CN114990128A

CN114990128A - Construction method and application of CD20 gene humanized non-human animal

Info

Publication number: CN114990128A
Application number: CN202210681774.XA
Authority: CN
Inventors: 赵磊; 刘畅; 李冲
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-06-16
Filing date: 2022-06-16
Publication date: 2022-09-02
Also published as: WO2022262808A1

Abstract

The invention provides a humanized CD20 gene, humanized CD20 protein, a targeting vector of a CD20 gene, a humanized non-human animal of a CD20 gene, a construction method thereof and application thereof in the field of biomedicine, wherein a nucleotide sequence for coding the human CD20 protein is introduced into the genome of the non-human animal by utilizing a homologous recombination mode, the human or humanized CD20 protein can be normally expressed in the animal body, and the humanized CD 3528 protein can be used as an animal model for researching the human CD20 signal mechanism and screening the drugs for tumors and autoimmune diseases, and has important application value for the research and development of new drugs of immune targets.

Description

Construction method and application of CD20 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 CD20 gene modified non-human animal model and application thereof in the field of biomedicine.

Background

CD20(Cluster of Differentiation 20) is a tetraspanin, localized on the surface of B lymphocytes. B lymphocytes are differentiated from pluripotent stem cells in the bone marrow and develop through several stages of progenitor B cells (Pro-B), Pre-B cells (Pre-B), Immature B cells (Immate B) and Mature B cells (Mature B). Mature B cells are released into lymphoid tissues and can continue to differentiate into Plasma cells (Plasma cells). CD20 appears at the pre-B cell to mature B cell stage as a surface antigen for B cells, but CD20 is not expressed on hematopoietic stem cells, progenitor B cells, and mature plasma cells. In humans, the surface antigen of CD20 is encoded by the MS4A1 (membrane-cloning 4-domains, subset A, member 1) gene. In addition to expression in normal B cells, CD20 is expressed in tumor cells of B cell-derived lymphomas, leukemias, and the like, as well as B cells involved in immune and inflammatory diseases, so the CD20 antigen is an ideal target for the treatment of lymphomas, leukemias, and certain autoimmune diseases. Because the original normal B cells are not affected by the anti-CD 20 monoclonal antibody, after the anti-CD 20 monoclonal antibody kills most of lymphoma cells and normal B cells expressing CD20 molecules, the B cell group can be reconstructed, and therefore, the anti-CD 20 monoclonal antibody is increasingly applied to the treatment of B cell related diseases.

Rituximab (Rituximab) is the first successful human-mouse chimeric anti-CD 20 monoclonal antibody and the first anti-tumor monoclonal antibody on the market worldwide, and is used for treating relapsed or refractory indolent lymphoma; ofatumumab (Ofatumumab) is a second generation anti-CD 20 humanized mab with a novel CD20 binding epitope, with stronger affinity for CD20 and more potent than rituximab in killing target cells;

obinutuzumab is a third generation humanized monoclonal antibody with an engineered Fc region to increase its binding affinity to the Fc γ RIIIa receptor. The Fc receptor binding capacity of the fusion protein is 50 times of that of rituximab, and the fusion protein is 10-100 times stronger than the antibody-dependent cell-mediated cytotoxicity effect of the rituximab in an NHL cell line expressing CD 20. And, due to the modified hinge region, it can induce stronger apoptotic effects in some NHL cell lines and B cells in early stages of deterioration. Since the time of introduction of rituximab, researchers began the study of the CD20 monoclonal antibody, and constructed more anti-CD 20 antibodies.

With the continuous development and maturation of genetic engineering technology, the replacement or substitution of animal homologous genes with human genes has been realized, and the development of humanized experimental animal models in this way is the future development direction of animal models. The gene humanized animal model is one animal model with normal or mutant gene replaced with homologous gene in animal genome and similar physiological or disease characteristics. The gene humanized animal not only has important application value, such as improving and promoting cell or tissue transplantation humanized animal model through gene humanization, but also can express or partially express the human protein in the animal body due to the insertion of the human gene segment, can be used as a target of a drug which can only identify the sequence of the human protein, and provides possibility for screening anti-human antibodies and other drugs at the animal level. However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of the genes, for example, the identity of human and mouse CD20 protein is 73%, how to construct an "effective" humanized animal model for new drug development remains the greatest challenge.

In view of the complex mechanism of action of CD20 and the huge application value in the field of tumor therapy, there is an urgent need in the art to develop a non-human animal model of CD 20-related signaling pathway in order to further explore its relevant biological properties, improve the effectiveness of preclinical drug efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

In a first aspect of the invention, there is provided a humanized CD20 gene, wherein the humanized CD20 gene comprises a portion of the human CD20 gene.

Preferably, the portion of the human CD20 gene comprises all or part of the coding region of the human CD20 gene.

Preferably, said portion of the human CD20 gene comprises a nucleotide sequence encoding all or part of a transmembrane, cytoplasmic and/or extracellular region of a human CD20 protein. Further preferably, said humanized CD20 gene comprises a nucleotide sequence encoding all or part of the transmembrane and/or extracellular region of human CD20 protein. Still more preferably, the humanized CD20 gene further comprises a nucleotide sequence encoding all or part of the cytoplasmic region of the human CD20 protein.

Preferably, said portion of the human CD20 gene comprises a nucleotide sequence encoding SEQ ID NO: 2 from position 1 to 297; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO: 2 from position 1 to 297, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO: 2 from position 1 to 297, which differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO: 2, 1-297, comprising a nucleotide sequence with one or more nucleotide substitutions, deletions and/or insertions.

In one embodiment, the portion of the human CD20 gene comprises all or part of the nucleotide sequence of exons 1 to 7 of human CD20 gene. Further preferably, the humanized CD20 gene comprises all or part of a nucleotide sequence of a combination of two or more consecutive exons among exons 1 to 7 of human CD20 gene. More preferably, the humanized CD20 gene comprises all or part of exon 2 to exon 7 of human CD20 gene, and even more preferably, all or part of the nucleotide sequence of exon 2 to exon 7 of human CD20 gene at least comprises part of exon 2, all of exon 3 to exon 6, and/or part of the nucleotide sequence of exon 7 of human CD20 gene. More preferably, the humanized CD20 gene further includes introns nos. 2-3, 3-4, 4-5, 5-6 and/or 6-7 of the human CD20 gene. Wherein, the part of exon 2 at least comprises 50bp nucleotide sequence, such as at least 50, 70, 90, 100, 110, 130, 150, 155, 156, 157, 158, 159, 160, 170, 175bp nucleotide sequence, preferably, the part of exon 2 at least comprises coding region, the part of exon 7 at least comprises 100bp nucleotide sequence, such as at least 100, 110, 130, 150, 170, 200, 210, 215, 216, 217, 218, 219, 220, 250, 300, 500, 1000, 1500, 2000, 2500, 2503bp nucleotide sequence, and the part of exon 7 at least comprises coding region.

In one embodiment, the portion of the human CD20 gene comprises a nucleotide sequence having a length of 100-6094bp starting at least from the first nucleotide of the coding region of exon 2 of the human CD20 gene and proceeding downstream.

In one embodiment, the portion of the human CD20 gene comprises at least the start codon of exon 2 and the stop codon of exon 7 of human CD20 gene.

In one embodiment, the portion of the human CD20 gene is a nucleotide sequence encoding SEQ ID NO: 2, 1-297.

In one embodiment of the present invention, the humanized CD20 gene comprises a partial nucleotide sequence of human CD20 gene comprising one of the following groups:

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

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

(C) and SEQ ID NO: 5 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 CD20 gene further includes a portion of a non-human animal CD20 gene.

Preferably, the portion of the non-human animal CD20 gene comprises at least the 5 'UTR and/or the 3' UTR of the non-human animal CD20 gene.

In a specific embodiment, the portion of the non-human animal CD20 gene further comprises all or part of exon 1 of the non-human animal CD20 gene.

In a specific embodiment, the portion of the non-human animal CD20 gene further comprises at least a non-coding region for exon 2 and/or a non-coding region for exon 7 of the non-human animal CD20 gene.

In a specific embodiment, the portion of the non-human animal CD20 gene comprises at least all or part of the nucleotide sequence encoding the transmembrane and/or cytoplasmic region of a non-human animal CD20 protein.

Preferably, the portion of the non-human animal CD20 gene includes a non-coding region of the non-human animal CD20 gene.

In a specific embodiment, said non-human animal CD20 gene further comprises a portion of exon 2 and/or all or a portion of exon 7; still more preferably, at least a non-coding region of exon 2 and/or exon 7 of the non-human animal CD20 gene is included.

Preferably, the humanized CD20 gene includes all of exon 1, part of exon 2 and part of exon 7 of the non-human animal CD20 gene.

In one embodiment, the nucleotide sequence of the humanized CD20 gene further comprises a nucleotide sequence identical to SEQ ID NO: 6 and/or SEQ ID NO: 7, or a nucleotide sequence comprising at least 60%, 70%, 80%, 90% or at least 95% identity to SEQ ID NO: 6 and/or SEQ ID NO: 7.

In one embodiment of the present invention, the mRNA transcribed from the nucleotide sequence of the humanized CD20 gene is selected from one of the following groups:

A) is 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 CD20 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.

Preferably, the specific inducer is selected from the tetracycline System (Tet-Off System/Tet-On System) or Tamoxifen System (Tamoxifen System).

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

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

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

In a second aspect of the invention, there is provided a human or humanized CD20 protein, wherein the human or humanized CD20 protein comprises all or part of a human CD20 protein.

Preferably, the human or humanized CD20 protein is expressed by cells of a non-human animal.

Preferably, the humanized CD20 protein comprises all or part of the extracellular domain of human CD20 protein.

In one embodiment, the humanized CD20 protein comprises an extracellular region encoded by exon 3 and/or an extracellular region encoded by exon 5 of human CD20 gene.

In one embodiment, the humanized CD20 protein comprises the amino acid sequence set forth in SEQ ID NO: 2 and/or the sequence from 79 to 83 and/or the sequence from 144-188.

Preferably, the humanized CD20 protein further comprises all or part of the transmembrane and/or cytoplasmic domain of human CD20 protein.

In one embodiment, the humanized CD20 protein comprises a portion of the transmembrane region of human CD20 protein that is encoded by exon 3, exon 4, exon 5, and/or exon 6 of the human CD20 gene.

In a specific embodiment, the humanized CD20 protein comprises a portion of the cytoplasmic region of human CD20 protein that is encoded by exon 2, exon 3, exon 4, exon 5, exon 6, and/or exon 7 of the human CD20 gene.

In a specific embodiment, the humanized CD20 protein comprises all or part of an amino acid sequence encoded by exons 1 to 7 of the human CD20 gene. Further preferably, the humanized CD20 protein comprises all or part of an amino acid sequence encoded by a combination of two or more consecutive exons from exons 1 to 7 of human CD20 gene. Still more preferably, the humanized CD20 protein comprises an amino acid sequence encoded by part of exon 2, all of exons 3 to 6, and part of exon 7 of human CD20 gene.

In one embodiment, the portion of human CD20 protein is identical to SEQ ID NO: 2 or an amino acid sequence having at least 90% identity to SEQ ID NO: 2 are identical.

Preferably, the humanized CD20 protein comprises all or part of at least the extracellular domain of human CD20 protein, further comprises all or part of at least the transmembrane domain of human CD20 protein, and further comprises all or part of the cytoplasmic domain of human CD20 protein.

The human or humanized CD20 protein can be recognized and/or bound by a human or humanized antibody targeted to CD 20.

In a specific embodiment, the amino acid sequence of the human or humanized CD20 protein derived from human CD20 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%.

In one embodiment, the amino acid sequence of the human or humanized CD20 protein is selected from one of the following groups:

a) is SEQ ID NO: 2 all or part of an amino acid sequence;

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

d) And SEQ ID NO: 2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized CD20 protein is encoded by the humanized CD20 gene as described above.

In a third aspect of the invention, there is provided a targeting vector comprising a donor nucleotide sequence.

Preferably, the donor nucleotide sequence comprises all or part of a human or humanized CD20 gene.

Preferably, the donor nucleotide sequence comprises all or part of the coding region of a human or humanized CD20 gene.

Preferably, the donor nucleotide sequence comprises all or part of a nucleotide sequence encoding a human or humanized CD20 protein.

Preferably, the donor nucleotide sequence comprises a nucleotide sequence encoding all or part of a transmembrane, cytoplasmic and/or extracellular region of a human CD20 protein. Further preferably, the humanized CD20 gene comprises a nucleotide sequence encoding all or part of the transmembrane region and/or extracellular region of human CD20 protein. Still further preferably, said humanized CD20 gene further comprises a nucleotide sequence encoding all or part of the cytoplasmic region of human CD20 protein.

In one embodiment, the donor nucleotide sequence comprises a nucleotide sequence encoding a nucleotide sequence identical to SEQ ID NO: 2 or an amino acid sequence having at least 90% identity to SEQ ID NO: 2, and (b) 2 is the nucleotide sequence of an amino acid sequence consistent with the amino acid sequence shown in the specification.

In a specific embodiment, the donor nucleotide sequence comprises all or part of the nucleotide sequence of exon 1 to exon 7 of the human CD20 gene. Further preferably, the humanized CD20 gene comprises all or part of a nucleotide sequence of a combination of two or more consecutive exons among exons 1 to 7 of human CD20 gene. More preferably, all or part of the nucleotide sequence of exon 2 to exon 7 of human CD20 gene comprises at least part of exon 2, all of exon 3 to exon 6 of human CD20 gene and/or part of the nucleotide sequence of exon 7. More preferably, the humanized CD20 gene further includes introns nos. 2-3, 3-4, 4-5, 5-6 and/or 6-7 of the human CD20 gene. Wherein, the part of exon 2 at least comprises 50bp nucleotide sequence, such as at least 50, 70, 90, 100, 110, 130, 150, 155, 156, 157, 158, 159, 160, 170, 175bp nucleotide sequence, preferably, the part of exon 2 at least comprises coding region, the part of exon 7 at least comprises 100bp nucleotide sequence, such as at least 100, 110, 130, 150, 170, 200, 210, 215, 216, 217, 218, 219, 220, 250, 300, 500, 1000, 1500, 2000, 2500, 2503bp nucleotide sequence, preferably, the part of human exon 7 at least comprises coding region.

Preferably, the donor nucleotide sequence comprises all or part of exons 2 to 7 of human CD20 gene, preferably at least comprises a nucleotide sequence with a length of 100-6094bp from the first nucleotide to the downstream of the coding region of exon 2 of human CD20 gene, and further preferably comprises the nucleotide sequence shown in SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment, the targeting vector further comprises a DNA fragment homologous to the 5 'end of the transition region to be altered, i.e.the 5' arm, selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD20 gene. Further preferred, said 5' arm is a nucleotide having at least 90% homology with NCBI accession No. NC — 000085.7. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 3 or SEQ ID NO: 22 or as shown in SEQ ID NO: 3 or SEQ ID NO: 22, respectively.

In a specific embodiment, the targeting vector further comprises a DNA fragment homologous to the 3 'end of the transition region to be altered, i.e.the 3' arm, selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the non-human animal CD20 gene. Further preferred, said 3' arm is a nucleotide having at least 90% homology with NCBI accession No. NC — 000085.7. Still more preferably, the 3' arm sequence is identical to SEQ ID NO: 4 or SEQ ID NO: 23, or as shown in SEQ ID NO: 4 or SEQ ID NO: shown at 23.

In one embodiment, the transition region to be altered is located at the CD20 locus of the non-human animal. Further preferably, the transition region to be altered is located on exons 1 to 7 of the non-human animal CD20 gene. Preferably located on exons 2 to 7 of the non-human animal CD20 gene, and preferably further comprises a flanking sequence upstream of exon 2.

Preferably, the targeting vector further comprises a 5' UTR of the non-human animal CD20 gene.

Preferably, the targeting vector further comprises a 3' UTR of the non-human animal CD20 gene.

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 fourth aspect of the invention, there is provided a non-human animal cell which expresses a humanised CD20 gene as described above, said cell expressing a human or humanised CD20 protein as described above, said cell comprising a humanised CD20 gene as described above.

Preferably, the cell is selected from a non-human animal primary cell or cell line, or from an embryonic stem cell, germ cell or somatic cell of a non-human animal, or from a primary cell or cell line of a non-human animal myeloid line.

In the fifth aspect of the invention, the invention provides an application of the cell in constructing humanized non-human animals with CD20 gene, relevant research on CD20 gene or protein function and preparation of CD20 related drugs.

In a sixth aspect of the invention, there is provided a sgRNA that targets a non-human animal CD20 gene, while the sequence of the sgRNA is unique on the target sequence on the CD20 gene to be altered.

Preferably, the target site of the sgRNA is located on exon 2 and/or exon 7 sequences of the CD20 gene.

Further preferably, the sequence of the target site at the 5' end targeted by the sgRNA is shown in SEQ ID NO: 24, the 3' end target site sequence is shown as SEQ ID NO: shown at 25.

In a seventh aspect of the invention, a DNA molecule encoding the sgRNA described above is provided.

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

In one embodiment of the present invention, the DNA molecule may be SEQ ID NO: 24 and SEQ ID NO: 27, SEQ ID NO: 26 and SEQ ID NO: 28, SEQ ID NO: 25 and SEQ ID NO: 30, or, SEQ ID NO: 29 and SEQ ID NO: 31.

an eighth aspect of the present invention provides a sgRNA vector including the sgRNA or the DNA molecule.

The ninth aspect of the invention provides a preparation method of an sgRNA vector, which includes:

(i) providing the sgRNA, and preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence, wherein the sgRNA targets a CD20 gene, the sgRNA is unique on a target sequence on a CD20 gene to be changed, and a target site of the sgRNA is positioned on a No. 2 exon and/or a No. 7 exon of a CD20 gene;

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

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

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

Preferably, the T7 promoter and sgRNA scaffold fragment DNA in step (ii) are as shown in SEQ ID NO: shown at 32.

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

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

In an eleventh aspect, the present invention provides an application of the targeting vector, the sgRNA, the DNA molecule or the sgRNA vector, the sgRNA vector obtained by the preparation method, or a cell containing the targeting vector or the sgRNA to CD20 gene modification. Preferably, the use includes but is not limited to knockout, insertion or replacement.

In the twelfth aspect of the invention, a method for constructing a humanized non-human animal of the CD20 gene is provided, wherein the human or humanized CD20 protein is expressed in the non-human animal.

Preferably, the genome of the non-human animal comprises the humanized CD20 gene.

Preferably, the method of construction comprises introducing a donor nucleotide sequence into the non-human animal CD20 locus to form a human or humanized CD20 gene.

Preferably, the humanized CD20 protein is a protein as described above.

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

A) all or part of a nucleotide sequence encoding a human or humanized CD20 protein;

B) a nucleotide sequence encoding all or part of the extracellular, transmembrane and/or cytoplasmic region of the human CD20 protein;

C) all or part of a human or humanized CD20 gene; or the like, or, alternatively,

D) all or part of exon 1 to exon 7 of the human CD20 gene, preferably comprising all or part of exon 2 to exon 7, preferably part of exon 2, exon 3 to exon 6 and/or part of exon 7 of the human CD20 gene, wherein part of exon 2 comprises at least a 50bp nucleotide sequence, e.g. at least a 50, 70, 90, 100, 110, 130, 150, 155, 156, 157, 158, 159, 160, 170, 175bp nucleotide sequence, preferably said part of exon 2 comprises at least a coding region, part of exon 7 comprises at least a 100bp nucleotide sequence, e.g. at least a 100, 110, 130, 150, 170, 200, 210, 215, 216, 217, 218, 219, 220, 250, 300, 500, 1000, 1500, 2000, 2500, 2503bp nucleotide sequence, said part of exon 7 comprises at least a coding region, preferably at least comprises a nucleotide sequence with the length of 100-6094bp from the first nucleotide of the coding region of the exon 2 of the human CD20 gene to the downstream, and further preferably comprises the nucleotide sequence shown in SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or, comprises a polypeptide having the sequence of SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment, the donor nucleotide sequence comprises a nucleotide sequence encoding SEQ ID NO: 2.

Preferably, the human or humanized CD20 gene in a humanized non-human animal of the CD20 gene is homozygous or heterozygous for the locus of the endogenous replaced gene.

Preferably, the humanized CD20 gene is regulated in a non-human animal by endogenous or exogenous regulatory elements. Further preferably, the regulatory element is a promoter.

Preferably, the method of construction comprises insertion or substitution into the non-human animal CD20 locus with the donor nucleotide sequence.

In one embodiment, a donor nucleotide sequence comprising a portion of the human CD20 gene described above is introduced into the corresponding region on the non-human animal CD20 locus to form a humanized CD20 gene.

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

Preferably, the construction method comprises introducing a nucleotide sequence comprising all or part of a nucleotide sequence encoding human CD20 protein into the non-human animal CD20 locus.

Preferably, the construction method comprises introducing a cDNA sequence encoding human CD20 protein into the CD20 locus of a non-human animal.

Preferably, the construction method comprises introducing a nucleotide sequence comprising the humanized CD20 gene as described above into the non-human animal CD20 locus.

Preferably, the construction method comprises introducing a nucleotide sequence encoding the humanized CD20 protein described above into the non-human animal CD20 locus.

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

The introduction is replacement or insertion, specifically, the introduced non-human animal CD20 locus is replacement of a corresponding region of a non-human animal, preferably replacement of all or part of a nucleotide sequence encoding endogenous CD20 protein in a genome of the non-human animal, and further preferably encoding a nucleotide sequence of SEQ ID NO: 1 is replaced.

Preferably, exon 2 through exon 7 of the non-human animal CD20 gene are replaced in whole or in part, and further preferably, exon 2, exon 3 through exon 6 and exon 7 of the non-human animal CD20 gene are replaced in part.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD20 gene in the non-human animal and then performing the insertion operation. Or the insertion step can cause frame shift mutation to the endogenous CD20 gene and can realize the step of inserting the human sequence.

Preferably, the nucleotide sequence encoding the humanized CD20 protein is substituted at the corresponding position in the non-human animal CD20 locus.

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

Preferably, the construction of the non-human animal is carried out by using a CRISPR/Cas9 technology, and comprises the steps of designing and synthesizing sgRNA targeting an endogenous CD20 gene of the non-human animal, constructing a targeting vector carrying all or part of a human CD20 gene, introducing the sgRNA and the targeting vector into cells of the non-human animal, and screening the cells carrying the human CD20 gene.

Preferably, the construction method comprises modifying the coding frame of the non-human animal CD20 gene, and inserting the coding frame containing the exogenous nucleotide sequence into the endogenous regulatory element of the non-human animal CD20 gene, wherein the coding frame of the modified non-human animal CD20 gene can adopt a method of knocking out the functional region of the non-human animal CD20 gene or a method of inserting a sequence so that the non-human animal CD20 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 CD20 gene can adopt a method of knocking out all or part of the nucleotide sequence from exon 2 to exon 7 of the non-human animal CD20 gene.

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

Preferably, the construction method comprises constructing a non-human animal by using the targeting vector and/or sgRNA of the present invention.

In a specific embodiment of the invention, the construction method comprises the steps of introducing the targeting vector, the sgRNA targeting the CD20 gene and the Cas9 into a non-human animal cell, culturing the cell (preferably a fertilized egg), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal humanized with the CD20 gene.

In another embodiment of the present invention, the construction method comprises introducing the targeting vector into embryonic stem cells of a non-human animal, introducing the targeting vector into a blastocyst isolated in advance after a short period of culture, transplanting the resulting chimeric blastocyst into an oviduct of a recipient female mouse, allowing the chimeric blastocyst to develop, and identifying and screening the non-human animal to obtain the humanized CD20 gene.

According to some embodiments of the invention, the constructing method further comprises: the CD20 gene-humanized non-human animal is mated with another genetically modified non-human animal, fertilized in vitro or directly subjected to gene editing, and then screened to obtain a polygene-modified non-human animal.

Preferably, the other gene is at least one gene selected from the group consisting of PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 and CD 226.

Preferably, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3, and CD226 protein.

According to some embodiments of the invention, the human or humanized CD20 gene and/or the other gene is homozygous or heterozygous for the endogenous modified locus.

In a thirteenth aspect of the present invention, there is provided a non-human animal obtained by the above-mentioned construction method.

In a fourteenth aspect of the invention, a non-human animal with a deletion of an endogenous CD20 gene is provided, wherein the non-human animal lacks all or part of the nucleotide sequence of the endogenous CD20 gene.

In a specific embodiment, the non-human animal lacks all or a portion of exons 2 to 7 of an endogenous CD20 gene. Further preferably, all or part of the nucleotide sequence of a combination of two or more consecutive exons among exons 1 to 7 is deleted. Even more preferably, all or part of the nucleotide sequence of exons 2 to 7 is deleted. Preferably, the non-human animal lacks the coding region for exon 2, exon 3, exon 4, exon 5, exon 6 and/or exon 7 of the CD20 gene. Preferably, the non-human animal is further deficient in intron 2-3, intron 3-4, intron 4-5, intron 5-6, and/or intron 6-7 of the CD20 gene. In a specific embodiment, the deleted portion is a nucleotide sequence beginning with the start codon of exon 2 and ending with the stop codon of exon 7.

Preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the cytoplasmic, transmembrane and/or extracellular domain of the endogenous CD20 protein. Further preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular domain and/or transmembrane domain of the endogenous CD20 protein. In one embodiment of the invention, the non-human animal lacks a nucleotide sequence encoding an extracellular domain of an endogenous CD20 protein.

Preferably, the non-human animal is constructed with sgrnas as described above.

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.

In a fifteenth aspect of the present invention, a method for constructing a CD20 knockout non-human animal is provided, the method comprising constructing the non-human animal using sgRNA. Wherein the sgRNA targets the non-human animal CD20 gene, while the sequence of the sgRNA is unique on the target sequence on the CD20 gene to be altered.

In a sixteenth aspect of the invention there is provided a humanized non-human animal that expresses human or humanized CD20 protein.

Preferably, the expression of endogenous CD20 protein in the non-human animal is reduced or absent.

Preferably, the humanized CD20 protein is selected from the humanized CD20 proteins described above.

Preferably, the genome of the non-human animal comprises a portion of the human CD20 gene. Further preferably, the genome of the non-human animal comprises the humanized CD20 gene as described above.

Preferably, the nucleotide sequence encoding the human or CD20 protein or the nucleotide sequence of the human or humanized CD20 gene is operably linked to endogenous regulatory elements at the endogenous CD20 locus in at least one chromosome of the non-human animal.

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

In a specific embodiment, the non-human animal is constructed using the targeting vector and/or sgRNA targeting the CD20 gene of the non-human animal.

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

Preferably, the non-human animal further comprises additional genetic modifications, the additional genes selected from at least one of PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3, or CD 226.

Preferably, the CD20 gene and the other gene are homozygous or heterozygous for the locus of the endogenous replaced gene.

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

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

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

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized with the genes PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 or CD 226.

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

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

In an eighteenth aspect of the present invention, there is provided a humanized non-human animal of the CD20 gene obtained by the above construction method, or a polygene-modified non-human animal or a progeny thereof.

In a nineteenth 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 twentieth aspect of the present invention, there is provided a method for producing an animal model, which comprises the step of constructing the above-mentioned non-human animal humanized with CD20 gene, non-human animal deficient in endogenous CD20 gene or multi-gene modified non-human animal or progeny thereof. Preferably, the animal model is a tumor-bearing or inflammation animal model, and more preferably, the preparation method of the tumor-bearing animal model further comprises the step of implanting tumor cells.

In a twenty-first aspect of the present invention, there is provided an application of the above-mentioned CD20 gene-humanized non-human animal, endogenous CD20 gene-deleted non-human animal, polygene-modified non-human animal or progeny thereof, or CD20 gene-humanized non-human animal, endogenous CD20 gene-deleted non-human animal, polygene-modified non-human animal or progeny thereof obtained by the above-mentioned construction method in preparing an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

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

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

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

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

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

In a twenty-fifth aspect of the invention, there is provided a cell humanised with the CD20 gene, said cell expressing a human or humanised CD20 protein.

Preferably, the humanized CD20 protein is the humanized CD20 protein of the first aspect of the invention.

Preferably, the expression of endogenous CD20 protein is reduced or absent in the cell.

Preferably, the genome of said cell comprises all or part of the human CD20 gene. Further preferably, the cell comprises a humanized CD20 gene as described above.

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

In a twenty-sixth aspect of the invention, there is provided a construct expressing the humanized CD20 protein described above. Preferably, the construct comprises a humanized CD20 gene.

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

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

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

In a twenty-ninth aspect of the present invention, there is provided a use of the humanized CD20 protein, the humanized CD20 gene, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, the above tumor-bearing or inflammatory model, the above cell or cell line or primary cell culture, the above tissue or organ or its culture, the above tumor-bearing tissue, the above cell, the above construct, the above cell or the above tissue, the use comprising:

(A) use in the development of products involving CD 20-related immune processes in human cells;

(B) use as a model system in pharmacological, immunological, microbiological and medical research associated with CD 20;

(C) to the production and use of animal experimental disease models for the research of CD 20-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

(D) the application of the human CD20 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo; alternatively, the first and second electrodes may be,

(E) the functions of the CD20 gene are researched, the medicine and the drug effect aiming at the target site of the human CD20 are researched, and the application in the aspects of the medicine for the immune-related diseases related to the CD20 and the anti-tumor medicine is researched.

Preferably, the use is for non-disease therapeutic and diagnostic purposes.

In a thirtieth aspect of the present invention, there is provided a screening method for a human CD 20-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 group consisting of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned tumor-bearing or inflammation model.

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

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

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

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

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

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

Preferably, the screening method for the human CD 20-specific modulator includes a therapeutic method and a non-therapeutic method.

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

In a thirty-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 non-human animal, the non-human animal obtained by the construction method, the non-human animal or its offspring, or the animal model.

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

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

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

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

In a thirty-second aspect of the present invention, there is provided a use of the non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the above animal model in the preparation of a human CD 20-specific modulator.

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

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

Preferably, the non-human animal 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 rodentThe animal is immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, 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, kidney 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 CD20 gene humanized non-human animal can normally express human or humanized CD20 protein in vivo, can be used for drug screening, drug effect evaluation, immune disease and tumor treatment aiming at a human CD20 target site, can accelerate the development process of a new drug, and can save time and cost. Provides effective guarantee for researching the functions of the CD20 protein and screening related disease drugs.

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

The humanized CD20 protein comprises a part derived from human CD20 protein and a part derived from non-human CD20 protein. Wherein, the "human CD20 protein" is identical to the whole human CD20 protein, namely the amino acid sequence of the "human CD20 protein" is identical to the full-length amino acid sequence of the human CD20 protein. The "part of human CD20 protein" is a continuous or spaced amino acid sequence of 5-297 and is identical with the amino acid sequence of human CD20 protein. Preferably, consecutive or spaced 5, 10, 20, 30, 40, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 255, 260, 270, 280, 290 or 297 amino acid sequences are identical to the amino acid sequence of the human CD20 protein.

The "whole transmembrane region of human CD20 protein", "whole cytoplasmic region of human CD20 protein" or "whole extracellular region of human CD20 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 CD20 protein, respectively.

The "humanized CD20 gene" of the present invention comprises a part derived from human CD20 gene and a part derived from non-human CD20 gene. Wherein, the "human CD20 gene" is the same as the whole "human CD20 gene", namely, the nucleotide sequence of the "human CD20 gene" is consistent with the full-length nucleotide sequence of the human CD20 gene. The 'part of the human CD20 gene' is a continuous or spaced nucleotide sequence of 20-14906bp (preferably 20-6094bp, 20-3293bp or 20-894bp) which is consistent with the nucleotide sequence of human CD20 or has more than 70% homology with the nucleotide sequence of human CD 20.

The whole or part and the whole are integrated; a "portion" is a part of the whole, or an individual in the whole. For example, "all or part of exon 1 to exon 7" and "all of exon 1 to exon 7" are the whole, i.e., the whole nucleotide sequence of exon 1 to exon 7; "part of exon 1 to exon 7" is a whole individual or a part of a whole individual, i.e., one or more consecutive or spaced nucleotide sequences of exon 2 to exon 7.

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

The term "two or more consecutive exons" as used herein means, for example, 2, 3 consecutive exons, 3, 4 consecutive exons, 4, 5 consecutive exons, 5, 6 consecutive exons, 6, 7 consecutive exons, 2, 3, 4 consecutive exons, 3, 4, 5 consecutive exons, 6, 5, 7 consecutive exons, 2, 3, 4, 5 consecutive exons, 3, 4, 5, 6 consecutive exons, 5, 6, 7 consecutive exons, 2, 3. 4, 5, 6, exons 3, 4, 5, 6, 7, exons 2, 3, 4, 5, 6, 7, exons 3, 4, 5, 6, 7, and exons 2, 3, 4, 5, 6, 7.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the portion of exon 2 of human CD20 gene comprises contiguous or spaced nucleotide sequences of 5-175bp, preferably 10-159 nucleotides identical to the nucleotide sequence of exon 2 of human CD20 gene. For example, the portion of exon 7 of human CD20 gene comprises consecutive or spaced nucleotide sequences of 5-2503bp, preferably 10-219bp, identical to the nucleotide sequence of exon 7 of human CD20 gene. In a specific embodiment of the present invention, the "part of exon 2" contained in the "human CD20 gene" at least includes the first nucleotide sequence of the coding region of exon 2 and the last nucleotide of exon 2. In one embodiment of the present invention, the "part of exon 7" contained in the "humanized CD20 gene" at least includes the nucleotide sequence from the first nucleotide sequence of exon 7 to the last amino acid encoded by the coding region.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, in one embodiment, the "CD 20 locus" represents a DNA fragment of an optional stretch of exons 1 to 7 of the CD20 gene. In one embodiment of the invention, the replaced CD20 locus may be a DNA fragment of an optional stretch of exon 2 to exon 7 of the CD20 gene.

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

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

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

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

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

In a particular embodiment, the non-human animal is a rodent selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola C57BL, C58 NOBr, A/Ca, PrCBA/34/CBA, PrCBA J, CBA/CBD, SCID-SCID strain ^scid IL-2rγ ^null Background mice.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); 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).

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

FIG. 1 is a schematic illustration (not to scale) of a comparison of the mouse CD20 locus and the human CD20 locus;

FIG. 2 is a schematic representation (not to scale) of humanization of the mouse CD20 gene;

FIG. 3 is a schematic representation (not to scale) of the CD20 gene targeting strategy and targeting vector design;

FIG. 4 is a schematic representation (not to scale) of the FRT recombination process for humanized mouse of the CD20 gene;

FIG. 5 shows the results of PCR identification of humanized mouse CD 20F 1 mouse tail, in which WT is wild type and H ₂ O is water control, PC is positive control, and M is marker;

FIG. 6 is a schematic diagram of the CD20 gene targeting strategy and targeting vector design;

FIG. 7 shows the Southern blot analysis of F1 mouse;

FIG. 8 shows the results of RT-PCR assay of C57BL/6 wild type mice (+/+) and CD20 gene humanized homozygote mice (H/H), wherein H is ₂ O is water control, and GAPDH is internal reference of glyceraldehyde-3-phosphate dehydrogenase.

Detailed Description

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

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

ScaI, NcoI, EcoRV enzymes were purchased from NEB under the respective accession numbers R3122S, R3193S, R3195S;

c57BL/6 mice and Flp tool mice were purchased from the national rodent experimental animal seed center of the Chinese food and drug assay institute;

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Biolegend, cat # 103138;

FITC anti-Mouse CD19 was purchased from Biolegend, cat # 115506;

v450 Rat Anti-mouse CD11b was purchased from Biolegend, cat # 8232657;

APC anti-mouse CD20 Antibody was purchased from Biolegend, cat # 152107;

PE anti-human CD20 Antibody was purchased from Biolegend, cat # 302305;

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

Example 1 humanized mouse with CD20 Gene

A comparison scheme between the mouse CD20 Gene (NCBI Gene ID: 12482, Primary source: MGI:88321, UniProt: P19437, located at positions 11227043 to 112435137 on chromosome 19 NC-000085.7, based on transcript NM-007641.6 and its encoded protein NP-031667.1 (SEQ ID NO: 1)) and the human CD20 Gene (NCBI Gene ID: 931, Primary source: HGNC:7315, UniProt ID: A0A024R507, located at positions 60455847 to 60470752 on chromosome 11 NC-000011.10, based on transcript NM-021950.4 and its encoded protein NP-068769.2 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purpose of the present invention, a nucleotide sequence encoding a human CD20 protein may be introduced at the endogenous CD20 locus of a mouse, so that the mouse expresses a human or humanized CD20 protein. Specifically, the humanized CD20 locus schematic diagram obtained by replacing the mouse CD20 genome coding region with the human CD20 genome coding region or replacing the partial sequence of the mouse exon 2 to the partial sequence of the exon 7 with about 6.1kb including the partial sequence of the exon 2 to the partial sequence of the exon 7 of the human CD20 gene to about 7.2kb under the control of mouse CD20 gene regulatory elements by using gene editing technology is shown in fig. 2, and realizes the humanized modification of the mouse CD20 gene.

The targeting strategy was designed as shown in FIG. 3, which shows the homology arm sequences on the targeting vector containing the upstream and downstream of the mouse CD20 gene, as well as a fragment A containing the sequence of human CD 20. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from position 11236186 to 11240359 of NCBI accession No. NC-000085.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence from position 11227043 to 11229028 of NCBI accession No. NC-000085.7. The nucleotide sequence (SEQ ID NO: 5) of the human CD20 fragment on the A fragment is identical to the nucleotide sequence from 60462375 to 60468468 of NCBI accession No. NC-000011.10; the ligation of the upstream human CD20 sequence to mice was designed as: 5' -CTTATTTTCAGGCGTTTGAAAA

ATGACAACACCCAGAAATTCAGTAAA-3' (SEQ ID NO: 6), wherein the sequence

Wherein "A" is the last nucleotide, sequence of mouse "ATGA"the first" A "in" is the first nucleotide in humans. The connection of the downstream of the human CD20 sequence with the mouse is designed to be 5' -AAAATGACAGCTCTCC

ACTCTTTTCTTTTCT-3' (SEQ ID NO: 7), wherein the sequence

Is the last of a personA nucleotide sequence "ACTC"A" in "is the first nucleotide of the mouse sequence.

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

GTCGACGGTATCGATAAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAAG-3' (SEQ ID NO: 8), wherein the sequence

Wherein "C" is the last nucleotide, sequence, of human "GTCG"the first" G "is the first nucleotide of the Neo cassette; the connection between the 3 'end of the Neo cassette and the human gene is designed to be 5' -AGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCACTAGTTCTAGAGCGGCCGCGTTAA

AAATCAGAACGAAAATCTAAACTCCTCTATTACTG-3' (SEQ ID NO: 9), wherein the sequence

Wherein "C" is the last nucleotide, sequence, of the Neo cassette "AAATThe "first" A "in" is the first nucleotide in humans. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CD20 after being transformed is shown as SEQ ID NO: 10, and the expressed protein sequence is shown as SEQ ID NO: 2, respectively.

Given that human CD20 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection 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 means of electroporation, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technology, screening correct positive clone cells, detecting clones identified as positive by the PCR, further carrying out Southern Blot (cell DNA is digested by NcoI, ScaI or EcoRV enzyme and hybridized by using 3 probes, the length of the probes and target fragments is shown in table 1), detecting the clones with positive Southern detection, and further carrying out next step of experiments on the clones which are verified as positive by sequencing and have no random insertion.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment	Recombinant sequence fragment
				NcoI	5’Probe	8.0kb	6.4kb
ScaI	3’Probe	9.2kb	12kb
				EcoRV	Neo Probe	—	12kb

Wherein the PCR assay comprises the following primers:

PCR-F1：5’-TGGAGGAAGCCTCAAGTGTCTCA-3’(SEQ ID NO:11)

PCR-R1：5’-CCTATACCGCATCAGCTTCTGTCA-3’(SEQ ID NO:12)；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

5’Probe-F：5’-ACTGTGCAGAAAAGGCAACAGGCTA-3’(SEQ ID NO：13)，

5’Probe-R：5’-TCCTGGGACCTACTCTCTCCTTGTG-3’(SEQ ID NO：14)；

3 'Probe (3' Probe):

3’Probe-F：5’-TTTACATGGCATGCCCACAATGGTT-3’(SEQ ID NO：15)，

3’Probe-R：5’-ATTCCACCTTCACTGAGTTCCCTCC-3’(SEQ ID NO：16)；

neo probe (NeoProbe):

NeoProbe-F：5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO：17)，

NeoProbe-R：5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO：18)；

the selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 4 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous mice for the CD20 gene. The somatic genotypes of the progeny mice were identified by PCR (primers shown in Table 2), and the results of identification of exemplary F1 generation mice (with the Neo marker gene removed) are shown in FIG. 5, in which three mice numbered F1-01, F1-02, and F1-03 were all positive heterozygous mice. This shows that using this method, humanized mice of the CD20 gene can be constructed which can be stably passaged and have no random insertion.

Table 2: name and specific sequence of primer

In addition, gene editing can be performed by using a CRISPR/Cas system, a schematic targeting strategy shown in FIG. 6 is designed, and homologous arm sequences containing the upstream and downstream of mouse CD20 and a human CD20 fragment nucleotide sequence (SEQ ID NO: 5) on a targeting vector are shown in FIG. 6. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 22) is identical to the nucleotide sequence from position 11236186 to 11237594 of NCBI accession No. NC-000085.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 23) is identical to the nucleotide sequence from position 11227619 to 11229028 of NCBI accession No. NC-000085.7. The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, direct synthesis and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The vector plasmid with the correct sequencing verification was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for constructing sgRNA expression vectors. A total of 14 sgRNA sequences of sgRNA1-sgRNA14 were designed and synthesized. The activity of the sgRNA is detected by using a UCA kit, the result shows that the sgRNA has different activities, the sgRNA-3 and the sgRNA-10 with the highest activity are selected, and the target site sequences are as follows:

sgRNA-3 target site sequence (SEQ ID NO: 24): 5'-TGGAGCAGGTTGCATGGCGAGGG-3'

sgRNA-10 target site sequence (SEQ ID NO: 25): 5'-GGAGCGATCTCATTTTCCACTGG-3'

The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide and a reverse oligonucleotide (the sequences are shown in a table 3), and after annealing, the annealing products are respectively connected to pT7-sgRNA plasmids (the plasmids are firstly linearized by BbsI), so as to obtain expression vectors pT7-CD20-3 and pT7-CD 20-10.

TABLE 3 sequence List of sgRNA-3 and sgRNA-10

sgRNA-3 sequence
		SEQ ID NO：24	Upstream: 5'-TGGAGCAGGTTGCATGGCGAGGG-3'
SEQ ID NO: 26 (Forward oligonucleotide)	Upstream: 5'-TAGG TGGAGCAGGTTGCATGGCGAGGG-3'
		SEQ ID NO：27	Downstream: 5'-CCCTCGCCATGCAACCTGCTCCA-3'
SEQ ID NO: 28 (reverse oligonucleotide)	Downstream: 5'-AAAC CCCTCGCCATGCAACCTGCTCCA-3'
		sgRNA-10 sequence
SEQ ID NO：25	Upstream: 5'-GGAGCGATCTCATTTTCCACTGG-3'
		SEQ ID NO: 29 (Forward oligonucleotide)	Upstream: 5'-TAGG GGAGCGATCTCATTTTCCACTGG-3'
SEQ ID NO：30	Downstream: 5'-CCAGTGGAAAATGAGATCGCTCC-3'
		The amino acid sequence of SEQ ID NO: 31 (reverse oligonucleotide)	Downstream: 5'-AAAC CCAGTGGAAAATGAGATCGCTCC-3'

Randomly selected clones were sent to a sequencing company for sequencing verification, and correctly ligated expression vectors pT7-CD20-3 and pT7-CD20-10 were selected for subsequent experiments.

pT7-sgRNA plasmid sources:

the plasmid backbone of the pT7-sgRNA vector was derived from Takara, cat # 3299. A fragment DNA containing a T7 promoter and sgRNA scaffold is synthesized by a plasmid synthesis company, is sequentially connected to a skeleton vector through enzyme digestion (EcoRI and BamHI), and is verified by sequencing of a professional sequencing company, so that a target plasmid is obtained. Fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 32):

gaattctaatacgactcactatagggggtcttcgagaagacctgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaggatcc

taking a C57BL/6 mouse prokaryotic stage fertilized egg, and injecting a premixed in-vitro transcription product (transcribed by using an Ambion in-vitro transcription kit according to a method of an instruction) of pT7-CD20-3 and pT7-CD20-10 plasmids and Cas9 mRNA into the cytoplasm or nucleus of the mouse fertilized egg by using a microinjection instrument. Microinjection of embryos is performed according to the method in the manual for mouse embryo manipulation experiments (third edition), fertilized eggs after injection are transferred to a culture solution for short-term culture, and then are transplanted to the oviduct of a recipient mother mouse to produce a genetically modified humanized mouse, so that a founder mouse (founder mouse, i.e., F0 generation) is obtained. The mRNA sequence of the humanized mouse CD20 after being transformed is shown as SEQ ID NO: 10, and the expressed protein sequence is shown as SEQ ID NO: 2, respectively.

The somatic cell genotype of the F0 mouse can be identified by conventional detection methods (e.g., PCR analysis) including the following primers: WT-F (SEQ ID NO: 19) and WT-R (SEQ ID NO: 20), and WT-F ((SEQ ID NO: 19) and Mut-R (SEQ ID NO: 21). CD20 humanized mice identified as positive by F0 were mated with C57BL/6 mice to give F1 generation mice.F 1 generation mice were genotyped using the same PCR method, and F1 generation mice identified as positive by PCR were subjected to Southern blot assay to confirm the presence of random inserts.

5 'Probe (5' Probe) Synthesis of primers:

5’Probe-F(SEQ ID NO：33)：5’-ACTGTGCAGAAAAGGCAACAGGCTA-3’

5’Probe-R(SEQ ID NO：34)：5’-TCCTGGGACCTACTCTCTCCTTGTG-3’

3 'Probe (3' Probe) Synthesis of primers:

3’Probe-F(SEQ ID NO：35)：5’-TTTACATGGCATGCCCACAATGGTT-3’

3’Probe-R(SEQ ID NO：36)：5’-ATTCCACCTTCACTGAGTTCCCTCC-3’

the Southern blot assay results are shown in FIG. 7. The results showed that 3 of 4 mice had no random insertions, and the 3 mice had accession numbers 1EA61-0001, 1EA61-0009 and 1EA 61-0010. These 3 mice were confirmed to be positive heterozygous mice and no random insertions were present. This shows that the method can construct the humanized gene engineering mouse of CD20 which can be stably passaged and has no random insertion.

Further detecting the expression of CD20mRNA in the mice by RT-PCR. Specifically, 1 mouse of 9-week-old female C57BL/6 wild-type mice and 1 mouse of humanized homozygote of CD20 gene obtained in this example were each taken, spleen tissue was harvested after cervical euthanasia, cellular RNA was extracted according to the instructions of Trizol kit, and after reverse transcription into cDNA, RT-PCR detection was performed (primers shown in table 4), and the detection results (shown in fig. 8) were shown: only murine CD20mRNA, no human CD20mRNA, was detected in C57BL/6 wild type mice (+/+); only human CD20mRNA was detected in humanized homozygous mice (H/H) of the CD20 gene.

TABLE 4 RT-PCR primer names and specific sequences

The expression of human or humanized CD20 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. Specifically, 1 mouse of 9-week-old female C57BL/6 wild-type mice and 1 mouse of 10-week-old female CD20 gene humanized homozygote was taken, spleen tissues were taken after cervical euthanization, and anti-mouse CD20 Antibody APC anti-mouse CD20 Antibody (mCD20-APC), anti-human CD20 Antibody PE anti-human CD20 Antibody (hCD20-PE) and mouse leukocyte recognition Antibody Brilliant Violet 510 were used ^TM Flow detection is carried out after recognition staining of anti-i-Mouse CD45, anti-Mouse CD19 antibody FITC anti-Mouse CD19(mCD19-BV605), anti-Mouse CD16/32 antibody Purified anti-Mouse CD16/32 and the like, and results show that B cells (characterized by mCD45+ mCD19+) in spleen of C57BL/6 mice have 48.1% of mCD20 positive cells (characterized by mCD45+ mCD19+ mCD20+), and 0.17% of hCD20 positive cells (characterized by mCD45+ mCD19+ hCD20 +). CD20 homozygous mice spleen B cells were 0.11% mCD20 positive cells (characterized by mCD45+ mCD19+ mCD20+) and 57.4% hCD20 positive cells (characterized by mCD45+ mCD19+ hCD20 +). The result shows that the spleen cells of the C57BL/6 mouse do not express the human CD20 protein, and the spleen cells of the CD20 humanized mouse can successfully express the human CD20 protein.

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

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared CD20 mouse. For example, in example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other gene modifications such as PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3, or CD226, or may be used to obtain a two-gene or multiple-gene modified mouse model of CD20 and other gene modifications based on humanized CD20 mice by using isolated mouse ES embryonic stem cells and gene recombination targeting techniques. The homozygote or heterozygote of the CD20 mouse obtained by the method can be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of humanized CD20 and heterozygote of double gene or multiple gene modification of other gene modification can be obtained with a certain probability according to Mendel genetic rule, the heterozygote is mated with each other to obtain homozygote of double gene or multiple gene modification, and the in vivo efficacy verification of targeted human CD20 and other gene regulators can be carried out by utilizing the mouse of double gene or multiple gene modification.

Example 3 verification of drug efficacy

The humanized mouse of CD20 prepared by the method can be used for evaluating the drug effect of a drug targeting human CD 20. For example, a CD20 humanized mouse homozygote is inoculated subcutaneously with mouse colon cancer cell MC38 (or mouse lymphoma cell EL4) until the tumor volume grows to about 100mm ³ The tumor volume was then divided into control group or treatment group, the treatment group randomly selected drugs targeting human CD20, and the control group injected with equal volume of saline or PBS. The tumor volume is measured periodically, the weight of the mouse is weighed, and the in-vivo safety and in-vivo efficacy of the medicament can be effectively evaluated by comparing the weight change of the mouse with the size of the tumor.

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 can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

Sequence listing

<110> Baiosai Picture (Beijing) pharmaceutical science and technology, Inc

Construction method and application of <120> CD20 gene humanized non-human animal

<130> 1

<150> CN202110668664.5

<151> 2021-06-16

<160> 42

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Pro Thr Gln Ser Phe Phe Met Arg Glu Ser Lys Ala Leu Gly Ala Val

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Gln Ile Met Asn Gly Leu Phe His Ile Thr Leu Gly Gly Leu Leu Met

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Met Ser Ser Leu Ser Leu Phe Ala Ala Ile Ser Gly Ile Ile Leu Ser

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Arg Leu Glu Leu Ile Gln Thr Ser Lys Pro Tyr Val Asp Ile Tyr Asp

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<213> Artificial Sequence (Artificial Sequence)

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tagaagtcaa aggggtttgt gggtttgtgg gagctgcagt gggtgataag atggaacggt 60

gggtgagcaa attaaatcat caattcagta tcagtattgt gaagctaaag cctatactgt 120

catattagac caatgtggtc cataccccta gtaatctacc aaacaatctt tgctgtgagc 180

tggagtctat tggagcaatt ttgtatgatt tttttaaaaa taaggcaatg atgcaaaaac 240

ctacatttgt gggaatatat ggaccagctc tagcttccgt tgccactaaa tgtaatttta 300

ttgcaccaac ctgtagtgct ctcacgtgtg tgagaaaggg agggagaaga gggagatcgt 360

tagtataaga tggcatataa aagttattat tttgactaca aaatattatt ttgtagtaat 420

agctattcaa cactttgtag atatacagtt actacaaaga tatgttctac aagagtgatg 480

ttcttctctt catattacta ctgccactag gccatacgga cacccccact gggctttgcc 540

taaataaaag gtttatctca gtaacaacac agacccaaga cttagtgagc agctgataac 600

tcaggtggaa ttatattaat ggtggtccaa tcatttcctt caccctgaac aattaatgac 660

cttgcatttg taagccacca tcactaaaat tagttcccta gaatggagtg aaatgacttc 720

tatctcattc catagttgcc aaaagatctt gggaaaataa cttatgtgaa cttctattga 780

cacatccact aaaaaggtga tacaatgcct cctgggaggg aggtatgaga aaaactggaa 840

ctagaacaac taataaccct gggacacagt acatgttcaa gtaaacaact gtggtcatac 900

aatctagcct ggaggtctac agagtgaggt ataaggtctt tgcaaaagga tgtcccgtgt 960

ctaggggaag gatccatatt tgtgatatga acaaactggt ttctggagat ttttctactt 1020

ctaaaccagt attcatttgc aaactccttg gtgggatata tatatgtata tatatatata 1080

tatatatata tatatatata tatatatata tatgtatatg tatatatata tatatatatt 1140

tttttttttt tttggttagt ggtctcatgg gatggtttgt ggtatgaaac agtcccccgg 1200

gccaagtgac agctgctaat ttaggacagg agggaaatga agtaagaaat gcttccctaa 1260

agcccatgat gttgcagaca ggtcacattc tgtgttgctt taagtcattc ggagtgtgca 1320

tattttgaag ggaaaggaag agaaatgaaa ctgttcccca cttgagtcag agcagtgagt 1380

ctgcccttca gttgacaagt tagggtcttc ttttcagatc attagtaatt acataacttc 1440

aagtgagcta cctaacctgt ctgcccttgt ccactcctct gtaaaacaga ggcaatgatt 1500

cacctcttgt ctcctctgct gggctcagaa gagatgtccc aggaggaatt tctttgtacg 1560

tataaaatag gacgaagtgt aagagatgca cccacccagc cctccatgtc ttagtaagat 1620

tcagcaacct gggtagacac tcttaattcc acagcttaca attagccaga tgtgtggccc 1680

tacagaggga tatccagggt gcagaaaata gcaatgaaat tcaaagacgt ggctgacata 1740

atcagtggat ggatattccc aacacaagac acataaagaa agctaaatag ccttagcctg 1800

tcagagctga aatgaatgtg actcattgtc tcatcacttc agaagcagct catgatcact 1860

ggcagatgat actctctacc ctctaccctg tcctgggtac atatgtccat tcacttctta 1920

tgataattct atgggtagga cactgttctc atttaataga tgcataaagt aaaaactaca 1980

gcttagaagt gtataacaag aatttcaaca aagcagctaa tatttattca gatatgggtg 2040

cttttcataa atctagcatc cctttgatgt taagaagcta taacacgtga gcattgtttt 2100

agatactcgt caaaaagcaa caacattgcc acggaccggg cctagtcgac ccccctcaat 2160

ccgtgtgaat cagggtctcg gacaaatggg catagagtcg gcgggaatgg ggtgacaaac 2220

agactcaaca caagggagtt tggatctgaa tgtaatgtca aattgaacat caaacctttt 2280

atacagaaga aaatagggaa gttaggtgac acatcagcaa ggtacaaatt aggttaccgg 2340

atgcttaatg gctcttacac aaaacaggaa tgtaaacaca aagactggca ggaactgggc 2400

aataaaacaa ctgagacaaa gtcagcccta tctaaggtca gctatagtct tagaagccag 2460

gtgtgaggtc tttacactcc tagggtaagg gctttcacac ccaagtcatg gttctaatta 2520

gggagttcag ctctagctaa ccatctcatg aataatgcaa tactctaaaa ccaccgctca 2580

atctacttcc taaaccattg taaattcctg tatatgggag cgacttggct tttattctaa 2640

gtgatagtgt ggggggactt ctactaataa gtaatgtagt ctgccataca taactataat 2700

aagaattcta aacttacatt gctaagcttg ccctctatag atttagaaat ctatagattt 2760

ctaactctat gcaggagata gtaatgcccg atttctttca ctatctctct tacaatacta 2820

gaagtaattc tgaatgtcac tgaataggca acattcttac tgaattccaa gcccagggtt 2880

ggctcaagga ctaccgaggg cattggtgaa agccagaaag caaagttcga ttttgcttag 2940

gtatttggca aatcattgct tggaggcacc tataataaaa caacactgaa aggaagcaca 3000

cagatccatt cacaaggaca aaattggagc atacgtgcta tggaatgcca cacttccagg 3060

agactaagtt tccgtgaaac tcttcgcctc aggactgcat ccaggctttt ggcctgtcat 3120

gcgtgtcact actgaagtgg atgtagcaca acatcatttg tttctgtcaa gagagaactg 3180

caataaactc gcatataagg taatttacca cagtcatgac tgaaatgagg taactagcac 3240

aggatgtgtt gaggcagggc gcatacacag ggcgcatagg ttgagggttt acagaaggcc 3300

tctgcggata gctggggttt aagctacacc tgggtgaaaa aaaacccaca ccaacatgaa 3360

ccgtcatcat caacctcctg taccacgttg tgagacaagg attcttacta ttttcagtct 3420

agagctgaag aatctgaggc ccaggtaact tggccaacac ctcagagtaa ttagaattac 3480

tgtttaaaac cagacagtct gactcacagg ctgtgattgt acatgtgata tcagagaata 3540

ttcttgaagt gacctgcaca acgtcagagt cagagggaga gagaggtcta gaccatccct 3600

cctgaatttc aggacagtcc tagccataat caagaatgac atgtctgggc tactacactg 3660

tccttctgaa catatgaaca agccaataaa aatatgcttt ggaatattaa atcataagaa 3720

catccaagga gggaccagct agacaaatgc atagagcaga gatgaagcgc ggtggtattt 3780

tcttagcatt cacaaagaga gtccaagccc caccagagaa aaactgcata tggacataaa 3840

tttatggtat ggctatgttc taagagtata gctcactggt aaactaattg actacattgt 3900

gatttcttga acattagcat gtgtccaaac atgattatca tcctagctca gattcctggg 3960

cccatgtgcc cttgcctgag atagcccctc cagatccttt cctataccac ggtagtagac 4020

cttgccttac atctgaagtt actaggaagt ggacatgact cagtggcacc atcaagggac 4080

tactaagaga agtattagat ggtgccagaa ggtaaagatt gccaaagact ctgatctcac 4140

ctcactgtct tattttcagg cgtttgaaaa ctca 4174

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

actcttttct tttctaagca ttattgttta gagagcttcc aagacacata gttaccctca 60

tctcttgtgg ccttccacaa tctattctcc atattttcac agcttaactt tgcatagaga 120

agccacatct agctctcctt cacatttgaa gaatgcagtg attataaaag attgtctttt 180

gccttgctta gggagtctta cactggcaga aacgctgaag aatccaattc tcattcacct 240

tttccttgga tgtgtgtctc agtagtggta atggtttttc cgcatttcct ccatcagcag 300

ttacagcaga gggaaaagac acatgactgt tctgttcatc tctgaactct ctgactcctt 360

cttcatgttt ggtggagtcc cttttgcatg attgtcttaa agaatatgag agaaattgtt 420

taatgagaat tgttttaata atgagagaaa atgagacacc tatatgcctg tggggaaggc 480

acaaaatatg gcatgttctt caagcttctg tgtcccactt gggaagctgt gaaaggccta 540

gtcctaagtc tgtctctgta caaaaagcat agcttacaca taagcaagtt tcatgtctta 600

actttgccca tctccagttt cttggcttct ggtcacatcg tttatgtatt tgtattcttt 660

agtcaagaga aaaaagaatg gtgagacaga aatgtaatgg gttcccagat ctcaaatcct 720

aagcaacagc tgtgctttga gattctttcc aaatggcaga gtagtttatt tggtctttca 780

tgattcacag aaaaaaaaca aaaaaacaaa aaacaaaaaa cctatcacta ttttgtaatt 840

tagttgaatg taagaagcaa gccaagatat tcaataatag ttgtggggac ttgagtcaaa 900

cccagctctt caaattctag aaatgtttga aataaccaaa ttatctatgc ccatatgcct 960

gtataagcta tatcaaagta tatactttgt gagcaatgtg ctgaatcaca aaagagtcag 1020

aattggattt gacaccatat gaataaagac acccagaatc ttacatcatt tggaaaacta 1080

gtcgtgacat taatatgcaa ttagctggaa agttataaga tgcaccacaa acacatctta 1140

tatgcactat aaattaccac acaaagatca tgcttaaaaa caacacagtt aaaatcctaa 1200

atctcaggcc accatacttt cccacccctg ttcctgagca gggatgctag cacctgacct 1260

agaaacactg ttagcctaaa aagatctcac catagcctat cattcagata ccatgacaag 1320

tctgcttcta ggcattatgg gtatatgaac agcttaagtt tcactccttg gaatatttct 1380

attttattag aggctaccgg tgggagttat ttcctgttat tacaaagaaa ctggaagtag 1440

caccttttcc ctgtgtggtc ttcaaaacaa gctctaagta aataaaactt tgtgtcagtg 1500

cctgcaatgg atactccacc ttggatgttt ctttcttctc cataacatca aacaatggga 1560

tgcttctgcc acataatact aacctgtgtt tgaatagtgg cacatccagt catattgagc 1620

tattcctacc tatctgacac atgctcccaa ggagtttact catccagtag gtctttacta 1680

agttctgccc acttacaagc cacagcacga ttctccatat ccattcttct ccatagaagg 1740

taccctaagc taaagaagat accgctatgc ctggagagga tggggaggtg ggaggaggag 1800

tgtcaagctg aaaagtttta aatgcagtat tcaatttgaa aagtttcccc tctctgaaat 1860

tcatttcctt aatgatttta cattagtcat cgtcagattc tcaaataatt agtctttacc 1920

aatttctcat gactaggaga taattttagt gtctatttaa aaagaataaa agttcttgtg 1980

agcctccccc aagaatcttc catcattgta gaatcaaatc tcattaccta atggcagaca 2040

tgtttatcaa agcagaaggt atttctctag cttcaagtta ggttttcctc ttaattttta 2100

ttcttataaa aacaattttg taaccataaa gaacaaaagc tttaaaaaaa atcactttca 2160

cccactggct agacacctac cccaccattg aagagactgt ttggctccat ggtttgggga 2220

ccaataatga gacagaagaa taccaagcaa ggaggcatgt ggtggtacaa aactgcccac 2280

cttgtgacag ctcggaagca aaacgatggc tgcaaatgtt tgggtcccaa tagcccttca 2340

agggcatttt gagcttaggc agtagatatt tagagggtat ttaagatcca aatcacaaca 2400

tatgataagg aatactgtta agaatgttat aaagacaaga cagagatctg caacttcaaa 2460

aatgttagtg cctgaatgaa gaaaataaaa atgtgttcaa agataattag ttggactcca 2520

agtaaggaat aaccatttca acaattgctc atgagactca tgatagccac acaaaattaa 2580

ataaataaac tggaagttta cttcacacag tataaaaaaa aaaaacccta gctcaaaatt 2640

tgatcagaag cataaatgta caagctacca ttaaaaaaat acctaaaaga aaactaaaca 2700

cttaaaatct gtgaccctga attagacaat agtttatttt ttattagata ttttcttttt 2760

ttacatttca aatgttatcc cctttcctca tttttccctc tgagaaactc ccatcccctc 2820

cctcctctcg ctgctcacca acccacccac tcctgcttcc tggtcctggc attcccctac 2880

actggggcat tgagccttca caggaccaag ggcctctcct cccattgatg cccaacaagg 2940

ccatcctctg ctatatatgt ggctagagcc atgaatccct ccatgtgtac tctttggttg 3000

gtggtttagt ccctaggtac tttgacaata gcttcttaaa taagcaaaag aataagcaac 3060

taacaaagat taaatgccat caaaacttta aaattttata atttttaaaa agattttaaa 3120

actattttac ttatgtgcac ctatgtgagc ttgtgtgagt ttagttgtac catatgtgtg 3180

ctggagcctg cagaggccag aggaaggggt gccaaatctc ctggaactaa agataaaagt 3240

ggcttttgag ctgccgtgtg gtgctaggaa ctaaacccag gtcctctgca agaacagtaa 3300

acactctttt tttttaagat ttatttattt atttatttta atatttatgt ttttatttta 3360

ttttacatat gtgagtacac tgtagctgta cagatggttg tgagccctgc atgtggttgt 3420

tgggaattga attttaggac ctctgctggc acttggtgga ctcactcgct ctggtcaacc 3480

ccgctcgttc actccctgct cactccagcc caaagattta ttttttttaa ttaggtattt 3540

tcttcattta catttccagt gctaccccaa aagtccccca aaccctctcc ccctccctcc 3600

actcacactt tttggccctg gctttccctt gtactggggc atataaagtt tgcacgacca 3660

atgggcctct ctttccactg atggctgact aggccatctt ctgatacata tgcagctaga 3720

gacaagagct ccgggggggg gggggggtac tggttagttc ataatgtggt tccacctata 3780

gggttgcata tccctttagc tccttgggta ctttccctag ctcctccatt ggaggccctg 3840

tgatccatcc aatagctgac tgtgagcatc cacttctgtg tttgctaggc cctggcatag 3900

tctcacaaga gacagctata tcagggtcca ttgagccatc tcctaggccc tgagaattta 3960

tgacttaaag gacacaaact ttaaaagtgt aaatacaact cacataaggg gaaaacattt 4020

acagaccctg aatctaacaa agatgctgtc accagaattt agaaagaaag cttaaattaa 4080

tttaacgtgg ggtgatggag agtagtctaa aattttaaat tagaaaatga tttgaacata 4140

ttcttctcca aacaaattgt ataaatggtc aataagcaaa taaaccattc actataatta 4200

ttcactagga aaatgtgaat aaaaacctca gtgtgagaca gcctcacatt catgagattg 4260

gctatcaaca gcaagaagca agaaagtgat gctgaggagc taatggaatt aaaataccca 4320

tagaatactg atacatatat ttatgtagtc tctttggaaa attattttcc aattactaaa 4380

aatactaaat gtaaagtttc catttgaccc aagagttcac taccaggcag atagatgccc 4440

aaggatagaa ctatgaacac acaaacttga gtacaattgt ccatagacaa aaataactaa 4500

aaaattggaa acaactcaaa tgtctaccaa ctaatgggtg aacaggaaaa attatgaaat 4560

tttcatctac caaaagaagg cactcctcat taaggcactc ctcatgttaa ggaacaagta 4620

gcc 4623

<210> 5

<211> 6094

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgacaacac ccagaaattc agtaaatggg actttcccgg cagagccaat gaaaggccct 60

attgctatgc aatctggtcc aaaaccactc ttcaggagga tgtcttcact ggtgggcccc 120

acgcaaagct tcttcatgag ggaatctaag actttggggg taagtcagtt gccttccatc 180

ccatgtcgta gggattctct ggctgacaga agctgatgcg gtataggcca catacagaat 240

tcaatccaat ttgaagaatt gggatccaac ctgatgtctt ctttatgtct aacacagtgg 300

gccaaatcag gggtgcatca gagaagttat cacttagatc acctctgggt gatcttatgt 360

caccttttgg ttttggggct tgtatatgca ggggtccccc atcccagtcc attgccagaa 420

tcccaggcat acctgctccc tggaaatgcc ccatgtggtt gaggaaacag attcgaacaa 480

gaaaaagaca aaattcttgg cacctccact gcttccttta ggcattcctc acagctccaa 540

gtcaggagcc agagcttcca accttgtctt tgcctgctag cagtgatgat ttcagctcat 600

ccactgctgc ctctgttctc tccccaggct gtccagatta tgaatgggct cttccacatt 660

gccctggggg gtcttctgat gatcccagca gggatctatg cacccatctg tgtgactgtg 720

tggtaccctc tctggggagg cattatggtg agtaaaagaa tagcagccat ttgggaaatg 780

gtgcagacaa aaatgttaaa aggctccaca gggatatgcc agattatttc tgtgttgagg 840

gaaatatatg agtaggaaat attattgggt taaagtaatt aagaagacag gttgaccaaa 900

ttgagtataa atcccatggt tgagagtcag tggtcctgtt tcatgtgaat tcagagaaag 960

gggccctgca tggatctcac agggactgtc caaagcaaga actctccaaa gtcagttctg 1020

gtggggaggg tggccctaga catttagact agatagcaag atgttttgga aagcaagagg 1080

cagcaggaac atccacttcc atctacccct tcttgcttac aattctgttt ggttactatg 1140

gtacctggtg aaacctgtcc catcacaagt cagtctcatt ttgcttatcg acagagcagc 1200

actcttttgg ctgttttacg tacatgtttt ccaaatctgt aaccctgtct gggtgtggca 1260

cataggacag tgatgtttat ttccccgtga tacttttcat agttgccact ataaaagata 1320

agtccaggat taaaattttc caaacaagta gagatgggtt agaacaagag tttcctccat 1380

tctacagcct gaatgtgaga aaaggaagat gagcaatagt catcatcact tcctgtaaca 1440

gccaatgttt tcatggagtg cctgtgccat tcaggtcaag tatttccttc tgcatcagtt 1500

cactcttcag agggcatcag agtcatttat gtcactgtga accccaaagg gcagttccac 1560

aagttaaaaa caaagaaaaa ctagaaataa aacttttaaa tttatggtat gagtattaat 1620

tgatgaggaa atttgagttc tgtctctttg gtcttactat attcctagtc acagatcccc 1680

agatgattga gtaaaaggca tgaatttagt gtcactgagc ctgaataaag gaggaatatg 1740

acagctgaaa aatgaataca actgataaaa atgggtggat ggttgtgtga aagttgctga 1800

aagtgtaggc ttctttctga ccagttatca atgttaaaaa gtgatctccc tctctcctct 1860

atctcctgtc ttgcccaccc cctctccatc tcccccacct ctctttttta cagtatatta 1920

tttccggatc actcctggca gcaacggaga aaaactccag gaagtgtttg gcaagtaacc 1980

atatgtcctt ctttcccaca tgtcagagaa gtacctattt ttttcggtta aaaactgaga 2040

cccttaaaaa gccaaggtat cacagcctct cagccctaaa aagcaaagac cctccacaat 2100

gttattgtga ttttatttat gaaaaactta gaagcgaggt ctatctgaag tatgttcatg 2160

ggaacagaac taaaagcaga tccatgaaaa ccatacctac agtcttaaga acgttaaatg 2220

ctgtgtgaaa ataatagacc tttctgaaag ccctatcatt tctcccagat caccatttag 2280

gaaaattatc tgatcaatgt catgattgat tcaaattcta gctaagccat tttttggccg 2340

taacattgaa caagtcagtt tacctctatg ttcctgagtt ttcacctaga aaggaagggt 2400

aacagtcctt gctaccatgt gacgtccaat ggagatgaaa ggcagtagag tgtgtgatgg 2460

tgcttcacag gctataaagt actacactgt ggtcttgccc ataaaacccc tagggactca 2520

tctagatcca gaggaaactg gcctgcagag ctgctgatgc tgtatggatg aaaagagttt 2580

agcagcaagt tcgctcccaa aaaattcctc ccccaacact gttactaaac tgtgtcactt 2640

tcataatcaa tgagggaatg ggtggattga gatggttcct gtcttaaagt ggcctgacac 2700

actcagtttg gggggaaaac tttttatgaa catcaaatta ttctctagat acagccagat 2760

ttactgactt gccatgtgta ggtcatagag ccaggaatga aatatgcgca gcataaaata 2820

ataatcaata atcccatatc attatggtac ttgttattta tattttcctg tttcaacctt 2880

ttatcatccc tgcaaggtag aacattcaca ctgatatttt cttacctatg ctacccaaag 2940

acatcagccc taattgtatt ttggaagata gctgactggg gctgattgca acctatgtca 3000

gcaggaatag atgttgttac tgttgttgct tctgcttttt ttatttccat ttatttgata 3060

gtacagatct agagggttct atctgaacct tcccaaccta tacttcataa taccatccca 3120

ctaaagtgtg atacaagaaa cttcttcact ctcttccctc tacctattta tgaaggcaga 3180

taataaactg gataatattt atcttcactt attcaacaaa catttattga gtgcctacta 3240

ggatggtggc agtggcagtg aaggaaatgc aaggatacaa gatatagaat caagggttac 3300

tcttagaatt tttgctttat aaaacagatg gatggtgaat gagataggga agactgagaa 3360

aagaacagga tagagacatg attttatttt atagtgacaa agaggctaaa aacaactgag 3420

agaacttcag tatatttagt tgtagttgct ttgtgagtca gggcagttgc atttggaatt 3480

ccctcccaga ttatgttttc caaagggaaa tcaaacccaa ttaataaatc tgtgtctcca 3540

tttcaggtca aaggaaaaat gataatgaat tcattgagcc tctttgctgc catttctgga 3600

atgattcttt caatcatgga catacttaat attaaaattt cccatttttt aaaaatggag 3660

agtctgaatt ttattagagc tcacacacca tatattaaca tatacaactg tgaaccagct 3720

aatccctctg agaaaaactc cccatctacc caatactgtt acagcataca atctctgttc 3780

ttggtaagtg ttcttggtaa gtgtgagatt ggatttctct ccagggagga aggatgactt 3840

gtttattatg agcatgaact ctggatccag accacctgag tttgctagtt actgtctgtg 3900

tggctttggg aaagaatttt aaccacactg tgcctcaatt tcttcaactg taaaatgggg 3960

atggtaacac tatttatcta atagggttgt tctgaggaat aaataagtta atatatacaa 4020

agcatttaga acagtgtttg gtacacagaa gtgctatata agcattggct ttaacattaa 4080

ttattctcaa catcattaat ggcattaata tttaccatta tattaactaa gatgagcata 4140

gaagaaattc ctaagatgtt atgaatatct tttgcccaag gacttttaaa tgtagagttt 4200

gaaagagata aaaaaaaatg catctcccaa ggatgaagag gtctcctagg catgatgcca 4260

caccaactaa ggctgaatac aatttaattt gagactcatt acttattctt tcctatacag 4320

aattgattct tcaactgatt attcatacct tactttctat cagcaataca cattaaccat 4380

ctgttgtgtg ccaaaagttg tgttaagagt tagggttata aagatgctgt ctcctgtact 4440

agcagttctc acagctattc attacttgtc taaagaattg atctcttaat cgttcaatta 4500

tagtcaacaa taacttactg aacaccaaca atgttctttg tgccattatt acattttcac 4560

cttcattctt ctgttgtttt tcagggcatt ttgtcagtga tgctgatctt tgccttcttc 4620

caggaacttg taatagctgg catcgttgag aatgaatgga aaagaacgtg ctccagaccc 4680

aaatctgtaa gtagtagccc ctctggccaa aacctccctc tagaaaatcc acatccacaa 4740

aggatcattt atggagaatg taatctgatg agttgttgaa actaggagct tgatttaaaa 4800

aaaaaaattg gtcttggcat atttctagaa agcaaataat atgatgtctt gaaatttgaa 4860

ataagtctgt agttagagcc tacagttcta tattctgtgc gtcttttttt tttttttttt 4920

tgagacagag tctcactttg ttgcccaggc tagagtgcaa tggcgcgatc ttggcccagt 4980

gcaacctctg cctccgggtt caggcgattc tcctgcctca ccctcccgag tagctgggat 5040

tacaggcacc cgccatcatg cctggccaat ttttgtattt ttgtagagac gggggttcac 5100

catgttggcc aggccagtct tgaactcccg acctcaggtg atcctcccac ctcagcctcc 5160

gaaagtgctg ggattgcagg cgtgagccac catgccccac ctctgtgcat cttcttatta 5220

accttccctg ctgcttgact tcaggcttcc atcctgattt catgattcca tatacagtgt 5280

gtaaaagcaa gatggttgaa ggttgaaggc taagtcacta gttctttggt ttcatttttc 5340

agtattcccc tcttccctcc ttttcaccta attcctcttc caaaacaaca ggttaaaggg 5400

ctccacaaat gggaaataaa aacagaccaa taatagatat attactaact gcttcagaac 5460

cagactgtta gatttagaat ttagaaatca tcgctaatta cttcattttg catttaaaga 5520

aaaatagtat caaagagaga aaattattct cccagagtta tattcactaa aggcaaatca 5580

gaacgaaaat ctaaactcct ctattactgc aatgttcttt tcccaatacc acgtggtcat 5640

tccaggcact gtggtcaatg tctgctgccc ttgaagattt attcagactt gagttttaat 5700

aaatgacttg ataaggatat aagcacctgc aaaaaaattt tggcatttaa aggcatataa 5760

taaatgacat aagtagcata aaaaccagga ggtatttgat aaatgtttgt ggagattgtt 5820

gacaaaggtg tcagtggtaa aagtaaagaa tggtttgttt aattttctgt tttagaacat 5880

agttctcctg tcagcagaag aaaaaaaaga acagactatt gaaataaaag aagaagtggt 5940

tgggctaact gaaacatctt cccaaccaaa gaatgaagaa gacattgaaa ttattccaat 6000

ccaagaagag gaagaagaag aaacagagac gaactttcca gaacctcccc aagatcagga 6060

atcctcacca atagaaaatg acagctctcc ttaa 6094

<210> 6

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cttattttca ggcgtttgaa aactcaatga caacacccag aaattcagta aa 52

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaaatgacag ctctccttaa actcttttct tttct 35

<210> 8

<211> 78

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagagttata ttcactaaag gcgtcgacgg tatcgataag cttgatatcg aattccgaag 60

ttcctattct ctagaaag 78

<210> 9

<211> 102

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agtataggaa cttcatcagt caggtacata atggtggatc cactagttct agagcggccg 60

cgttaacaaa tcagaacgaa aatctaaact cctctattac tg 102

<210> 10

<211> 3002

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

actcatcttc aagtacttga gatagaagag gccaactgat ctcagctgtg agtggctaat 60

ttggccctta agccttggag ccttggagcc ttggagaccc aggcgtttga aaactcaatg 120

acaacaccca gaaattcagt aaatgggact ttcccggcag agccaatgaa aggccctatt 180

gctatgcaat ctggtccaaa accactcttc aggaggatgt cttcactggt gggccccacg 240

caaagcttct tcatgaggga atctaagact ttgggggctg tccagattat gaatgggctc 300

ttccacattg ccctgggggg tcttctgatg atcccagcag ggatctatgc acccatctgt 360

gtgactgtgt ggtaccctct ctggggaggc attatgtata ttatttccgg atcactcctg 420

gcagcaacgg agaaaaactc caggaagtgt ttggtcaaag gaaaaatgat aatgaattca 480

ttgagcctct ttgctgccat ttctggaatg attctttcaa tcatggacat acttaatatt 540

aaaatttccc attttttaaa aatggagagt ctgaatttta ttagagctca cacaccatat 600

attaacatat acaactgtga accagctaat ccctctgaga aaaactcccc atctacccaa 660

tactgttaca gcatacaatc tctgttcttg ggcattttgt cagtgatgct gatctttgcc 720

ttcttccagg aacttgtaat agctggcatc gttgagaatg aatggaaaag aacgtgctcc 780

agacccaaat ctaacatagt tctcctgtca gcagaagaaa aaaaagaaca gactattgaa 840

ataaaagaag aagtggttgg gctaactgaa acatcttccc aaccaaagaa tgaagaagac 900

attgaaatta ttccaatcca agaagaggaa gaagaagaaa cagagacgaa ctttccagaa 960

cctccccaag atcaggaatc ctcaccaata gaaaatgaca gctctcctta aactcttttc 1020

ttttctaagc attattgttt agagagcttc caagacacat agttaccctc atctcttgtg 1080

gccttccaca atctattctc catattttca cagcttaact ttgcatagag aagccacatc 1140

tagctctcct tcacatttga agaatgcagt gattataaaa gattgtcttt tgccttgctt 1200

agggagtctt acactggcag aaacgctgaa gaatccaatt ctcattcacc ttttccttgg 1260

atgtgtgtct cagtagtggt aatggttttt ccgcatttcc tccatcagca gttacagcag 1320

agggaaaaga cacatgactg ttctgttcat ctctgaactc tctgactcct tcttcatgtt 1380

tggtggagtc ccttttgcat gattgtctta aagaatatga gagaaattgt ttaatgagaa 1440

ttgttttaat aatgagagaa aatgagacac ctatatgcct gtggggaagg cacaaaatat 1500

ggcatgttct tcaagcttct gtgtcccact tgggaagctg tgaaaggcct agtcctaagt 1560

ctgtctctgt acaaaaagca tagcttacac ataagcaagt ttcatgtctt aactttgccc 1620

atctccagtt tcttggcttc tggtcacatc gtttatgtat ttgtattctt tagtcaagag 1680

aaaaaagaat ggtgagacag aaatgtaatg ggttcccaga tctcaaatcc taagcaacag 1740

ctgtgctttg agattctttc caaatggcag agtagtttat ttggtctttc atgattcaca 1800

gaaaaaaaac aaaaaaacaa aaaacaaaaa acctatcact attttgtaat ttagttgaat 1860

gtaagaagca agccaagata ttcaataata gttgtgggga cttgagtcaa acccagctct 1920

tcaaattcta gaaatgtttg aaataaccaa attatctatg cccatatgcc tgtataagct 1980

atatcaaagt atatactttg tgagcaatgt gctgaatcac aaaagagtca gaattggatt 2040

tgacaccata tgaataaaga cacccagaat cttacatcat ttggaaaact agtcgtgaca 2100

ttaatatgca attagctgga aagttataag atgcaccaca aacacatctt atatgcacta 2160

taaattacca cacaaagatc atgcttaaaa acaacacagt taaaatccta aatctcaggc 2220

caccatactt tcccacccct gttcctgagc agggatgcta gcacctgacc tagaaacact 2280

gttagcctaa aaagatctca ccatagccta tcattcagat accatgacaa gtctgcttct 2340

aggcattatg ggtatatgaa cagcttaagt ttcactcctt ggaatatttc tattttatta 2400

gaggctaccg gtgggagtta tttcctgtta ttacaaagaa actggaagta gcaccttttc 2460

cctgtgtggt cttcaaaaca agctctaagt aaataaaact ttgtgtcagt gcctgcaatg 2520

gatactccac cttggatgtt tctttcttct ccataacatc aaacaatggg atgcttctgc 2580

cacataatac taacctgtgt ttgaatagtg gcacatccag tcatattgag ctattcctac 2640

ctatctgaca catgctccca aggagtttac tcatccagta ggtctttact aagttctgcc 2700

cacttacaag ccacagcacg attctccata tccattcttc tccatagaag gtaccctaag 2760

ctaaagaaga taccgctatg cctggagagg atggggaggt gggaggagga gtgtcaagct 2820

gaaaagtttt aaatgcagta ttcaatttga aaagtttccc ctctctgaaa ttcatttcct 2880

taatgatttt acattagtca tcgtcagatt ctcaaataat tagtctttac caatttctca 2940

tgactaggag ataattttag tgtctattta aaaagaataa aagttcttgt gagcctcaaa 3000

aa 3002

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggaggaagc ctcaagtgtc tca 23

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cctataccgc atcagcttct gtca 24

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

actgtgcaga aaaggcaaca ggcta 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tcctgggacc tactctctcc ttgtg 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tttacatggc atgcccacaa tggtt 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

attccacctt cactgagttc cctcc 25

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggatcggcca ttgaacaaga t 21

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cagaagaact cgtcaagaag gc 22

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaagtggaca tgactcagtg gcacc 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aacctgcctg tagaaggcag actga 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aggtgacata agatcaccca gaggt 25

<210> 22

<211> 1409

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tctatgcagg agatagtaat gcccgatttc tttcactatc tctcttacaa tactagaagt 60

aattctgaat gtcactgaat aggcaacatt cttactgaat tccaagccca gggttggctc 120

aaggactacc gagggcattg gtgaaagcca gaaagcaaag ttcgattttg cttaggtatt 180

tggcaaatca ttgcttggag gcacctataa taaaacaaca ctgaaaggaa gcacacagat 240

ccattcacaa ggacaaaatt ggagcatacg tgctatggaa tgccacactt ccaggagact 300

aagtttccgt gaaactcttc gcctcaggac tgcatccagg cttttggcct gtcatgcgtg 360

tcactactga agtggatgta gcacaacatc atttgtttct gtcaagagag aactgcaata 420

aactcgcata taaggtaatt taccacagtc atgactgaaa tgaggtaact agcacaggat 480

gtgttgaggc agggcgcata cacagggcgc ataggttgag ggtttacaga aggcctctgc 540

ggatagctgg ggtttaagct acacctgggt gaaaaaaaac ccacaccaac atgaaccgtc 600

atcatcaacc tcctgtacca cgttgtgaga caaggattct tactattttc agtctagagc 660

tgaagaatct gaggcccagg taacttggcc aacacctcag agtaattaga attactgttt 720

aaaaccagac agtctgactc acaggctgtg attgtacatg tgatatcaga gaatattctt 780

gaagtgacct gcacaacgtc agagtcagag ggagagagag gtctagacca tccctcctga 840

atttcaggac agtcctagcc ataatcaaga atgacatgtc tgggctacta cactgtcctt 900

ctgaacatat gaacaagcca ataaaaatat gctttggaat attaaatcat aagaacatcc 960

aaggagggac cagctagaca aatgcataga gcagagatga agcgcggtgg tattttctta 1020

gcattcacaa agagagtcca agccccacca gagaaaaact gcatatggac ataaatttat 1080

ggtatggcta tgttctaaga gtatagctca ctggtaaact aattgactac attgtgattt 1140

cttgaacatt agcatgtgtc caaacatgat tatcatccta gctcagattc ctgggcccat 1200

gtgcccttgc ctgagatagc ccctccagat cctttcctat accacggtag tagaccttgc 1260

cttacatctg aagttactag gaagtggaca tgactcagtg gcaccatcaa gggactacta 1320

agagaagtat tagatggtgc cagaaggtaa agattgccaa agactctgat ctcacctcac 1380

tgtcttattt tcaggcgttt gaaaactca 1409

<210> 23

<211> 1410

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

actcttttct tttctaagca ttattgttta gagagcttcc aagacacata gttaccctca 60

tctcttgtgg ccttccacaa tctattctcc atattttcac agcttaactt tgcatagaga 120

agccacatct agctctcctt cacatttgaa gaatgcagtg attataaaag attgtctttt 180

gccttgctta gggagtctta cactggcaga aacgctgaag aatccaattc tcattcacct 240

tttccttgga tgtgtgtctc agtagtggta atggtttttc cgcatttcct ccatcagcag 300

ttacagcaga gggaaaagac acatgactgt tctgttcatc tctgaactct ctgactcctt 360

cttcatgttt ggtggagtcc cttttgcatg attgtcttaa agaatatgag agaaattgtt 420

taatgagaat tgttttaata atgagagaaa atgagacacc tatatgcctg tggggaaggc 480

acaaaatatg gcatgttctt caagcttctg tgtcccactt gggaagctgt gaaaggccta 540

gtcctaagtc tgtctctgta caaaaagcat agcttacaca taagcaagtt tcatgtctta 600

actttgccca tctccagttt cttggcttct ggtcacatcg tttatgtatt tgtattcttt 660

agtcaagaga aaaaagaatg gtgagacaga aatgtaatgg gttcccagat ctcaaatcct 720

aagcaacagc tgtgctttga gattctttcc aaatggcaga gtagtttatt tggtctttca 780

tgattcacag aaaaaaaaca aaaaaacaaa aaacaaaaaa cctatcacta ttttgtaatt 840

tagttgaatg taagaagcaa gccaagatat tcaataatag ttgtggggac ttgagtcaaa 900

cccagctctt caaattctag aaatgtttga aataaccaaa ttatctatgc ccatatgcct 960

gtataagcta tatcaaagta tatactttgt gagcaatgtg ctgaatcaca aaagagtcag 1020

aattggattt gacaccatat gaataaagac acccagaatc ttacatcatt tggaaaacta 1080

gtcgtgacat taatatgcaa ttagctggaa agttataaga tgcaccacaa acacatctta 1140

tatgcactat aaattaccac acaaagatca tgcttaaaaa caacacagtt aaaatcctaa 1200

atctcaggcc accatacttt cccacccctg ttcctgagca gggatgctag cacctgacct 1260

agaaacactg ttagcctaaa aagatctcac catagcctat cattcagata ccatgacaag 1320

tctgcttcta ggcattatgg gtatatgaac agcttaagtt tcactccttg gaatatttct 1380

attttattag aggctaccgg tgggagttat 1410

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tggagcaggt tgcatggcga ggg 23

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ggagcgatct cattttccac tgg 23

<210> 26

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

taggtggagc aggttgcatg gcgaggg 27

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccctcgccat gcaacctgct cca 23

<210> 28

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

aaacccctcg ccatgcaacc tgctcca 27

<210> 29

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

taggggagcg atctcatttt ccactgg 27

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ccagtggaaa atgagatcgc tcc 23

<210> 31

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aaacccagtg gaaaatgaga tcgctcc 27

<210> 32

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 33

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

actgtgcaga aaaggcaaca ggcta 25

<210> 34

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

tcctgggacc tactctctcc ttgtg 25

<210> 35

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tttacatggc atgcccacaa tggtt 25

<210> 36

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

attccacctt cactgagttc cctcc 25

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

actcctggca gcaacggaga a 21

<210> 38

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

tgggtctgga gcacgttctt tt 22

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gtcccctcgc catgcaacct 20

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gcatcgccga cagaatgccc 20

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

tcaccatctt ccaggagcga ga 22

<210> 42

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

gaaggccatg ccagtgagct t 21

Claims

1. A humanized CD20 gene, wherein the humanized CD20 gene comprises a portion of the human CD20 gene.

2. The humanized CD20 gene according to claim 1, wherein the portion of human CD20 gene comprises all or part of exons 1 to 7 of human CD20 gene, preferably wherein the humanized CD20 gene comprises all or part of exons 2 to 7 of human CD20 gene, more preferably wherein all or part of exons 2 to 7 of human CD20 gene comprise at least part of exons 2, 3 to 6 and/or part of the nucleotide sequence of exons 7 of human CD20 gene, wherein part of exons 2 comprises at least a nucleotide sequence of 50bp, and part of exons 7 comprises at least a nucleotide sequence of 100 bp.

3. The humanized CD20 gene of claim 1 or 2, wherein the portion of the human CD20 gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

4. The humanized CD20 gene according to any one of claims 1 to 3, wherein the humanized CD20 gene further comprises a portion of a non-human animal CD20 gene, preferably wherein the portion of the non-human animal CD20 gene comprises all or part of exon 1 of the non-human animal CD20 gene, further preferably wherein the portion of the non-human animal CD20 gene further comprises part of exon 2 and/or part of exon 7.

5. The humanized CD20 gene of any one of claims 1 to 4, wherein the nucleotide sequence of the humanized CD20 gene comprises any one of the following group:

A) the transcribed mRNA is SEQ ID NO: 10;

B) the transcribed mRNA is identical to SEQ ID NO: 10 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

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

D) the transcribed mRNA has the sequence of SEQ ID NO: 10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted; or the like, or, alternatively,

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

6. A humanized CD20 protein encoded by the humanized CD20 gene of any one of claims 1-5.

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

C) a human or humanized CD20 gene; or the like, or, alternatively,

D) all or part of exon 1 to exon 7 of the human CD20 gene, preferably comprising all or part of exon 2 to exon 7, further preferably part of exon 2, all of exon 3 to exon 6 and/or part of exon 7 of the human CD20 gene, wherein part of exon 2 comprises at least a 50bp nucleotide sequence and part of exon 7 comprises at least a 100bp nucleotide sequence, further preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

8. The targeting vector according to claim 7, wherein said targeting vector further comprises a 5 'arm and/or a 3' arm, preferably wherein said 5 'arm has at least 90% homology to the sequence of NCBI accession number NC-000085.7, further preferably wherein said 5' arm sequence is as set forth in SEQ ID NO: 3 or as shown in SEQ ID NO: 22, said 3' arm having at least 90% homology to the sequence having NCBI accession number NC _ 000085.7; further preferably, the 3' arm sequence is as set forth in SEQ ID NO: 4 or as shown in SEQ ID NO: shown at 23.

9. A construction method of a humanized non-human animal of a CD20 gene is characterized in that the human or humanized CD20 protein is expressed in the non-human animal body.

10. The method of claim 9, wherein the humanized CD20 protein is the humanized CD20 protein of claim 6.

11. The method of claim 9 or 10, wherein the genome of the non-human animal comprises the humanized CD20 gene of any one of claims 1 to 5.

12. The construct of any of claims 9-11, comprising introducing a donor nucleotide sequence into the non-human animal CD20 locus, preferably wherein the donor nucleotide sequence comprises one of the following groups:

C) a human or humanized CD20 gene; or the like, or, alternatively,

13. The construct of claim 12, wherein the donor nucleotide sequence is regulated in the non-human animal by endogenous regulatory elements.

14. The method of construction of any one of claims 12-13, wherein said introducing is a substitution or insertion, optionally said introducing into the non-human animal CD20 locus is a substitution of a corresponding region of a non-human animal, preferably a substitution of part of exon 2, all of exons 3 to 6 and/or part of exon 7 of a non-human animal CD20 gene.

15. The method of any one of claims 9 to 14, wherein the targeting vector of any one of claims 7 to 8 is used for the construction of a non-human animal.

16. The method of constructing a recombinant human animal of any one of claims 9-15, further comprising mating the humanized non-human animal with another genetically modified non-human animal, in vitro fertilization or directly performing gene editing, and screening to obtain a polygenetically modified non-human animal, preferably wherein the other gene is at least one selected from the group consisting of PD-1, PD-L1, TIGIT, TNFA, 41BB, 41BBL, CTLA4, CD47, SIRPA, OX40, TIM3 and CD 226.

17. A cell, tissue or organ which expresses the humanized CD20 protein of claim 6, wherein the cell, tissue or organ comprises the humanized CD20 gene of any one of claims 1-5, or wherein the cell, tissue or organ is derived from a non-human animal obtained by the construction method of any one of claims 9-16.

18. A tumor tissue after tumor loading, wherein the tumor tissue expresses the humanized CD20 protein of claim 6, the tumor tissue comprises the humanized CD20 gene of any one of claims 1 to 5, or the tumor tissue is derived from the non-human animal obtained by the construction method of any one of claims 9 to 16.

19. Use of the humanized CD20 gene according to any one of claims 1 to 5, the humanized CD20 protein according to claim 6, the non-human animal obtained by the construction method according to any one of claims 9 to 16, the cell, tissue or organ according to claim 17 or the neoplastic tissue according to claim 18, wherein the use comprises:

A) use in the development of products involving CD 20-related immune processes in human cells;

B) use as a model system associated with CD20 for pharmacological, immunological, microbiological and medical studies;

C) to the production and use of animal experimental disease models for the research of CD 20-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) the application of the human CD20 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo; alternatively, the first and second electrodes may be,

E) the functions of the CD20 gene are researched, the medicine and the drug effect aiming at the target site of the human CD20 are researched, and the application in the aspects of the medicine for the immune-related diseases related to the CD20 and the anti-tumor medicine is researched.

20. The humanized CD20 gene according to any one of claims 4 to 5, the humanized CD20 protein according to claim 6, the method of construction according to any one of claims 9 to 16, the cell, tissue or organ according to claim 17, the neoplastic tissue according to claim 18 or the use according to claim 19, wherein the non-human animal is a non-human mammal, preferably wherein the non-human mammal is a rodent, more preferably wherein the rodent is a rat or a mouse.