CN114230657A

CN114230657A - B7H4 gene humanized non-human animal and construction method and application thereof

Info

Publication number: CN114230657A
Application number: CN202111501094.7A
Authority: CN
Inventors: 吕锐利; 刘重慧; 赵素曼
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2020-12-09
Filing date: 2021-12-09
Publication date: 2022-03-25

Abstract

The invention provides a B7H4 gene humanized non-human animal and a construction method thereof, wherein a part of nucleotide sequence coding human B7H4 protein is introduced into the genome of the non-human animal by utilizing a homologous recombination mode, and the humanized B7H4 protein can be normally expressed in the animal body, can be used as an animal model for researching the signal mechanism of human B7H 4and screening drugs for tumors and immune related diseases, and has important application value for the research and development of new drugs for immune targets. The invention also provides a humanized B7H4 protein, a humanized B7H4 gene, a targeting vector, a non-human animal obtained by the construction method and application thereof in the field of biomedicine.

Description

B7H4 gene humanized non-human animal and construction method and application thereof

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a B7H4 gene humanized non-human animal, a construction method thereof and application thereof in the field of biomedicine.

Background

In recent years, tumor immunotherapy has been vigorously developed, bringing about a first line of eosin for human beings to overcome cancer. CD8+ T cells are the primary contributors to the immune system's attack on cancer cells. However, in the tumor microenvironment, CD8+ T cells often enter a dysfunctional or depleted state and are therefore not effective in preventing the progression of cancer. The interaction of the tumor cell surface up-regulated inhibitory ligand PD-L1 with PD-1 on CD8+ T cells is one of the mechanisms mediating T cell depletion; drugs that block the PD-1/PD-L1 pathway have superior efficacy to conventional therapies in cancer patients and have been FDA approved for the treatment of a variety of cancers. However, in most cancer types, only a small fraction (20-30%) of patients respond to anti-PD-1/PD-L1 immunotherapy, and non-responders typically experience a transient activation of T cell function in vivo before disease progression. Thus, the tumor microenvironment PD-1 may synergistically promote T cell failure with other molecules.

B7H4, also known as B7x, B7S1 and VTCN1, was discovered almost simultaneously in 2013 as a negative T cell co-stimulatory molecule in three experiments, listed in japan, James p. mRNA of B7H4 molecule is widely expressed in human peripheral tissues, but protein thereof is not expressed or is low expressed in normal organism, and is high expressed in a plurality of tumor cells of breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, liver cancer and the like. In the group of subjects in the morning, the phenotype of wild mice and B7H4 gene knockout mice is compared, and the fact that the blocking of a B7H4 signal channel can obviously improve the number and the effector function of tumor infiltrating CD8+ T cells, inhibit the T cell failure process and cause compensatory up-regulation of PD-1 is found; in a mouse tumor model, the combined administration of the anti-B7H 4 antibody and the anti-PD-1 antibody obviously inhibits the growth of tumors, provides important basis for the clinical application of PD-1 and B7H4 antibody medicaments, and is expected to further improve the current situation of immunotherapy. B7H4 has been identified as a new target for tumor immunotherapy.

Early studies found that B7H4 belongs to a type I transmembrane glycoprotein, binding to the corresponding receptor on the surface of lymphocytes negatively regulates immune responses. However, intracellular expression of B7H4 was also found in some tumor cells. The analysis result of clinical tissue samples shows that the membrane expression B7H4 is negatively related to tumor-infiltrating T lymphocytes, while the intracellular expression B7H4 molecules are not obviously related to the tumor-infiltrating T lymphocytes, so that the biological functions of the intracellular B7H4 molecules are different from those of the membrane expression B7H4 molecules, but the specific biological significance is still not quite clear.

The experimental animal disease model is an indispensable research tool for researching etiology and pathogenesis of human diseases, developing prevention and treatment technologies and developing medicines. However, due to the differences between the physiological structures and metabolic systems of animals and humans, the traditional animal models cannot reflect the real conditions of human bodies well, and the establishment of disease models closer to the physiological characteristics of human bodies in animal bodies is an urgent need of the biomedical industry. However, because of the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, how to construct "efficient" humanized animal models that approach the physiological characteristics of humans remains the greatest challenge for new drug development.

In view of the huge application value of B7H4 in the field of tumor immunotherapy and the current situation of research on the current signal pathway and biological function thereof, in order to further explore the relevant biological characteristics, improve the effectiveness of the preclinical pharmacodynamic test, improve the success rate of research and development, make the preclinical test more effective and minimize the research and development failure, there is an urgent need in the art to develop a non-human animal model of the B7H 4-related signal pathway. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

The present invention utilizes gene editing technology to replace homologous genes in non-human animal genome with human normal or mutant genes and to create non-human animals with normal or mutant genes that more closely approximate the physiological or disease characteristics of humans. The cell or tissue transplantation can be improved and promoted through gene humanization, and more importantly, due to the insertion of human gene segments, human proteins can be expressed or partially expressed in an animal body and can be used as targets of drugs only capable of recognizing human protein sequences, so that the possibility of screening anti-human antibodies and other drugs at the animal level is provided.

In a first aspect of the invention, there is provided a humanized B7H4 protein, wherein the humanized B7H4 protein comprises all or part of a human B7H4 protein.

Preferably, the humanized B7H4 protein comprises all or part of the signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of the human B7H4 protein. Further preferably, the humanized B7H4 protein comprises all or part of the extracellular domain of human B7H4 protein.

Preferably, the humanized B7H4 protein further comprises a portion of a non-human animal B7H4 protein, more preferably a signal peptide, transmembrane region and/or cytoplasmic region of a non-human animal B7H4 protein. Further preferably, the composition may further comprise a part of the extracellular domain of a non-human animal.

Preferably, the humanized B7H4 protein comprises at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 217, 220, 226, 230, 235 consecutive amino acids of the extracellular region of the human B7H4 protein.

Preferably, the humanized B7H4 protein comprises an extracellular region of human B7H4 protein having an amino acid sequence comprising an extracellular region of human B7H4 protein with 0-20, preferably 5-15, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues removed from the N-terminus and/or C-terminus. Further preferably, the human B7H4 protein extracellular region contains 9 amino acid residues removed from the N terminal and/or C terminal. Still further preferably, the humanized B7H4 protein further comprises all or part of a signal peptide of human B7H4 protein.

In one embodiment of the invention, the humanized B7H4 protein comprises a sequence identical to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, or an amino acid sequence having at least 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259.

Preferably, the humanized B7H4 protein comprises a signal peptide, a transmembrane region, a cytoplasmic region, and an extracellular region.

In one embodiment of the present invention, the humanized B7H4 protein comprises a signal peptide of non-human animal B7H4 protein, a transmembrane region of non-human animal B7H4 protein, a cytoplasmic region of non-human animal B7H4 protein, and a portion of the extracellular region of human B7H4 protein (preferably, the extracellular region of human B7H4 protein with 0 to 20 amino acid residues removed from the N-terminus and/or C-terminus) and a portion of the extracellular region of non-human animal (0 to 20 amino acid residues from the N-terminus and/or C-terminus).

Preferably, the humanized B7H4 protein comprises all or part of the amino acid sequence encoded by exons 1 to 6 of the human B7H4 gene. Further preferably, all or part of an amino acid sequence encoded by any one, two, three or more, two or three or more consecutive exons among exons 1 to 6 is contained. Even more preferably, it comprises all or part of the amino acid sequence encoded by exon 3 to 5. Still further preferably, the nucleotide sequence encoding part of exon 3, all of exon 4and part of exon 5 of the human B7H4 gene, wherein part of exon 3 of human B7H4 gene comprises the nucleotide sequence of exon 3 excluding the coding for first 0-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably the 1 st amino acid, or alternatively, part of exon 3 of human B7H4 gene comprises a nucleotide sequence of at least 20, 50, 100, 150, 200, 250, 300, 310, 320, 330, 340, 346 or 348bp contiguous with exon 3, and part of exon 5 of human B7H4 gene comprises a nucleotide sequence of exon 5 coding for first 0-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, more preferably 9 amino acids, alternatively, the portion of exon 5 of the human B7H4 gene comprises a nucleotide sequence of at least 20, 26, 30, 40, 50, 100, 150, 160, or 170bp contiguous to exon 5. Most preferably, the polypeptide comprises a sequence identical to SEQ ID NO: 7 or a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

Preferably, the humanized B7H4 protein further comprises all or part of an amino acid sequence encoded by a non-human animal B7H4 gene, preferably an amino acid sequence encoded by exon 1 and/or exon 2 of a non-human animal B7H4 gene, and further preferably an amino acid sequence encoded by a part of exon 3 and/or a part of exon 5.

In one embodiment of the present invention, the amino acid sequence of the humanized B7H4 protein comprises one of the following groups:

A) SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259;

B) and SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 4, positions 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid difference; or

D) And SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, and includes an amino acid sequence in which one or more amino acid residues are substituted, deleted, and/or inserted.

I) SEQ ID NO: 13 amino acid sequence, in whole or in part;

II) and SEQ ID NO: 13 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical in amino acid sequence;

III) and SEQ ID NO: 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

IV) and SEQ ID NO: 13, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a second aspect of the invention, there is provided a humanized B7H4 gene, said humanized B7H4 gene comprising part of a human B7H4 gene.

Preferably, the humanized B7H4 gene comprises all or part of the nucleotide sequence encoding a signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of human B7H4 protein. Further preferably, the recombinant human B7H4 protein comprises all or part of the nucleotide sequence of the extracellular region of the human B7H4 protein. Still further preferred is a nucleotide sequence comprising an extracellular region encoding human B7H4 protein with the N-and/or C-terminal being deleted from 0-20, preferably 5-15, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues. Still further preferably, the recombinant human B7H4 protein comprises a nucleotide sequence coding for an extracellular domain of human B7H4 protein with 9 amino acid residues removed from both the N-terminus and/or the C-terminus. Still further preferred is a nucleotide sequence comprising at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 217, 220, 226, 230, 235 consecutive amino acids of the extracellular region encoding human B7H4 protein. Preferably also comprises a nucleotide sequence encoding a human B7H4 signal peptide. Most preferably, the polypeptide comprises a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, or an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259.

Preferably, the humanized B7H4 gene further comprises all or a portion encoding a non-human animal B7H4 protein. For example, a partial nucleotide sequence encoding a cytoplasmic, signal peptide, a transmembrane, and/or an extracellular region of a non-human animal B7H4 protein.

Preferably, the humanized B7H4 gene encodes the humanized B7H4 protein described above.

Preferably, the humanized B7H4 gene comprises all or part of exons 1 to 6 of a human B7H4 gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 6 is contained. Even more preferably, all or part of exons 3 to 5 are included. Still further preferably, the part comprising exon 3, all of exon 4and part of exon 5, preferably further comprises intron 3-4 and/or intron 4-5, wherein the part of exon 3 of human B7H4 comprises the nucleotide sequence of exon 3 excluding the coding for the first 0-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably the 1 st amino acid, or the part of exon 3 of human B7H4 gene comprises a nucleotide sequence of at least 20, 50, 100, 150, 200, 250, 300, 310, 320, 330, 340, 346 or 348bp contiguous to exon 3; the part of exon 5 of the human B7H4 gene comprises a nucleotide sequence encoding the first 0-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, more preferably 9 amino acids, exon 5, or the part of exon 5 of the human B7H4 gene comprises a nucleotide sequence of at least 20, 26, 30, 40, 50, 100, 150, 160, or 170bp contiguous to exon 5.

Preferably, the humanized B7H4 gene further comprises a portion of a non-human animal B7H4 gene; preferably, it is the exon 1 and/or 2 of the B7H4 gene of the non-human animal; further preferably, the non-human animal further comprises a part of exon 3 and/or a part of exon 5.

In one embodiment of the invention, the humanized B7H4 gene comprises a sequence identical to SEQ ID NO: 8 and/or 9, or a nucleotide sequence comprising at least 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 8 and/or 9.

In one embodiment of the invention, the humanized B7H4 gene comprises a sequence identical to SEQ ID NO: 10 and/or 11, or a nucleotide sequence comprising at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 10 and/or 11.

In one embodiment of the present invention, the humanized B7H4 gene comprises one of the following groups:

(A) SEQ ID NO: 7, or a portion or all of the nucleotide sequence set forth in seq id no;

(B) and SEQ ID NO: 7 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

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

(D) Has the sequence shown in SEQ ID NO: 7, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment of the present invention, the nucleotide sequence of the humanized B7H4 gene comprises one of the following groups:

(i) the transcribed mRNA is SEQ ID NO: 12, or a portion or all of the nucleotide sequence set forth in seq id no;

(ii) the transcribed mRNA is identical to SEQ ID NO: 12 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) the transcribed mRNA is identical to SEQ ID NO: 12 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

(iv) The transcribed mRNA has the sequence of SEQ ID NO: 12, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In a third aspect of the invention, a non-human animal humanized with a B7H4 gene is provided, wherein the non-human animal expresses a human or humanized B7H4 protein, or wherein the genome of the non-human animal comprises a portion of a human B7H4 gene.

Preferably, the non-human animal has reduced or absent expression of endogenous B7H4 protein.

Preferably, a portion of the human B7H4 gene or the nucleotide sequence of the humanized B7H4 gene is operably linked to a non-human animal endogenous regulatory element.

Preferably, the non-human animal body contains the humanized B7H4 gene.

In a fourth aspect of the present invention, there is provided a targeting vector, said targeting vector comprising any one of:

A) part of the human B7H4 gene, preferably part of exon 3, all of exon 4and part of exon 5 of the human B7H4 gene, preferably further comprising the nucleotide sequence of intron 3-4 and/or intron 4-5, wherein the part of exon 3 comprises the nucleotide sequence of exon 3 excluding the coding first 0-10 amino acids, alternatively, part of exon 3 of the human B7H4 gene comprises a nucleotide sequence of at least 20, 50, 100, 150, 200, 250, 300, 310, 320, 330, 340, 346, or 348bp contiguous to exon 3, and part of exon 5 comprises a nucleotide sequence of exon 5 encoding the first 0-15 amino acids, alternatively, the portion of exon 5 of the human B7H4 gene comprises a nucleotide sequence of at least 20, 26, 30, 40, 50, 100, 150, 160, or 170bp contiguous to exon 5; further preferred comprises SEQ ID NO: 7;

B) a nucleotide sequence encoding all or part of the human B7H4 protein, preferably comprising all or part of an extracellular region encoding the human B7H4 protein, preferably comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, 1-250, 1-256, 1-259, 15-250, 15-256, 15-259, 25-250, 25-259, 34-250, or 34-259;

C) a nucleotide sequence encoding the humanized B7H4 protein described above; or the like, or, alternatively,

D) the humanized B7H4 gene described above.

Preferably, the targeting vector further comprises a5 'arm (5' homology arm) and/or a 3 'arm (3' homology arm)). The 5 'arm and the 5' end of the switching region to be changed are homologous DNA fragments selected from 100-10000 nucleotides in length of the non-human animal B7H4 gene genome DNA. Preferably, the 5' arm has at least 90% homology with NCBI accession number NC _ 000069.6. Further preferably, the 5' arm sequence is identical to SEQ ID NO: 5 or as shown in SEQ ID NO: 5, respectively. The 3 'arm and the 3' end of the switching region to be changed are homologous DNA fragments selected from 100-10000 nucleotides in length of the non-human animal B7H4 gene genome DNA. Preferably, the 3' arm has at least 90% homology with NCBI accession number NC _ 000069.6. Further preferably, the 3' arm sequence is identical to SEQ ID NO: 6 or as shown in SEQ ID NO: and 6.

Preferably, the transition region to be altered is located at the B7H4 locus of the non-human animal. More preferably, it is located on exons 1 to 6, and even more preferably, exons 3 to 5 of the B7H4 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 one embodiment of the present invention, the targeting vector further comprises a targeting sequence substantially identical to SEQ ID NO: 8 and/or 9, or a nucleotide sequence comprising at least 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 8 and/or 9.

In one embodiment of the present invention, the targeting vector further comprises a targeting sequence substantially identical to SEQ ID NO: 10 and/or 11, or a nucleotide sequence comprising at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 10 and/or 11.

In a fifth aspect of the invention, there is provided a sgRNA that targets a non-human animal B7H4 gene, while the sequence of the sgRNA is on a target sequence on the B7H4 gene to be altered.

Preferably, the target site of the sgRNA is located on exon 1 to exon 6 sequences of the B7H4 gene. Preferably, the target site of the sgRNA is located on exon 3 to exon 5 sequences of the B7H4 gene. Preferably, the target site of the sgRNA is located on exon 3 and/or exon 5 sequences of the B7H4 gene.

In a sixth aspect of the present 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.

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

In an eighth aspect of the invention, a cell comprising the targeting vector, sgRNA, DNA molecule, and/or sgRNA vector described above is provided.

In a ninth aspect, the present invention provides use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector, or the cell for modification of the B7H4 gene. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In the tenth aspect of the invention, a method for constructing a B7H4 gene humanized non-human animal, wherein the non-human animal expresses human or humanized B7H4 protein; or the genome of the non-human animal comprises a part of the human B7H4 gene, and preferably, the non-human animal expresses the human or humanized B7H4 protein in vivo.

Preferably, the genome of the non-human animal comprises all or part of exons 1 to 6 of the human B7H4 gene. Further preferably, all or part of an exon comprising any one, two, three, two consecutive or three of exons 1 to 6 of the human B7H4 gene is contained. Still further preferably, it comprises all or part of exon 3 to 5 of the human B7H4 gene. In one embodiment of the present invention, the portion of human B7H4 gene comprising exon 3, all of exon 4and part of exon 5, preferably further comprising intron 3-4 and/or intron 4-5, wherein the portion of exon 3 of human B7H4 gene comprises a nucleotide sequence that excludes exon 3 encoding the first 0-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably the 1 st amino acid, or the portion of exon 3 of human B7H4 gene comprises a nucleotide sequence of at least 20, 50, 100, 150, 200, 250, 300, 310, 320, 330, 340, 346 or 348bp contiguous to exon 3; the part of exon 5 of the human B7H4 gene comprises a nucleotide sequence encoding the first 0-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, more preferably 9 amino acids, exon 5, or the part of exon 5 of the human B7H4 gene comprises a nucleotide sequence of at least 20, 26, 30, 40, 50, 100, 150, 160, or 170bp contiguous to exon 5. In one embodiment of the invention, the polypeptide comprises a sequence identical to SEQ ID NO: 7 or a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity to a nucleotide sequence comprising SEQ ID NO: 7.

Preferably, the genome of the non-human animal comprises a nucleotide sequence encoding the human or humanized B7H4 protein. Further preferably, it comprises all or part of the extracellular region, signal peptide, cytoplasmic region and/or transmembrane region encoding human B7H4 protein. In one embodiment of the invention, the polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 4, 1-250, 1-256, 1-259, 15-250, 15-256, 15-259, 25-250, 25-259, 34-250 or 34-259. In another embodiment of the invention, the polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 13, or a nucleotide sequence of the amino acid shown in 13.

Preferably, the genome of the non-human animal comprises the humanized B7H4 gene. In one embodiment of the invention, the polypeptide comprising SEQ ID NO: 12.

Preferably, the method of construction comprises introducing into a non-human animal at the B7H4 locus a nucleotide sequence comprising any one of:

A) a portion of the human B7H4 gene, preferably a portion of exon 3, all of exon 4, and a portion of exon 5 of the human B7H4 gene, preferably further comprising intron 3-4 and/or intron 4-5 nucleotide sequences, wherein the portion of exon 3 comprises the nucleotide sequence of exon 3 excluding the first 0-10 amino acids and the portion of exon 5 comprises the nucleotide sequence of exon 5 encoding the first 0-15 amino acids; further preferred comprises SEQ ID NO: 7;

D) the humanized B7H4 gene described above.

Preferably, the introduction is insertion or substitution. Preferably, the site of insertion or substitution is after the endogenous regulatory elements of the B7H4 gene.

Wherein, the insertion is to directly place the target fragment between two adjacent bases without deleting the nucleotide. Among them, the target fragment is, for example, a human B7H4 gene, a humanized B7H4 gene, a nucleotide sequence encoding a human or humanized B7H4 protein, a nucleotide sequence obtained by splicing a human B7H4 gene with a non-human B7H4 gene. Of course, the partial nucleotide sequence of the human B7H4 gene may be also included, for example, in the case of inserting exon 1 of the B7H4 gene of a non-human animal immediately adjacent to exon 2 to 6 or exon 2 to 5 of the B7H4 gene, in the case of inserting exon 2 of the B7H4 gene of a non-human animal immediately adjacent to exon 3 to 5 or exon 3 to 6 of the B7H4 gene of a human animal, in the case of inserting exon 3 of a non-human animal immediately adjacent to exon 4 to 6 or exon 4 to 5 of the B7H4 gene of a human animal, in the case of inserting exon 4 of a non-human animal immediately adjacent to exon 5 or exon 6 of the B7H4 gene of a human animal, and so on.

Preferably, the insertion may further comprise disruption of the coding frame of the endogenous B7H4 gene of the non-human animal or disruption of the coding frame of the endogenous B7H4 gene following the insertion sequence, followed by the insertion procedure, as desired for a particular embodiment. Or the insertion step can cause frame shift mutation to the endogenous B7H4 gene and can realize the step of inserting the human sequence.

Further preferably, according to the requirement of specific embodiment, the insertion may further include auxiliary sequences (e.g., stop codon or sequence containing stop function, etc.) or other methods (e.g., sequence inversion or sequence knockout) after the inserted target fragment, so that the endogenous B7H4 protein of the non-human animal after the insertion site cannot be normally expressed.

Wherein the replacement includes replacement of a corresponding position or replacement of a non-corresponding position. The substitution of the corresponding position not only represents a mechanical substitution directly corresponding to the base site of the human and non-human animal B7H4 gene but also includes a substitution of a corresponding functional region such as a substitution of a nucleotide sequence encoding the extracellular region of the non-human animal B7H4 protein with a nucleotide sequence encoding the extracellular region of the human B7H4 protein, a substitution of a nucleotide sequence encoding the signal peptide of the non-human animal B7H4 protein with a nucleotide sequence encoding the signal peptide of the human B7H4 protein, a substitution of a nucleotide sequence encoding the transmembrane region of the non-human animal B7H4 protein with a nucleotide sequence encoding the transmembrane region of the human B7H4 protein, a substitution of a nucleotide sequence encoding the signal peptide of the non-human animal B7H4 protein with a nucleotide sequence encoding the cytoplasmic region of the human B7H4 protein, a substitution of a nucleotide sequence encoding the signal peptide of the non-human animal B7H4 protein and the extracellular region with a nucleotide sequence encoding the signal peptide of the non-human animal B7H4 protein and the extracellular region, a substitution of the signal peptide of the human B4 protein with a nucleotide sequence encoding the extracellular region of the non-human B7H4 protein, The nucleotide sequences of the extracellular region and the cytoplasmic region replace the nucleotide sequences encoding the signal peptide, the extracellular region and the cytoplasmic region of the non-human animal B7H4 protein, the nucleotide sequences encoding the signal peptide, the extracellular region, the cytoplasmic region and the transmembrane region of the non-human animal B7H4 protein replace the nucleotide sequences encoding the signal peptide, the extracellular region, the cytoplasmic region and the transmembrane region of the human B7H4 protein, and the nucleotide sequences encoding the extracellular region and the cytoplasmic region of the human B7H4 protein replace the nucleotide sequences encoding the extracellular region and the transmembrane region of the non-human animal B7H4 protein.

Preferably, said introduction into the B7H4 locus of a non-human animal is a replacement for the corresponding region of the non-human animal. It is further preferred that all or part of exons 3 to 5 of the non-human animal B7H4 gene be replaced.

Preferably, the construction method comprises insertion or substitution of a nucleotide sequence comprising all or part of the nucleotide sequence encoding human B7H4 protein into the non-human animal B7H4 locus. Further preferably, the non-human animal B7H4 locus is inserted or substituted with a nucleotide sequence comprising all or part of the extracellular region encoding human B7H4 protein. Still further preferably, the insertion or substitution into the B7H4 locus of the non-human animal is with a nucleotide sequence comprising an extracellular region encoding a human B7H4 protein with 0 to 20, preferably 5 to 15, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues removed from the N-terminus and/or C-terminus. Still further preferably, the insertion or substitution is made at the B7H4 locus of a non-human animal with a nucleotide sequence comprising an extracellular region encoding human B7H4 protein from which 9 amino acid residues have been removed at the N-terminus and/or C-terminus. Most preferably, the polypeptide is produced by a polypeptide comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, or an amino acid sequence that is at least 85%, 90%, 95%, or at least 99% identical to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, with a nucleotide sequence identical to the amino acid sequence shown in positions 1-259, 1-256, 1-250, 34-250, or 34-259 being inserted or substituted into the B7H4 locus of the non-human animal.

In one embodiment of the invention, the method of construction comprises contacting the nucleic acid sequence comprising the nucleic acid sequence encoding SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, by substituting the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259.

Preferably, the construction method comprises insertion or substitution of a partial nucleotide sequence comprising the human B7H4 gene at the non-human animal B7H4 locus. Further preferably, the non-human animal B7H4 locus is inserted or substituted with a nucleotide sequence comprising all or part of exons 1 to 6 of the human B7H4 gene. Still further preferably, all or part of the nucleotide sequence comprising any one, two, three or more, two or more consecutive, or a combination of three or more consecutive exons of human B7H4 gene from exon 1 to exon 6 is inserted or substituted into the non-human animal B7H4 locus. Still further preferably, the non-human animal B7H4 locus is inserted or substituted with a nucleotide sequence comprising all or part of exons 3 to 5 of the human B7H4 gene. Most preferably, the non-human animal B7H4 locus is inserted or substituted with a nucleotide sequence comprising part of exon 3, all of exon 4and part of exon 5 of the human B7H4 gene, preferably further comprising intron 3-4 and/or intron 4-5 of the human B7H4 gene, wherein the part of exon 3 of the human B7H4 gene comprises the nucleotide sequence of exon 3 excluding the coding for first 0-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more preferably 1 amino acid, and the part of exon 5 of the human B7H4 gene comprises the nucleotide sequence of exon 5 coding for first 0-15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, more preferably 9 amino acids. In one embodiment of the invention, the polypeptide comprising a sequence identical to SEQ ID NO: 7 or a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 7 into or replaces the B7H4 locus of a non-human animal.

In one embodiment of the invention, the method of construction comprises replacing all or part of exons 3 to 5 of a non-human animal B7H4 gene with an exon comprising all or part of exons 3 to 5 of a human B7H4 gene.

In a specific embodiment of the invention, the construction method comprises replacing part of exon 3, all of exon 4and part of exon 5 of the non-human animal B7H4 gene with a recombinant vector comprising part of exon 3, all of exon 4and part of exon 5 of the human B7H4 gene.

In a specific embodiment of the present invention, the construction method comprises replacing part of exon 3, intron 3-4, exon 4, intron 4-5 and exon 5 of the non-human animal B7H4 gene with a gene comprising part of exon 3, intron 3-4, all of exon 4, intron 4-5 and part of exon 5 of the human B7H4 gene.

In one embodiment of the invention, the method of construction comprises the step of using a nucleic acid comprising SEQ ID NO: 7 replaces all or part of exons 3 to 5 of a non-human animal B7H4 gene.

In one embodiment of the invention, the method of construction comprises the step of using a nucleic acid comprising SEQ ID NO: 7 replaces the nucleotide sequence shown in the non-human animal B7H4 gene to code SEQ ID NO: 2, and (3) a nucleotide sequence of amino acids 34-250.

In one embodiment of the invention, the cDNA sequence encoding human B7H4 protein is inserted or substituted into the non-human animal B7H4 locus.

In a specific embodiment of the invention, the non-human animal B7H4 locus is inserted or substituted with a nucleotide sequence comprising a sequence encoding a humanized B7H4 protein.

In a specific embodiment of the invention, the nucleotide sequence comprising the humanized B7H4 gene is inserted or substituted into the non-human animal B7H4 locus.

Preferably, the human or humanized B7H4 gene is regulated in the non-human animal by endogenous regulatory elements.

Preferably, the non-human animal is homozygous or heterozygous.

Preferably, the genome of the non-human animal comprises a humanized B7H4 gene on at least one chromosome.

Preferably, at least one cell in the non-human animal expresses a human or humanized B7H4 protein.

Preferably, the non-human animal is constructed using gene editing techniques including gene targeting using embryonic stem cells, regular clustered spacer short palindromic repeats (CRISPR/Cas9) techniques, Zinc Finger Nucleases (ZFNs) techniques, transcription activator-like effector nucleases (TALENs) techniques, homing endonucleases (megabase megaribozymes), or other molecular biology techniques.

Preferably, the targeting vector described above is used for the construction of non-human animals.

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector into a cell of a non-human animal, culturing the cell (preferably an embryonic stem cell), 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 B7H4 gene.

Preferably, to improve the recombination efficiency, the sgRNA may be used together with the targeting vector to construct a non-human animal.

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector, the sgRNA and the Cas9 into a cell of a non-human animal, culturing the cell (preferably an embryonic stem cell), 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 to obtain the humanized B7H4 gene.

The construction method also comprises the steps of mating the B7H4 gene humanized non-human animal with other gene modified non-human animals, in vitro fertilization or directly carrying out gene editing, and screening to obtain the multi-gene modified non-human animal. Preferably, the other genes comprise one or more than two of CD3, PD-1, PD-L1, CD40, OX40, TIGIT, CD27, CD28, CD47, GITR or SIRPA.

Preferably, the non-human animal can be selected from any non-human animal such as rodent, pig, rabbit, monkey, etc. which can be genetically modified by gene editing.

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

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

In an eleventh aspect of the present invention, there is provided a non-human animal in which the B7H4 gene is deleted, the non-human animal being deleted of all or a part of the nucleotide sequence of the B7H4 gene.

Preferably, the non-human animal lacks all or part of exons 1 to 6 of the B7H4 gene. Further preferably, all or part of exon 3 to 5 of the B7H4 gene is deleted. Even more preferably, part of exon 3, all of exon 4and part of exon 5 are deleted, and preferably the nucleotide sequence of intron 3-4 and/or intron 4-5 is also deleted.

Preferably, a non-human animal with a deletion of the B7H4 gene is prepared using the targeting vector and/or sgRNA described above.

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

providing the non-human animal and the non-human animal obtained by the construction method;

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

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized by a combination of one or more of the genes CD3, PD-1, PD-L1, CD40, OX40, TIGIT, CD27, CD28, CD47, GITR, or SIRPA.

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

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

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

The non-human animal according to any of the above aspects may be selected from any non-human animal such as rodents, pigs, rabbits, monkeys, etc., which can be genetically engineered to humanize the gene.

In a fourteenth aspect of the present invention, an animal tumor-bearing or inflammation model is provided, wherein the tumor-bearing or inflammation 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.

In a fifteenth aspect of the present invention, there is provided a method for constructing a tumor-bearing or inflammatory model in an animal, comprising the above-described method for constructing a non-human animal, a non-human animal or a progeny thereof, a gene-deleted animal or a polygene-modified non-human animal.

In a sixteenth aspect, the present invention provides a non-human animal derived from the above non-human animal, a non-human animal obtained by the above construction method, the above non-human animal or its progeny, or the above constructed polygene-modified non-human animal, for use in preparing a tumor-bearing or inflammation model of the animal.

In a seventeenth aspect of the present invention, there is provided a cell, a tissue or an organ, wherein the cell, the tissue or the organ expresses human or humanized B7H4 protein, or a genome of the cell, the tissue or the organ includes a part of human B7H4 gene, preferably the humanized B7H4 gene, or the cell, the tissue or the organ is derived from the non-human animal, the non-human animal obtained by the above construction method, the non-human animal or a progeny thereof, the tumor-bearing cell or the inflammation model thereof.

In an eighteenth aspect of the present invention, there is provided a tumor-bearing tissue obtained 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 tumor-bearing or inflammation model.

In a nineteenth aspect of the present invention, there is provided a cell humanized with a B7H4 gene, the cell expressing a human or humanized B7H4 protein.

Preferably, the cell expresses the humanized B7H4 protein described above.

Preferably, the genome of said cell comprises part of the human B7H4 gene. More preferably, the cell comprises the humanized B7H4 gene described above.

In a twentieth aspect of the present invention, there is provided a cell in which the B7H4 gene is deleted, wherein the cell is deleted of all or part of the nucleotide sequence of the B7H4 gene.

Preferably, the cell lacks all or part of exon 1 to exon 6 of the B7H4 gene. Further preferably, all or part of exon 3 to 5 of the B7H4 gene is deleted. Even more preferably, part of exon 3, all of exon 4and part of exon 5 are deleted, and preferably the nucleotide sequence of intron 3-4 and/or intron 4-5 is also deleted.

Preferably, a B7H4 gene-deleted cell is prepared using the targeting vector and/or the sgRNA.

In a twenty-first aspect of the present invention, there is provided a humanized B7H4 protein derived from the above, a humanized B7H4 gene derived from the above, a non-human animal derived from the above construction method, a non-human animal derived from the above or a progeny thereof, a tumor-bearing or inflammation model derived from the above, a cell, a tissue or an organ derived from the above, a tumor tissue derived from the above, or an application of the above cell, wherein the application comprises:

A) use in the development of products involving the immunological process of human cells; the product is preferably an antibody;

B) as model systems for pharmacological, immunological, microbiological or medical research;

C) to the production and use of animal experimental disease models for the study of etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) screening, validating, evaluating or studying B7H4 pathway function; preferably human B7H4 pathway signaling mechanism;

or E) screening and evaluating the application of human medicine and drug effect research. The drug is preferably an antibody or an immune-related drug.

Preferably, the application is related to B7H4 gene or protein.

In a twenty-second aspect of the invention, there is provided a method of screening for a human B7H 4-specific modulator, said method comprising administering the modulator to an individual implanted with tumour cells, and detecting tumour suppressive activity; wherein the individual is selected from the group consisting of the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or a progeny thereof, or the above tumor-bearing or inflammation model.

Preferably, the modulator is selected from CAR-T, a drug; 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 method of screening for a human B7H 4-specific modulator is not a therapeutic method. The method is used for screening or evaluating drugs, and detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary and is only a possibility.

In a twenty-third aspect of the present invention, there is provided a method for evaluating an intervention program, comprising implanting tumor cells into an individual, applying an intervention program to the individual in which the tumor cells are implanted, and detecting and evaluating a tumor-suppressing effect of the individual after applying the intervention program; 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 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 method of assessing the intervention regimen is not a method of treatment. The evaluation method detects and evaluates the effect of the intervention program to determine whether the intervention program has a therapeutic effect, i.e. the therapeutic effect is not necessarily but only a possibility.

In a twenty-fourth aspect, the present invention provides a use of the non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, the above tumor-bearing or inflammation model, for preparing a human B7H 4-specific modulator.

The twenty-fifth aspect of the invention provides a use of the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the above tumor-bearing or inflammation model in the preparation of a medicament for treating tumor or immune-related diseases.

The B7H4 gene humanized non-human animal can normally express human or humanized B7H4 protein in vivo, can be used for drug screening, drug effect evaluation, immune-related diseases and tumor treatment aiming at human B7H4 access target sites, can accelerate the development process of new drugs, and can save time and cost.

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 "tumor" according to the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein 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. In one embodiment of the present invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, liver cancer.

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 B7H4 protein of the invention comprises a part derived from human B7H4 protein and a part of non-human B7H4 protein.

Wherein, the 'humanized B7H4 protein' comprises 5 to 282 amino acid sequences which are continuous or alternate and are consistent with the amino acid sequence of the human B7H4 protein, preferably 10 to 217, 10 to 226, 10 to 235, 10 to 250, 10 to 256 and 10 to 259, more preferably 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 217, 226, 235, 250, 256, 259 or 282 amino acid sequences which are continuous or alternate and are consistent with the amino acid sequence of the human B7H4 protein.

The "humanized B7H4 gene" of the present invention comprises a part derived from a human B7H4 gene and a part derived from a non-human B7H4 gene.

Wherein, the humanized B7H4 gene comprises 20bp-67000bp nucleotide sequences which are continuous or spaced and are consistent with the nucleotide sequence of the human B7H4 gene, preferably 20-9163 nucleotides which are continuous or spaced, and 20-651 more preferably 20, 50, 100, 200, 300, 400, 500, 600, 651, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9163, 9500, 10000, 20000, 30000, 40000, 50000, 60000 or 67000bp nucleotide sequences which are consistent with the nucleotide sequence of the human B7H4 gene.

The "xx to xxx exon" or "all of xx to xxx exons" of the present invention comprise nucleotide sequences of exons and introns therebetween, for example, the "3 to 5 exons" comprise all nucleotide sequences of exon 3, intron 3-4, exon 4, intron 4-5 and exon 5.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 3-4" means an intron between exon 3 and exon 4.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "B7H 4 locus" refers to a DNA fragment of an optional stretch of exons 1 to 6 of the B7H4 gene. In one embodiment of the invention, the replaced B7H4 locus may be a DNA fragment of an optional stretch of exon 1 to 6 of the B7H4 gene. In one embodiment of the invention, the replaced B7H4 locus may be a DNA fragment of an optional stretch of exon 3 to exon 5 of the B7H4 gene.

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

The term "more than three" as used herein includes, but is not limited to, three, four, five, six or seven, etc.

The term "three or more in succession" in the present invention includes, but is not limited to, three in succession, four in succession, five in succession, six in succession, seven in succession, and the like. Wherein "three or more consecutive exons 1 to 6" includes three, four, five or six, etc. consecutive exons, and also includes intron nucleotide sequences in between.

The term "treating" (or "treatment") as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treatment" or the like refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

All combinations of items described herein as "and/or" including "are to be understood as meaning that each combination has been individually listed herein. For example, "A and/or B" includes "A", "A and B", and "B". As another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

The term "comprising" or "comprises" as used herein is open-ended, and when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity of the invention. Furthermore, it is clear to the skilled person that the methionine at the N-terminus of the polypeptide encoded by the start codon may be retained in certain practical cases (e.g.during expression in a particular expression system), but does not substantially affect the function of the polypeptide. Thus, in describing a particular polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by the start codon at the N-terminus, the sequence containing the methionine is also encompassed herein, and accordingly, the encoding nucleotide sequence may also contain the start codon; and vice versa.

The term "homology" as used herein refers to the fact that, in the aspect of using an amino acid sequence or a nucleotide sequence, a person skilled in the art can adjust the sequence according to the actual working requirement, so that the used sequence has (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. In one aspect, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal to which the gene is humanized 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, a strain of C57BL, C58, a/Br, CBA/Ca, CBA/J, CBA/CBA/mouse selected from 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/10 sn, C57BL/10Cr and C57 BL/Ola.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mulliserial.u.s.pat.no. 4, 683, 195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds. inchief, Academic Press, Inc., New York), specific, Vols.154and 155(Wuetal. eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Caloseds, 1987, Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.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

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

FIG. 1: schematic comparison of mouse B7H4 locus to human B7H4 locus (not to scale);

FIG. 2: a schematic representation of humanization of the mouse B7H4 gene (not to scale);

FIG. 3: B7H4 gene targeting strategy and targeting vector design schematic (not to scale);

FIG. 4: B7H4 recombined cell Southern blot result, wherein WT is wild type control, 1-A02, 1-E01, 1-F02, 2-G03 and 3-G07 are clone numbers;

FIG. 5: schematic representation (not to scale) of the FRT recombination process for humanized B7H4 mouse;

FIG. 6: B7H4 humanized mouse F1 generation rat tail genotype identification result, wherein, WT is wild type, H₂O is water control, PC is positive control, F1-01, F1-02 and F1-03 are rat tail numbers;

FIG. 7: the comparison result of the amino acid sequences of the human B7H4 protein and the mouse B7H4 protein, wherein Query is the partial amino acid sequence of the human B7H4 protein, and Sbjct is the partial amino acid sequence of the mouse B7H4 protein;

FIG. 8: western blot detection results, wherein +/-is wild type control, and H/H is a B7H4 gene humanized homozygote mouse;

FIG. 9: c57BL/6 wild type mouse (+/+) and B7H4 gene humanized homozygote mouse (H/H) in ovary B7H4mRNA detection results, wherein H7H 4mRNA detection results₂O is water control.

Detailed Description

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

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

XmnI, AseI and ScaI enzymes are purchased from NEB, and the cargo numbers are R0194, R0526 and R0122 respectively;

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

B7-H4 Polyclonal Antibody, available from Invitrogen under the code PA 5-86039;

beta-actin Mouse mAb was purchased from Biyunyan, cat # AF 0003.

Example 1B7H4 Gene humanized mouse

This example describes the engineering of a non-human animal (e.g., a mouse) to include a nucleotide sequence encoding a humanized B7H4 protein in the non-human animal, resulting in a genetically modified non-human animal that expresses a humanized B7H4 protein. A comparative scheme of the mouse B7H4 Gene (NCBI Gene ID: 242122, Primary source: MGI: 3039619, Unit prot: Q7TSP5, located at positions 100825459 to 100896922 of chromosome 3 NC-000069.6, based on transcript NM-178594.3 (SEQ ID NO: 1) and its encoded protein NP-848709.2 (SEQ ID NO: 2)) and the human B7H4 Gene (NCBI Gene ID: 79679, Primary source: HGNC:28873, Unit prot ID: Q7Z7D3, located at positions 117143587 to 117210985 of chromosome 1 NC-000001.11, based on transcript NM-024626.4 (SEQ ID NO: 3) and its encoded protein NP-078902.2 (SEQ ID NO: 4)) is shown in FIG. 1.

To achieve the object of the present invention, a nucleotide sequence partially encoding a human B7H4 protein may be introduced at the endogenous B7H4 locus in a mouse, so that the mouse expresses a humanized B7H4 protein. Specifically, the sequence of the humanized B7H4 gene (schematic diagram is shown in FIG. 2) can be obtained by replacing the 8828bp sequence from the No. 3 exon partial sequence to the No. 5 exon partial sequence of the mouse B7H4 gene with the corresponding human DNA sequence through a gene editing technology, so that the humanized transformation of the mouse B7H4 gene is realized.

In the schematic of the targeting strategy shown in FIG. 3, the homology arm sequences upstream and downstream of the mouse B7H4 gene are shown on the targeting vector, along with an A fragment comprising the human B7H4 sequence. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 5) is the same as the nucleotide sequence from position 100879248 to 100883445 of NCBI accession No. NC-000069.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is the same as the nucleotide sequence from position 100892575 to 100896653 of NCBI accession No. NC-000069.6; a genomic DNA sequence (SEQ ID NO: 7) comprising from the No. 3 exon part sequence to the No. 5 exon part sequence of the human B7H4 gene on the A fragment, which is identical to the nucleotide sequence of from 117147757 to 117156919 of NCBI accession No. NC-000001.11; the connection between the upstream of the human B7H4 sequence in the A fragment and the mouse B7H4 gene is designed

5’-accagggcctcactggcatgggtccttcctgaaccgcaggc

ctccatcacagtcactactgtcgcctcagctggga-3' (SEQ ID NO: 8), wherein the last "c" in the sequence "caggc" is the last nucleotide in the mouse, sequence

The first "a" in (a) is the first nucleotide of a human; the connection of the downstream of the human B7H4 sequence and the mouse B7H4 gene is designed

5’-ctctcacttcacagaatcggagatcaaaaggcgga

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

The last "c" in (a) is the last nucleotide, sequence, of a human "ctgcaThe first "c" of "is the first nucleotide of the mouse.

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

5’-gagagccagaatagtcacatgacttgtttaagattaaatgG

ACCATTATGTACCTGACTGATGAAGTTCCTATAC-3' (SEQ ID NO: 10), wherein the sequence "aaatgGThe second G is the last nucleotide, sequence, of the mouse

"G" in (1) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo box and the mouse gene is designed as

5’-AAGTTCCTATACTTTCTAGAGAATAGGAACTTCGG

gctgttcaggaacaaagccgggatgtgaatattcacact c-3' (SEQ ID NO: 11), wherein the sequence

"C" in (A) is the last nucleotide, sequence, of the Neo cassette "gctgtThe first "g" in "is the first nucleotide in the mouse. 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 B7H4 after being transformed is shown as SEQ ID NO: 12, the expressed protein sequence is shown as SEQ ID NO: shown at 13.

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 by using PCR and Southern Blot technologies to confirm the integration condition of an exogenous gene, screening correct positive clone cells, detecting clones identified as positive by the PCR by using Southern Blot (cell DNA is digested by XmnI or AseI or ScaI respectively and hybridized by using 3 probes, the lengths of the probes and target fragments are shown in table 1), and detecting the result as shown in figure 4, wherein the detection result shows that 5 clones which are positive by the PCR are not randomly inserted, and the 5 clones are further verified to be positive by sequencing and are not randomly inserted, and are specifically numbered as 1-A02, 1-E01, 1-F02, 2-G03 and 3-G07.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				XmnI	5’Probe	8.1kb	11.8kb
AseI	3’Probe	16.0kb	7.0kb
				ScaI	Neo Probe	—	9.0kb

Wherein the PCR assay comprises the following primers:

F1：5’-CTACACGCCCATTTTCCTCTTTGGC-3’(SEQ ID NO：14)，

R1：5’-GCTCGACTAGAGCTTGCGGA-3’(SEQ ID NO：15)；

F2：5’-ACCAGTCATGTGAAGAAGACATCCAG-3’(SEQ ID NO：16)，

R2：5’-GAAGGCCTATTTTCTGTGGGAGGCA-3’(SEQ ID NO：17)；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

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

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

3 'Probe (3' Probe):

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

3’Probe-R：5’-TTCCCTGATAGGAACAGGCAAGGCT-3’(SEQ ID NO：21)；

neo Probe (Neo Probe):

Neo Probe-F：5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO：22)，

Neo Probe-R：5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO：23)。

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. 5 for a schematic diagram of the process), and then mated with each other to obtain humanized B7H4 gene homozygous mice. 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. 6, in which 3 mice numbered F1-01, F1-02, and F1-03 were all positive heterozygous mice. This shows that the humanized B7H4 gene engineering mouse which can be stably passaged and has no random insertion can be constructed by using the method.

Table 2: primer name and specific sequence

In addition, based on the amino acid sequence structures of human B7H 4and mouse B7H4 proteins (as shown in FIG. 7), the method of the embodiment is adopted to replace the corresponding sequences of mice with nucleotide sequences encoding amino acid sequences of 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250 and 34-259 of human B7H4 proteins, so as to achieve the purpose of the invention.

Western Blot was used to detect the expression of B7H4 protein in mice. Specifically, 1 mouse of the wild type C57BL/6 male mice of 8 weeks old and 1 mouse of the humanized homozygote of the B7H4 gene prepared in this example were selected, and testis tissues were taken after cervical-free euthanasia, and Western Blot assay was carried out using a B7H4 Polyclonal Antibody (B7-H4 Polyclonal Antibody: purchased from Invitrogen, cat. No.: PA5-86039), and the assay results are shown in FIG. 8. As can be seen from the figure, the B7H4 protein was detected in both wild-type C57BL/6 mice and B7H4 gene humanized homozygote mice.

Since the polyclonal antibody B7H4 can recognize human B7H4 protein and mouse B7H4 protein, in order to further verify that the B7H4 gene humanized homozygote mouse detects that the B7H4 protein is humanized B7H4 protein in vivo, the expression of B7H4mRNA in the mouse can be detected by a conventional detection method, such as RT-PCR and the like. Specifically, 1 mouse of the C57BL/6 wild-type mice and 1 mouse of the B7H4 gene humanized homozygote obtained in this example were taken, and after deported, ovarian tissues of the mice were taken, cellular RNA was extracted according to the instructions of Trizol kit, and after reverse transcription into cDNA, RT-PCR detection was performed (primers are shown in table 3), and the detection results are shown in fig. 9: only murine B7H4mRNA, no humanized B7H4mRNA was detected in C57BL/6 wild type mice; the humanized B7H4mRNA was only detectable in B7H4 humanized homozygous mice. The results of Western Blot analysis showed that only the humanized B7H4 protein could be detected in the humanized homozygote mouse of B7H4 gene.

TABLE 3 RT-PCR primer names and specific sequences

Example 2 drug efficacy verification

The B7H4 humanized mouse prepared by the method can be used for evaluating the drug effect of an antibody drug targeting human B7H 4. For example, a B7H4 humanized homozygote mouse is subcutaneously inoculated with tumor cells (or tumor cells overexpressing B7H 4), after the tumor grows to a certain volume, the tumor is divided into a control group or a treatment group according to the tumor volume, the treatment group is injected with an antibody drug targeting human B7H4, and the control group is injected with an equal volume of physiological saline or PBS. The tumor volume is measured periodically, the body weight of the mice is weighed, and the safety and the in-vivo efficacy of the antibody drug in the humanized B7H4 mice can be effectively evaluated by comparing the change of the body weight of the mice with the tumor volume.

Example 3 preparation of double-or multiple-humanized mice

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared B7H4 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 CD3, PD-1, PD-L1, CD40, OX40, TIGIT, CD27, CD28, CD47, GITR, SIRPA, etc., or may be obtained from a mouse model modified with two or more genes such as B7H 4and other genes by using an isolated mouse ES embryonic stem cell and a gene recombination targeting technique on the basis of a humanized B7H4 mouse. The homozygote or heterozygote of the B7H4 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 B7H 4and double-gene or multi-gene modified heterozygote of other gene modification can be obtained with a certain probability according to Mendelian genetic law, the heterozygote is mated with each other to obtain double-gene or multi-gene modified homozygote, and the in vivo efficacy verification of targeted human B7H 4and other gene regulators can be carried out by using the double-gene or multi-gene modified mice.

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 within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

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

Sequence listing

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

<120> B7H4 gene humanized non-human animal and construction method and application thereof

<130> P0102020040248Z

<150> 2020114313238

<151> 2020-12-09

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2622

<212> DNA

<213> Mus musculus

<400> 1

gtgagtcaca acacccagga gggcagcagc aggcaggcag ctccactcac caaaatctgg 60

ccccacacac agcaggactg tgggaaggaa ctccctctcc atggcttcct tggggcagat 120

catcttttgg agtattatta acatcatcat catcctggct ggggccatcg cactcatcat 180

tggctttggc atttcaggca agcacttcat cacggtcacg accttcacct cagctggaaa 240

cattggagag gacgggaccc tgagctgcac ttttgaacct gacatcaaac tcaacggcat 300

cgtcatccag tggctgaaag aaggcatcaa aggtttggtc cacgagttca aagaaggcaa 360

agacgacctc tcacagcagc atgagatgtt cagaggccgc acagcagtgt ttgctgatca 420

ggtggtagtt ggcaatgctt ccctgagact gaaaaacgtg cagctcacgg atgctggcac 480

ctacacatgt tacatccgca cctcaaaagg caaagggaat gcaaaccttg agtataagac 540

cggagccttc agtatgccag agataaatgt ggactataat gccagttcag agagtttacg 600

ctgcgaggct cctcggtggt tcccccagcc cacagtggcc tgggcatctc aagtcgacca 660

aggagccaat ttctcagaag tctccaacac cagctttgag ttgaactctg agaatgtgac 720

catgaaggtc gtatctgtgc tctacaatgt cacaatcaac aacacatact cctgtatgat 780

tgaaaacgac attgccaaag ccaccgggga catcaaagtg acagattcag aggtcaaaag 840

gcgaagtcag ctgcagttgc tgaactctgg gccttccccg tgtgtttttt cttctgcctt 900

tgtggctggc tgggcactcc tatctctctc ctgttgcctg atgctaagat gaggggccct 960

ggctacacaa aagcatgcaa cgttgctggt ccaacagaat cccggagaac tacagaaata 1020

ttttcctcaa gacatgacct agttttatat ttctagaaga agatgaaatc atgtctagaa 1080

gtctggagag agcagacagg aacaagatgt ggaaggaaaa caaaagtaac ccacagacac 1140

ccccgatcgg aacaagatgg acctagaaaa taattcaacc aaactagagt atactaagtg 1200

tgctgttaca atgtgtgtag ggtaggtgtc ctcccacatc tcaggggcct cccctggtcc 1260

accagctcct gagttaggat gggctgttat gatgtcactc tgaaggttcc tggatggttc 1320

ctactgccat atactcattt tatattcagc acattaaacc atagtgaatg ctatgaaaag 1380

ctgctaatca gctgccactc cgagattcgg aggtggcaac gtctgagtga caggtccagt 1440

gattcgcttc tccttaggat gcttttacaa gctctttggc gtctcctccc acctggcaaa 1500

tgccaaatgc ataggggagg gtgatcatca ttctagggca aacaaaatag ttgagggatg 1560

ctgatttccc aaatcatccg aatcacttct cccttgagca aacaagcgcc ctgttatttc 1620

tcaaatgctg ctttgtgaat cagtccaggg caaggcgctc tcctcatccc gctatgtggc 1680

cttaagtcat cgtaaggttt gaagtttcta ctttcgatcc tgcatggaga gctataatct 1740

cagctccccc gcccccccca cacacacctc tgcacacaca ccccccccca acactgggag 1800

taaaccagga tgatgtccgt cttctcattc cccatgtgac cgttggcagt gtagagagac 1860

tgattgtcac agctaaagga agagggacaa cagggtcact ggtgtctaca gagattatat 1920

tctacgtgtc tcactgaatt tacacaactc caagtgccaa ccacatcaag gtcaggaaat 1980

cctgaactgg aataagaaag acccagaaga tgaatgtgaa cagatccatt tgcttcccga 2040

cagtgggcac agacttcagt ctctggctac tgttccaaga cccagggctc tgcaattgtg 2100

tgacatcctt cagtgaaccc acatgggaaa ttctccatgg aattatcttc agcccactgt 2160

acttctgaat ccctcttcct tccttctgtg ccacacagca agtctggctt aaatgctgcc 2220

tgatctccat ttcaagtttt ctgcctctgg atttttagat ctcaagacca tggacgaaac 2280

atcagttaca gcaacaaaag tgaattttcc gtgcagagac ttctaggggt tctgtttgtt 2340

ttcagggtgc tagagatcac actcagatgc tcatatatgt taggtaaatg ttctcccact 2400

gagttacagc ccagctcaca cagagacttc taaaagaaaa tacggccatg ctctttgaaa 2460

tggagcattg agggatgaag tttggatggc gaagaaaact tctcaccagc tctctcccca 2520

cattcgtgcc aagcactgcc tccctagact tcgggtcacc atatctgtac tacgttttga 2580

tacagaaggc tcgagaccat tcaagagaat tatttagtac ac 2622

<210> 2

<211> 283

<212> PRT

<213> Mus musculus

<400> 2

Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser Ile Ile Asn Ile Ile

1 5 10 15

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

20 25 30

Gly Lys His Phe Ile Thr Val Thr Thr Phe Thr Ser Ala Gly Asn Ile

35 40 45

Gly Glu Asp Gly Thr Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu

50 55 60

Asn Gly Ile Val Ile Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val

65 70 75 80

His Glu Phe Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met

85 90 95

Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn

100 105 110

Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr

115 120 125

Thr Cys Tyr Ile Arg Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu

130 135 140

Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn Val Asp Tyr Asn

145 150 155 160

Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln

165 170 175

Pro Thr Val Ala Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser

180 185 190

Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met

195 200 205

Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser

210 215 220

Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val

225 230 235 240

Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Gln Leu Leu Asn Ser

245 250 255

Gly Pro Ser Pro Cys Val Phe Ser Ser Ala Phe Val Ala Gly Trp Ala

260 265 270

Leu Leu Ser Leu Ser Cys Cys Leu Met Leu Arg

275 280

<210> 3

<211> 2605

<212> DNA

<213> Homo sapiens

<400> 3

gtgagtcacc aaggaaggca gcggcagctc cactcagcca gtacccagat acgctgggaa 60

ccttccccag ccatggcttc cctggggcag atcctcttct ggagcataat tagcatcatc 120

attattctgg ctggagcaat tgcactcatc attggctttg gtatttcagg gagacactcc 180

atcacagtca ctactgtcgc ctcagctggg aacattgggg aggatggaat cctgagctgc 240

acttttgaac ctgacatcaa actttctgat atcgtgatac aatggctgaa ggaaggtgtt 300

ttaggcttgg tccatgagtt caaagaaggc aaagatgagc tgtcggagca ggatgaaatg 360

ttcagaggcc ggacagcagt gtttgctgat caagtgatag ttggcaatgc ctctttgcgg 420

ctgaaaaacg tgcaactcac agatgctggc acctacaaat gttatatcat cacttctaaa 480

ggcaagggga atgctaacct tgagtataaa actggagcct tcagcatgcc ggaagtgaat 540

gtggactata atgccagctc agagaccttg cggtgtgagg ctccccgatg gttcccccag 600

cccacagtgg tctgggcatc ccaagttgac cagggagcca acttctcgga agtctccaat 660

accagctttg agctgaactc tgagaatgtg accatgaagg ttgtgtctgt gctctacaat 720

gttacgatca acaacacata ctcctgtatg attgaaaatg acattgccaa agcaacaggg 780

gatatcaaag tgacagaatc ggagatcaaa aggcggagtc acctacagct gctaaactca 840

aaggcttctc tgtgtgtctc ttctttcttt gccatcagct gggcacttct gcctctcagc 900

ccttacctga tgctaaaata atgtgcctcg gccacaaaaa agcatgcaaa gtcattgtta 960

caacagggat ctacagaact atttcaccac cagatatgac ctagttttat atttctggga 1020

ggaaatgaat tcatatctag aagtctggag tgagcaaaca agagcaagaa acaaaaagaa 1080

gccaaaagca gaaggctcca atatgaacaa gataaatcta tcttcaaaga catattagaa 1140

gttgggaaaa taattcatgt gaactagaca agtgtgttaa gagtgataag taaaatgcac 1200

gtggagacaa gtgcatcccc agatctcagg gacctccccc tgcctgtcac ctggggagtg 1260

agaggacagg atagtgcatg ttctttgtct ctgaattttt agttatatgt gctgtaatgt 1320

tgctctgagg aagcccctgg aaagtctatc ccaacatatc cacatcttat attccacaaa 1380

ttaagctgta gtatgtaccc taagacgctg ctaattgact gccacttcgc aactcagggg 1440

cggctgcatt ttagtaatgg gtcaaatgat tcacttttta tgatgcttcc aaaggtgcct 1500

tggcttctct tcccaactga caaatgccaa agttgagaaa aatgatcata attttagcat 1560

aaacagagca gtcggcgaca ccgattttat aaataaactg agcaccttct ttttaaacaa 1620

acaaatgcgg gtttatttct cagatgatgt tcatccgtga atggtccagg gaaggacctt 1680

tcaccttgtc tatatggcat tatgtcatca caagctctga ggcttctcct ttccatcctg 1740

cgtggacagc taagacctca gttttcaata gcatctagag cagtgggact cagctggggt 1800

gatttcgccc cccatctccg ggggaatgtc tgaagacaat tttggttacc tcaatgaggg 1860

agtggaggag gatacagtgc tactaccaac tagtggatag aggccaggga tgctgctcaa 1920

cctcctacca tgtacaggac gtctccccat tacaactacc caatccgaag tgtcaactgt 1980

gtcagggcta agaaaccctg gttttgagta gaaaagggcc tggaaagagg ggagccaaca 2040

aatctgtctg cttcctcaca ttagtcattg gcaaataagc attctgtctc tttggctgct 2100

gcctcagcac agagagccag aactctatcg ggcaccagga taacatctct cagtgaacag 2160

agttgacaag gcctatggga aatgcctgat gggattatct tcagcttgtt gagcttctaa 2220

gtttctttcc cttcattcta ccctgcaagc caagttctgt aagagaaatg cctgagttct 2280

agctcaggtt ttcttactct gaatttagat ctccagaccc tgcctggcca caattcaaat 2340

taaggcaaca aacatatacc ttccatgaag cacacacaga cttttgaaag caaggacaat 2400

gactgcttga attgaggcct tgaggaatga agctttgaag gaaaagaata ctttgtttcc 2460

agcccccttc ccacactctt catgtgttaa ccactgcctt cctggacctt ggagccacgg 2520

tgactgtatt acatgttgtt atagaaaact gattttagag ttctgatcgt tcaagagaat 2580

gattaaatat acatttccta cacca 2605

<210> 4

<211> 282

<212> PRT

<213> Homo sapiens

<400> 4

Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile

1 5 10 15

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

20 25 30

Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile

35 40 45

Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu

50 55 60

Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val

65 70 75 80

His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met

85 90 95

Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn

100 105 110

Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr

115 120 125

Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu

130 135 140

Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn

145 150 155 160

Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln

165 170 175

Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser

180 185 190

Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met

195 200 205

Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser

210 215 220

Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val

225 230 235 240

Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser

245 250 255

Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu

260 265 270

Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys

275 280

<210> 5

<211> 4198

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgcattgtg tcaaaggaca gttcatttcc taacttttat ccagtgctcc cccatcacaa 60

tttctatgaa atgtttacct catgtttctt atacccaccc ccaaatatcc ctgcttctac 120

aggggagaag caattaaaca tttctcacct gtgtggctat gttgagacta ctacttgctt 180

acctatgatg tctcccatcc ctttcctcat gtgcccagag ctatggatgg taggaattgc 240

tccattgtca tagcagtgct agctgtcaac ttgactcaag cccagagtca ccaacaaaga 300

gggaaattca actgaagaat tgtccaggtc agaggccatg tctgttaggc atttccttaa 360

ttgctatttg atgtaagagc tcactatgag tggtaccatc caggggcagg tggactatat 420

aacaaagata gctaaggggc tggagatatg acgctgcagt tgagagcact ggctgctttt 480

gcagagaacc cagctttggt tgctagaccc cacacagtac atacatcgtc acaaccaccc 540

gtagctccag ctctagcaga tctcatattt tcttctggcc tcctagagca tcagatatgt 600

atatagcata cacaggcaaa cactcatata caataaaaat aaatgcatat atgttttagt 660

tataaaagga aaagaagtag ccgagcaaac agaacaagtg agccaggaag cagtggttct 720

caacttgtgg gtcgtagccc taactggatc aaatgaccct ttcaagagac tcacctaaga 780

ccataggaaa acacagataa ttatattatg attcataaca gtagcaaaat tacagttatg 840

aagaaacagt gaaaataatt ttatgaccag gggtcaccac cacatgagga actgtattaa 900

agagtcgtag cattaagaag tttgagagcg gggctggaga gatggctcag cggttaagag 960

cactgactgc tcttccagag gtcctgagtt caaatcccag caaccacatg gtggctcaca 1020

accatccata gtgaaatctg atgccctctt ctgggaggtc tgaagacagc tacagtgtac 1080

ttacatataa taaataaata aatctttaaa aaaataaaga agaagaagaa gtttgagagc 1140

cactcatcta tggtctctgc ttcagttcct gcctccaggt tcctgccttg agctcttgct 1200

ctggcctccc tcaacagtgg aaagtgacgt gaaagcaaag cccagtaacc cctccctcct 1260

ccaggccact tctggacact gttttcagaa accaagctag ggtagcctct ttagcaaact 1320

atgtattttc agcaaattcc cctgtcttcc aatttctgag gagtttgggg acaaccttgg 1380

aggccaatga gactgtttaa atctgtgtca gtcacagaat tctcgatcaa cagacagtgg 1440

cttctaacct atctaaaagg gactcttgtc ccttttctac ctgtcaccta ggagcctctg 1500

gtggtcagaa tgcctggaga attgcatcgc accaggctac gtaatgttcc ttgcaccagg 1560

atgtgtttgt acatttgagt acattaagtt ctgttcagtc acttaactcc ttatttattc 1620

atgtaacaag catttgttga ctctggctgg atgcaaagca agccttcctc ttcgtgatgg 1680

ctgaggttct cactggtttt attctcctca tatttcctat ggatggggat tcattcagtg 1740

aattcatacc acatcatgag tgtgacctca aatgacctga agctgcagca ggtccccaag 1800

gcatgccaga actgcctatt tttaattacc aagttctcta ggagatgtgt cagtggattc 1860

cctacctgtg ggctcaaggc tgatgataca ttaaaatgaa ttccgtaaca caattaccaa 1920

ccgaggtatg tcattatcat ttcatttcct ccgtaattgt gctaataatg tgggttcttt 1980

tctgaaaagt aattttccag attaacttcg atgacacttt ataaagtacg gtcaccttct 2040

ctctagctct gactgagtcc catttgttct gtggtttcat tgagaaaccg tatatatttt 2100

tattgtcctt ttttttaaag gctaatgttt ctcccaaatt taactgcagt gaccttggca 2160

cgtgctcttc accctggcaa tggatagatt atttaagcag tgctttgatt tgtgcttagc 2220

cccatttatt cataaactca taaaatatta tttctctgta gaacaggaca gtaaacgtga 2280

tcaggcctgt agttactact gaaacagttt cccccttgaa caaagcttgc agatgagtgc 2340

aaggtccaga cgtgagtcaa ggaggaggaa ggcggttgcc atggtaaagg gaaaagaaga 2400

gctatggggg caggtgtatc tgcctattct tccttagcag gctggaaagg gcctcccaga 2460

aggactggca tcccagctgg ttccaataag agtagatggt gggaaagcgg ggacatcgag 2520

ccagaactcc tgacaggaga aaggagcagg gtggtgacag tgcagagtgt gaccacacag 2580

cccatgcacg cgtgggtatt ttaataaaca aagataaatg gaaggaagcc tgaattttca 2640

aagcagagtt caagaaaaag cctggcagga gacgggcgcc gtgttcttat catagtcttc 2700

ttttgccatc tttcctagtt agttttccaa atctctgtca caaaaggcca cacatggcag 2760

ctgctcccca aatccctttt cttccacatt gccaaacctc caaagcgtct tccagcccag 2820

cctaggcacg cctgtttcct ccatctcctt tttccagcta tacttcctct tcgatcccta 2880

gccctctgtt ctttttttgt gcagatctgt ttagcttgat gtctttgggt ctattttcca 2940

tgtactacac tcatctcttt tacaaaggtc ttggagtcaa gtagactatc ccgagcctga 3000

atgagctaga agtgccaagg acacccttct gtctagaatg tttgcttctg tatttgaaca 3060

tgaacatgga ctaattgttt atgactcttt aagttgaaga aagaaaatca acaaactatt 3120

ggacacagac aatgaaagag gacagtggga gaggaaactg ccaggcagaa acaaaaggca 3180

ttcactgtgt gccaggcagt gcctggcact gaaggccagg cagggaaatc tgcgactcat 3240

tctagagccc atagtaacca tgggaaatta gtgcctaggt ggcagatgta tttggcttgt 3300

actcttaaga aagccacctg caagagcaat gcctttaaaa tgtgctggat ggaggaagat 3360

atgcacggta ggaaaaccag taaagaagct ggcactgtcc aagacccttc acagtgatgt 3420

ctgtgggcgt cagaaaggcc tgtatggacc atcttaaatg cttaaatcag gtgtctatgt 3480

gaagaatatg cggaggttgg gacagcatct cctgccactg tcctcagcat gactcaggat 3540

gagactctcc atagttctct tcctcaccat tccttgtaac tggtcggtaa tcctcttggg 3600

attgacagac attttcttag cttcacatct gtgtcatggg agccactaaa gatatttaat 3660

taattgagtt accttaaggt gccatcagtc agaacaactg gcacaaagaa cccttgactg 3720

ggtgggctgt aggctggaca accaaaggag aggccgtaga atggagatac tcccatagtc 3780

aatgtttctc ctgctgatag gatcaaacgg gtgaggagag tagtttctat cttggaccaa 3840

aacatcacta agtaccactc ttcattatga tgtagataac actataactt cagccacagc 3900

acttcccttc tccatgcctg agttccttca tcattccaag aaagacaaag tcccagcatg 3960

ttagaattga actttctttg gaattactag ccttattagt taacatgaag tatcgctttg 4020

tgtcacttct gattagctaa gtataatacc ttatctgtgt aactgccgtt cagcaagtca 4080

agtttatacc tacttgattt gttcctcaca ataacctctc aacgttgaca agatggtagt 4140

gttccctcca ttcccctgag agccagaata gtcacatgac ttgtttaaga ttaaatgg 4198

<210> 6

<211> 4079

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctgcagttgc tgaactctgg gccttccccg tgtgtttttt cttctgcctt tgtggctggc 60

tgggcactcc tatctctctc ctgttgcctg atgctaagat gaggggccct ggctacacaa 120

aagcatgcaa cgttgctggt ccaacaggtg aggtcaagca caaagagcgt gctggatggg 180

tcatagccca gcaagcgtac ctagccagtt ctcctgccct agactgttat gaaacagaat 240

cacacagcct tcagcaccag ccacagggag gaatatacac tgatctcagg tcagggactt 300

tgtcctagag gtgtgaaagg taaaatttta gtgtccagca ctaaccaagg gcagatacaa 360

aaaccaaccc cacacaatcc aatcatttat gtaacttcaa acacctattc atttgctgag 420

tcattagcgg attccaaatt gccttttcaa aaagtaagaa ctgtttggtc ctgactaatt 480

accctgggct tcactgttat tcacactgag agccttgtaa tccctgggta aacgaccttt 540

tccccacccc catcctagtt tgtccacctt gggccctggt gactcaccag ctagccctcc 600

tgctgagatc tgttctctgg ctccaatgac tgattggaca tttccaccta aatattcttc 660

tgcaagtaga atttgcacag aactgagctg tggagttctg gtcagctccc acataaggaa 720

ggcttcctaa ggctcacagg tccagtgttt tttctgaccg tcctctgtcc ttcatgtcag 780

ctgttgccat gaaccctaca gtgttctctc agactgatcc atttgtatca ccttatatcc 840

aagctaattc taatttaaaa ttttgtgttt tattattttt gtatgtacat gggtgttttg 900

tcatatgtat gtctatgccc caaatatgtg ctcagtgccc atataaacca gaagagtata 960

tcagattccc tggaacagga gctatagaca gttgtgagcc accatgtgag ttctgggaat 1020

cgagcctgga tcctttggaa gagcaaccag ggctcttaat cactaagtga tctctttaac 1080

ccaaagtcta gttctaaaac attgtgtttg cacctccccc catctctctg tctgtctctg 1140

tctctctgtc tctgtctctg tctctgtctc tgtctctgtc tctctctctc tctctctctc 1200

tctctcttat aggtgatagg aaactaggta tcaggaaaaa aaatacacta ttacactcta 1260

gtgtctcctt gctctggaac ccagaatgag tctagacaca agtgtctcag agctactgtc 1320

ttcagctgag caatcacagt cctctatctt cttccaggca tgtgcacgtg tacacacaca 1380

cacacacact cactcccagc cccattctgg gaaatgtagt ctacagaagt ttctgtccat 1440

gtgttccttt atcctttaca gggaaatgct cttcagccta agaccccttt cctgagggcc 1500

cctgaacccc acattcagct ggcctgggta taggcacacc ctaccctctc tgaggcttgt 1560

gtgtgccttg cccagatagg taagatctgg aggccggcaa gacctgtgca tttgtttctt 1620

atccctcctt ctctcgtttt ctgagctctt aagacagtgc agtgtaagaa gtggattcta 1680

tgtaagggag cctgccagct gccctcattt ctgaccccat ggctttctgc agagggagat 1740

gtcccccaag gagccactcc ttagggattt cacaggtagc cagaggagct cgaccttcct 1800

cccttgattc ctgcttacac tataaatgag ttctttaaat aagcagcaat taggcactgt 1860

ggcctgtaga atatagatta cttatccttt tttcttattc ccttggagca ataactttca 1920

gctttgtaaa aagtcatgtc aagggctgga gagatggctc catggttaaa agcaccaatg 1980

gctcttccaa aggtcctgag ttcaaatccc agcaaccaca tggtggctca caaccatctg 2040

taatgagatc tgacaccctc ttctgatgca tcagaagaca gctacagtgt acttagatat 2100

aataataaat aaatcttttt taaaagccat gtcatacatg taaaaagtga tggtcacaca 2160

tagcacacac agaattggac acatactcag ctgccacttt gttgtctgta gaaagtaact 2220

ttctctatag gagaataact ttaaaccaaa tagatttttc tttgaagagg tttttggttt 2280

ggtttggttc aggttttttg ttgttgtttt tgttcttcac taagatttct aaagtaaagg 2340

gacagatcat tatttcagat acacactatt gttggccaag acacactagg tcccatacca 2400

tccagctagg aaaagaggca gatggtgtct tccaacctca ttccagaacc ctgcctatgt 2460

ctattgctct gaggacccac ttctctcctg agactagaac tgcaaggtct tctcttaata 2520

taaaatttaa gccatcacct tcccagagta gacccagatc atggaccatg ggccacaggc 2580

cacagtagaa cagcagaata cagaaaaata agcccatttc aaaacaggag gtacactcac 2640

acaaaggcta gggaatgacg actctgggtt ctcagtaaac atcagccaaa tgcctatcat 2700

gatatcttat tgtgttcttt cagaatcccg gagaactaca gaaatatttt cctcaagaca 2760

tgacctagtt ttatatttct agaagaagat gaaatcatgt ctagaagtct ggagagagca 2820

gacaggaaca agatgtggaa ggaaaacaaa agtaacccac agacaccccc gatcggaaca 2880

agatggacct agaaaataat tcaaccaaac tagagtatac taagtgtgct gttacaatgt 2940

gtgtagggta ggtgtcctcc cacatctcag gggcctcccc tggtccacca gctcctgagt 3000

taggatgggc tgttatgatg tcactctgaa ggttcctgga tggttcctac tgccatatac 3060

tcattttata ttcagcacat taaaccatag tgaatgctat gaaaagctgc taatcagctg 3120

ccactccgag attcggaggt ggcaacgtct gagtgacagg tccagtgatt cgcttctcct 3180

taggatgctt ttacaagctc tttggcgtct cctcccacct ggcaaatgcc aaatgcatag 3240

gggagggtga tcatcattct agggcaaaca aaatagttga gggatgctga tttcccaaat 3300

catccgaatc acttctccct tgagcaaaca agcgccctgt tatttctcaa atgctgcttt 3360

gtgaatcagt ccagggcaag gcgctctcct catcccgcta tgtggcctta agtcatcgta 3420

aggtttgaag tttctacttt cgatcctgca tggagagcta taatctcagc tcccccgccc 3480

cccccacaca cacctctgca cacacacccc cccccaacac tgggagtaaa ccaggatgat 3540

gtccgtcttc tcattcccca tgtgaccgtt ggcagtgtag agagactgat tgtcacagct 3600

aaaggaagag ggacaacagg gtcactggtg tctacagaga ttatattcta cgtgtctcac 3660

tgaatttaca caactccaag tgccaaccac atcaaggtca ggaaatcctg aactggaata 3720

agaaagaccc agaagatgaa tgtgaacaga tccatttgct tcccgacagt gggcacagac 3780

ttcagtctct ggctactgtt ccaagaccca gggctctgca attgtgtgac atccttcagt 3840

gaacccacat gggaaattct ccatggaatt atcttcagcc cactgtactt ctgaatccct 3900

cttccttcct tctgtgccac acagcaagtc tggcttaaat gctgcctgat ctccatttca 3960

agttttctgc ctctggattt ttagatctca agaccatgga cgaaacatca gttacagcaa 4020

caaaagtgaa ttttccgtgc agagacttct aggggttctg tttgttttca gggtgctag 4079

<210> 7

<211> 9163

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agacactcca tcacagtcac tactgtcgcc tcagctggga acattgggga ggatggaatc 60

ctgagctgca cttttgaacc tgacatcaaa ctttctgata tcgtgataca atggctgaag 120

gaaggtgttt taggcttggt ccatgagttc aaagaaggca aagatgagct gtcggagcag 180

gatgaaatgt tcagaggccg gacagcagtg tttgctgatc aagtgatagt tggcaatgcc 240

tctttgcggc tgaaaaacgt gcaactcaca gatgctggca cctacaaatg ttatatcatc 300

acttctaaag gcaaggggaa tgctaacctt gagtataaaa ctggaggtga gttacttttg 360

gagagagatt ttttaaagca ccaaaagtat ttgaggctaa agattatgag ttgcttatga 420

aatatgttgt gaccaatatc agacaatgat gctcccaatt ctgctaacag ctgtttttgc 480

cattttatgg ccaagactca caccaacccc aaaagtagcc aactataaga gaggccttaa 540

aaatcttttg ttctgggttc tctctgcttc agaatttcca ggtgtgttct ctattcaaca 600

aaataaatgt agcctctttt gtggtagccc ctggtacagc taataagagc agcctagcct 660

gggacatttc cacgctaagt agacccaagc gcaatcctga aatcagtatc ttcagagact 720

catgacagtg agtgcatttt agaagttcaa agggaactaa ccaaaacgaa aacaacattg 780

tcctataaac tcattaattc cccttcccct gcaacaatga aatccaccct ttgttgccaa 840

cccaatttaa catatgtttt ctgaatttat atacaaggga ctcaggtact gtgagagtat 900

aaagatgatt aaacagattt cccctaccct tacagagctc ataatataga tctcacagag 960

ttaggggtca ggacagagat aataaaagtc tgtaaatgat aataattagt aaagtgtatc 1020

aaatgcttta agagaaatat aaatggagtg attgagaaga agagagagat cccgttcaaa 1080

tgcataggag ggaagggatg ggaaagcccc ctggagaagc tggcatttga ggggagcctt 1140

gaaggatagg cagaaaccgg gcctttccag cctaaactag tatgcacagg tctttcttgc 1200

acacctcata acatgcatat gcaatgattc gaagctcaga tctagaatca gacctgtgta 1260

agtacagtgg cctgggcaaa gcaatctctc tgagcctcag tttcttcatc ttaaaatggg 1320

gtcatgtcaa agggatactg gagccaactg aaagagcttc caatggcaaa gctggaacaa 1380

tttaagcaac aaaataagta acatagtatt gaattataac tcaaaggtat agaataaata 1440

catatgactt catacttatt taaataaatg agggatatga aacaaattcc ccttacagat 1500

gaattccaaa taatttattt agatctcttc cctccaaaaa gtggagcttg aaaccttccc 1560

tatcccactc agggtatatt ggacttagtg aagtccagct tccaaagaaa tccggcaaac 1620

actgctttag ccaggtgatc caggttaaca tcattaataa gtcatgttaa cagcatgtac 1680

cttctgatat aatgtgatgg gaagggcact ttacctcctg tgctcttcct ctctaaaacc 1740

tgtaacctca gtctaatcag gagaaaacat cagatgaatt ctaacagagg ggcaacctac 1800

agaataccta accagtactc ctcagaactg tcaaggtcat ggaaaatacc taaccggtac 1860

tcctcaaaat acctaaccag tactcctcaa aactgtcaac gtcatggaat atacagaaaa 1920

ttgggaaaac atgacagact agaagaaact caagcaacat gatgactaaa agcatggtgg 1980

tgttctggaa ggaatcttgg aacagaaaaa gaacatcagt ggaaaaactg ttgaaataca 2040

aatcaaatct ggaatttagt tgatagtaat ataccattgg cggtttctcc gttttttctt 2100

ttttttcttt tctttctttt tttttttttt ttttttgaga tggagtttca ctcttgttgc 2160

ccaggctgga gtacaacagc acgatctcgg ctcacagcaa cctgcgcctc ccaggttcaa 2220

gtgattctcc tgcctcagcc tcccaagtag ctgggattac aggcatgcac ctccacagcc 2280

ggctaatttt gtatttttag tagagactgg gtttcttcat gttggtcagg ctggtctcaa 2340

gctcccgacc tcaggtgatc tgcccacctc agcctcccaa agtgcttgga ttacaggcgt 2400

gtgccactgc gcctggcctc cagtttctca gttttgacta atgcacattg gttaagatgt 2460

tgacattaga ggaaactggg tgaggcctat gccagaactc tataccttct atgcaacttt 2520

tctgtacacg tgaaattatt tcaaaattaa aagttgattt ttttttaatg gggatgatag 2580

tattcacagg agtgttgtga ggatcaaatg aaataaggtc tggaatacag tactggcaca 2640

gaatataccc tcaatgaatg gtggttcatt gtcatgtaaa cttagtttcc cacatcatcg 2700

cccaagagcg tgttccctgc ctgctccgag catgagtctt gtctcccctg ctagagaaca 2760

gactccttaa gggttaggca agggctttcc ttttactact tactcagcac cagctcagtc 2820

ccaggtaaag gaagacgcca aataaattaa taaattatat tttaccccaa taaaaagtcc 2880

aggcttaaac attttcccac aactgtctct ctgcctggat atggggggaa gctaagtctt 2940

aatttcaaaa tgcagttaac tctcctgcga ggagaattac cttggaggaa aacaaatgtc 3000

tgtttctatc cctggatacc agagctggac aatctctacc caagcaaact ccaaatccct 3060

gtgatgacaa agcttgtgaa tggaatttct gtgggtgata gatgaatgct gtttgtttct 3120

tctttctcct tctccctacc caccctaact ttatcctcca gggagaaaaa tacataaaat 3180

ccagaagctt cacagatccc taggaaaatg cctaattccc taagggatag gcttttttat 3240

gaccactgaa gttcctctta cttggagaaa attttctatt gtaaccataa tatacctaga 3300

atttaggtcc ttggggtttg tgattggcca aagggttgtg atgtatagca atactatgag 3360

caaataatta ttcgcaccta taatacaatg ggctatacac aggtggacct ctgaggaagt 3420

actgagtggc ttcaatgata aaaaaaaaaa aaaaaaaatg actgcatttt taaggcctag 3480

cgcttcaaag gctatattgt cttacgtgtc tgactttggc atctgccctt tctgactttt 3540

gaccctgcag ccttcagcat gccggaagtg aatgtggact ataatgccag ctcagagacc 3600

ttgcggtgtg aggctccccg atggttcccc cagcccacag tggtctgggc atcccaagtt 3660

gaccagggag ccaacttctc ggaagtctcc aataccagct ttgagctgaa ctctgagaat 3720

gtgaccatga aggttgtgtc tgtgctctac aatgttacga tcaacaacac atactcctgt 3780

atgattgaaa atgacattgc caaagcaaca ggggatatca aagtgacagg tgggttcctg 3840

catgcttttg tatggattta ctggggaaag agtaaaatct aaattaaaat ttaacttcat 3900

taatagatat ataccaaggc acaaaaatct ctagagtcct cttcactaag aatttgatca 3960

ctcaattcca gttgagattg tattacataa gagaaagccc aacacaaaac aatatgtatg 4020

gcatataggt caaagatggc aattaggttt catcttgtat gctttctcat tgactggttg 4080

tctggagaac agtgtttagg attctgaggc tctgtgtaac tcagtaggac taccttgcta 4140

cagcagtgag gaagtgataa ttacttgaca tctttgatat gttcagctgg acttcaagcc 4200

ctccaaaact taggctagaa accaaggttt gcttccaccc tgcctttgag aaggctgtga 4260

atctgaagac taaaagtctg gagtctgact tagttttatt catccctcat gagactgact 4320

catgatccca ctaatattgg ctcaagatta atctgaaacc cctagatgga actggtctcc 4380

cagcacccta gctgagagct ggctgagaag acccagcacc ctagtctccc tgcagtgctt 4440

tagtgcatac aaaaccttag gttgagctag tatcaagaaa atgcatagac tgaattctga 4500

cttttaagac catccttcct ggggcgaaat tggattcaaa ctgtagtgaa aggagtacgt 4560

caggaggctg ccatttaaat caagcctagc actgggcaca aaatacctta cacattataa 4620

gtaccctata aatgtttgcc gaatctaact gaataaatca tattcttatg attttttaat 4680

ttgaaattat acatatgaac ccaaaattat ctacaactca gatatatcaa tttaaaaaat 4740

aaattacact cacacacaca aaagtaacct atggaatggg agaaaatatt tgtaaatcat 4800

tttaaggggt ttcaatccag aatatattaa gaactcctac aactcaacaa caacaaaaat 4860

caaataacct cattttaaaa tgggcaaagt acttgaagag acattcaacc caagatgata 4920

taaaaatagc taaaaagcaa atgaaaagat gctcaacatc actaatcatt ggagaaatgc 4980

aagtcaaaac cacaatgaga tatcacctaa tgcccattag ggtggctact ctaaaaacaa 5040

aataaggcaa aaacagaaaa taacatgttg gtgaggatgc ggaaaaattg gaaccctcat 5100

gcactgttgg tgggactgta aaatggtgca acttctatag aaaatagtat aaagtgtcat 5160

caaaacaatt aaaaaaaaga actactgaga tatggagcca gccggacttc gtgggtcgag 5220

tggggacttg gagaactttt ctgtctagct agaggattgc aaatgcacca atcagcactc 5280

tgtctagcta aaggattgta aatgcaccaa tcagcactct gtaaaaatga accaatcagc 5340

actctgtaaa atggaccaat cagcgctctg taaaatggac caataggcag gaagtgggcg 5400

gggccaaata agggaataaa agctggccac tggagccagc agcagcaacc cggtagggct 5460

gctgtctgtg atgtgcaggg tttggtgttt tgctgttcac aataaatctt gctgctgctc 5520

actctttggg tccacactag ctttaagagc tgtgactctc actgcgaagg tctgcagctt 5580

cattcctgaa gtcagtgaga ccacaaaccc actgggagga acaaacaact ccagatgcac 5640

catatctgaa catctgaagg aacaaactcc ggacacacca tctttaagaa ttgtaacact 5700

ctccaaaaaa aaaaaaaaag aactgtaaca ctcaggttgg gcatggtggc tcacacttac 5760

actatcatat gatctaacag tcctgcttct gggtatgcct tcaaaagaat tcagagtctg 5820

aatgagatat ttgaacaccc atggtcacag tagcactatt caccaaagtc aagagataga 5880

atcaacccaa aatgtccatt agtagatgag tagataaaca aaatatggta tacacataca 5940

atagaatacc attcaggctc aaaaaggaag gaaatcctat catatgctat gacatgaatg 6000

aactttcagg acattatcta tgtgaaataa gccagtcaca aaaagacaaa tactatatga 6060

tttaaacaaa gcatctaaag cagtcaaatg cacagaaaca gaaagtagaa tggtggttag 6120

cagagggcag ggtggaagag ttgaaggaga aaggtttttg tttaatgagt atagagtttc 6180

agatttgcaa aatgaaaaag ttctaatgat ttgttttata acaacgtgga tatacttaac 6240

actactgaac tgtacactta aaaatgctta agatggtaaa ttttgttgtt tttactgcaa 6300

tttttaaaaa gtatctaact aaaaaattac atatgagagc gagagctctt gcaccatctt 6360

gaggaacaaa ttggcaacta tcctggttag gattattagt tgcctgtaac agaaaaccaa 6420

tataaataaa tctagtctga aagggaagtg taggaagtgt actagaatgt tgtggaacca 6480

aagacaggaa gttcagttga cttcaggatc aactggaacc aggaacttaa atcccattga 6540

gattcttttt ctctcatttc tacttaattc tctctttact ctcttgtttt agtccatatg 6600

gcaagacgtg attgccaagc attcctatat ttaaaaatct cttactcaag agagtggtga 6660

gactgaacct caattcatca gtctcattct acattcccaa gaaggacttt gactccactt 6720

gggtcagctg cccataccta ggccaatgag gcagtatctg atgaaaggcc ttcccagaag 6780

agaaggaggg actgaagtat tggtcattta aaacaatggg tgctgacaac agtatctcag 6840

tccagttccc agtaaaaagc tccaagctgt gtaaataatg tcttctagta gcaagggcca 6900

caactgaaca agacatagac attgagcaca ccttttattc aattaacaga tctgtcttct 6960

ttacatgttt gttggataca tcaaaacaag tgttcgaagt gtcaggctag gatgaaatac 7020

aacttttggt agctcaaaat taagaaaata gcagaatttc ttattgttgt agacaaagta 7080

ggtgctccat gcatgttttt aaacaagtag ggccatggga tttacatatt gtgttctctg 7140

cctatactgc taatttaggt cattcattta acaaatatct attgtgtgcc aagtatgttc 7200

agagtacttt tttaagcccc ctgagatact gaaatgaata gcataagtat caaaagagaa 7260

gttaatcaca gtgtagattt agtacctatg ggaaagagtt gttaattatg actgaagtgg 7320

atccatagat aaggcagagt tccggctggg ttttgaagaa gaaaaaaaat ggtttggata 7380

aaatgggaag acctgaagtc aggagcttac agtaatggtc tgggggagaa attatgagag 7440

tattaactgg agcaggggca atgggaaaga aaagggggaa gaatcaaaag ttgcataaac 7500

aaaataatgt acagtccttg gtgatgcaaa tgtgaaagtc aagagaaatc acaagtcaaa 7560

gtgggtttga tatttctacc caggatactt aggaggttgg tgatgctacc agaaaaaaaa 7620

aaaaaaccca ccccaaaaca taggagcaat atgtgtgggg agtagataag ggttttagag 7680

atgtcgagtt tgaaatgccc atagtgtacc caggtagaga tgcccagtgc ttagcacaat 7740

agccagcatg tgataggttt tggggagaac actacaccct tactccatat atttcctttt 7800

atgagaaggg atccccacca gtgtcatctt tttagcctct ctcttcctcc ctctttcact 7860

cttcctgaga gaacaaggaa ttaatttatc tgattgatga gtatattagg agcagctgga 7920

cataagtagc tcagctcctc ctttttcttt ttgtttttgc taactctcag gaagcccgtg 7980

tacattctgc tgtgctcctg caggccgtca gttgttcagg ggtagaatct gctcctgcag 8040

gagacttgca ccagtctggt tctgtctgac agtggggagt ctcggctgta gcccacttgc 8100

ctaactacaa agcctcattc tctattaaca tattttcagc actaaaggtg atattttgat 8160

cttccagttg ccttattttt ttgtccttct ctttaattgg ttcagagcta agctggggaa 8220

gaggctgtta tttattggac ttctttgaac cccaatagta ggtactcaga aatatttact 8280

gaataattat gtttttaaat gacatatcta gcagcaactg gaaatctaga tgtggaactc 8340

aagacagagt tcaaactaag gaggtagact tggaaatgac tagcatgaaa gtgatattta 8400

ttaggcttga gtgatgttgg ctttattgtc gtttttacag ccagtactta tggagtgctt 8460

attataggcc agacactgtt ctaagtgctt tgcatgcact aactcattaa tcctcataag 8520

aaccctatga agtaggttat tagtattcta gccctattta tagatgaaga aactggagca 8580

catacattga gtaacttgcc taaagtcaca cagctggtaa gtgacagagc tgaaatgaaa 8640

taaaagagat aggattgtct agggaggaaa ttgagcaaaa gaagtatgat ttcatttctt 8700

tacatctacc attctctgct tcagaatgag aataatactt cctagacact ttatagtggt 8760

atgatgaaag ctaatgtaat gacgtggatc ctgtaaagat cactgcctgg agggaaaatg 8820

cgtctcaagc caagaactgg ccatggctgt ttgtttagcc tattctttat ttggtaaccc 8880

ataacaatct cttattggta tagtttttca ttcggctgag tgttcactgg agtggaccac 8940

agaatggaat tttctttgaa gaaagagaaa gctctacttt taagccagtt acggagaggt 9000

gggcaaaggg gtaaaaaatt gcctgaatta ggaacaactt gttctgtttt tcaggtactt 9060

ttctttgcta accagtcatg tgaagaagac atccagcttc tcctgtatga ccctaaactt 9120

tttctctcac ttcacagaat cggagatcaa aaggcggagt cac 9163

<210> 8

<211> 81

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

accagggcct cactggcatg ggtccttcct gaaccgcagg cagacactcc atcacagtca 60

ctactgtcgc ctcagctggg a 81

<210> 9

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctctcacttc acagaatcgg agatcaaaag gcggagtcac ctgcagttgc tgaactctgg 60

gccttccccg tgtgtttttt 80

<210> 10

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gagagccaga atagtcacat gacttgttta agattaaatg ggatccacca ttatgtacct 60

gactgatgaa gttcctatac 80

<210> 11

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aagttcctat actttctaga gaataggaac ttcggaattc gctgttcagg aacaaagccg 60

ggatgtgaat attcacactc 80

<210> 12

<211> 2622

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtgagtcaca acacccagga gggcagcagc aggcaggcag ctccactcac caaaatctgg 60

ccccacacac agcaggactg tgggaaggaa ctccctctcc atggcttcct tggggcagat 120

catcttttgg agtattatta acatcatcat catcctggct ggggccatcg cactcatcat 180

tggctttggc atttcaggca gacactccat cacagtcact actgtcgcct cagctgggaa 240

cattggggag gatggaatcc tgagctgcac ttttgaacct gacatcaaac tttctgatat 300

cgtgatacaa tggctgaagg aaggtgtttt aggcttggtc catgagttca aagaaggcaa 360

agatgagctg tcggagcagg atgaaatgtt cagaggccgg acagcagtgt ttgctgatca 420

agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg caactcacag atgctggcac 480

ctacaaatgt tatatcatca cttctaaagg caaggggaat gctaaccttg agtataaaac 540

tggagccttc agcatgccgg aagtgaatgt ggactataat gccagctcag agaccttgcg 600

gtgtgaggct ccccgatggt tcccccagcc cacagtggtc tgggcatccc aagttgacca 660

gggagccaac ttctcggaag tctccaatac cagctttgag ctgaactctg agaatgtgac 720

catgaaggtt gtgtctgtgc tctacaatgt tacgatcaac aacacatact cctgtatgat 780

tgaaaatgac attgccaaag caacagggga tatcaaagtg acagaatcgg agatcaaaag 840

gcggagtcac ctgcagttgc tgaactctgg gccttccccg tgtgtttttt cttctgcctt 900

tgtggctggc tgggcactcc tatctctctc ctgttgcctg atgctaagat gaggggccct 960

ggctacacaa aagcatgcaa cgttgctggt ccaacagaat cccggagaac tacagaaata 1020

ttttcctcaa gacatgacct agttttatat ttctagaaga agatgaaatc atgtctagaa 1080

gtctggagag agcagacagg aacaagatgt ggaaggaaaa caaaagtaac ccacagacac 1140

ccccgatcgg aacaagatgg acctagaaaa taattcaacc aaactagagt atactaagtg 1200

tgctgttaca atgtgtgtag ggtaggtgtc ctcccacatc tcaggggcct cccctggtcc 1260

accagctcct gagttaggat gggctgttat gatgtcactc tgaaggttcc tggatggttc 1320

ctactgccat atactcattt tatattcagc acattaaacc atagtgaatg ctatgaaaag 1380

ctgctaatca gctgccactc cgagattcgg aggtggcaac gtctgagtga caggtccagt 1440

gattcgcttc tccttaggat gcttttacaa gctctttggc gtctcctccc acctggcaaa 1500

tgccaaatgc ataggggagg gtgatcatca ttctagggca aacaaaatag ttgagggatg 1560

ctgatttccc aaatcatccg aatcacttct cccttgagca aacaagcgcc ctgttatttc 1620

tcaaatgctg ctttgtgaat cagtccaggg caaggcgctc tcctcatccc gctatgtggc 1680

cttaagtcat cgtaaggttt gaagtttcta ctttcgatcc tgcatggaga gctataatct 1740

cagctccccc gcccccccca cacacacctc tgcacacaca ccccccccca acactgggag 1800

taaaccagga tgatgtccgt cttctcattc cccatgtgac cgttggcagt gtagagagac 1860

tgattgtcac agctaaagga agagggacaa cagggtcact ggtgtctaca gagattatat 1920

tctacgtgtc tcactgaatt tacacaactc caagtgccaa ccacatcaag gtcaggaaat 1980

cctgaactgg aataagaaag acccagaaga tgaatgtgaa cagatccatt tgcttcccga 2040

cagtgggcac agacttcagt ctctggctac tgttccaaga cccagggctc tgcaattgtg 2100

tgacatcctt cagtgaaccc acatgggaaa ttctccatgg aattatcttc agcccactgt 2160

acttctgaat ccctcttcct tccttctgtg ccacacagca agtctggctt aaatgctgcc 2220

tgatctccat ttcaagtttt ctgcctctgg atttttagat ctcaagacca tggacgaaac 2280

atcagttaca gcaacaaaag tgaattttcc gtgcagagac ttctaggggt tctgtttgtt 2340

ttcagggtgc tagagatcac actcagatgc tcatatatgt taggtaaatg ttctcccact 2400

gagttacagc ccagctcaca cagagacttc taaaagaaaa tacggccatg ctctttgaaa 2460

tggagcattg agggatgaag tttggatggc gaagaaaact tctcaccagc tctctcccca 2520

cattcgtgcc aagcactgcc tccctagact tcgggtcacc atatctgtac tacgttttga 2580

tacagaaggc tcgagaccat tcaagagaat tatttagtac ac 2622

<210> 13

<211> 283

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser Ile Ile Asn Ile Ile

1 5 10 15

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

20 25 30

Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile

35 40 45

Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu

50 55 60

Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val

65 70 75 80

His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met

85 90 95

Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn

100 105 110

Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr

115 120 125

Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu

130 135 140

Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn

145 150 155 160

Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln

165 170 175

Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser

180 185 190

Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met

195 200 205

Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser

210 215 220

Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val

225 230 235 240

Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser

245 250 255

Gly Pro Ser Pro Cys Val Phe Ser Ser Ala Phe Val Ala Gly Trp Ala

260 265 270

Leu Leu Ser Leu Ser Cys Cys Leu Met Leu Arg

275 280

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctacacgccc attttcctct ttggc 25

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctcgactag agcttgcgga 20

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

accagtcatg tgaagaagac atccag 26

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gaaggcctat tttctgtggg aggca 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttcttacctc ccaaggcctc gatct 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agccaaagag gaaaatgggc gtgta 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgagggatga agtttggatg gcgaa 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttccctgata ggaacaggca aggct 25

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggatcggcca ttgaacaaga t 21

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cagaagaact cgtcaagaag gc 22

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

acaaatctgc ccttgttggg aacca 25

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttgtgcttga cctcacctgt tggac 25

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tcactggagt ggaccacaga atgga 25

<210> 27

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

agtgttccct ccattcccct gagag 25

<210> 28

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tgactggaaa gactagaaag agcagga 27

<210> 29

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gacaagcgtt agtaggcaca tatac 25

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gctccaattt cccacaacat tagt 24

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gcaagcactt catcacggtc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcagctgact tcgccttttg 20

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

caaagatgag ctgtcggagc agg 23

<210> 34

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gcacagacac aaccttcatg gtcac 25

<210> 35

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tcaccatctt ccaggagcga ga 22

<210> 36

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gaaggccatg ccagtgagct t 21

Claims

1. A humanized B7H4 protein, wherein the humanized B7H4 protein comprises all or part of a human B7H4 protein.

2. The humanized B7H4 protein of claim 1, wherein the humanized B7H4 protein comprises at least 50 contiguous amino acids of the extracellular region of human B7H4 protein; preferably, the humanized B7H4 protein comprises a sequence identical to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259, or an amino acid sequence having at least 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 4, 1-259, 1-256, 1-250, 15-256, 15-259, 25-250, 34-250, or 34-259;

and/or, the humanized B7H4 protein comprises an amino acid sequence encoded by part of exon 3, all of exon 4and part of exon 5 of the human B7H4 gene; preferably, the polypeptide comprising SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

3. The humanized B7H4 protein of claim 1 or 2, wherein the amino acid sequence of the humanized B7H4 protein comprises one of the following group:

I) SEQ ID NO: 13 amino acid sequence, in whole or in part;

III) and SEQ ID NO: 13 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or the like, or, alternatively,

4. A humanized B7H4 gene, wherein the humanized B7H4 gene comprises a portion of the human B7H4 gene; preferably, the humanized B7H4 gene encodes the humanized B7H4 protein of any one of claims 1-3.

5. The humanized B7H4 gene according to claim 4, wherein the humanized B7H4 gene comprises part of exon 3, all of exon 4and part of exon 5 of human B7H4 gene, preferably further comprises intron 3-4 and/or intron 4-5; preferably, the humanized B7H4 gene comprises SEQ ID NO: 7.

6. The humanized B7H4 gene of claim 4 or 5, wherein the nucleotide sequence of the humanized B7H4 gene comprises one of the following group:

(iii) the transcribed mRNA is identical to SEQ ID NO: 12 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

(iv) the transcribed mRNA has the sequence of SEQ ID NO: 12, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted;

preferably, the polypeptide comprising SEQ ID NO: 8 and/or 9.

7. A targeting vector, wherein said targeting vector comprises any one of:

A) a portion of the human B7H4 gene, preferably a portion of exon 3, all of exon 4, and a portion of exon 5 of the human B7H4 gene, preferably further comprising intron 3-4 and/or intron 4-5 nucleotide sequences; further preferred comprises SEQ ID NO: 7;

C) a nucleotide sequence encoding the humanized B7H4 protein of any one of claims 1-3; or the like, or, alternatively,

D) the humanized B7H4 gene of any one of claims 4-6;

preferably, the targeting vector further comprises a5 'arm and/or a 3' arm; the 5' arm has at least 90% homology of nucleotide with NCBI accession number NC-000069.6; preferably, the 5' arm is identical to SEQ ID NO: 5 or as shown in SEQ ID NO: 5 is shown in the specification; the 3' arm has at least 90% homology of nucleotide with NCBI accession number NC-000069.6; preferably, the 3' arm sequence is identical to SEQ ID NO: 6 or as shown in SEQ ID NO: and 6.

8. A construction method of a B7H4 gene humanized non-human animal, which is characterized in that the human or humanized B7H4 protein is expressed in the non-human animal; and/or, the genome of said non-human animal comprises a portion of the human B7H4 gene, preferably, said non-human animal expresses the humanized B7H4 protein of any one of claims 1-3 in vivo.

9. The method of claim 8, wherein the genome of said non-human animal comprises part of exon 3, all of exon 4, and part of exon 5 of human B7H4 gene, preferably further comprises nucleotide sequences of intron 3-4 and/or intron 4-5; further preferred comprises SEQ ID NO: 7; more preferably, the genome of the non-human animal comprises the humanized B7H4 gene of any one of claims 4-6.

10. The method of construction according to claim 8 or 9, comprising introducing into the non-human animal B7H4 locus a nucleotide sequence comprising any one of:

A) a portion of the human B7H4 gene, preferably a portion of exon 3, all of exon 4, and a portion of exon 5 of the human B7H4 gene, preferably further comprising intron 3-4 and/or intron 4-5 nucleotide sequences; further preferred comprises SEQ ID NO: 7; or the like, or, alternatively,

preferably, said introduction is an insertion or a substitution, and further preferably, said introduction is at the B7H4 locus of a non-human animal to replace the corresponding region of the non-human animal; it is further preferred that all or part of exons 3 to 5 of the non-human animal B7H4 gene be replaced;

11. The method of any one of claims 8 to 10, wherein the targeting vector of claim 7 is used to construct a non-human animal.

12. The method according to any one of claims 8 to 11, further comprising the steps of mating the B7H4 gene-humanized non-human animal with another genetically modified non-human animal, in vitro fertilization, or directly performing gene editing, and screening the animal to obtain a multi-genetically modified non-human animal,

preferably, the other genes comprise one or more than two of CD3, PD-1, PD-L1, CD40, OX40, TIGIT, CD27, CD28, CD47, GITR or SIRPA.

13. The method of any one of claims 8-12, wherein the non-human animal is a mouse or rat.

14. A cell, tissue or organ which expresses the humanized B7H4 protein of any one of claims 1-3, or which comprises the humanized B7H4 gene of any one of claims 4-6 in its genome, or which is derived from a non-human animal obtained by the construction method of any one of claims 8-13.

15. Use of the humanized B7H4 protein of any one of claims 1 to 3, the humanized B7H4 gene of any one of claims 4 to 6, the non-human animal obtained by the construction method of any one of claims 8 to 13, or the cell, tissue or organ of claim 14, wherein said use comprises:

A) use in the development of products involving the immunological process of human cells;

D) screening, validating, evaluating or studying B7H4 pathway function; or the like, or, alternatively,

E) screening and evaluating the application of human medicine and drug effect research.