagggat gaagctgggt gttgttcaga gccaggatct 900

gtccaggagc cagaggctta ctcttcagtc tagactggct ggctagtgag ctgccagggt 960

ccctcctgtg tccacctccc cagcatggga cacagacagg catgccatca gacacaaagt 1020

ttacatggct gccggagctc tgaacgcagg tttcccctga ctgtcttgtt cctatccaac 1080

ggtctgtcca attccaatgt gtgaactctg ggaacacgtt catttaggct gtagcaaata 1140

ttcacaggca gaaacggtga gtggactata aggaaactct gttctctgct atgcaaacca 1200

taaaaggatg tgttcttaag taaaataaaa atgacctcag tggaaatata ttgtgacggg 1260

aaaggactga ttttcaaggt ttgagcacgg ggggtattat aaagctgcta gggggaatct 1320

ccaccatcca cataggatga tgtcaacagg tgcagccttg aaacttcccc ttcaatgaca 1380

ctcaactgtg tagtaacttg atttatggta ctaagtgaca actgagagga gacctttaat 1440

cccagcactt gggaggcaga ggcaggcaga tctgaattca aagccagcct gatctacaga 1500

gtgattttca gggctataca gtgaaacctt gtgagagctg gggaagcgat ggctactctt 1560

ccagaggacc tgggtttgag tccccgtgct ttatggactg aaccatgcct ggacctttaa 1620

ggttcatttc ccctcaggat gtaggctcca gctctattct gtttctgaag gtctgtgcgt 1680

ataaagttgg gcatcatggc acacacctgt catcccaaca ttcccaggaa gctgaggcag 1740

gaggtttgca agtttgaggc caatcttagc tacatagtgt gctcttatct ggaaaaacaa 1800

aacaaacagt gggaatggga gggagaggaa gaaagggggt gtaaggggag agagagatag 1860

ggaagatggg ggcgggcatg gcagggaaga gagagagaag gaagaatggc gaaggaaatg 1920

caccacctct ctgttcgact ctattgtttg ttccgggtct catttacaaa acagaatagt 1980

tacacatatg tgcttttgaa actcgggatc tggacacaca cactgcacaa tggtcacttc 2040

tattcaacaa gttgaggtct cccgtctcct cagttgaccg tttctcctgc ctgcatgttt 2100

ctctttctgt ggaaatgcta tcaacagtcg atattattac cattgttact gctgaaagtt 2160

cctacagtct cggtcagggc ctccaactga ctcagataca caaccacaac ttctgagtaa 2220

ggtctcacac atttcacagg ggtctaggga gttcagcctt cttatttccc acgtctaagt 2280

aacaatatgc agtagcatcg ttcagggctg aataacactt cattgggtct ggatacccca 2340

ttagttgatt tggctagagc tgaaaataga ggatttcatt atgacatgtt cctacatggg 2400

gctcacattc acccccacaa tattctcttg tccccccttc ctcctacccc cttccccttt 2460

cctctaggcc tcatcaaatt gccccttctt gtatatttct ctctccaaat gcctaatatt 2520

agagaaaagg atggctttgt ctgagtctgt ctatgtctgt cttgttttaa taaaatggcc 2580

tctgtttcta tccataatag aaacatgatt taattccttt ttaaaaggtt ttaaagaatg 2640

tgatttattt attattgatg tgtatgtgat tctcacagtg catgtgcagg tccagggata 2700

gaacctagag tctgccttta cccactggat cagctcaatg cccccccccc accttttctt 2760

taacaggttt tgcacagtcc agtctggcct cacttcactc ttttttacag ttcaataaga 2820

cagcttcatt ttgtgcaatg tgtatctaga ccaaattttc tttttttgaa gacttattta 2880

tgtgtgaaag cccatagaag ccagaaatgg atatagtaac ctctggggag ggagttacag 2940

atggttgtga gtgccagtga gtgcagaatc gaattcagtc ctctgcaaga gctgcccagt 3000

gcgctaacca ctgggccact gctccagcct ctcagccttt tttaagagtc atcctcacag 3060

atatgaggtt tgttgggact ctggttagca ctttctgcca ttgctgcagt tggactcctt 3120

ttgcatactc tctgcccatt tctagtgctt acgaaacaga ttgaacttca ccaagaaagg 3180

aattttgctt ctactgaaaa gacaagccag ggcagcctct ctggggggtc ccattcttca 3240

gagcccaaaa ttaggacggt ggtgtgacct cagcatggac ccaaagaatg ttcaactgac 3300

tcaggacaga cacctgctca tggtactcac tgctgctccc ttcgcagtag ccaagaagtg 3360

tgccagcctg tgtgcctaac agcagattcg tgatgaggaa aacgtgacag cctggtggga 3420

atttctctat ttttgcaggc aatggagaca ggtaatgatg taagctgttg ctaggtccat 3480

gagaagagac tagtcacaat tctattccat tgatatatgt gtctatgcat atatccacgc 3540

ctcagtgttt attttgtctt tgagatagat gagagacaca cagagaatga agtatctatg 3600

gatggataaa tgaatgatag acagacagac aaacagatag atacctagat acatacatag 3660

acacagatat atagatatat agatagatct tagatagata catagatcaa aagacaggtg 3720

atagatgatg atagatatat agacagataa taatacatat agatgcatac atacacatat 3780

atagagagat acatacatac atacatacat atacatacat atgtgcatag ttacatacat 3840

ggaaacatag ataataattg tcctataaga cagcctgggc tcacctccac catcctgttt 3900

tcataagtga acttccagct ttcaactgtt ttctgatgga tttatcttga ttttgtttgt 3960

ttatttgacc aattggttgg ttgagcaact tttgagacaa ggtctcatgt agtctaagcc 4020

tcaaactatc tgtaaagctg aatacatcct tcaactcctg cttcttcc 4068

<210> 5

<211> 4842

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgccgctcg agtcacttgg ggtgagttga gatggaaaag ttgggaagaa aacatagaga 60

ggcgcgtgac cgaaaagaca gaatgagatg ggtacaaaga ggccagagag gaagatctgg 120

tagggcagag acagagacca gaacagggag gcgaggcggg gaccaggctg cccggtgtag 180

gggctacgag acaggcagcc ctgccaggag gtacagggag atcccgggat gggaaaggta 240

ggcacacatg gaaatggaag atgactcggc tctggtgttc ccccggcagg ctgactcaga 300

ggctgctggg ggcttcacaa ggctgggcgt gggggcttcc tggggcctcc taggacggga 360

tggccccagc cactcgctcc gggtggggga ggggtccctt tggggaccgc gccgggcgcc 420

tttgcagcgt agagagtccg ctgcgcgcgg tgctctcgcg cccagtgaca tccaggaaaa 480

cgattcggga aacgaagaag ttcttttgaa ggtctcgact tcacgttccc cgctggttca 540

gacctgcttc ctctttaaga agtcttaaga gtaaaaaaaa ataaaatgaa ataaaatcac 600

cagtgcgcgc cgtgggatga gaggtggaaa ggaggatgga cagagaaaag agagctcctg 660

gcacagggga cacatagaac ctctctgctt acgtccgtgc cctgttttct ggtcttttct 720

tccagtggga cgtagctgag ctgcagctga atcacacagg taacacgggg gacgtggagg 780

gacggggaga agaagaggca cagagagaga aggaaggaga ggtagaaaga caagtgggga 840

gagacagaga gaaagagaca cagacagaga cggagggaga gagggaggga gagataggga 900

gggaaacgga gagggggaga cagagagaag acagagaggg agggagaggc ccagagaaag 960

ggagggagag acagaaaaaa gaacagagag agggacagag gggacagacg ggacaggaga 1020

agaaggggaa agaaagaaac agggagagac gcagggagag tggaagaggg agggagggag 1080

acaggcgggg gagacagaac gagagagagg gagagagacg gagaaagaga aaaaagacag 1140

atagaggaag agagaataaa aacgagagag gcgacctggc gtggttgctg gagcctgtaa 1200

tcccagcgct ttcggagggc gcggcgggtg gatcacttga gcccaggagt ttgagaccag 1260

cctggccaac atggtgaaac cccgtctcta ctaaaattac aaaaattagc cgggtgtggt 1320

ggcaggcacc tgtaatccca gctactcagg aggccgaggc aggagaatcg cctgaacctg 1380

ggaggcagaa gttgcagtga gccaagataa tggcactgct ctccagcctg ggtgacagag 1440

ggagattctg tcaaataaat aaataaaaag aagaaaggaa aagaaaaaga aagaaaggaa 1500

ggaaggaaaa gaaagagaag aaggaaggaa ggagagaggg agggaggtga gagagagaga 1560

aaggaaaaag aaagaagaaa gagaaaaaaa gaaataagag aaagaaaaga aagaaagcca 1620

gagagaggaa aagagaagga gaataacaga gaggaagaga cagagacaga gagggggaag 1680

aagaaaaaca gggcggggga gaaagagatc attcccaaga gaaaaaggaa aagggagaga 1740

acgagagaga gagagaggag agagagagcg agcaggtttg tgtttctggg catccactac 1800

ctttctctcc ctttccctct cagtccttca cattcgatct cctcccttgg aagaatcaaa 1860

ggggttttgt tagtaaacga aaacgctcac aggaaaggct acccttcctc cattcatgct 1920

acaagcacta attatacatc tgcagtgggc aggcactgtt ccggtactgc agtcgcgcac 1980

aaaacagaca gaaatccctg cctggtgagc ttcccttcta ctatcactcc taccgaggag 2040

gagatgacca aaaaaataaa aacgaacatg tgaaaaacag agcaatgcca cgtagagtaa 2100

aacaggccaa gagcaggttt tagagaaaaa atcaggcaag gaggtgggtg gggaatccgg 2160

gcatgccagg gttggggatt ttcatttaaa gtggagtcgc ggcggtcggg tgggggggtg 2220

gtatctgagt gggtggcatt ggcaagaagc cttgagggag aagaaatgaa cagttctgct 2280

ctaggtggaa gtctctacct cctcccagcc tctctcgaac gcttcttcct cttctgtttt 2340

tccacggttc atccccagag actaaaatta ctgcacgctt gcgtgttttg ttccttccat 2400

tttgtaatgg aagggacctt tgttgctctc tcttctcctt gggtctctga cccctgatat 2460

atcttccccc cctccccggc ctttgccata tttttcctgt aatttggtct tgaggcccct 2520

ttctggagag tggctgtcca gccccacatc gatggggcac caggggtggt ggccacaggt 2580

gttgacagtg tctttcacag gacagctctt tcttctggtg gatgatatca tgcctaagtg 2640

tgtgacaggg gaccaggtgt cactcttaca ggaaatttgc aaagcctggt aaacaccctt 2700

ggggctcctg tttgaccccg ttcagttcct tcctaacaag atagccagtc tccaggagag 2760

ccttgagtga ggaggagagt gaggctcagg tgtgtcagtc agataagttg cggagacagg 2820

agacgctgag atgaaacacg tgaaatatag ccgaggacct tggctcccgg ctgtgatccc 2880

agcgctttgg gaggctgagg tgggaggatc gcttgagccc aggagttcta gactatgttt 2940

cttgggaaac atagcaagac caccatctct agaacaaaat tttgaaaatt agctggtcat 3000

ggtggcgcat gccacctccc agtgactcgg gaggctgagg tgggagatag cttgagcccc 3060

agaggctgag gctgcagtga gctatgattg caccactgca ctccagcctg ggtgacagga 3120

ccagaccctg tctctaaaga aaataaagat cctagagaga agagggccaa cgggaaggta 3180

ggggagctct ctgagacaca cactcaacca gcgggtggaa agcaagaaaa aaaaagggtc 3240

ctgggagcca aagcttttat tggcgttcag ggtgtgaccc gagcaggcgt cccttgggga 3300

attctctcgt gcactctctt gcgcgctgtc tcgtgcgcac gctgtctctc tcgtgctgtc 3360

tgtctcactc tctcaggcac tctctcgctc actcccgtgc actctcgagc tctgtagctc 3420

tcatcctctc tcacacacac actctcccat gcactctctc gagctcactc tcacactctg 3480

tctcgtgcac tctctctcaa gctctctcgt cctctctcac acacacactc tctctcttgc 3540

tctctctctc atgctgtctc tcgtactctc tcctgctctc tcgtgctctc tctcactctc 3600

tcgtgcactc tccctcaagc tctctcgtcc tctctctcac actctctcgc tgtctcatgc 3660

tctctctccc tcatgatctc tctctctctc atgctgtctc ttgcactctc tcctgctctc 3720

tcgtgctctc tcgcactctc tcgtgcactc tccctcaagc tctctcgtcc tctcacacac 3780

tctctcgctc tctcatgctc tctctctcat gctctctgtc gcactctctc gtgcactctc 3840

gtcctctctc acacatacac tctctctctt gctctctctc tgcattctct ctctatatat 3900

ctctccctcc ccctctatct ttacccttgt ctgtttctgt tttttttttt tttttttttg 3960

tctctctctc tcctggtgtc tctctctccc tgtctctacc cctttcccct tacccctctc 4020

tccttccctt tagatctcta accttctatc cccctccctt ttttttgaga cagggtttct 4080

gtgaccaagg ctggagtgca gtggtgtgat catagctcac tgcagtctca gcctcccagg 4140

ctcaagcgat cctccagcct cagcctccca agagtagcca ggaccagagg cgtgcaccac 4200

cacacctggc tgacttttta ttttttgtag agatggggtc tcgccctgtt gtccaggctg 4260

gtcttgaact gctgacctca ggtgatccac ccgccttggc ctcctaaact gttaggattt 4320

caggcatgag ccaccacgtc caacctattc cttgatctct aactctcgat ctctatatct 4380

gactttctct atgaccccct gtgcctcagt ttccctaaac ctccatcctc tcatccctct 4440

aaccctgtgc ccccaggacc tcagcaggac cccaggctat actggcaggg gggcccagca 4500

ctgggccgct ccttcctgca tggaccagag ctggacaagg ggcagctacg tatccatcgt 4560

gatggcatct acatggtaca catccaggtg acgctggcca tctgctcctc cacgacggcc 4620

tccaggcacc accccaccac cctggccgtg ggaatctgct ctcccgcctc ccgtagcatc 4680

agcctgctgc gtctcagctt ccaccaaggt tgtaccattg cctcccagcg cctgacgccc 4740

ctggcccgag gggacacact ctgcaccaac ctcactggga cacttttgcc ttcccgaaac 4800

actgatgaga ccttctttgg agtgcagtgg gtgcgcccct ga 4842

<210> 6

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttggtttcat tgtagcggac tactcagtaa gcagcaacag ctgccgctcg agtcacttgg 60

ggtgagttga gatggaaaag 80

<210> 7

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgatgagacc ttctttggag tgcagtgggt gcgcccctga ccacaactcc aggatgactt 60

gtgaatattt tttttctttt 80

<210> 8

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

taaaatttta attctgttct tagtaacact cctcacgcag cacccagtgg atatcgaatt 60

ccgaagttcc tattctctag 80

<210> 9

<211> 121

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaagttccta ttctctagaa agtataggaa cttcatcagt caggtacata atggtggatc 60

cactagttct agagcggccg ctactatgga ggaggcctgt gcagaagcta taggcctcta 120

g 121

<210> 10

<211> 842

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaaggtgcca aaagctccag gggatttccc tgccctccga gaagaggccc agttcttccc 60

ctgcatcgga catccccgag gttctaaggg caggtcaagg caggcagaag cttcaaaagc 120

tcggctgagg aggctacagc ttcccgctgc cttcaggccg ctgcttccgt gcagggatgc 180

cggaggaagg tcgcccttgc ccctgggttc gctggagcgg gaccgcgttc cagcgccaat 240

ggccatggct gctgctggtg gtgtttatta ctgtgttttg ctgttggttt cattgtagcg 300

gactactcag taagcagcaa cagctgccgc tcgagtcact tgggtgggac gtagctgagc 360

tgcagctgaa tcacacagga cctcagcagg accccaggct atactggcag gggggcccag 420

cactgggccg ctccttcctg catggaccag agctggacaa ggggcagcta cgtatccatc 480

gtgatggcat ctacatggta cacatccagg tgacgctggc catctgctcc tccacgacgg 540

cctccaggca ccaccccacc accctggccg tgggaatctg ctctcccgcc tcccgtagca 600

tcagcctgct gcgtctcagc ttccaccaag gttgtaccat tgcctcccag cgcctgacgc 660

ccctggcccg aggggacaca ctctgcacca acctcactgg gacacttttg ccttcccgaa 720

acactgatga gaccttcttt ggagtgcagt gggtgcgccc ctgaccacaa ctccaggatg 780

acttgtgaat attttttttc ttttcaagtt ctacgtattt ataaatgtat atagtacaca 840

ta 842

<210> 11

<211> 195

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Pro Glu Glu Gly Arg Pro Cys Pro Trp Val Arg Trp Ser Gly Thr

1 5 10 15

Ala Phe Gln Arg Gln Trp Pro Trp Leu Leu Leu Val Val Phe Ile Thr

20 25 30

Val Phe Cys Cys Trp Phe His Cys Ser Gly Leu Leu Ser Lys Gln Gln

35 40 45

Gln Leu Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu

50 55 60

Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly

65 70 75 80

Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly

85 90 95

Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val

100 105 110

Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr

115 120 125

Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu

130 135 140

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

145 150 155 160

Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr

165 170 175

Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp

180 185 190

Val Arg Pro

195

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cactgccacg gaattcttga ggtga 25

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcattctgtc ttttcggtca cgcg 24

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctcgactag agcttgcgga 20

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acagtctgga cctccgaggc a 21

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agaagcaact tgcagaaggg acagg 25

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttagaggcc ctcggagatt ggtct 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tctcctctct ctttgttggc tctgg 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aaggccttct agcacaggat ctcca 25

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggatcggcca ttgaacaaga t 21

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cagaagaact cgtcaagaag gc 22

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gtctgtggga tggcagaagg tgcca 25

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctcatgccac ccttcccact acttc 25

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccacaggtgt tgacagtgtc tttcac 26

<210> 25

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tctagagatg gtggtcttgc tatgttt 27

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tacaacccag ctccctctca gcac 24

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gggaccagga ttggaatgtt caggt 25

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gacaagcgtt agtaggcaca tatac 25

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gctccaattt cccacaacat tagt 24

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tgctggtggt gtttattact gtg 23

<210> 31

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

cagggtgagg ttggtacaga g 21

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tagcggacta ctcagtaagc a 21

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cagtgtttcg ggaaggcaaa 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

accacagtcc atgccatcac 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tacagcaaca gggtggtgga 20

Claims

1. A humanized CD70 protein, wherein the humanized CD70 protein comprises all or a portion of a human CD70 protein.

2. The humanized CD70 protein of claim 1, wherein the portion of the human CD70 protein comprises all or part of an extracellular region of the human CD70 protein, preferably comprises the amino acid sequence of SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; or, comprising a nucleotide sequence identical to SEQ ID NO:2 at positions 39-193 or 48-193 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.

3. The humanized CD70 protein of any one of claims 1 to 2, wherein the humanized CD70 protein further comprises a portion of a non-human animal CD70 protein, wherein the portion of the non-human animal CD70 protein comprises a cytoplasmic region and a transmembrane region of the non-human animal CD70 protein, and further preferably comprises a partial extracellular region; more preferably comprises SEQ ID NO:1, 1-49 or 1-44; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at positions 1-49 or 1-44 of at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%.

4. The humanized CD70 protein of any one of claims 1 to 3, wherein the amino acid sequence of the humanized CD70 protein comprises any one of the following groups:

a) The amino acid sequence of SEQ ID NO:11, and (b) the amino acid sequence shown in the figure;

b) And SEQ ID NO:11 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;

D) And SEQ ID NO:11, comprising the amino acid sequence of substitution, deletion and/or insertion of one or more amino acid residues.

5. A nucleic acid encoding the humanized CD70 protein of any one of claims 1 to 4.

6. A humanized CD70 gene comprising a portion of a human CD70 gene.

7. The humanized CD70 gene of claim 6, wherein the portion of the human CD70 gene comprises a portion of exon 1, a portion of exon 2, and a portion of exon 3 of the human CD70 gene, wherein the portion of exon 1 comprises at least 10bp of nucleotide sequence and the portion of exon 3 comprises at least 100bp of nucleotide sequence; preferably, the gene also comprises an intron 1-2 and/or an intron 2-3;

further preferably, said portion of the human CD70 gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.

8. The humanized CD70 gene according to any of claims 6 to 7, further comprising a portion of a non-human animal CD70 gene, preferably comprising all or part of exon 1 and/or all or part of exon 3 of a non-human animal CD70 gene; further preferred comprises a nucleic acid sequence encoding SEQ ID NO:1, or 1-44, or a nucleotide sequence that hybridizes with a nucleic acid sequence encoding SEQ ID NO:1 or 1-44 of at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;

even more preferably, said humanized CD70 gene encodes a humanized CD70 protein according to any of claims 1 to 4.

9. The humanized CD70 gene of any one of claims 6 to 8 wherein the mRNA transcribed from the humanized CD70 gene comprises any one of the group consisting of:

a) SEQ ID NO: 10;

b) And SEQ ID NO:10 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;

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

d) Has the sequence shown in SEQ ID NO:10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

10. A targeting vector, said targeting vector comprising a donor nucleotide sequence, said donor nucleotide sequence comprising one of the following groups:

a) A nucleotide sequence encoding the humanized CD70 protein of any one of claims 1 to 4;

b) A nucleotide sequence encoding all or part of the extracellular region of the human CD70 protein, preferably encoding a polypeptide comprising SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; alternatively, the coding sequence comprises a nucleotide sequence identical to SEQ ID NO:2, positions 39-193 or 48-193, or at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99% of the amino acid sequence identity;

c) The humanized CD70 gene of any one of claims 6-9; or,

d) Part of the No. 1 exon, the whole No. 2 exon and part of the No. 3 exon of the human CD70 gene, wherein the part of the No. 1 exon at least comprises 10bp nucleotide sequence, and the part of the No. 3 exon at least comprises 100bp nucleotide sequence; preferably comprises SEQ ID NO:5, or a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.

11. The targeting vector according to claim 10, wherein said targeting vector further comprises a 5 'arm and/or a 3' arm; the 5' arm has at least 90% homology with the sequence having NCBI accession number NC _ 000083.7; preferably, the 5' arm sequence is as set forth in SEQ ID NO:3 is shown in the figure;

the 3' arm has at least 90% homology with the sequence having NCBI accession number NC _ 000083.7; preferably, the 3' arm sequence is as shown in SEQ ID NO:4, respectively.

12. A method for constructing a non-human animal humanized with a CD70 gene, wherein the non-human animal expresses a human or humanized CD70 protein in vivo, and/or the genome of the non-human animal comprises the human or humanized CD70 gene;

preferably, the humanized CD70 protein is selected from the group consisting of the humanized CD70 protein of any one of claims 1 to 4;

preferably, the humanized CD70 gene is selected from the group consisting of the humanized CD70 gene of any one of claims 6 to 9.

13. The method of claim 12, wherein the non-human animal has reduced or absent expression of endogenous CD70 protein.

14. The method of constructing a recombinant vector according to any one of claims 12-13, which comprises introducing into the CD70 locus of a non-human animal any one of the following nucleotide sequences:

b) A nucleotide sequence encoding all or part of the extracellular region of the human CD70 protein, preferably encoding a polypeptide comprising SEQ ID NO:2 from position 39 to 193 or from position 48 to 193; alternatively, the code comprises a nucleotide sequence identical to SEQ ID NO:2 from position 39-193 or 48-193, is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98%, or at least 99%;

c) The humanized CD70 gene of any one of claims 6-9; or,

d) Part of No. 1 exon, all of No. 2 exon and part of No. 3 exon of human CD70 gene, wherein the part of No. 1 exon at least comprises 10bp nucleotide sequence, and the part of No. 3 exon at least comprises 100bp nucleotide sequence; preferably comprises SEQ ID NO:5, or a nucleotide sequence identical to SEQ ID NO:5 is at least 85%, 90%,91%,92%,93%,94%,95%,96%,97%,98% or at least 99%.

15. The method of any one of claims 12-14, wherein the human or humanized CD70 gene, nucleotide sequence encoding human or humanized CD70 protein, is regulated in the non-human animal by endogenous regulatory elements.

16. The method of any one of claims 14-15, wherein said introducing is substitution or insertion; preferably, said introduced non-human animal CD70 locus replaces a corresponding region of a non-human animal; preferably, part of exon 1, all of exon 2, and part of exon 3 of the non-human animal CD70 gene are replaced; it is further preferred that introns 1-2 and/or introns 2-3 of the non-human animal are also replaced.

17. The method of any one of claims 12 to 16, wherein the targeting vector of any one of claims 10 to 11 is used for the construction of a non-human animal.

18. The method according to any one of claims 12 to 17, wherein the method comprises mating a non-human animal humanized with a CD70 gene with another genetically modified non-human animal, in vitro fertilization or direct gene editing, and screening to obtain a polygenetically modified non-human animal;

preferably, the additional gene is selected from at least one of PD-1, PD-L1, CD27, CD40, OX40, 4-1BB and/or LAG 3.

19. Construction method according to any one of claims 12 to 18, characterized in that the human or humanized CD70 gene or other gene is homozygous or heterozygous for the endogenous modified locus.

20. A cell, tissue or organ which expresses the humanized CD70 protein of any one of claims 1 to 4 or which comprises the humanized CD70 gene of any one of claims 6 to 9 in its genome or which is derived from a non-human animal obtained by the construction method of any one of claims 12 to 19.

21. A tumor-bearing tumor tissue, wherein the tumor tissue expresses the humanized CD70 protein of any one of claims 1 to 4, or the genome of the tumor tissue comprises the human or humanized CD70 gene of any one of claims 6 to 9, or the tumor tissue is derived from the non-human animal obtained by the construction method of any one of claims 12 to 19.

22. Use of a humanized CD70 protein according to any of claims 1 to 4, a humanized CD70 gene according to any of claims 6 to 9, or a non-human animal obtained by the construction method according to any of claims 12 to 19, comprising:

a) Use in the development of products involving CD 70-related immune processes in human cells;

b) Use as a model system in association with CD70 for pharmacological, immunological, microbiological and medical research;

c) To the production and use of animal experimental disease models for the study of CD 70-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

d) The application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human CD70 signal channel regulator is studied in vivo; or,

e) The research on the CD70 gene function, the research on the medicine and the drug effect aiming at the human CD70 target site, and the research on the application of the medicine for the immune-related diseases related to the CD70 and the anti-tumor medicine.

23. The humanized CD70 protein according to any one of claims 3 to 4, the humanized CD70 gene according to any one of claims 8 to 9, or the method of construction according to any one of claims 12 to 19, wherein the non-human animal is a rat or a mouse.