CN114316025A

CN114316025A - VEGFR2 gene humanized non-human animal and construction method and application thereof

Info

Publication number: CN114316025A
Application number: CN202111596716.9A
Authority: CN
Inventors: 赵磊; 尚诚彰
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2020-12-25
Filing date: 2021-12-24
Publication date: 2022-04-12

Abstract

The invention provides a VEGFR2 gene humanized non-human animal and a construction method thereof, wherein a part of nucleotide sequence for coding human VEGFR2 protein is introduced into the genome of the non-human animal in a homologous recombination mode, and the humanized VEGFR2 protein can be normally expressed in the animal body, can be used as an animal model for researching the signal mechanism of human VEGFR2 and screening drugs for tumors and immune related diseases, and has important application value for the research and development of new drugs for immune targets. The invention also provides a humanized VEGFR2 protein, a humanized VEGFR2 gene, a targeting vector, a non-human animal obtained by the construction method and application of the non-human animal in the field of biomedicine.

Description

VEGFR2 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 VEGFR2 gene humanized non-human animal, a construction method thereof and application thereof in the field of biomedicine.

Background

Angiogenesis refers to the process of formation of new blood vessels by the pre-existing capillaries in a budding manner, and various important physiological and pathological processes of a human body all relate to the formation of new blood vessels, including embryonic development, wound healing, diabetic retinopathy, tumor infiltration and metastasis, and the like. This process is triggered and regulated by a variety of factors, including basic fibroblast growth factor (bFGF), Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor (PDGF), interleukin 6(IL6), and the like. Among them, VEGF is one of the most important factors for regulating the growth and proliferation of vascular endothelial cells, and it induces capillary proliferation, promotes endothelial cell proliferation and migration, enhances vascular permeability, and inhibits apoptosis by acting specifically on glycosylated cells of endothelial cells, including mitogen. The action of the compound depends on the combination with vascular endothelial cell specific receptors (VEGFRs), and further activates in vivo signal transduction to exert biological effects.

VEGF family members include VEGFA, VEGFB, VEGFC, VEGFD and VEGFE, of which VEGFA is the most important member and one of the most potent vasopermeability-promoting factors known to date. There are 3 members of the VEGF receptor family: VEGFR1, VEGFR2 and VEGFR3, of which VEGFR2 is one of the most important regulatory factors, is mainly distributed in vascular endothelial cells and lymphatic endothelial cells, and plays a leading role in VEGF signal transduction and vascular endotheliogenesis. VEGFR2 forms a dimer by binding with VEGFA, causes autophosphorylation, activates and transmits a cell membrane/cytoplasmic kinase cascade reaction signal to a nucleus, initiates a series of reactions such as endothelial cell proliferation, invasion and migration, regulates endothelial cells through mechanisms such as anti-apoptosis, promotes angiogenesis and maintains the integrity of the angiogenesis. The VEGFA/VEGFR2 signal pathway is the most important rate-limiting step of physiological and pathological angiogenesis, is also an important pathway of tumor angiogenesis, not only directly leads to the generation of tumor vessels, but also indirectly promotes the formation of lymphatic metastasis. High expression of VEGFR2 has been detected in various tumor tissues, such as non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, gastric cancer, brain glioma, etc., and its expression is associated with tumor staging, recurrence and prognosis. Therefore, VEGFR2 has been considered as an important target of antitumor drug action, and can be used as a tumor prognosis index to assist clinical medication, providing important basis for determination of treatment scheme, efficacy evaluation and prognosis.

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, and the complexity of genes, it remains the greatest challenge to construct an "effective" humanized animal model that approximates the physiological characteristics of humans for the development of new drugs.

In view of the fact that the VEGFA/VEGFR2 signal pathway has great application value in the field of tumor immunotherapy, in order to further research relevant biological characteristics, improve the effectiveness of preclinical pharmacodynamic tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure, there is an urgent need in the art to develop a non-human animal model involving the VEGFA/VEGFR2 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.

Specifically, in the first aspect of the present invention, a humanized VEGFR2 protein is provided, wherein the humanized VEGFR2 protein comprises all or part of a human VEGFR2 protein.

Preferably, the humanized VEGFR2 protein comprises all or part of the signal peptide, transmembrane region, cytoplasmic region, and/or extracellular region of the human VEGFR2 protein. Further preferably, the humanized VEGFR2 protein comprises all or part of the extracellular domain of human VEGFR2 protein.

Preferably, the humanized VEGFR2 protein further comprises a portion of a non-human animal VEGFR2 protein, more preferably all or a portion of the signal peptide, transmembrane region, and/or cytoplasmic region of the non-human animal VEGFR2 protein. Further preferably, the VEGFR2 protein may also comprise a portion of the extracellular domain of a non-human animal VEGFR2 protein.

In one embodiment of the present invention, the humanized VEGFR2 protein comprises a signal peptide, a transmembrane region, a cytoplasmic region, and an extracellular region. Wherein the extracellular domain comprises all or part of the extracellular domain of human VEGFR2 protein. Preferably, the portion of the extracellular domain of human VEGFR2 protein comprises an extracellular domain of human VEGFR2 protein with 0-20, preferably 5-15, specifically 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 preferred, the extracellular domain of human VEGFR2 protein comprises 6 amino acid residues removed from the N-terminus and 13 amino acid residues removed from the C-terminus. The signal peptide, transmembrane region and cytoplasmic region are derived from a non-human animal.

Preferably, the humanized VEGFR2 protein comprises at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 726, 730, 732, 738, 739, 740, 741 or 745 consecutive amino acids of the extracellular domain of the human VEGFR2 protein.

In one embodiment of the invention, the humanized VEGFR2 protein comprises a sequence identical to SEQ ID NO: 4, amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 or comprising the amino acid sequence of SEQ ID NO: 4, positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751. In a specific embodiment of the present invention, the humanized VEGFR2 protein comprises a non-human animal VEGFR2 protein signal peptide, a non-human animal VEGFR2 protein transmembrane region, a non-human animal VEGFR2 protein cytoplasmic region, and a portion of the extracellular domain of the human VEGFR2 protein (preferably, the extracellular domain of the human VEGFR2 protein with 0 to 20 amino acid residues removed from the N-terminus and/or C-terminus) and a portion of the extracellular domain of the non-human animal (0 to 20 amino acid residues from the N-terminus and/or C-terminus).

Preferably, the humanized VEGFR2 protein comprises all or part of an amino acid sequence encoded by exon 1 through exon 30 of the human VEGFR2 gene. Further preferably, the VEGFR gene comprises all or part of an amino acid sequence encoded by any one, two, three or more, two or more consecutive exons or a combination of three or more consecutive exons of human VEGFR2 gene from exon 1 to exon 30. Even more preferably, all or part of the amino acid sequence encoded by the exons 2 to 15 of the human VEGFR2 gene. Still further preferably, the VEGFR gene comprises an amino acid sequence encoded by part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, wherein part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence of exon 2 with 0-3 amino acids N-terminal removed, or part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous exons 2; the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of exon 15 with the C-terminal 0-4 amino acids removed, or the portion of exon 15 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 50, 80, 100, 110, 119, 120, 130, or 132bp contiguous to exon 15. Most preferably, the polypeptide comprises a sequence identical to SEQ ID NO: 7 or a nucleotide sequence having at least 80%, 85%, 90%, 95% or at least 99% identity to the sequence of SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

Preferably, the humanized VEGFR2 protein further comprises all or part of an amino acid sequence encoded by a non-human animal VEGFR2 gene, preferably exons 1, 16-30, preferably further comprises part of exon 2 and/or part of exon 15 of a non-human animal VEGFR2 gene.

In one embodiment of the present invention, the amino acid sequence of the humanized VEGFR2 protein comprises one of the following groups:

A) SEQ ID NO: 4, 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751;

B) and SEQ ID NO: 4 at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% amino acid sequence identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751;

C) and SEQ ID NO: 4, positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751, differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, D) is substantially identical to SEQ ID NO: 4, 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751, including substitutions, deletions and/or insertions of one or more amino acid residues.

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

II) and SEQ ID NO: 13 is at least 80%, 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 present invention, a humanized VEGFR2 gene is provided, wherein the humanized VEGFR2 gene comprises a portion of the human VEGFR2 gene.

Preferably, the humanized VEGFR2 gene comprises all or part of a nucleotide sequence encoding a signal peptide, extracellular, transmembrane and/or cytoplasmic region of human VEGFR2 protein. Further preferably, the human VEGFR2 protein comprises all or part of the nucleotide sequence of the extracellular domain. Still further preferred is a nucleotide sequence comprising an extracellular domain of human VEGFR2 protein encoding N-terminal and/or C-terminal deletion of 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 human VEGFR2 protein comprises a nucleotide sequence encoding an extracellular domain of human VEGFR2 protein with 6 amino acid residues removed from the N-terminus and 13 amino acid residues removed from the C-terminus. Still further preferred is a nucleotide sequence comprising at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 726, 730, 732, 738, 739, 740, 741 or 745 consecutive amino acids of the extracellular domain encoding human VEGFR2 protein.

Preferably, the human VEGFR2 further comprises a nucleotide sequence encoding a signal peptide of human VEGFR 2. Most preferably, the polypeptide comprises a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 4, amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 or to the amino acid sequence of SEQ ID NO: 4, 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751, and a nucleotide sequence identical to the amino acid sequence shown in position 1-764, 14-764, 20-764, 26-751.

Preferably, the humanized VEGFR2 gene further comprises all or a portion of a protein encoding non-human animal VEGFR 2. For example, a partial nucleotide sequence encoding a cytoplasmic, signal peptide, a transmembrane, and/or an extracellular region of a non-human animal VEGFR2 protein.

Preferably, the humanized VEGFR2 gene encodes the humanized VEGFR2 protein described above.

Preferably, the humanized VEGFR2 gene comprises all or part of exons 1 to 30 of human VEGFR2 gene. Further preferably, all or part of a combination of any one, two, three or more, two consecutive or three or more exons from exon 1 to exon 30 of human VEGFR2 gene is contained. Even more preferably, all or part of exons 2 to 15 of human VEGFR2 gene are included. Still further preferably, the VEGFR gene comprises part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprises intron 2-3 and/or intron 14-15, wherein part of exon 2 comprises a nucleotide sequence of exon 2 excluding the nucleotide sequence encoding the N-terminal 0-3 amino acids, or the part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous of exon 2; part of exon 15 comprises the nucleotide sequence of exon 15 with the C-terminal 0-4 amino acids removed, or part of exon 15 of the human VEGFR2 gene comprises a nucleotide sequence of at least 20, 50, 80, 100, 110, 119, 120, 130 or 132bp contiguous to exon 15.

Preferably, the humanized VEGFR2 gene further comprises a portion of a non-human animal VEGFR2 gene; preferably exons 1, 16-30, preferably also part of exon 2 and/or part of exon 15 of the non-human animal VEGFR2 gene.

Preferably, the humanized VEGFR2 gene comprises a nucleotide sequence identical to SEQ ID NO: 8 and/or 9, or a nucleotide sequence comprising at least 80%, 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 8 and/or 9.

Preferably, the humanized VEGFR2 gene comprises a nucleotide sequence identical to SEQ ID NO: 10 and/or 11, or a nucleotide sequence comprising at least 80%, 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 VEGFR2 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 80%, 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 VEGFR2 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 80%, 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.

Preferably, the humanized VEGFR2 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a conventionally induced or repressed substance.

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

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

A) a portion of the human VEGFR2 gene, preferably comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprising intron 2-3 and/or intron 14-15, wherein the portion of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 2 encoding the N-terminal 0-3 amino acids, or the portion of exon 2 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous exons 2; the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of exon 15 with the C-terminal 0-4 amino acids removed, or the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of at least 20, 50, 80, 100, 110, 119, 120, 130, or 132bp contiguous to exon 15; further preferably, said humanized VEGFR2 gene comprises SEQ ID NO: 7;

B) a nucleotide sequence encoding all or a portion of a human VEGFR2 protein, preferably comprising all or a portion of an extracellular region encoding a human VEGFR2 protein, further preferably comprising a nucleotide sequence encoding at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 726, 730, 732, 738, 739, 740, 741, or 745 consecutive amino acids of an extracellular region of a human VEGFR2 protein, more preferably comprising a nucleotide sequence encoding SEQ ID NO: 4 at position 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, or 26-751;

C) a nucleotide sequence encoding the humanized VEGFR2 protein described above; or the like, or, alternatively,

D) the humanized VEGFR2 gene described above.

Preferably, the targeting vector further comprises a 5 '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 genome DNA of the VEGFR2 gene of the non-human animal. Preferably, the 5' arm has at least 90% homology with NCBI accession number NC _ 000071.6. 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 comprises a DNA fragment homologous to the 3' end of the transition region to be altered selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the VEGFR2 gene of a non-human animal. Preferably, the 3' arm has at least 90% homology with NCBI accession number NC _ 000071.6. 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 of the targeting vector is located at the non-human animal VEGFR2 locus. Further preferably, it is located on exon 1 to 30 of the non-human animal VEGFR2 gene. Even more preferably, it is located on exon 2 to exon 15 of the VEGFR2 gene of a non-human animal.

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

In a fifth aspect of the present invention, there is provided the use of the targeting vector or the cell comprising the targeting vector described above for the modification of the VEGFR2 gene. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In a sixth aspect of the present invention, there is provided a non-human animal humanized with VEGFR2 gene, said non-human animal expressing human or humanized VEGFR2 protein; alternatively, the genome of the non-human animal comprises a portion of the human VEGFR2 gene.

Preferably, the non-human animal has reduced or absent expression of endogenous VEGFR2 protein.

Preferably, the genome of the non-human animal comprises a humanized VEGFR2 gene.

Preferably, a portion of the human VEGFR2 gene or the nucleotide sequence of the humanized VEGFR2 gene is operably linked to a non-human animal endogenous regulatory element.

In a seventh aspect of the present invention, a method for constructing a non-human animal humanized with VEGFR2 gene is provided, wherein the non-human animal expresses the human or humanized VEGFR2 protein in vivo; alternatively, the genome of the non-human animal comprises a portion of the human VEGFR2 gene.

Preferably, the genome of the non-human animal comprises a nucleotide sequence encoding a human VEGFR2 protein. Further preferably comprises all or part of the nucleotide sequence encoding the extracellular, signal, cytoplasmic, and/or transmembrane regions of the human VEGFR2 protein. Even more preferably, the recombinant human VEGFR2 comprises all or part of the nucleotide sequence of an extracellular domain encoding a human VEGFR2 protein. Still further preferred is a nucleotide sequence comprising an extracellular domain of human VEGFR2 protein encoding an N-terminal and/or C-terminal removed 0-20, preferably 5-15, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues. In one embodiment of the present invention, the present invention comprises a nucleotide sequence encoding an extracellular domain of human VEGFR2 protein with 6 amino acid residues removed from the N-terminus and 13 amino acid residues removed from the C-terminus. Still further preferred is a nucleotide sequence comprising at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 710, 720, 726, 730, 732, 738, 739, 740, 741 or 745 consecutive amino acids of the extracellular domain encoding human VEGFR2 protein.

In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence that encodes a nucleotide sequence identical to SEQ ID NO: 4, amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 or to the amino acid sequence of SEQ ID NO: 4, 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751, and a nucleotide sequence identical to the amino acid sequence shown in position 1-764, 14-764, 20-764, 26-751.

In one embodiment of the invention, the genome of the non-human animal 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 all or part of exons 1 to 30 of human VEGFR2 gene. Further preferably, all or part of a combination of any one, two, three or more, two consecutive or three or more exons from exon 1 to exon 30 of human VEGFR2 gene is contained. Even more preferably, all or part of exons 2 to 15 of human VEGFR2 gene are included. Still further preferably, the VEGFR gene comprises part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprises intron 2-3 and/or intron 14-15, wherein the part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 2 encoding the N-terminal 0-3 amino acids, or the part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that is at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous to exon 2; the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of exon 15 with the C-terminal 0-4 amino acids removed, or the portion of exon 15 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 50, 80, 100, 110, 119, 120, 130, or 132bp contiguous to exon 15. In one embodiment of the invention, the genome of the non-human animal comprises SEQ ID NO: 7.

In a specific embodiment of the present invention, the genome of the non-human animal comprises the human or humanized VEGFR2 gene described above.

Preferably, the method of construction comprises introducing into the non-human animal VEGFR2 locus a nucleotide sequence comprising any one of:

A) a portion of the human VEGFR2 gene, preferably comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprising intron 2-3 and/or intron 14-15, wherein the portion of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 2 encoding the N-terminal 0-3 amino acids, or the portion of exon 2 of human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous exons 2; the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of exon 15 with the C-terminal 0-4 amino acids removed, or the portion of exon 15 of human VEGFR2 gene comprises the nucleotide sequence of at least 20, 50, 80, 100, 110, 119, 120, 130, or 132bp contiguous to exon 15; further preferred, comprises SEQ ID NO: 7;

B) a nucleotide sequence encoding all or a portion of a human VEGFR2 protein, preferably comprising all or a portion of an extracellular region encoding a human VEGFR2 protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 4 at position 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, or 26-751;

D) the humanized VEGFR2 gene described above.

The introduction is insertion or replacement. Preferably, the site of insertion or substitution is after the endogenous regulatory elements of the VEGFR2 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 VEGFR2 gene, a humanized VEGFR2 gene, a nucleotide sequence encoding a human or humanized VEGFR2 protein, a nucleotide sequence obtained by splicing a human VEGFR2 gene with a non-human VEGFR2 gene. Of course, a partial nucleotide sequence of the human VEGFR2 gene may be used, preferably, the exon x +1 to the exon 30 of the human VEGFR2 gene is inserted immediately adjacent to the exon x of the non-human animal VEGFR2 gene; for example, exon 1 of the VEGFR2 gene of non-human animal is inserted next to exon 2 to exon 30 of VEGFR2 gene of human VEGFR, exon 2 of VEGFR2 gene of non-human animal is inserted next to exon 3 to exon 30 of VEGFR2 gene of human VEGFR, exon 3 of non-human animal is inserted next to exon 4 to exon 30 of VEGFR2 gene of human VEGFR, exon 4 of non-human animal is inserted next to exon 30 of VEGFR2 gene of human VEGFR, and the like.

Preferably, the insertion may further comprise disruption of the coding frame of the endogenous VEGFR2 gene in the non-human animal or disruption of the coding frame of the endogenous VEGFR2 gene following the insertion, followed by the insertion procedure, as desired for a particular embodiment. Or the insertion step may be performed to cause a frame shift mutation in the endogenous VEGFR2 gene or to effect insertion of a human sequence.

Further preferably, the insertion may be followed by addition of auxiliary sequences (e.g., stop codons or sequences with termination functions, etc.) or other methods (e.g., flipping sequences, or knocking out sequences) after the insertion of the desired fragment, as desired for a particular embodiment, such that the endogenous VEGFR2 protein in the non-human animal is not normally expressed after the insertion site.

Wherein the replacement includes replacement of a corresponding position or replacement of a non-corresponding position. The substitution of the corresponding position does not merely represent a mechanical substitution corresponding directly to the base site of the human and non-human animal VEGFR2 gene, but also includes a substitution of the corresponding functional region such as a substitution of the nucleotide sequence of the extracellular region encoding the non-human animal VEGFR2 protein with the nucleotide sequence of the extracellular region encoding the human VEGFR2 protein, a substitution of the nucleotide sequence of the signal peptide encoding the non-human animal VEGFR2 protein with the nucleotide sequence of the signal peptide encoding the human VEGFR2 protein, a substitution of the nucleotide sequence of the transmembrane region encoding the non-human animal VEGFR2 protein with the nucleotide sequence of the transmembrane region encoding the human VEGFR2 protein, a substitution of the nucleotide sequence of the cytoplasmic region encoding the non-human animal VEGFR2 protein with the nucleotide sequence of the cytoplasmic region encoding the human VEGFR2 protein, a substitution of the nucleotide sequence of the signal peptide and the extracellular region encoding the human VEGFR2 protein with the nucleotide sequence of the extracellular region encoding the non-human VEGFR2 protein, a substitution of the nucleotide sequence of the signal peptide and extracellular region encoding the human VEGFR2 protein, The nucleotide sequences of the extracellular and cytoplasmic regions replaced the nucleotide sequences encoding the signal peptide, extracellular and cytoplasmic regions of the non-human animal VEGFR2 protein, the nucleotide sequences encoding the signal peptide, extracellular, cytoplasmic and transmembrane regions of the human VEGFR2 protein replaced the nucleotide sequences encoding the signal peptide, extracellular, cytoplasmic and transmembrane regions of the non-human animal VEGFR2 protein, and the nucleotide sequences encoding the extracellular and cytoplasmic regions of the human VEGFR2 protein replaced the nucleotide sequences encoding the extracellular and transmembrane regions of the non-human animal VEGFR2 protein.

Preferably, said introducing into the VEGFR2 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 2 to 15 of non-human animal VEGFR2 gene be replaced.

Preferably, the method of construction comprises insertion or substitution of a nucleotide sequence comprising a sequence encoding a human VEGFR2 protein into the non-human animal VEGFR2 locus. Further preferably, the non-human animal VEGFR2 locus is inserted or substituted with a nucleotide sequence comprising all or part of the extracellular region encoding human VEGFR2 protein. Even more preferably, the nucleotide sequence encoding the extracellular domain of human VEGFR2 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-and/or C-terminus is inserted or substituted into the non-human animal VEGFR2 locus. Still further preferably, the insertion or substitution is at the non-human animal VEGFR2 locus with a nucleotide sequence comprising an extracellular region encoding human VEGFR2 protein with 6 amino acid residues removed from the N-terminus and 13 amino acid residues removed from the C-terminus. Most preferably, the polypeptide is produced by a polypeptide comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 4, amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 or to the amino acid sequence of SEQ ID NO: 4, 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751, or a substitution into the non-human animal VEGFR2 locus.

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, the nucleotide sequence of amino acids 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, or 26-751 of the non-human animal VEGFR2 gene encoding the amino acid sequence of SEQ ID NO: 2, 1-762, 14-762, 20-762, 26-762, 23-758, 20-749 or 26-749.

Preferably, this comprises an insertion or substitution into the non-human animal VEGFR2 locus with a partial nucleotide sequence comprising the human VEGFR2 gene. Further preferably, the non-human animal VEGFR2 locus is inserted or substituted with a full or partial nucleotide sequence comprising exons 1 to 30 of human VEGFR2 gene. Still further preferably, all or part of the nucleotide sequence comprising any one, two, more than three, two consecutive or a combination of three consecutive or more exons of human VEGFR2 gene from exon 1 to exon 30 is inserted or substituted into the non-human animal VEGFR2 locus. Still further preferably, the non-human animal VEGFR2 locus is inserted or substituted with a full or partial nucleotide sequence comprising exons 2 to 15 of human VEGFR2 gene. Even more preferably, the non-human animal VEGFR2 locus is inserted or substituted with a partial nucleotide sequence comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprising intron 2-3 and/or intron 14-15, wherein the part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 2 encoding the N-terminal 0-3 amino acids, or the part of exon 2 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 2 encoding the C-terminal 0-4 amino acids, at least 20, 30, 40, 50, 60, 70, 80, 86, 90, or 94bp contiguous with exon 2, the part of exon 15 of human VEGFR2 gene comprises a nucleotide sequence that excludes exon 15 encoding the C-terminal 0-4 amino acids, or the part of exon 15 of human VEGFR2 gene comprises at least 20 contiguous exons 20, at least 20 contiguous exons with exon 15, 50. 80, 100, 110, 119, 120, 130 or 132bp nucleotide sequence. Most preferably, the polypeptide is produced using a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 7 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 7, inserted or substituted into the non-human animal VEGFR2 locus.

In a specific embodiment of the invention, the method of construction comprises replacing all or part of exons 2 to 15 of non-human animal VEGFR2 gene with all or part of exons 2 to 15 of human VEGFR2 gene.

In a specific embodiment of the invention, the method of construction comprises replacing part of exon 2, all of exon 3 to 14, and part of exon 15 of the VEGFR2 gene of a non-human animal with a construct comprising part of exon 2, all of exon 3 to 14, and part of exon 15 of the human VEGFR2 gene.

In a specific embodiment of the invention, the constructing method comprises replacing part of exon 2, all of intron 2-3, all of exon 3-14, all of intron 14-15, and part of exon 15 of VEGFR2 gene in a non-human animal with a construct comprising part of exon 2, all of intron 2-3, all of exon 3-14, all of intron 14-15, and part of exon 15 of VEGFR2 gene.

In one embodiment of the invention, the method of construction comprises the step of using a nucleic acid comprising SEQ ID NO: 7 for replacing all or part of exons 2 to 15 of a non-human animal VEGFR2 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 non-human animal VEGFR2 gene encoding SEQ ID NO: 2, amino acid sequence from 26 to 749.

In a specific embodiment of the invention, the cDNA sequence encoding the human VEGFR2 protein is inserted or substituted into the non-human animal VEGFR2 locus.

In a specific embodiment of the invention, the non-human animal VEGFR2 locus is inserted or substituted with a nucleotide sequence comprising a sequence encoding a humanized VEGFR2 protein.

In a specific embodiment of the invention, the nucleotide sequence comprising the humanized VEGFR2 gene is inserted or substituted at the non-human animal VEGFR2 locus.

Preferably, the human or humanized VEGFR2 gene is regulated in a non-human animal by endogenous regulatory elements.

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

Preferably, the non-human animal comprises a humanized VEGFR2 gene on at least one chromosome in its genome.

Preferably, at least one cell in the non-human animal expresses a human or humanized VEGFR2 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 non-human animal cell, 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 VEGFR2 gene.

The construction method also comprises the steps of mating the VEGFR2 gene humanized non-human animal with other gene modified non-human animals, carrying out 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-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR or TIGIT.

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 humanise a gene.

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.

The eighth aspect of the present invention provides 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 includes, but is not limited to, non-human animals modified by a combination of one or more of the genes CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR or TIGIT.

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

In a tenth aspect of the present invention, there is provided a tumor-bearing or inflammation model of an animal, wherein the tumor-bearing or inflammation model is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned non-human animal or its progeny.

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

In a twelfth 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 thirteenth aspect of the present invention, there is provided a cell, a tissue or an organ, wherein the cell, the tissue or the organ expresses the above-mentioned humanized VEGFR2 protein, or a genome of the cell, the tissue or the organ comprises the above-mentioned humanized VEGFR2 gene, or the cell, the tissue or the organ is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned tumor-bearing or inflammation model. Preferably, the cell, tissue or organ is incapable of developing into an individual.

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

In a fifteenth aspect of the present invention, there is provided a cell humanized of the VEGFR2 gene, said cell expressing a human or humanized VEGFR2 protein; alternatively, the genome of the cell comprises a portion of the human VEGFR2 gene. Preferably, the cell expresses the above-described humanized VEGFR2 protein.

Preferably, the cell comprises a humanized VEGFR2 gene as described above.

In a sixteenth aspect, the present invention provides a humanized VEGFR2 protein derived from the above-mentioned VEGFR2, the above-mentioned humanized VEGFR2 gene, the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method or its progeny, the above-mentioned tumor-bearing or inflammation model, the above-mentioned cell, tissue or organ, the above-mentioned tumor-bearing tissue, or an application of the above-mentioned cell, wherein the application comprises a) an application in the development of a product relating to the immune 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 VEGFR2 pathway function; preferably human VEGFR2 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 an application related to VEGFR2 gene or protein.

Preferably, said use is a non-disease diagnostic and therapeutic method.

In a seventeenth aspect of the present invention, there is provided a method of screening for a modulator specific for human VEGFR2, the method comprising administering the modulator to an individual implanted with tumor cells, and detecting tumor suppression; wherein the individual is selected from the group consisting of the above 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. Further preferably, the drug is an antibody.

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

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

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

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

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

Preferably, the method of screening for a modulator specific for human VEGFR2 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 the eighteenth aspect of the present invention, an evaluation method of an intervention program is provided, the evaluation method comprises implanting tumor cells into an individual, applying the intervention program to the individual implanted with the tumor cells, and detecting and evaluating a tumor inhibition 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 nineteenth aspect of the present invention, there is provided a use of the non-human animal derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, the above-mentioned tumor-bearing or inflammation model for preparing a human VEGFR 2-specific modulator.

In a twentieth aspect, the invention provides a non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the use of the above tumor-bearing or inflammation model in the preparation of a medicament for treating tumor, angiogenesis-related diseases, embryonic development or immune-related diseases.

The VEGFR2 gene humanized non-human animal can normally express human or humanized VEGFR2 protein in vivo, can be used for drug screening, drug effect evaluation, immunity-related diseases and tumor treatment aiming at human VEGFR2 pathway target sites, can accelerate the development process of new drugs, and can save time and cost.

The "angiogenesis-related diseases" described in the present invention include, but are not limited to, wound healing, diabetes, retinopathy, tumor infiltration and metastasis, and the like.

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 non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, gastric cancer, brain glioma.

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 VEGFR2 protein" of the present invention comprises a portion derived from the human VEGFR2 protein and a portion of the non-human VEGFR2 protein.

Wherein the humanized VEGFR2 protein comprises 5 to 1356 amino acid sequences which are continuous or spaced and are consistent with the amino acid sequence of the human VEGFR2 protein, preferably 10 to 726 amino acids which are continuous or spaced, more preferably 5, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 726, 750, 800, 900, 1000, 1100, 1200, 1300 or 1356 amino acid sequences which are continuous or spaced and are consistent with the amino acid sequence of the human VEGFR2 protein.

The "humanized VEGFR2 gene" of the present invention includes a portion derived from the human VEGFR2 gene and a portion derived from the non-human VEGFR2 gene.

Wherein, the humanized VEGFR2 gene comprises 20bp-47000bp nucleotide sequences which are continuous or separated and are consistent with the nucleotide sequence of the human VEGFR2 gene, preferably 20-19273 nucleotides which are continuous or separated, and 20-2178 nucleotides, more preferably 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2178, 2500, 3000, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 19273, 20000, 30000, 40000 and 47000bp nucleotide sequences which are consistent with the nucleotide sequence of the human VEGFR2 gene.

The "exon nos. xx to xxx" or the entire "exon nos. xx to xxx" of the present invention include nucleotide sequences of exons and introns therebetween, for example, the "exon nos. 2 to 15" includes exon nos. 2, intron nos. 2 to 3, exon nos. 3, intron nos. 3 to 4, exon nos. 4, intron nos. 4 to 5, exon nos. 5, intron nos. 5 to 6, exon nos. 6 to 7, exon nos. 7, intron nos. 7 to 8, exon nos. 8, intron nos. 8 to 9, exon nos. 9, intron nos. 9 to 10, exon nos. 10, intron nos. 10 to 11, exon nos. 11, intron nos. 11 to 12, intron nos. 12 to 13, exon nos. 13 to 14, intron nos. 13 to 14 and intron nos. 14, The complete nucleotide sequence of intron 14-15 and exon 15.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 2-3" means an intron between exon 2 and exon 3.

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 "VEGFR 2 locus" refers to a DNA fragment of an optional stretch of exon 1 to exon 30 of VEGFR2 gene. In one embodiment of the present invention, the VEGFR2 locus that is replaced may be a DNA fragment of an optional stretch of exon 1 to exon 30 of VEGFR2 gene. In one embodiment of the present invention, the VEGFR2 locus that is replaced may be a DNA fragment of an optional segment of the exon nos. 2 to 15 of the VEGFR2 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 "three or more" as used herein includes, but is not limited to, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, or thirty, etc.

"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, eight in succession, nine in succession, ten in succession, eleven in succession, twelve in succession, thirteen in succession, fourteen in succession, fifteen in succession, sixteen in succession, seventeen in succession, eighteen in succession, nineteen in succession, twenty-one in succession, twenty-two in succession, twenty-three in succession, twenty-four in succession, twenty-five in succession, twenty-six in succession, twenty-seven in succession, twenty-eight in succession, twenty-nine in succession, thirty in succession, and the like. Wherein "three or more consecutive exons from exon 2 to exon 15" includes three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, 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 context of using an amino acid sequence or a nucleotide sequence, a person skilled in the art can adjust the sequence to have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity.

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

In one aspect, the non-human animal is a mammal. 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 VEGFR2 gene and human VEGFR2 locus (not to scale);

FIG. 2: schematic representation of humanization of mouse VEGFR2 gene (not to scale);

FIG. 3: VEGFR2 gene targeting strategy and targeting vector design schematic (not to scale);

FIG. 4: VEGFR2 recombinant cell Southern blot result, wherein WT is wild type control, 1-A04, 1-A05, 1-C08, 1-E02, 2-B02, 2-C06, 2-F02, 2-H06, 3-A02, 3-B12, 3-E05, 3-F07 are clone numbers;

FIG. 5: schematic representation of the humanized VEGFR2 mouse FRT recombination process (not to scale);

FIG. 6: VEGFR2 humanized mouse F1 genotype identification, where WT is wild type and H₂O is water control, and PC is positive control;

FIG. 7: the results (part) of the alignment of human and mouse VEGFR2 protein sequences, where Query is the amino acid sequence of human VEGFR2 protein and Sbjct is the amino acid sequence of mouse VEGFR2 protein;

FIG. 8: results of detection of VEGFR2 mRNA in the lung of embryo of C57BL/6 wild type mice (+/+) and VEGFR2 gene humanized homozygote mice (H/H).

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, ScaI and HindIII enzymes are purchased from NEB, and the product numbers are R0194, R0122 and R0104 respectively;

Heraeus^TMFresco^TM21Microcentrifuge purchased from Thermo Fisher Scientific, model Fresco 21;

mouse VEGFR2/Flk-1 Quantikine ELISA Kit was purchased from R & D under Cat No. MVR 200B;

human VEGF Receptor 2ELISA kit purchased from Abcam, cat No. ab 213476;

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;

BV480 Rat Anti-Mouse CD31 from BD Horizon^TMItem number 565629;

PE/Cyanine7 anti-mouse CD34 Antibody, available from Biolegend under Cat 128617;

APC anti-mouse CD309(VEGFR2, Flk-1) Antibody, available from Biolegend under Cat 136405;

human VEGFR2/KDR/Flk-1 PE-conjugated Antibody available from R & D under the accession number FAB 357P-025;

Zombie NIR^TMfixable visual Kit available from Biolegend under cat # 423105;

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

trizol kit was purchased from Takara, cat # 6110A.

Example 1VEGFR2 Gene humanized mice

This example modifies a non-human animal (e.g., a mouse) to include a nucleotide sequence encoding a humanized VEGFR2 protein in the non-human animal, resulting in a genetically modified non-human animal that expresses a humanized VEGFR2 protein. A comparative scheme of mouse VEGFR2 Gene (NCBI Gene ID: 16542, Primary source: MGI: 96683, located at positions 75932827 to 75979072 of chromosome 5 NC-000071.6, based on transcript NM-010612.3 (SEQ ID NO: 1) and its encoded protein NP-034742.2 (SEQ ID NO: 2)) and human VEGFR2 Gene (NCBI Gene ID: 3791, Primary source: HGNC:6307, UniProt ID: P35968, located at positions 55078481 to 55125595 of chromosome 4 NC-000004.12, based on transcript NM-002253.3 (SEQ ID NO: 3) and its encoded protein NP-002244.1 (SEQ ID NO: 4)) is shown in FIG. 1.

For the purposes of the present invention, a portion of the nucleotide sequence encoding the human VEGFR2 protein may be introduced at the endogenous VEGFR2 locus in mice, such that the mice express the humanized VEGFR2 protein. Specifically, the 18321bp sequences from the sequence of the exon part 2 to the sequence of the exon part 15 of the VEGFR2 gene of the mouse can be replaced by corresponding human DNA sequences by using a gene editing technology to obtain a humanized VEGFR2 gene sequence (a schematic diagram is shown in fig. 2), so that humanized modification of the VEGFR2 gene of the mouse can be realized.

In the schematic of the targeting strategy shown in fig. 3, the homology arm sequences on the targeting vector containing the upstream and downstream of the mouse VEGFR2 gene are shown, as well as the a fragment containing the human VEGFR2 sequence. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 5) is the same as the nucleotide sequence from position 75974488 to 75978469 of NCBI accession No. NC-000071.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is the same as the nucleotide sequence from position 75951590 to 75955601 of NCBI accession No. NC-000071.6; a human VEGFR2 sequence (SEQ ID NO: 7) comprising the sequence of the No. 2 exon portion through the No. 15 exon portion of the human VEGFR2 gene on the A fragment, the human VEGFR2 sequence being identical to the nucleotide sequence from position 55101910 to 55121182 of NCBI accession No. NC-000004.12; the connection between the upstream of the human VEGFR2 sequence in fragment A and the mouse VEGFR2 gene was designed to be 5' -ttggatccaactcttttctaatttgtttctaggtttgcctagtgtttctcttgatctgcccaggctcagcatacaaaaag-3' (SEQ ID NO: 8), sequence thereofColumn'tgcctThe last "t" in "is the last nucleotide in the mouse, and" a "in the sequence" agtgt "is the first nucleotide in the human; the connection between the downstream of the human VEGFR2 sequence and the mouse VEGFR2 gene was designed to

5’-ggcatgcagtgttcttggctgtgcaaaagtggaggcatttttcataatagaaggtcagtgtgatgtcataggctcatcag-3' (SEQ ID NO: 9), wherein the sequence

The last "t" in (a) is the last nucleotide, sequence, of a human "ttcatThe first "t" in "is the first nucleotide in 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’-ctggggaaaaatagatttggtgttgggcattgctattcaa

TGATATCGAATTCCGAAGTTCCTATTCTCTAGAAA-3' (SEQ ID NO: 10), wherein the sequence "ttcaaThe last "a" in "is the last nucleotide, sequence, of the mouse

The first "a" in (a) is the first nucleotide of the Neo cassette; the connection between the 3 'end of the Neo box and the mouse gene is designed to be 5' -GAACTTCATCAGTCAGGTACATAATGGTGGATCCG

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

"C" in (A) is the last nucleotide, sequence, of the Neo cassette "ctagt"in" c "is a mouseThe first nucleotide of (a). 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 modified humanized mouse VEGFR2 is shown in SEQ ID NO:12, the expressed protein sequence is shown as SEQ ID NO: shown at 13.

Furthermore, based on the amino acid sequence structures of the human VEGFR2 protein and the mouse VEGFR2 protein (as shown in fig. 7), the VEGFR2 sequence derived from human includes, in addition to SEQ ID NO: 4, the same amino acid residues may also be from an extracellular region and/or a signal peptide, e.g. including SEQ ID NO: 4, or the signal peptide and the extracellular region at the positions of 20-764, 26-764, 23-760, 20-751 or 26-751, and the mRNA sequence of the humanized mouse VEGFR2 after being modified is shown in SEQ ID No. 12, and the expressed protein sequence is shown in 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 performing electroporation transfection on a targeting vector which is verified to be correct by sequencing into embryonic stem cells of a C57BL/6 mouse, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technology, screening correct positive clone cells, detecting clones which are verified to be positive by PCR by using Southern Blot (cell DNA is digested by XmnI, ScaI or HindIII and hybridized by using 3 probes respectively, 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 12 clones which are verified to be positive by PCR are detected, and the sequencing shows that the rest 11 clones are positive clones except 3-F07 and have no random insertion, and the specific numbers are 1-A04, 1-A05, 1-C08, 1-E02, 2-B02, 2-C06, 2-F02, 2-H06, 3-A02, 3-B12 and 3-E05.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				XmnI	5’Probe	7.0kb	11.6kb
ScaI	3’Probe	7.4kb	12.6kb
				HindIII	Neo Probe	—	5.9kb

Wherein the PCR assay comprises the following primers:

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

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

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

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

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

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

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

3 'Probe (3' Probe):

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

3’Probe-R：5’-GCAGAATTCCACAATCACCATGAGA-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 representation of the process), and then mated with each other to obtain humanized VEGFR2 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 2 mice numbered F1-01, F1-02 were all positive heterozygous mice. This indicates that using this method, it is possible to construct a humanized mouse of the VEGFR2 gene that can be stably passaged without random insertions.

Table 2: primer name and specific sequence

Expression of the humanized VEGFR2 protein in the humanized mouse of the VEGFR2 gene can be detected by conventional methods, such as ELISA. The expression conditions of VEGFR2 protein in 11-week-old male C57BL/6 wild-type mice and VEGFR2 gene humanized homozygote mice are detected according to the operation procedures of Mouse VEGFR2/Flk-1 Quantikine ELISA Kit and human VEGF Receptor 2ELISA specification. The results show that the expression of the murine VEGFR2 protein is detected in the C57BL/6 wild-type mouse, and the humanized VEGFR2 protein is not detected; expression of the humanized VEGFR2 protein was detected in the VEGFR2 gene humanized homozygous mouse, and no expression of murine VEGFR2 was detected.

The expression of the humanized VEGFR2 protein in the VEGFR2 gene humanized mice was also confirmed by flow cytometry. Specifically, 1 female C57BL/6 wild-type Mouse and 1 Mouse of VEGFR2 gene humanized homozygote each of which is 12-14 days old at 11 weeks gestation are taken, and lung tissues of Mouse embryos are taken and treated with Anti-Mouse CD31 Antibody BV480 Rat Anti-Mouse CD31, Anti-Mouse CD34 Antibody PE/Cyanine7 Anti-Mouse CD34 Anti-body, Anti-Mouse VEGFR 2Antibody APC Anti-Mouse CD309(VEGFR2, Flk-1) Anti-body, Anti-Human 2Antibody Human VEGFR2/KDR/Flk-1 PE-conjugated Anti-body, Zombie NIR^TMAnd (3) carrying out flow detection after recognition and staining by using a Fixable visualization Kit, a Purified anti-mouse CD16/32Antibody anti-mouse CD16/32Antibody and the like, and detecting the expression condition of the VEGFR2 protein, wherein the detection result is shown in Table 3.

TABLE 3 flow cytometry detection of VEGFR2 protein assay results

The results showed (table 3) that only murine VEGFR2 protein, no human or humanized VEGFR2 protein was detected in embryonic lung tissue derived from C57BL/6 wild type mice, and humanized VEGFR2 protein was detected only in mouse embryonic lung tissue of VEGFR2 humanized homozygote mice (hVEGFR 2). However, about 15% of the endothelial cells (CD31+ cells or CD34+ cells) of hVEGFR2 mice detected mouse VEGFR2 protein, and it was presumed that the anti-Human VEGFR 2Antibody Human VEGFR2/KDR/Flk-1 PE-conjugated Antibody might be a Human-mouse cross Antibody.

To further verify the expression of VEGFR2 in the humanized homozygote mouse of VEGFR2 gene, the expression of human VEGFR2 mRNA in the mouse can be confirmed by a conventional detection method, such as RT-PCR. Specifically, 1 mouse of each of the 11-week-old, 12-14-day-pregnant female C57BL/6 wild-type mice and the VEGFR2 gene humanized homozygote mouse prepared in this example was taken, cells of embryonic lung of the mice were extracted after cervical dislocation, cellular RNA was extracted according to the instructions of Trizol kit, and reverse transcription was performed to cDNA for RT-PCR (primers shown in table 4), and the results of the detection are shown in fig. 8: only murine VEGFR2 mRNA, no human VEGFR2 mRNA was detected in C57BL/6 wild type mice; human VEGFR2 mRNA was only detectable in VEGFR2 humanized homozygous mice. Combined with the flow cytometry results, it was shown that only the humanized VEGFR2 protein could be detected in the VEGFR2 gene humanized homozygous mouse.

Table 4: name and specific sequence of RT-PCR primer

Example 2 drug efficacy verification

(Ramucirumab with full-length Heavy (HC) sequence of SEQ ID NO: 37 and full-length Light (LC) sequence of SEQ ID NO: 38) is a humanized monoclonal antibody that binds to VEGFR2 that was developed pharmaceutically by rituximab.

The VEGFR2 gene humanized mouse prepared by the method is used for constructing a tumor model and can be used for testing the drug effect of a drug targeting human VEGFR 2. Specifically, the humanized homozygote mouse of VEGFR2 gene prepared in example 1 was selected and inoculated subcutaneously with mouse colon cancer cell MC38 (5X 10)⁵One), the tumor volume is about 100-³Thereafter, the tumor volume was divided into a control group to which PBS was injected or a treatment group (n ═ 6/group) to which Ramucirumab was administered. The administration mode comprises the following steps: intraperitoneal (i.p.) injection, the administration is started on the same day, 2 times per week and 6 times in total. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of a single mouse reached 3000mm³And performing euthanasia. Specific groupingAnd dosing is shown in table 5.

Table 5: grouping and administration of drugs

The results show that the health of the animals in the experimental process of each group is good, and the weight of the animals in all treatment groups (G2 and G3) is increased compared with the weight of the animals in a control group (G1), and no obvious difference exists. From the result of tumor volume, the tumor of the control group mice continuously grows in the experimental period, and compared with the control group mice, the tumor volumes of all the treatment group mice are reduced and/or disappeared to different degrees, which shows that the anti-human VEGFR 2antibody Ramucirumab with different concentrations has a certain tumor inhibition effect, does not generate obvious toxic effect on animals, and has better safety. VEGFR2 humanized mice are demonstrated to be useful for screening human VEGFR2 targeted drugs (e.g., antibody drugs) and in vivo drug efficacy testing. Example 3 preparation of double humanized or multiple double humanized mice

The VEGFR2 mouse prepared by the method can also be used for preparing a double-gene humanized or multi-gene humanized mouse model. For example, in example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing gene modifications such as CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR, TIGIT, etc., or may be obtained from humanized VEGFR2 mice by using isolated mouse ES embryonic stem cells and gene recombination targeting techniques to obtain a two-gene or multi-gene modified mouse model of VEGFR2 and other gene modifications. The homozygote or heterozygote of the VEGFR2 mouse obtained by the method can be mated with other homozygote or heterozygote mouse modified by genes, the offspring of the homozygote or heterozygote mouse can be screened, the homozygote or heterozygote mouse modified by humanized VEGFR2 and other genes and double genes or multiple genes can be obtained with a certain probability according to Mendel genetic rules, the heterozygote can be mated with each other to obtain homozygote modified by double genes or multiple genes, and the in vivo efficacy verification of targeted human VEGFR2 and other gene regulators can be carried out by utilizing the mouse modified by double genes or multiple genes.

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> VEGFR2 gene humanized non-human animal and construction method and application thereof

<130> BHVEGFR2.002CN1-P0102020040247Z

<150> 202011564564X

<151> 2020-12-25

<160> 38

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5921

<212> DNA

<213> Mus musculus

<400> 1

gagtcctcag gaccccaaga gagtaagctg tgtttcctta gatcgcgcgg accgctaccc 60

ggcaggactg aaagcccaga ctgtgtcccg cagccgggat aacctggctg acccgattcc 120

gcggacaccg ctgcagccgc ggctggagcc agggcgccgg tgccccgcgc tctccccggt 180

cttgcgctgc gggggcgcat accgcctctg tgacttcttt gcgggccagg gacggagaag 240

gagtctgtgc ctgagaactg ggctctgtgc ccagcgcgag gtgcaggatg gagagcaagg 300

cgctgctagc tgtcgctctg tggttctgcg tggagacccg agccgcctct gtgggtttgc 360

ctggcgattt tctccatccc cccaagctca gcacacagaa agacatactg acaattttgg 420

caaatacaac ccttcagatt acttgcaggg gacagcggga cctggactgg ctttggccca 480

atgctcagcg tgattctgag gaaagggtat tggtgactga atgcggcggt ggtgacagta 540

tcttctgcaa aacactcacc attcccaggg tggttggaaa tgatactgga gcctacaagt 600

gctcgtaccg ggacgtcgac atagcctcca ctgtttatgt ctatgttcga gattacagat 660

caccattcat cgcctctgtc agtgaccagc atggcatcgt gtacatcacc gagaacaaga 720

acaaaactgt ggtgatcccc tgccgagggt cgatttcaaa cctcaatgtg tctctttgcg 780

ctaggtatcc agaaaagaga tttgttccgg atggaaacag aatttcctgg gacagcgaga 840

taggctttac tctccccagt tacatgatca gctatgccgg catggtcttc tgtgaggcaa 900

agatcaatga tgaaacctat cagtctatca tgtacatagt tgtggttgta ggatatagga 960

tttatgatgt gattctgagc cccccgcatg aaattgagct atctgccgga gaaaaacttg 1020

tcttaaattg tacagcgaga acagagctca atgtggggct tgatttcacc tggcactctc 1080

caccttcaaa gtctcatcat aagaagattg taaaccggga tgtgaaaccc tttcctggga 1140

ctgtggcgaa gatgtttttg agcaccttga caatagaaag tgtgaccaag agtgaccaag 1200

gggaatacac ctgtgtagcg tccagtggac ggatgatcaa gagaaataga acatttgtcc 1260

gagttcacac aaagcctttt attgctttcg gtagtgggat gaaatctttg gtggaagcca 1320

cagtgggcag tcaagtccga atccctgtga agtatctcag ttacccagct cctgatatca 1380

aatggtacag aaatggaagg cccattgagt ccaactacac aatgattgtt ggcgatgaac 1440

tcaccatcat ggaagtgact gaaagagatg caggaaacta cacggtcatc ctcaccaacc 1500

ccatttcaat ggagaaacag agccacatgg tctctctggt tgtgaatgtc ccaccccaga 1560

tcggtgagaa agccttgatc tcgcctatgg attcctacca gtatgggacc atgcagacat 1620

tgacatgcac agtctacgcc aaccctcccc tgcaccacat ccagtggtac tggcagctag 1680

aagaagcctg ctcctacaga cccggccaaa caagcccgta tgcttgtaaa gaatggagac 1740

acgtggagga tttccagggg ggaaacaaga tcgaagtcac caaaaaccaa tatgccctga 1800

ttgaaggaaa aaacaaaact gtaagtacgc tggtcatcca agctgccaac gtgtcagcgt 1860

tgtacaaatg tgaagccatc aacaaagcgg gacgaggaga gagggtcatc tccttccatg 1920

tgatcagggg tcctgaaatt actgtgcaac ctgctgccca gccaactgag caggagagtg 1980

tgtccctgtt gtgcactgca gacagaaata cgtttgagaa cctcacgtgg tacaagcttg 2040

gctcacaggc aacatcggtc cacatgggcg aatcactcac accagtttgc aagaacttgg 2100

atgctctttg gaaactgaat ggcaccatgt tttctaacag cacaaatgac atcttgattg 2160

tggcatttca gaatgcctct ctgcaggacc aaggcgacta tgtttgctct gctcaagata 2220

agaagaccaa gaaaagacat tgcctggtca aacagctcat catcctagag cgcatggcac 2280

ccatgatcac cggaaatctg gagaatcaga caacaaccat tggcgagacc attgaagtga 2340

cttgcccagc atctggaaat cctaccccac acattacatg gttcaaagac aacgagaccc 2400

tggtagaaga ttcaggcatt gtactgagag atgggaaccg gaacctgact atccgcaggg 2460

tgaggaagga ggatggaggc ctctacacct gccaggcctg caatgtcctt ggctgtgcaa 2520

gagcggagac gctcttcata atagaaggtg cccaggaaaa gaccaacttg gaagtcatta 2580

tcctcgtcgg cactgcagtg attgccatgt tcttctggct ccttcttgtc attgtcctac 2640

ggaccgttaa gcgggccaat gaaggggaac tgaagacagg ctacttgtct attgtcatgg 2700

atccagatga attgcccttg gatgagcgct gtgaacgctt gccttatgat gccagcaagt 2760

gggaattccc cagggaccgg ctgaaactag gaaaacctct tggccgcggt gccttcggcc 2820

aagtgattga ggcagacgct tttggaattg acaagacagc gacttgcaaa acagtagccg 2880

tcaagatgtt gaaagaagga gcaacacaca gcgagcatcg agccctcatg tctgaactca 2940

agatcctcat ccacattggt caccatctca atgtggtgaa cctcctaggc gcctgcacca 3000

agccgggagg gcctctcatg gtgattgtgg aattctgcaa gtttggaaac ctatcaactt 3060

acttacgggg caagagaaat gaatttgttc cctataagag caaaggggca cgcttccgcc 3120

agggcaagga ctacgttggg gagctctccg tggatctgaa aagacgcttg gacagcatca 3180

ccagcagcca gagctctgcc agctcaggct ttgttgagga gaaatcgctc agtgatgtag 3240

aggaagaaga agcttctgaa gaactgtaca aggacttcct gaccttggag catctcatct 3300

gttacagctt ccaagtggct aagggcatgg agttcttggc atcaaggaag tgtatccaca 3360

gggacctggc agcacgaaac attctcctat cggagaagaa tgtggttaag atctgtgact 3420

tcggcttggc ccgggacatt tataaagacc cggattatgt cagaaaagga gatgcccgac 3480

tccctttgaa gtggatggcc ccggaaacca tttttgacag agtatacaca attcagagcg 3540

atgtgtggtc tttcggtgtg ttgctctggg aaatattttc cttaggtgcc tccccatacc 3600

ctggggtcaa gattgatgaa gaattttgta ggagattgaa agaaggaact agaatgcggg 3660

ctcctgacta cactacccca gaaatgtacc agaccatgct ggactgctgg catgaggacc 3720

ccaaccagag accctcgttt tcagagttgg tggagcattt gggaaacctc ctgcaagcaa 3780

atgcgcagca ggatggcaaa gactatattg ttcttccaat gtcagagaca ctgagcatgg 3840

aagaggattc tggactctcc ctgcctacct cacctgtttc ctgtatggag gaagaggaag 3900

tgtgcgaccc caaattccat tatgacaaca cagcaggaat cagtcattat ctccagaaca 3960

gtaagcgaaa gagccggcca gtgagtgtaa aaacatttga agatatccca ttggaggaac 4020

cagaagtaaa agtgatccca gatgacagcc agacagacag tgggatggtc cttgcatcag 4080

aagagctgaa aactctggaa gacaggaaca aattatctcc atcttttggt ggaatgatgc 4140

ccagtaaaag cagggagtct gtggcctcgg aaggctccaa ccagaccagt ggctaccagt 4200

ctgggtatca ctcagatgac acagacacca ccgtgtactc cagcgacgag gcaggacttt 4260

taaagatggt ggatgctgca gttcacgctg actcagggac cacactgcgc tcacctcctg 4320

tttaaatgga agtggtcctg tcccggctcc gcccccaact cctggaaatc acgagagagg 4380

tgctgcttag attttcaagt gttgttcttt ccaccacccg gaagtagcca catttgattt 4440

tcatttttgg aggagggacc tcagactgca aggagcttgt cctcagggca tttccagaga 4500

agatgcccat gacccaagaa tgtgttgact ctactctctt ttccattcat ttaaaagtcc 4560

tatataatgt gccctgctgt ggtctcacta ccagttaaag caaaagactt tcaaacagtg 4620

gctctgtcct ccaagaagtg gcaacggcac ctctgtgaaa ctggatcgaa tgggcaatgc 4680

tttgtgtgtt gaggatgggt gagatgtccc agggccgagt ctgtctacct tggaggcttt 4740

gtggaggatg cgggctatga gccaagtgtt aagtgtggga tgtggactgg gaggaaggaa 4800

ggcgcaagct cgctcggaga gcggttggag cctgcagatg cattgtgctg gctctggtgg 4860

aggtgggctt gtggcctgtc aggaaacgca aaggcggccg gcagggtttg gttttggaag 4920

gtttgcgtgc tcttcacagt cgggttacag gcgagttccc tgtggcgttt cctactccta 4980

atgagagttc cttccggact cttacgtgtc tcctggcctg gccccaggaa ggaaatgatg 5040

cagcttgctc cttcctcatc tctcaggctg tgccttaatt cagaacacca aaagagagga 5100

acgtcggcag aggctcctga cggggccgaa gaattgtgag aacagaacag aaactcaggg 5160

tttctgctgg gtggagaccc acgtggctgc cctggtggca gtgtctgagg gttctctgtc 5220

aagtggcggt aaaggctcag gctggtgttc ttcctctatc tccactcctg tcaggccccc 5280

aagtcctcag tattttagct ttgtggcttc ctgatggcag aaaaatctta attggttggt 5340

ttgctctcca gataatcact agccagattt cgaaattact ttttagccga ggttatgata 5400

acatctactg tatcctttag aattttaacc tataaaacta tgtctactgg tttctgcctg 5460

tgtgcttatg ttaaaaaaaa aaagaaagaa agaaactgtt cttttcattt ggtaccatag 5520

tgtgaagagc tgggagcaat gactgttaaa catgctatgg cacatctatt tatagtctgt 5580

tatgtagaac aaatgtaata tattaaaacg ttatattata tataatgaac tttgtactac 5640

ccaccttttg tatcagtatt atgtaccact agagagatta caaggctttc agcagccgct 5700

gttgttttgt taaagacttt gagaaactcg aaggaatcct ttcatggaat atgcagctat 5760

ataccctacc gtctctctca tctcaaacgg aggaggagga ggaggagtca ggtataatgt 5820

gagtgtgttc tacgtgtcct tgttctctgt tcttaggagg aatgatttca tcaaatgttt 5880

atatgcttta taaaccaata aacgtattct gagtaaagag a 5921

<210> 2

<211> 1345

<212> PRT

<213> Mus musculus

<400> 2

Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu

1 5 10 15

Thr Arg Ala Ala Ser Val Gly Leu Pro Gly Asp Phe Leu His Pro Pro

20 25 30

Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly

65 70 75 80

Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val

85 90 95

Gly Asn Asp Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile

100 105 110

Ala Ser Thr Val Tyr Val Tyr Val Arg Asp Tyr Arg Ser Pro Phe Ile

115 120 125

Ala Ser Val Ser Asp Gln His Gly Ile Val Tyr Ile Thr Glu Asn Lys

130 135 140

Asn Lys Thr Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn

145 150 155 160

Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly

165 170 175

Asn Arg Ile Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr

180 185 190

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

195 200 205

Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg

210 215 220

Ile Tyr Asp Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala

225 230 235 240

Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val

245 250 255

Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys

260 265 270

Lys Ile Val Asn Arg Asp Val Lys Pro Phe Pro Gly Thr Val Ala Lys

275 280 285

Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp Gln

290 295 300

Gly Glu Tyr Thr Cys Val Ala Ser Ser Gly Arg Met Ile Lys Arg Asn

305 310 315 320

Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser

325 330 335

Gly Met Lys Ser Leu Val Glu Ala Thr Val Gly Ser Gln Val Arg Ile

340 345 350

Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg

355 360 365

Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met Ile Val Gly Asp Glu

370 375 380

Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val

385 390 395 400

Ile Leu Thr Asn Pro Ile Ser Met Glu Lys Gln Ser His Met Val Ser

405 410 415

Leu Val Val Asn Val Pro Pro Gln Ile Gly Glu Lys Ala Leu Ile Ser

420 425 430

Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr

435 440 445

Val Tyr Ala Asn Pro Pro Leu His His Ile Gln Trp Tyr Trp Gln Leu

450 455 460

Glu Glu Ala Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys

465 470 475 480

Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu

485 490 495

Val Thr Lys Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val

500 505 510

Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys

515 520 525

Glu Ala Ile Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His

530 535 540

Val Ile Arg Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr

545 550 555 560

Glu Gln Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe

565 570 575

Glu Asn Leu Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His

580 585 590

Met Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp

595 600 605

Lys Leu Asn Gly Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile

610 615 620

Val Ala Phe Gln Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys

625 630 635 640

Ser Ala Gln Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln

645 650 655

Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu

660 665 670

Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala

675 680 685

Ser Gly Asn Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr

690 695 700

Leu Val Glu Asp Ser Gly Ile Val Leu Arg Asp Gly Asn Arg Asn Leu

705 710 715 720

Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln

725 730 735

Ala Cys Asn Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile

740 745 750

Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Val Ile Ile Leu Val Gly

755 760 765

Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile Val Leu

770 775 780

Arg Thr Val Lys Arg Ala Asn Glu Gly Glu Leu Lys Thr Gly Tyr Leu

785 790 795 800

Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg Cys Glu

805 810 815

Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp Arg Leu

820 825 830

Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val Ile Glu

835 840 845

Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr Val Ala

850 855 860

Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg Ala Leu

865 870 875 880

Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu Asn Val

885 890 895

Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu Met Val

900 905 910

Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu Arg Gly

915 920 925

Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg Phe Arg

930 935 940

Gln Gly Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys Arg Arg

945 950 955 960

Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly Phe Val

965 970 975

Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Ser Glu Glu

980 985 990

Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr Ser Phe

995 1000 1005

Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys Ile His

1010 1015 1020

Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn Val Val

1025 1030 1035 1040

Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp

1045 1050 1055

Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met Ala Pro

1060 1065 1070

Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val Trp Ser

1075 1080 1085

Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr

1090 1095 1100

Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys Glu Gly

1105 1110 1115 1120

Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr

1125 1130 1135

Met Leu Asp Cys Trp His Glu Asp Pro Asn Gln Arg Pro Ser Phe Ser

1140 1145 1150

Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala Gln Gln

1155 1160 1165

Asp Gly Lys Asp Tyr Ile Val Leu Pro Met Ser Glu Thr Leu Ser Met

1170 1175 1180

Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met

1185 1190 1195 1200

Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala

1205 1210 1215

Gly Ile Ser His Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro Val

1220 1225 1230

Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu Val Lys

1235 1240 1245

Val Ile Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu Ala Ser

1250 1255 1260

Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro Ser Phe

1265 1270 1275 1280

Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala Ser Glu Gly

1285 1290 1295

Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp Asp Thr

1300 1305 1310

Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu Leu Lys Met Val

1315 1320 1325

Asp Ala Ala Val His Ala Asp Ser Gly Thr Thr Leu Arg Ser Pro Pro

1330 1335 1340

Val

1345

<210> 3

<211> 5849

<212> DNA

<213> Homo sapiens

<400> 3

actgagtccc gggaccccgg gagagcggtc aatgtgtggt cgctgcgttt cctctgcctg 60

cgccgggcat cacttgcgcg ccgcagaaag tccgtctggc agcctggata tcctctccta 120

ccggcacccg cagacgcccc tgcagccgcg gtcggcgccc gggctcccta gccctgtgcg 180

ctcaactgtc ctgcgctgcg gggtgccgcg agttccacct ccgcgcctcc ttctctagac 240

aggcgctggg agaaagaacc ggctcccgag ttctgggcat ttcgcccggc tcgaggtgca 300

ggatgcagag caaggtgctg ctggccgtcg ccctgtggct ctgcgtggag acccgggccg 360

cctctgtggg tttgcctagt gtttctcttg atctgcccag gctcagcata caaaaagaca 420

tacttacaat taaggctaat acaactcttc aaattacttg caggggacag agggacttgg 480

actggctttg gcccaataat cagagtggca gtgagcaaag ggtggaggtg actgagtgca 540

gcgatggcct cttctgtaag acactcacaa ttccaaaagt gatcggaaat gacactggag 600

cctacaagtg cttctaccgg gaaactgact tggcctcggt catttatgtc tatgttcaag 660

attacagatc tccatttatt gcttctgtta gtgaccaaca tggagtcgtg tacattactg 720

agaacaaaaa caaaactgtg gtgattccat gtctcgggtc catttcaaat ctcaacgtgt 780

cactttgtgc aagataccca gaaaagagat ttgttcctga tggtaacaga atttcctggg 840

acagcaagaa gggctttact attcccagct acatgatcag ctatgctggc atggtcttct 900

gtgaagcaaa aattaatgat gaaagttacc agtctattat gtacatagtt gtcgttgtag 960

ggtataggat ttatgatgtg gttctgagtc cgtctcatgg aattgaacta tctgttggag 1020

aaaagcttgt cttaaattgt acagcaagaa ctgaactaaa tgtggggatt gacttcaact 1080

gggaataccc ttcttcgaag catcagcata agaaacttgt aaaccgagac ctaaaaaccc 1140

agtctgggag tgagatgaag aaatttttga gcaccttaac tatagatggt gtaacccgga 1200

gtgaccaagg attgtacacc tgtgcagcat ccagtgggct gatgaccaag aagaacagca 1260

catttgtcag ggtccatgaa aaaccttttg ttgcttttgg aagtggcatg gaatctctgg 1320

tggaagccac ggtgggggag cgtgtcagaa tccctgcgaa gtaccttggt tacccacccc 1380

cagaaataaa atggtataaa aatggaatac cccttgagtc caatcacaca attaaagcgg 1440

ggcatgtact gacgattatg gaagtgagtg aaagagacac aggaaattac actgtcatcc 1500

ttaccaatcc catttcaaag gagaagcaga gccatgtggt ctctctggtt gtgtatgtcc 1560

caccccagat tggtgagaaa tctctaatct ctcctgtgga ttcctaccag tacggcacca 1620

ctcaaacgct gacatgtacg gtctatgcca ttcctccccc gcatcacatc cactggtatt 1680

ggcagttgga ggaagagtgc gccaacgagc ccagccaagc tgtctcagtg acaaacccat 1740

acccttgtga agaatggaga agtgtggagg acttccaggg aggaaataaa attgaagtta 1800

ataaaaatca atttgctcta attgaaggaa aaaacaaaac tgtaagtacc cttgttatcc 1860

aagcggcaaa tgtgtcagct ttgtacaaat gtgaagcggt caacaaagtc gggagaggag 1920

agagggtgat ctccttccac gtgaccaggg gtcctgaaat tactttgcaa cctgacatgc 1980

agcccactga gcaggagagc gtgtctttgt ggtgcactgc agacagatct acgtttgaga 2040

acctcacatg gtacaagctt ggcccacagc ctctgccaat ccatgtggga gagttgccca 2100

cacctgtttg caagaacttg gatactcttt ggaaattgaa tgccaccatg ttctctaata 2160

gcacaaatga cattttgatc atggagctta agaatgcatc cttgcaggac caaggagact 2220

atgtctgcct tgctcaagac aggaagacca agaaaagaca ttgcgtggtc aggcagctca 2280

cagtcctaga gcgtgtggca cccacgatca caggaaacct ggagaatcag acgacaagta 2340

ttggggaaag catcgaagtc tcatgcacgg catctgggaa tccccctcca cagatcatgt 2400

ggtttaaaga taatgagacc cttgtagaag actcaggcat tgtattgaag gatgggaacc 2460

ggaacctcac tatccgcaga gtgaggaagg aggacgaagg cctctacacc tgccaggcat 2520

gcagtgttct tggctgtgca aaagtggagg catttttcat aatagaaggt gcccaggaaa 2580

agacgaactt ggaaatcatt attctagtag gcacggcggt gattgccatg ttcttctggc 2640

tacttcttgt catcatccta cggaccgtta agcgggccaa tggaggggaa ctgaagacag 2700

gctacttgtc catcgtcatg gatccagatg aactcccatt ggatgaacat tgtgaacgac 2760

tgccttatga tgccagcaaa tgggaattcc ccagagaccg gctgaagcta ggtaagcctc 2820

ttggccgtgg tgcctttggc caagtgattg aagcagatgc ctttggaatt gacaagacag 2880

caacttgcag gacagtagca gtcaaaatgt tgaaagaagg agcaacacac agtgagcatc 2940

gagctctcat gtctgaactc aagatcctca ttcatattgg tcaccatctc aatgtggtca 3000

accttctagg tgcctgtacc aagccaggag ggccactcat ggtgattgtg gaattctgca 3060

aatttggaaa cctgtccact tacctgagga gcaagagaaa tgaatttgtc ccctacaaga 3120

ccaaaggggc acgattccgt caagggaaag actacgttgg agcaatccct gtggatctga 3180

aacggcgctt ggacagcatc accagtagcc agagctcagc cagctctgga tttgtggagg 3240

agaagtccct cagtgatgta gaagaagagg aagctcctga agatctgtat aaggacttcc 3300

tgaccttgga gcatctcatc tgttacagct tccaagtggc taagggcatg gagttcttgg 3360

catcgcgaaa gtgtatccac agggacctgg cggcacgaaa tatcctctta tcggagaaga 3420

acgtggttaa aatctgtgac tttggcttgg cccgggatat ttataaagat ccagattatg 3480

tcagaaaagg agatgctcgc ctccctttga aatggatggc cccagaaaca atttttgaca 3540

gagtgtacac aatccagagt gacgtctggt cttttggtgt tttgctgtgg gaaatatttt 3600

ccttaggtgc ttctccatat cctggggtaa agattgatga agaattttgt aggcgattga 3660

aagaaggaac tagaatgagg gcccctgatt atactacacc agaaatgtac cagaccatgc 3720

tggactgctg gcacggggag cccagtcaga gacccacgtt ttcagagttg gtggaacatt 3780

tgggaaatct cttgcaagct aatgctcagc aggatggcaa agactacatt gttcttccga 3840

tatcagagac tttgagcatg gaagaggatt ctggactctc tctgcctacc tcacctgttt 3900

cctgtatgga ggaggaggaa gtatgtgacc ccaaattcca ttatgacaac acagcaggaa 3960

tcagtcagta tctgcagaac agtaagcgaa agagccggcc tgtgagtgta aaaacatttg 4020

aagatatccc gttagaagaa ccagaagtaa aagtaatccc agatgacaac cagacggaca 4080

gtggtatggt tcttgcctca gaagagctga aaactttgga agacagaacc aaattatctc 4140

catcttttgg tggaatggtg cccagcaaaa gcagggagtc tgtggcatct gaaggctcaa 4200

accagacaag cggctaccag tccggatatc actccgatga cacagacacc accgtgtact 4260

ccagtgagga agcagaactt ttaaagctga tagagattgg agtgcaaacc ggtagcacag 4320

cccagattct ccagcctgac tcggggacca cactgagctc tcctcctgtt taaaaggaag 4380

catccacacc cccaactcct ggacatcaca tgagaggtgc tgctcagatt ttcaagtgtt 4440

gttctttcca ccagcaggaa gtagccgcat ttgattttca tttcgacaac agaaaaagga 4500

cctcggactg cagggagcca gtcttctagg catatcctgg aagaggcttg tgacccaaga 4560

atgtgtctgt gtcttctccc agtgttgacc tgatcctctt tttcattcat ttaaaaagca 4620

tttatcatgc cccctgctgc gggtctcacc atgggtttag aacaaagacg ttcaagaaat 4680

ggccccatcc tcaaagaagt agcagtacct ggggagctga cacttctgta aaactagaag 4740

ataaaccagg caatgtaagt gttcgaggtg ttgaagatgg gaaggatttg cagggctgag 4800

tctatccaag aggctttgtt taggacgtgg gtcccaagcc aagccttaag tgtggaattc 4860

ggattgatag aaaggaagac taacgttacc ttgctttgga gagtactgga gcctgcaaat 4920

gcattgtgtt tgctctggtg gaggtgggca tggggtctgt tctgaaatgt aaagggttca 4980

gacggggttt ctggttttag aaggttgcgt gttcttcgag ttgggctaaa gtagagttcg 5040

ttgtgctgtt tctgactcct aatgagagtt ccttccagac cgttacgtgt ctcctggcca 5100

agccccagga aggaaatgat gcagctctgg ctccttgtct cccaggctga tcctttattc 5160

agaataccac aaagaaagga cattcagctc aaggctccct gccgtgttga agagttctga 5220

ctgcacaaac cagcttctgg tttcttctgg aatgaatacc ctcatatctg tcctgatgtg 5280

atatgtctga gactgaatgc gggaggttca atgtgaagct gtgtgtggtg tcaaagtttc 5340

aggaaggatt ttaccctttt gttcttcccc ctgtccccaa cccactctca ccccgcaacc 5400

catcagtatt ttagttattt ggcctctact ccagtaaacc tgattgggtt tgttcactct 5460

ctgaatgatt attagccaga cttcaaaatt attttatagc ccaaattata acatctattg 5520

tattatttag acttttaaca tatagagcta tttctactga tttttgccct tgttctgtcc 5580

tttttttcaa aaaagaaaat gtgttttttg tttggtacca tagtgtgaaa tgctgggaac 5640

aatgactata agacatgcta tggcacatat atttatagtc tgtttatgta gaaacaaatg 5700

taatatatta aagccttata tataatgaac tttgtactat tcacattttg tatcagtatt 5760

atgtagcata acaaaggtca taatgctttc agcaattgat gtcattttat taaagaacat 5820

tgaaaaactt gaaaaaaaaa aaaaaaaaa 5849

<210> 4

<211> 1356

<212> PRT

<213> Homo sapiens

<400> 4

Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu

1 5 10 15

Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro

20 25 30

Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser

65 70 75 80

Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn

85 90 95

Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser

100 105 110

Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser

115 120 125

Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys

130 135 140

Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser

145 150 155 160

Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg

165 170 175

Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile

180 185 190

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

195 200 205

Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr

210 215 220

Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu

225 230 235 240

Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile

245 250 255

Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu

260 265 270

Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe

275 280 285

Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu

290 295 300

Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr

305 310 315 320

Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met

325 330 335

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

340 345 350

Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly

355 360 365

Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr

370 375 380

Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu

385 390 395 400

Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val

405 410 415

Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val

420 425 430

Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr

435 440 445

Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu

450 455 460

Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr

465 470 475 480

Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys

485 490 495

Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys

500 505 510

Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr

515 520 525

Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser

530 535 540

Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln

545 550 555 560

Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser

565 570 575

Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro

580 585 590

Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr

595 600 605

Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile

610 615 620

Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr

625 630 635 640

Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val

645 650 655

Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn

660 665 670

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

675 680 685

Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn

690 695 700

Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg

705 710 715 720

Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr

725 730 735

Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe

740 745 750

Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Ile Ile Ile Leu

755 760 765

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

770 775 780

Ile Leu Arg Thr Val Lys Arg Ala Asn Gly Gly Glu Leu Lys Thr Gly

785 790 795 800

Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His

805 810 815

Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp

820 825 830

Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val

835 840 845

Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg Thr

850 855 860

Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg

865 870 875 880

Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu

885 890 895

Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu

900 905 910

Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu

915 920 925

Arg Ser Lys Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg

930 935 940

Phe Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys

945 950 955 960

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

965 970 975

Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Pro

980 985 990

Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr

995 1000 1005

Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys

1010 1015 1020

Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn

1025 1030 1035 1040

Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp

1045 1050 1055

Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met

1060 1065 1070

Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val

1075 1080 1085

Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser

1090 1095 1100

Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys

1105 1110 1115 1120

Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr

1125 1130 1135

Gln Thr Met Leu Asp Cys Trp His Gly Glu Pro Ser Gln Arg Pro Thr

1140 1145 1150

Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala

1155 1160 1165

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

1170 1175 1180

Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser

1185 1190 1195 1200

Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn

1205 1210 1215

Thr Ala Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg

1220 1225 1230

Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu

1235 1240 1245

Val Lys Val Ile Pro Asp Asp Asn Gln Thr Asp Ser Gly Met Val Leu

1250 1255 1260

Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Thr Lys Leu Ser Pro

1265 1270 1275 1280

Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser Val Ala Ser

1285 1290 1295

Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp

1300 1305 1310

Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu Ala Glu Leu Leu Lys

1315 1320 1325

Leu Ile Glu Ile Gly Val Gln Thr Gly Ser Thr Ala Gln Ile Leu Gln

1330 1335 1340

Pro Asp Ser Gly Thr Thr Leu Ser Ser Pro Pro Val

1345 1350 1355

<210> 5

<211> 3982

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tctctttctg ccctgagtcc tcaggacccc aagagagtaa gctgtgtttc cttagatcgc 60

gcggaccgct acccggcagg actgaaagcc cagactgtgt cccgcagccg ggataacctg 120

gctgacccga ttccgcggac accgctgcag ccgcggctgg agccagggcg ccggtgcccc 180

gcgctctccc cggtcttgcg ctgcgggggc gcataccgcc tctgtgactt ctttgcgggc 240

cagggacgga gaaggagtct gtgcctgaga actgggctct gtgcccagcg cgaggtgcag 300

gatggagagc aaggcgctgc tagctgtcgc tctgtggttc tgcgtggaga cccgagccgc 360

ctctgtgggt aagaagccca ctctttagta gtaaggcgga gaagtagggt gcgggcggag 420

agtgggaata gaagaggacc taactcgtag agctctagag accctcctcc cttgggtgtt 480

ctttcactta ccaatgggga aactgaggtt caaagactct tccgaaatga ctcagccagg 540

attctactct cccccgggca tcggttggag cgtgtcctgc ggagccgtca cagcccctgg 600

cgctaggtag gcaggagtgg aaaggcggcc tgagccgggg caggagatgc tcccactggc 660

aggaacaggc ggtcaaacgc tgggaagcca gctcaagcca agcggcccgg ctggcatcaa 720

tcactccgtg ctgttgccca ccgccctagt gtggggcagg gaatccgcct ctggctccgc 780

tcccctttag ctccagcgtg taagcgcacg gactatgtga ggtgaggtct cttcatagag 840

caacactttc ctccctcaac tttctttgat gcagaatgct atttttgctg gtaggaggaa 900

gacgcggctt tctcttctgt gacagcttct ccaggtgtat taaactaaat aactctccac 960

ttaccgactc caaagcgctg gtcctggggt aaactctgaa agtctcagaa actcttgagc 1020

ttggcaccta gttataggtc acttttcttg ttttaaaatg ccctctgctt caaggttagg 1080

cccacactcg ctcttgggct tttgtgcaat aatttccctt cccttccctt cccttccctt 1140

cccttccctt cccttccctt cccttccctt cccttccctt tccctcttcc ttttcctcct 1200

cctcttcctc ctctatttct ctgtcatttc ctttttgaag ccacagtttg cagatttcca 1260

atctccaccc attggagaat ggagaatcag gaaaaaagaa gtcaattctg cagaaacatt 1320

ccttgcgccc taagagaatc gcatggctta aaagcattgg cactgacata cggcgccaag 1380

atcgcctgtc tagagctatt gagttttcct cataatgact tggttcatca ggctagctcc 1440

accacgagtg ccctcttgtt cctgagaagg ccgcactctc cccctttctg ggaagagaaa 1500

gacagcctgg aacatgtgct tgccctgggt tccatagaga agcaagttgc tttaaagccc 1560

agagaattcc tagtgtagca gcttaacagc gtcccgttct ctgaataaga tggaggttgc 1620

ccttttggag tgtgtgactt gcttaattgg attgggctat aattggtgcc atccaagtct 1680

cgagacagag ccgctgttgt ttttccttct ggtctttgag cgggaaggat aacagtgcac 1740

aaattaatta atgttggtta tcggatttga acataaaagg gcttttattg tatagtagca 1800

tatgtacctc ttgcagtcag aatgagctgt ctaaagaaca gaacccaaac ttgccgatga 1860

aaatgaatga ggtttaataa aggcgatgga tgagcattag tcactgatgt aaatctccag 1920

ttattgataa cctcattgac tggatttgat tgcagacatg tattggtatg gggcatcctt 1980

taaagatgag catagccaac gtgcctgcac tctaagagaa tctatggctg tatgttatta 2040

cagagacagt tgagaagctc ttagtggctc tggcgtgtag atcagcggta gagcgctgag 2100

gctctgcgct cgcttcctgg cactgaagaa taaaggccat ttactgtggt ggtgcagtgg 2160

gcgcagtttg tgacgagtta ctactacatt ttcctcacac atctgcctga ctaatgagtt 2220

catcagatga gcgtatccag tgattgtttg caggttaatg gttctcagtc atgtttagaa 2280

tctacttatc aaacaaattg ttttctcatt tcctgcttct tctcaaacaa agtaagattc 2340

cattattgaa aggcttgttt aagagcattt taactgcttg cctatgttag ggacagtgac 2400

ttatttcata ttgacaaata ttatgccgat taattgaata tgactaccca gttctatagc 2460

tgtctcaggg cagaccaaga gcatctgtga tccagtcact ttaaatgcca tttaaaatgc 2520

ataatttgtt ggtctaggaa taaacacact gtaaagttta gaatcacggc ccaaacacaa 2580

gtctttaaca atgccaacta gcttctgaga ttcattaatg tcatttaatt accaatgttt 2640

taaaaatatg tcattaatta ctaaatctat agttgtaaca gcaacacatg tacatcttat 2700

taggttgggt atattcaggg tggcatagct gtagactatt gcacatctgt gtaggtgagc 2760

cagtggacag ctgcctctgg ctgttctcag agggccacag tgtcacggca ttggctattt 2820

gccttggctc tttgctaata ctttattgac atggcctcat cttcgttcac gttcacttat 2880

ttgcccaaca acgtcaatgc cagctgaggc cttaggagtc atctgttctt agtcagtgtg 2940

aattagaaag cctggatgcc tgcctgctat taattagtta ttcttctctt ctgagacaga 3000

gtctcactgt gtggcccagg ctagtctcaa acttgcggtc catttgtctc actcatcaga 3060

atgctgggct tccaggtgtg tgcaccacac taggtagctc gcgttttaag ctaagagctg 3120

gaagatcctg atgtccttta ccatggtggg catgttacag gttagttgac tgaaaactag 3180

ttatctcgct gtgtaatgac ctgcagtggt atgtatctct caagatgctt ttttgcattt 3240

caatcagtta ggtaacaagt gtcttatgtc tccagctttg tattggtatg agctcagagc 3300

tttgattaat gagttgggac cccctagcta ttgctcatta gacttacact atttttagtt 3360

ttgctctgag tttatgaata tgcatgtatg catgaacttg ggagatattt ctcttcccca 3420

attccttttc ctccatttaa atgtgctgtc tttagaagcc actgcctcag cttctgcagc 3480

tcagatacca aaggaagtct ggtacacagc atgataaaag acaatgggac ggggtcacag 3540

tggctcccgt ccctttcagg ggtatggaga cgagctgtag agagatgtct ccagggagtt 3600

ttcattaatc agcaatttag tcagatctgt gcatcctatg ctttacaaga aatgtcagtg 3660

ggcctgagat catcagatgg aggttcatcg ggtttcaatg tcccgtatcc ttttgtaaga 3720

ccttgaagtt ggcaacgcag gaaaacagga actccaccct ggtgccgtga attgcagagc 3780

tgttgtgttg gtttgtgacc atctgcccat tcttcctgtt atgacagagc ttgtgaactt 3840

taactgggac tggggcaaag tcaatcccac ctttatacaa tgaattgctg aagaggcctt 3900

ttaaaacttg gagtgtgcat tgtttatgga agggctttcc tattggatcc aactcttttc 3960

taatttgttt ctaggtttgc ct 3982

<210> 6

<211> 4012

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctagtgacaa ggatcaaagg ttcaggattt tccccagagc agagacaaga ttcactcata 60

aacaatagat gattaatagg tgattgcttt tgtgatatgg acaatccggg gttataattg 120

aaatatgaaa ggaaacttcg tttcccctgg ctgtgcgtgt gcgagagagt gtgtgtgtgt 180

atgagtgtgt tttgtgtgtg tgtgtgtgta tgggtgtgtt atgtgtgtgt gtgtgtatga 240

tcgatatgaa ccctaacctt tgtttttcag tcagtattct gtgtcagaac atttcatctt 300

cagaagagcg ttcctggatc ttgtttttgt aatacactaa gggaacaatg tgactctatt 360

gtgtgagtga aggagtggtg tgcaaggggg tgactcaggg tggttagaga ggtaggtttg 420

gcagaattct aacatctcca agtctaaatg ttggcccctt tcatctgccc aactcaccat 480

gtccagtgct gcatctagag ccactagcat gctctcaaat ctctaagatg gttaggattc 540

tataaactgc acctgcaact gaagatgcca aagtgcatgg tagttctgtc actaccaaag 600

tggttcatgg cttggtgcca aatccaggga ggatgcaccc gccagttctc tctggtactc 660

tgcctccacc cactagccat ctgcattagg ttatcatgcc tatgtgggca cagtggcata 720

gatactatgg agacaggaaa ggggtggggg ggagcgagag agagagagag agagagagag 780

agagagagag agagagagag agagagagag agaggaagag aggaagagag gaagagagga 840

agagaggaag agagaggaga gggtagacat tgaatgcttc tacagaagtt cagctgtcag 900

atgttaatat gagactattc acacataaac tgattgagac cattgaatct ttgacctttc 960

ttctgtctct tacatgagta ggcttttaag cctttacata caggtagaac cattacttct 1020

ctggaaacat cagagaaacc aaatccactt acagatgcac tcagagctca ctcccaactg 1080

gtaactctta ttttgccaac taattaattg cttgtcttct ggcttctggc ttcaagcctc 1140

ccagcttgag gctcatttca actgacactg cacagtgtga gtggagagac tgagacccta 1200

agtgataatt gttttctctt aatcacctgg tgctaagcat ttggtgctgc gagcctcctg 1260

cccgtatctc tccacttgga atgtgttctg gagagcaacc tacagtctta cttgctagcc 1320

tttcccttgc tccctccaca cataaggcct ttatcttggc gctctctgtc tctgcctgtc 1380

tctctgtgtg agtctctgtc tgtctgtcta tctgtctatc tctgtgttcg tctttctgtc 1440

tgcctgcctg cctgcctgcc tgtctgtctg tctgtctgtc tgtctgtctc tccctctctt 1500

tatttctgtg tctctccctc acctatcctt cccttctctg tcttcctctc ttttctcctc 1560

tttcaggctt ctgccacata gggacaaggt ggtcagctca atttggacaa atccagtata 1620

acccttgaac agaccaccaa gttcctgtat ctcacagaca agtagcttaa ccttttaagc 1680

cttatttctt catctgaaaa ctgaaggtta ctgaaaaaat aaagaagggc gctatattta 1740

aagtgaaggg cgctatattt aaagtgacca gtgcatagta ggtgtgtggt cacaagtagc 1800

tgtctgcgca aagagcttta cttatctcct tgtgtagatt cctaaacaat caattttccc 1860

atgccacata agcaaagatg ttgactatca tcacaaaatc agactcaagt acccatgaat 1920

agaagtatat tttttcctta gagttctctc accagacaca tccctataat gtatctcttt 1980

tttaaaatat ttttattaca tattttcctc aattacattt ccaatgctat ctccaaagtc 2040

ccccataccc tccccccctt ccctacccag ccattcccat ttttttggcc ctggcattcc 2100

cctgtactgg ggcatataca gtttgcgtgt ccaatgggcc tctctttcca gtgatggccg 2160

actaggccat cttttgatac atatgcagct agagtcaaga gctccggggt actggttagt 2220

tcataatgtt gttccaccta tagggttgca gatcccttta gctccttggg aactttctct 2280

agctcctcct ttgggagccc tgtgatccat ccaatagctg actgagcatc cacttctgtg 2340

tttgctaggc cctggcatag tctcacaaga gacagctaca tctgggtcct ttcgataaaa 2400

tcttgctata taatgtatct cttaagacac gtgtaagcac ctgtggttca ttgctggatg 2460

tcttcatgag tggtgtgttt ctgttgttct ttaaaagtat gaactctatc cagcttaagg 2520

agctattggt ccctaggaat cttaaaagtg ggtgacatga ggtcctgcct tttctccctt 2580

catgtcaccc actttgtcac cttctaccat aggctatgaa tttccagctg ttgatcactc 2640

aaacatgtct tccaggtgcc caggaaaaga ccaacttgga agtcattatc ctcgtcggca 2700

ctgcagtgat tgccatgttc ttctggctcc ttcttgtcat tgtcctacgg accgttaagc 2760

gggtaacaac ataatttccc tcctgtccct tgtgtcttgg tttttgtgat taatggaagc 2820

tgactgggtt tctttcagcg gcttcttccc attgttattg gctcaatggg cacattttta 2880

cctcaataca ataacattct tgtccatttt ctttgggtgg actgtgggca ttaattgatg 2940

ggagccacca gaggggtgaa aggtttggac tgtccactgt aattaagatt tagaaacctt 3000

attctgaact cttttttgga aactgaagtg gcactaaagg caggattata atagcagcgc 3060

tctcaaacac catgagtttt attggaaaat gagattatca tgaatgaggc ccttattaaa 3120

caacaatatg tatacaaaac aacaaccaaa caagaggccg tgtgtgtatg acggggagga 3180

tttatgcttt ccaggccaat gaaggggaac tgaagacagg ctacttgtct attgtcatgg 3240

atccagatga attgcccttg gatgagcgct gtgaacgctt gccttatgat gccagcaagt 3300

gggaattccc cagggaccgg ctgaaactag gtgagttgtc aactgctatt aacttgatat 3360

aagtttttac ccgctcatct ggctctctgt taagacaatg acagatctgg tctatttaga 3420

tgatgtattc tgatttataa atattaattt tatctcttga ctttgggtaa tcatccattt 3480

agctttctag tagtaaggag cctgtgcaac catccaaagc agggcatttt gaaaagcaaa 3540

tggaaaaacc agaacaaagg aggaaagtgc tctgtgggtc taactgggat gcaccatcct 3600

cttgaccaat tggaatcttc atatgccttt tgacagtgtg acaatcaaag tgtattttgt 3660

aataccactg tcattggttc taggaaaacc tcttggccgc ggtgccttcg gccaagtgat 3720

tgaggcagac gcttttggaa ttgacaagac agcgacttgc aaaacagtag ccgtcaagat 3780

gttgaaaggt aaaagggaaa atcattatgt ttctctttct gttttgtccc tcactaaaca 3840

tgagtcttgt gtcagtggcc ctttgcatag gtaggcttta agcccctggc ttatattgcc 3900

tttgcacaaa ttgaaaacag ttttctcctt cagagcctca tgacctcaaa acaagggcta 3960

acttgcacaa ttctatttag tgatcctggt tacacagagg aggggcaata tc 4012

<210> 7

<211> 19273

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agtgtttctc ttgatctgcc caggctcagc atacaaaaag acatacttac aattaaggct 60

aatacaactc ttcaaattac ttgcaggtaa ggattcattc tagatctaga tttcttgtgt 120

taagtaactg attgtttatt gagtggaaat aatttccagt agagcagaat tataatagag 180

cttgtagtaa ttgttcataa gtggtgaggt ttctaagaac tgatgtaata atggaaaatg 240

agaagaattt tctctcaaaa attctgtaca attttgctgg tgtttttata ctattctctg 300

ccaacatgca tacacacaca cacacacaca cgcacacaaa tacacaccca cacccacatt 360

ccaataacca gtacagccac ctggcgtata gtagacatac gctcaataaa tatgaatgaa 420

taaatgaagt tgagggcata catttaagga atagagttga aaaaatttgg gactatattt 480

attatgcttg gtatgattct tgaacactta ttatcccttt ccaaaaactt tgctttataa 540

gaaatttatt actataatta cttaggcagt aatatttaat agcaatttaa tatttagtgg 600

gtaatattac tgagcgcatg atctacataa ataatggact tcgggccctg ccttgatatt 660

ctggaatgca tctttcccca cttgctagca agaagtcatg ctattgattt ttgataactg 720

gagaagtaga cttctttgtc aagaagaaga ggcctttaaa ttttgccttt caacccttac 780

cccaggacga aagatagaag acccttgggt ttaacatagt gatcacacac gaaaggcatg 840

gagccttctt aggacctgtg tgtttttggt agagactgtg acaagtggag gtgatgttac 900

cctcctggaa gagtgctggg ggtccacaaa ggaccttggg taggttattg ccattgcttc 960

atacttgttg aatactaagc attaaaccga atgacataca tctattttag actgcagtat 1020

aaagaatacc ctagcccctt accaataccc agcccttggg aaaaaacaca gtagcaggtg 1080

ctgtttctct agctttactt gtttaagaca catttcccat tagattttcc ttttaccgac 1140

cctcgataac aaggttattt gaaatcccca aggatcccat gctccctttt taaaactctg 1200

cataaacatt tcttatgttc tgaaaaaaac catggagtgt gttaaaagta acttcattga 1260

tttagctgca acttcctgga aattttaagt tctttgaatg aagggccaat aatgttacat 1320

tcttcttgat gttgactatc ttcttatctt ccttggggcc ttgtagagaa atgctgcagt 1380

acaagccatc tatgttttaa tgcgaggtcc ttacaaggtc ctgagggact cttacttgca 1440

cctccttcct tcctaacctc acttcttact cccctttgct cactcttacc tggctgctct 1500

ggtttcctgg ctgttccctt aatactccag atatgcacct gctccagggc ctttccatgt 1560

gctgtttttg ctcctgtaat actgctcttc atgatgttcc tatggctagc tttatcaaga 1620

ccacctcctg caaaattctt tactcttttc tttgtatctt ctatattttt ctccatagta 1680

ctaaacacta tcttttatac aataaacttt ccttactttt taattgcctg ttttctccag 1740

ttagactgag gttccataaa ggcattgatt tttgtctgat ttgttcactg ctctttctct 1800

agtccttaac aagtttggca catagtagat gcttaataga tatttgttga aagaaagaat 1860

gcattaatta atggaaaact caggaatctt tataagtgac ttctgaagct gagtttataa 1920

cttttcatca tatgtcaatc tgacttgttg gtagaagact ttgttttttt ttttttgagg 1980

cagggttgcc ctcttgccca ggctgaagtg cagtggtgtg attttggctc actgcaacct 2040

ccacctcccg ggttcaagca attctcatgc ctcagcctcc tgagtagctg ggattacagg 2100

catgcgccac cacacctggc tcatttttgt atttttagta gagacagggt tttaccatgt 2160

tgcccagcct ggtctcgaac tcctggcctc aggtgatcca tccgccttgg cctcccaaag 2220

tgctgggatt ataggcatga gccaccatgc ctggccggta gaagactgac tgtgtctgtt 2280

gaagagttta tttaagtttc aaaaccaaat tttctctttt cttagaaata gcctcacagt 2340

ctggcacttc atattaatac ctccctgaaa ttaatttttc aggggacaga gggacttgga 2400

ctggctttgg cccaataatc agagtggcag tgagcaaagg gtggaggtga ctgagtgcag 2460

cgatggcctc ttctgtaaga cactcacaat tccaaaagtg atcggaaatg acactggagc 2520

ctacaagtgc ttctaccggg aaactgactt ggcctcggtc atttatgtct atgttcaagg 2580

taagtggtga aataaaattc atttcccacg tctctttacc agttataaaa gacaataggc 2640

tcaaagaaga attgagtaca acaaagggct tgctctaaag gctgtttgcc aagaggaata 2700

cacacaattc ttctctcctg aggctttctc tgagaaataa gactcattga ttctggagct 2760

tgggccgtgt tacctctttt ttgcccagtt agtttgggtc tgatctttgt ttccaaggta 2820

aatctgtgtt cactgttggc cattgagact tataaaaagt cttcctatgt ttgagaagaa 2880

aacctaaaat tcttgaaatc gaggaagatt tgggggtgaa ttatggagaa atttctgtgg 2940

agagataagt tatctacagc agagtaggag attttcccaa gaatgcatag gaaagcattt 3000

tttgccaagg gctctggagt tttttgcaca taggaacctt tttttcttac tagtatttca 3060

taaaaaacaa ttcccatact catgtgcaaa taaagacatt gcttcagact cttttcagga 3120

caatgtttct ttcctttgct tgtttggtct gagatcttgg atgatatgct gtatctttct 3180

aggatgtgca gtttgggatt gatattatga aggctgactt aacatccata tagtataaaa 3240

taaatgtcac acatattctg catttataat gagttatgca ttcttttgtg tttcaaaaat 3300

cttacactat cttatctttt ctgtgaaaac ctaacttaac taatgagatc cctatgatat 3360

aaatttaagg aatgtaaggg ctgcatcata gtttggttgg atgtaccaaa tatttttctt 3420

ttcagtgaag ataaacagac attttatgta tttacgtata tgccttttta catcccagag 3480

tatttgagac aggtgaagat gacttagact tttttcccag aagcagcttt tacagggcaa 3540

gaatttcatc agctttggga aacacacttg catatctctg cttacatttc agtagtgtaa 3600

tatggtcagt gcaatgaaaa agtggagacc acatcaaaat aacctatgcc actggattca 3660

caatgtttga gaaatatctt tgcccagagt aagcactgtc aaagatagaa ttctgtgccc 3720

tcctccttcc ctccacaaga tttgaaagag acaaggctca catcttggag aatttctggc 3780

tccttttgac ctggcagtct tgagagatgc agctcggtca gaagattgca aggatttcct 3840

gctttcagcc tgtctagaaa tactacaaga tgaacatccc ccatatctca ttatttactt 3900

cttcctaagt caggaaactt ggagacatgt gaaaattcat ttcatgagtt tcagtaaata 3960

ttttattttg agaggctggg tggtggtttg ggtttctttt gtttatttcc tttttttgag 4020

ataccgaaat agaattgatt tactaaatag gtttagtctt acgtcaaagg gttaatttag 4080

cttccaaagg cttgctctgt aagcaagtta tgtaatattt cataacatgt ggatgaaagg 4140

taggcaatat taagaagtgg caatccctag cactgtttat tggtacactg cctgtctttg 4200

ggtataccat taaattctgc ttcctgtcta agcttaaagt tctaggagtt gggctgtcca 4260

agattttggc catgaagtta aacaatggga aaggaaacac tgaagtattc tctatggata 4320

ggtgtttaat gtcccctctg gtcgccacct tacttcccta gtcttctgac cccattctct 4380

tcagcaatgg atggagccag gaagtgagcc ctggcctcat aagataatgg ctatggcatg 4440

tggtgggcta gattggctgc ttttctgtgc tttccagctg ggaaggaaat caaacttctg 4500

ctgttgcagg gaattagctg cctttgtccc ctgtggttta attaactctt tcttcacttt 4560

gactgactat tatgaagcac tctgagaatg cttgatggga tgtgttgggc atagcaatgt 4620

gaaatgttat ctctctgaga tttcaagcat gactccacac cacatcatct ctatctctga 4680

ggaatggact aggtttccag cagcatgtta acattgtatg agtaatgttt gattggcctt 4740

gaaatctttt tttttttttt tttttgagac ggagttttgc tcttgttgcc caggctaaag 4800

tgcagtggtg ctatctcagc tcactgcaac ttctgccccc cggttcaaat gattctcctg 4860

cctcagcctc tgaaatagct gggactacag gtgcgtgcca tcatgcctgg ctaatttttt 4920

gtatttttcg tagagatggg gttttgccac gttggtcagg ctggtctcaa actcctgacc 4980

tcaagtgatc cacctgcctc agcctcccaa agtgctggga ttacaggcgt gagccaagaa 5040

cccagtcaga atctcttcag ttttcttctc agtctttgga gtggtgactt ttcaaatgtt 5100

tgtcattgaa gatatcaatg actgctaaat gttaaactaa atgcaaaaac aattaaacat 5160

ggttttagaa agaatcatat ccctagtctt cagaatctta aaatgctcac atgaatggtc 5220

ctcttgaata accaaattca aaagtgttag ctgtttcctg ttaatctaaa gatcctttgg 5280

gatccattca tttattttca tggaatttac attatttacc taaagagaga gcacatgagt 5340

attttaaata ttagtaaaac ttgtcggtaa agtgtataga tttaacttta aattttaaag 5400

taaatattat ccttcatttt gaaaaaatta taatgattaa tcttttaaaa tgtgaaatct 5460

ataaaaatat attctgcttg tcaataaacc ttgtgaaagg agtcaatctc aattgggagt 5520

tttttttcaa aatttttata cacacagata tatacacatg catgtgcatg cacaaacaca 5580

cacacacaca tacacacaca ccctcatgta gcacagatat ctatcagcag aataatctgt 5640

ggatgccttt ggttgtgtga ggtgtccctt ccagtcattc acttgtctgg ttagagttta 5700

ggaacctgaa aaatgaccaa cttttctagt aaatactatt aactcattaa taaaactaaa 5760

ttttcttcta gattacagat ctccatttat tgcttctgtt agtgaccaac atggagtcgt 5820

gtacattact gagaacaaaa acaaaactgt ggtgattcca tgtctcgggt ccatttcaaa 5880

tctcaacgtg tcactttgtg cagtaagttg catctcctcc aatcgtctct taagttttta 5940

taattttaag ctaatattaa gatgggtaac ctgtttataa tattcacaat gagttttaag 6000

gatcctttag gaagggtcaa atgcaatgaa taaaactaat tagtattctt aaaaataaga 6060

tgaattcttc agtgatcatt gtacatggct ctcatttttg gtactggatt aaatatttga 6120

tatgtctttt tattacccag agatacccag aaaagagatt tgttcctgat ggtaacagaa 6180

tttcctggga cagcaagaag ggctttacta ttcccagcta catgatcagc tatgctggca 6240

tggtcttctg tgaagcaaaa attaatgatg aaagttacca gtctattatg tacatagttg 6300

tcgttgtagg taagaggaca tttcctttcc atatcattaa taacatatcc ttgtattaag 6360

atcttggaga taacaacata gagtgaagaa ggatattgaa aagtatagga actcaggata 6420

tggtgttggg caattcatct gctcttctct accaaataaa cccatgtgca attgaggttg 6480

tctcttttct tgccaagatt aaggaagaaa aagaaaactt tttaaaaaaa ggatgaaagc 6540

gaatggtatt actcgagcac attttatgaa gaattcaatg ttcagagcat tgcttgctat 6600

caattatttc aattatgact attttatgga aacttcagca atttgctaaa gctggcccta 6660

ctggcctagg gctactgacc actgaaagtt tactactttt ctgtccactg ggttacaaca 6720

tctttgagat ctgtgaaggt agtgctttgt aaacctctgt tggccatttt cctgggagct 6780

accaagtatt ggtgaggcct gcagggaaaa acaatgtggc atgttttaaa gttgcattac 6840

tttaaaaaat aaatctgtgc aaagttatag gcttatttgc tctctcatgt tctgtttttt 6900

caatttactt gctctagggt ataggattta tgatgtggtt ctgagtccgt ctcatggaat 6960

tgaactatct gttggagaaa agcttgtctt aaattgtaca gcaagaactg aactaaatgt 7020

ggggattgac ttcaactggg aatacccttc ttcgaaggta acgctaatga ttcaaagcca 7080

gacctccaaa tacttagata ataagcccca gtgaagtttg cttgagagat aggggcctct 7140

ttggccagat aaaatgtaag agccttaaac acacacacat acacacccac tcacacacac 7200

atacacacac acacaattta agggaattgc agaacagata gcacccacca aaaggtgaaa 7260

taccaggaat tttgtcctat tctgcaatag ccaggctatg aatattagtt ttctctaggt 7320

gattacatct ttccacatta tgtcatttct ctgttctcca aagtttttga tctacattcc 7380

ttttaaggga atttctcttt aagaggtggc atgagataca ctgctcctta aacagtggtc 7440

acatttactt gtgtttctgc agtttatatc catctcactt tcaccacgtg aggttttaaa 7500

aatcctaatt cagttggttc catttatttc tcctgaaaca aaatatattt gttgtctgca 7560

tgaggttaaa agttctggtg tccctgtttt tagcattaaa taatgtttac caaagcccag 7620

atttaattct gtgtgttact agaagttatt gggtaatgtt atatgctgtg ctttggaagt 7680

tcagtcaact ctttttttca gcatcagcat aagaaacttg taaaccgaga cctaaaaacc 7740

cagtctggga gtgagatgaa gaaatttttg agcaccttaa ctatagatgg tgtaacccgg 7800

agtgaccaag gattgtacac ctgtgcagca tccagtgggc tgatgaccaa gaagaacagc 7860

acatttgtca gggtccatgg taagctatgg tcttggaaat tattctgtgc cttgacaagt 7920

gagataattt aaataaattt aggtcactta gtgattccta ttttgttcat tcagaagata 7980

gtttctagtt tttcttgtta gggaggccac atgacctaga ggtcaagagc atagctttgt 8040

agtcaggaac ttgggttcaa acctcaactt taaagatgag atgtgctgat atacagtaag 8100

agttcattta gtattactta ttatagttat tgctgctatt aggattgtta ctatgataaa 8160

tagtattagc taaggtagtt tttaaatttt cattttattg caaggctgag aggcctactt 8220

gaataagcat gagctttgca aactggggaa acatttagca atatacagtt gacctgtgag 8280

caactcaggg attgggggaa ctcaggggag ttcccctaac tttccctcct ctgcagtcaa 8340

aaatccatgt ataggccggg cgcggtggct cacgcctgta atcccaacac tttgggagtc 8400

tgaggtgggt ggatcacctg agatcaggag ttcgaaacca gcctggtcaa catggtggaa 8460

ccccatctct actaaaaatc caaaaaatta gcctggtgtg gtggtgggag cttgtaatcc 8520

cagctactca ggaggctgag gcaggagaat tgcttgaacc caggaggtgg aggttgcagt 8580

gagccaagat cgtgccattg taccccagcc tgggcaacaa gagtgaaact ccttctcaaa 8640

aaaaaaaaaa aaaaaaaaat caaggtataa cttttgactt ccacaaaaca taactaatgg 8700

cctactgttg actggaagcc ctactgataa cataaacagt caattaacac atattttata 8760

tgttatatgt attatatact gtattcttcc aataaagcta gagaaaagaa aatgttatta 8820

agaaaattgt aaggaagaga aaatatattt actattcatt aagtgtaagt ggatcatcat 8880

aaaggtcttc atccttgtct tcacgttgag taggctgagg aaaaggggga agaggagggg 8940

gtggttttgc tgtctcaggg gtggcagagg tggaagaaaa tctgcttata agtggactca 9000

tgtagttcaa gtttgtgtta tttaagggtc aactgtaatt gaactggaat taaattgaac 9060

tggccttgag aaaatcacct taattttttg tttattctct ttcatttaca taaatgtctg 9120

agtttacatg gtaatttgtg tggcatccta cttataagcc ttggaaagga ttttggagtt 9180

tatattatga gaatgcatca atacagtgaa attttaaaaa taccttagat aatgctattt 9240

attagagttg taatcataaa agtggcaaca actataacaa gtatgattta gtgagcactt 9300

actttattag ctcatctcat ctttgaagct gagattggaa ctcaagttcc tgactacaaa 9360

gctatgctct tgacctctag gtcacgtggc atccctagca agaacttgaa aatttcttct 9420

gaatgaacaa aatagaaatc actaagtgtc ctaaatttat ttaaattatt tcacttgcca 9480

agatgcactt gtcaaaatac acagagagag atgtgctctg gcttatgttt ttatagaatt 9540

acttttgttt tccagaatac ttcagggaaa taggggcaga aataaggagg tcagttggga 9600

ggctaattgc agttatccaa gtgagagttg aggggtggct tagacaaggg tagttgaggt 9660

ggaggtagtg agaggtgatc tgcttctgga tatattttga aggtagagtc aacagggtcc 9720

gctgatcaat tcattggttg tggagtataa gagaaaaaga gtggaagatg actcgagcgt 9780

tagcatgagc aactgagtaa atgatggtgt tatttactga gatggcaaag atcgagaagg 9840

cagtgagatt tagggaaaca gtgttagata tgtttatctg gagatgcctg ttaaacatcc 9900

aagtggagat atttaacata tcaacccgga acccagagga gtcagggcag aagataacac 9960

atttaggagg tacgtgaatg atactttaaa cctgaggcta gaggaaggtg taaataaaga 10020

ggaggtctga ggactgagtc ctggggcctc atggtggaag aggtgtgtgg aggctgtcat 10080

gggagcagag gagaaggagc acccaagcat ccctggggga cttagagaaa gctgcacaga 10140

ggagcaagtg tttgagttga gacttgagca atcactaggc ttgtgggagt gcactagcgg 10200

ggagagaaaa gcaaatgcaa acacaggagg tgtgggagaa acacgggagg tgtgggagaa 10260

gctgaaaagt gacccactga aagatagtac aggaaatctt ggaactgcag ctactcagac 10320

cctcaaggtc tttgacgttt cacttgaaat gaaaaactaa atcaaatgac catttacagt 10380

aagttgacct tttttttttt ttattttctt ccagaaaaac cttttgttgc ttttggaagt 10440

ggcatggaat ctctggtgga agccacggtg ggggagcgtg tcagaatccc tgcgaagtac 10500

cttggttacc cacccccaga aataaaatgg taactactgg aaataaatgc aaagcatcat 10560

ttcgtgtgag agcaaatcct ttgactatac taattcctga gaattttttt tcataggtat 10620

aaaaatggaa taccccttga gtccaatcac acaattaaag cggggcatgt actgacgatt 10680

atggaagtga gtgaaagaga cacaggaaat tacactgtca tccttaccaa tcccatttca 10740

aaggagaagc agagccatgt ggtctctctg gttgtgtatg gtgagtccat tcaattttcc 10800

tctctgccca agatttatta tgatacattg tcttccaaat cagccaaacc accgttcctc 10860

tgcctcctgc tgcttcactc atatcatggc tgggcctgcg tacaaaagtc atctggcgtg 10920

gtgaagctga agtgaaacgt aggaccatgt gctctggcca tgtttgttta agaggccgtg 10980

taaatgagct ttgtggtgga caaatgcaag attaaagtag tgataccctc gatagctaaa 11040

tgttgtgaaa taagaatgcc cacagggaca gttgtcaagc taagttatac taccatgttc 11100

ccctctcatg gaattgccca cctggtacac agatgtgtaa gacccttctc cttagatttt 11160

gtgcaaagct tctagtttga tgttgtagtt gatgtatcag agatgtgcag gcacgttcca 11220

actctgaagg cttttgaagt tgacactgtt ggcttggttg ggagcttttc ttttttcctt 11280

tttgacagga gttcaggatc tgattttgag tctgtaaagg aaagatagta agtttttgat 11340

gtaaagataa tttgaacttt gttttctgaa actgaaaggt acaaataagt gtttggaatg 11400

gagtggggag aagggtgcca tggtcaagtg agtgtgagag gtgctaaggt gatgtgtaga 11460

tgtgtaacag gtttctttat tgcaggactt cgcagaacct tttatatgct aatgtatatt 11520

ggtattctcc aggaggagag acatagagta ttcaaggttt aacaaaccta tttgaccaga 11580

gcaccttttt tcccctgagc aaattcatta atctctcact ccaaacagtt tgagaaatgc 11640

ttctctgttg taattctttg ttcccccttc tggtacggca tattaaaact tcaggatatt 11700

ttcccatgac attaaggtgc ttccctacgt gtcctgatac tcttctgtag gccgctgaac 11760

ttggctttat tatttttttt cagggaatat tttaaagata ggctgggtgc cgtggtttgc 11820

atctgtaatc ccagcacttt gggaggccga ggcggatgga tcacctgagg tcaggagttc 11880

gagaccagcc tggccaacat gatgaaaacc cgtctctact aaaaatataa aaattagcca 11940

ggcatggtgg tgggcacctg taatcccagc tacttgggag gctgaggcag gagaatcact 12000

tgaacccagg aggtggaggt tgcagatagc cgagatcgca ccattgtact ccagcctggt 12060

gacaagagca aaactccgtc tcaaaaaaaa agttaacagg ttccaaaaag gttgtttaga 12120

agcagcatag gtgtagggga ctggggagag gagaaactgg aaagtgtata agtaggatgg 12180

gaggaggaaa tgaacaggaa ataaaaacaa aacacggaca gcaaatagcc catttcatca 12240

gttcatgaag ccactaaata ttttattcac tttagcaaat tctctgctat atgaaataaa 12300

cataaaaaag aagtcaagtc ttcaaagcat aatctgaggc tttaggttga cagtaataag 12360

gaaatagttt tgactttgga gtcaaaaaag aaagaaagga aaaagggaga gaagaaagaa 12420

ggaagtgaga gaagggagaa ggaagaaagg ggaagaggga aagggagtgg agagggaggg 12480

agggaggaag agggagagag aatgaaaaac tcagatgatg gtggcaggaa tgcattctct 12540

aaagatttac accttccttt aacatgaggt ggtttacgtg tttgggttca gaagtcagag 12600

tgtctaggtt tgttccaggt tttgccgttc gttaactgag tgaccttggg cgagtcattt 12660

ttttctgttt catttttttc tcacgtataa agctgtggac agtaatagtg gttgtgagga 12720

ttaagtgaat gaattcatgc aaagcacttc aaacaatgct tggcacataa taaatgtatt 12780

tactgtgcta tttcagctgt tttctgtagc ctttccctga tctcctaaac ttgagaggac 12840

agagagaact atctctgtaa tacagatgag aggcacagga tttcaacact tccataaagt 12900

cattcagctt gttagtttat tattattatt agcttattgt catttttatt ttatttcgtt 12960

actttattcc tttttttttt ttttggtaga gatggggtct caccatgtgg cccaggctgg 13020

tcttgatctc ctgggcttaa gcgatccacc taccttggcg tcccaaaata ctgagattac 13080

aggcataagc ccccatgcct ggctagttgt tatttttatg agtatcacta gaactcaggt 13140

ctcttgtttc cacatctagg tgttcttcga aaaagaaagt ggaagcaaaa tcatatgctt 13200

aaagaaagtc agctttagtt gctaaaatcc tctatttccc attcttcaaa gctgactgac 13260

aattcaaaag ttgtttttcc catcttcagt cccaccccag attggtgaga aatctctaat 13320

ctctcctgtg gattcctacc agtacggcac cactcaaacg ctgacatgta cggtctatgc 13380

cattcctccc ccgcatcaca tccactggta ttggcagttg gaggaagagt gcgccaacga 13440

gcccaggtga gtaaggccac atgctctttg ctttcctgcc atcttgcatt tcttacagct 13500

gagctatgat atgactccat cctaaatgga gaagcctaaa ccaaaaaaag ttttctctca 13560

agaggtagcc tgaatctcca tccatctttc tctgtgtctt acattttagg ggatgtcttt 13620

gcttggagta tcctcctttg gggttagcta agctcagcct tgttaggtta gccgtgaggt 13680

acacttctcc aaacacaggc tatttgctca gtttgctaat tgccagtctt tggtttttct 13740

cccgatacca atcggctggt gaataccaca tccctccttc ttgtgtgtgt gaagatccat 13800

ctctcagagg aaatgctgat agatgagagg cagtgataga cccagcccca gtcctcaggg 13860

tctcaggccc agcttatcat gctctgacac aagtccagac atccttaggg aaaaacacaa 13920

caacagcagc caacccacca ccaccctaag cagtccactt cctgttgttg tttttgaaat 13980

ggccactatg agcttcttcc tcagctgctg atcatttcct tcacagagac catggtccca 14040

gagaaattac tttaaggagc ccagtggctt ctaagtttcc ttgccttcct ttgaactaaa 14100

ttaacttgaa ttgtcttgtc gatccaattt atgaatgaag gtttattccc agaatagctg 14160

cttccctcct gtatcctgaa tgaatctacc tagaaccttt tccttcattg tcaatgccta 14220

tttttaattg gcgccaagtc ttgtaccatg gtaggctgcg ttggaagtta tttctaagaa 14280

cagaataacc aaagtctgaa tcttttcctt actcttgact ctaattaaag aaaaattaaa 14340

tcataatatg cgctgttatc tctttcttat agccaagctg tctcagtgac aaacccatac 14400

ccttgtgaag aatggagaag tgtggaggac ttccagggag gaaataaaat tgaagttaat 14460

aaaaatcaat ttgctctaat tgaaggaaaa aacaaagtga gtttgaagtt ttaaaatttg 14520

aaaatctctc tctctttaat ggaaggatgg tacaataata tgtgaggcat attggagatt 14580

aataatcaaa tagtctggat gattaaatag agcgtattaa gtcactttga aaataccatt 14640

gacttttagc agtaccatta acttattaat agcttatcag agaaaaataa aaacatctat 14700

gacattaaat ctatgcatct gtgtagggtg attctgattt tataaacatg agaatgaaaa 14760

aatgtgtatc atatcatatt aaaacacatc attagtttca tggcttccaa agcccttttt 14820

atataatgtg tgagctccac agcagcataa ttatacaaat tgagtaaata tcccaaacct 14880

aaaaacccca aatccaaaat gctccagatt ctgaaccttt ttgagtgccg acatggtgct 14940

caaaggaaac gctcgttgga gcattttgga ttttcagatt agggatgctc aactggtaag 15000

tatacaatgc aaatattcca aaatccaaaa aaaaaaatcc aaaatccaaa ccacttttgg 15060

tcccaagcgt tttgagtaag ggatactcaa cctgcaattg cataaatttg agcgtgtcca 15120

accgctgcag aagtgggaat ggcataggca ggttggagtg attgtggaga ctgctggact 15180

gagtgcttgt gcacaaacag ccgcgttgtt tatggcctgg gatttgtttt ttccccgcac 15240

agactgtaag tacccttgtt atccaagcgg caaatgtgtc agctttgtac aaatgtgaag 15300

cggtcaacaa agtcgggaga ggagagaggg tgatctcctt ccacgtgacc agtaagtact 15360

cttctctgga ggtttgggtt ggatcactca cacagtgggt actaagctat gtaattccct 15420

gttgtttttg ccattcatgt gagtggcatg gcatttagga aagaggactt ggattgatca 15480

ttgatgcttt cattcataaa ttacaacttc tcaggtatct cctgggctta tgtgaagtca 15540

gtgcgtctaa ctacactgga gagagaatgg tttcacagat gctttaaacc acaagctctg 15600

tgtggtattt acatctcagt cttcagagtc tggcacagtg cctggcttat tgagcttcag 15660

tacatattgg tgggcttgct gtggaacagt tgatgagggt gggctttatg gaggcaatca 15720

gaaggacata ggagcagtgc cctcccaatg ctgccgattt tgcctgtgca tcttagtttt 15780

atggataagc tttagctgat tgtgctgaat ggaatattat agccagggct aattcattgg 15840

cataaatgta gctttcatat cattgagtgt tagtgttaat gaagacctaa ttttaaaatt 15900

ctgttagaat tagagatttt gctttggatt tttaatatat taaacattgc gtagagctca 15960

tagtggagat gtggtaaata tctgaggaat tcgtttacat tttcaagtaa tgtgtttggc 16020

caaataagat attttgggac ctgaattgtc tagtttgttt gtcaagttgt agtacatcac 16080

ctggaacgga tagagcttca tttcttttgg tactttgtag tagtctgaaa gcagcaagat 16140

gatagtgagc tgtaccaagt taaatcacca ttcaataact atggcctctt cattttaggg 16200

ggtcctgaaa ttactttgca acctgacatg cagcccactg agcaggagag cgtgtctttg 16260

tggtgcactg cagacagatc tacgtttgag aacctcacat ggtacaagct tggcccacag 16320

cctctgccaa tccatgtggg agagttgccc acacctgttt gcaagaactt ggatactctt 16380

tggaaattga atgccaccat gttctctaat agcacaaatg acattttgat catggagctt 16440

aagaatgcat ccttgcagga ccaaggagac tatgtctgcc ttgctcaaga caggaagacc 16500

aagaaaagac attgcgtggt caggcagctc acagtcctag gtagggagac aattctggat 16560

cattgtgcag aggcagttgg aatgccttaa atgtagtgca attcaggtgc tatgcaaaga 16620

ttactgtcct ctaggagatt atgttgtaaa ctggtgcaca cttcttcacc gaaagtcctt 16680

gaggaagaaa gaagctaata ataatgaaat gatatatcga aaggagaaaa taacaaaacc 16740

tgatgatgga gtaattcact agtatatgca agggatatta gcttgaacca gggaaacttc 16800

tgccttatct tgggcatcca tttatttaaa tagacaaata tttgtggaat gcctgctatg 16860

agctaggaga gtgtcagaaa ttcacagtgg taaacatgaa ggaaaggagg agaacatagg 16920

caaccactgg gaagtcacag cacagtgagg tctctgtgtc catgagaaca ggaattgttc 16980

tctgttttgc tccctgctat agctctagtc atagagcata gcagcatata ctaactgctc 17040

aataaggcac ctgctgcatg aagagtggga tgatgggctg cgtttaagac ctagaagact 17100

ccatgggaag gaagctacat tcactgtctg tacctctggg tcatcccaca tgatccagcg 17160

tagcccaagg tcaatgggac gatcacttca gtgagcagat agctctgtaa attcctccat 17220

agaggcactg tctacccctt gtctaacctc atgccttgtg caaaagctgg gcagccatgg 17280

ctttgtctgt gggaaaatca ggcaaatttg gggagcgtct ctttgtgcca cttctctcca 17340

ttttctcctc ttgtggtgtc cctttccaat tcctaggata tatgtgccct ctgttttttt 17400

tttactgtta ggaaggaaat tgcccaagta aattcatcta taccacagtt ttagagggta 17460

acgtcttcat cagaggcctt ggcgtatttg aagaggcacc ttctgacaga cactagcata 17520

aagttcgcta gttttaagac tcaggtgtca taataagaga tactttgggg tcaagtcatc 17580

cccagcatcc ttcaagtcac accacataga tcacatggat tttctgttgg cttgtctggc 17640

ttcaaggtta tggcagaatt gagaaagaga tgtgaagtag gctcctggcc tagctgtgcc 17700

cagaaaatat gtgctcgcag ttagctgctt tgcttcccta aggactccta acttgttttc 17760

ctaaaaccta ttcttagaaa taggctagaa tccagtacat ttgcttagac ttcaatgtag 17820

tacgctgttg aggtaatctc attttgctaa gtgttgacgt ggattttttc agcatgattc 17880

cttttgatgt tcagttggtt gggacaagat atttccacag cactttgatg atctgaagaa 17940

agaataaatc taaagtgttc ttgtacactt aaacaaatac tcatgggctt cattttcttt 18000

aaatccaaga cttcccttag ggtattgttg ttttgtttgt gttttagtgg aaatagcact 18060

gaactggtct tttagcctca ccagattctg taaacagttc aactgtttac ttagttgcag 18120

ggacatggac aagtggttta atgtcgctga acatcattta tttcatctgt gagataacgc 18180

taacagtcct attctgctca ttacataaga tcactagtga ggaacacaaa ttgtgtaaac 18240

aagttttata agaattgcca aataaatgta aggcattatt ggttgaatga tactaaaatt 18300

tggcacttcc aagagaaatt tgaagggatt ctagggtatt attgactaga atcttcatgg 18360

gagggaagtt ttcacctggg gaggctgtgt ctaattagag gaaaaatcca taaaggtgac 18420

cctgaacctt tcttttgtga tgggattacc agctagtatc actaatatga atgttaaaag 18480

ccattaatct gtttgcagtg tcctgactga cttgtttcat ttaactttac ccagtgacca 18540

gtgtattttc ccagaagtta atatatcaac aagttccttt ttactaaatt taaactgttt 18600

aaaagtttgc tgataccaga accatttcaa aagttataat tccatgttct gtgattttct 18660

ttttgtgtgt ctagagcgtg tggcacccac gatcacagga aacctggaga atcagacgac 18720

aagtattggg gaaagcatcg aagtctcatg cacggcatct gggaatcccc ctccacagat 18780

catgtggttt aaagataatg agacccttgt agaagactca ggtaaataga atttggctat 18840

cactcttggg ttgcagaact ttcccaggga tgttatctaa aaagccatat tatttcttga 18900

tgtaatgtag aaaaaaagca gtattggtgt ccatgacctg gctcatttca cagacttaga 18960

attggagtat ggggccctgt tgaattttca tgaaagccat ataggagatt agtcagcagt 19020

agatcccatg tgactctaca gagttagata atagaacaag atgaagggca gcatttatat 19080

tttctaaatt tccctgaaaa acttcacaga ctacatcatc ataaatgaga atgatcgttt 19140

tcttcctctg ttaggcattg tattgaagga tgggaaccgg aacctcacta tccgcagagt 19200

gaggaaggag gacgaaggcc tctacacctg ccaggcatgc agtgttcttg gctgtgcaaa 19260

agtggaggca ttt 19273

<210> 8

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttggatccaa ctcttttcta atttgtttct aggtttgcct agtgtttctc ttgatctgcc 60

caggctcagc atacaaaaag 80

<210> 9

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggcatgcagt gttcttggct gtgcaaaagt ggaggcattt ttcataatag aaggtcagtg 60

tgatgtcata ggctcatcag 80

<210> 10

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctggggaaaa atagatttgg tgttgggcat tgctattcaa aagcttgata tcgaattccg 60

aagttcctat tctctagaaa 80

<210> 11

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaacttcatc agtcaggtac ataatggtgg atccgatatc ctagtgacaa ggatcaaagg 60

ttcaggattt tccccagagc 80

<210> 12

<211> 5927

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gagtcctcag gaccccaaga gagtaagctg tgtttcctta gatcgcgcgg accgctaccc 60

ggcaggactg aaagcccaga ctgtgtcccg cagccgggat aacctggctg acccgattcc 120

gcggacaccg ctgcagccgc ggctggagcc agggcgccgg tgccccgcgc tctccccggt 180

cttgcgctgc gggggcgcat accgcctctg tgacttcttt gcgggccagg gacggagaag 240

gagtctgtgc ctgagaactg ggctctgtgc ccagcgcgag gtgcaggatg gagagcaagg 300

cgctgctagc tgtcgctctg tggttctgcg tggagacccg agccgcctct gtgggtttgc 360

ctagtgtttc tcttgatctg cccaggctca gcatacaaaa agacatactt acaattaagg 420

ctaatacaac tcttcaaatt acttgcaggg gacagaggga cttggactgg ctttggccca 480

ataatcagag tggcagtgag caaagggtgg aggtgactga gtgcagcgat ggcctcttct 540

gtaagacact cacaattcca aaagtgatcg gaaatgacac tggagcctac aagtgcttct 600

accgggaaac tgacttggcc tcggtcattt atgtctatgt tcaagattac agatctccat 660

ttattgcttc tgttagtgac caacatggag tcgtgtacat tactgagaac aaaaacaaaa 720

ctgtggtgat tccatgtctc gggtccattt caaatctcaa cgtgtcactt tgtgcaagat 780

acccagaaaa gagatttgtt cctgatggta acagaatttc ctgggacagc aagaagggct 840

ttactattcc cagctacatg atcagctatg ctggcatggt cttctgtgaa gcaaaaatta 900

atgatgaaag ttaccagtct attatgtaca tagttgtcgt tgtagggtat aggatttatg 960

atgtggttct gagtccgtct catggaattg aactatctgt tggagaaaag cttgtcttaa 1020

attgtacagc aagaactgaa ctaaatgtgg ggattgactt caactgggaa tacccttctt 1080

cgaagcatca gcataagaaa cttgtaaacc gagacctaaa aacccagtct gggagtgaga 1140

tgaagaaatt tttgagcacc ttaactatag atggtgtaac ccggagtgac caaggattgt 1200

acacctgtgc agcatccagt gggctgatga ccaagaagaa cagcacattt gtcagggtcc 1260

atgaaaaacc ttttgttgct tttggaagtg gcatggaatc tctggtggaa gccacggtgg 1320

gggagcgtgt cagaatccct gcgaagtacc ttggttaccc acccccagaa ataaaatggt 1380

ataaaaatgg aatacccctt gagtccaatc acacaattaa agcggggcat gtactgacga 1440

ttatggaagt gagtgaaaga gacacaggaa attacactgt catccttacc aatcccattt 1500

caaaggagaa gcagagccat gtggtctctc tggttgtgta tgtcccaccc cagattggtg 1560

agaaatctct aatctctcct gtggattcct accagtacgg caccactcaa acgctgacat 1620

gtacggtcta tgccattcct cccccgcatc acatccactg gtattggcag ttggaggaag 1680

agtgcgccaa cgagcccagc caagctgtct cagtgacaaa cccataccct tgtgaagaat 1740

ggagaagtgt ggaggacttc cagggaggaa ataaaattga agttaataaa aatcaatttg 1800

ctctaattga aggaaaaaac aaaactgtaa gtacccttgt tatccaagcg gcaaatgtgt 1860

cagctttgta caaatgtgaa gcggtcaaca aagtcgggag aggagagagg gtgatctcct 1920

tccacgtgac caggggtcct gaaattactt tgcaacctga catgcagccc actgagcagg 1980

agagcgtgtc tttgtggtgc actgcagaca gatctacgtt tgagaacctc acatggtaca 2040

agcttggccc acagcctctg ccaatccatg tgggagagtt gcccacacct gtttgcaaga 2100

acttggatac tctttggaaa ttgaatgcca ccatgttctc taatagcaca aatgacattt 2160

tgatcatgga gcttaagaat gcatccttgc aggaccaagg agactatgtc tgccttgctc 2220

aagacaggaa gaccaagaaa agacattgcg tggtcaggca gctcacagtc ctagagcgtg 2280

tggcacccac gatcacagga aacctggaga atcagacgac aagtattggg gaaagcatcg 2340

aagtctcatg cacggcatct gggaatcccc ctccacagat catgtggttt aaagataatg 2400

agacccttgt agaagactca ggcattgtat tgaaggatgg gaaccggaac ctcactatcc 2460

gcagagtgag gaaggaggac gaaggcctct acacctgcca ggcatgcagt gttcttggct 2520

gtgcaaaagt ggaggcattt ttcataatag aaggtgccca ggaaaagacc aacttggaag 2580

tcattatcct cgtcggcact gcagtgattg ccatgttctt ctggctcctt cttgtcattg 2640

tcctacggac cgttaagcgg gccaatgaag gggaactgaa gacaggctac ttgtctattg 2700

tcatggatcc agatgaattg cccttggatg agcgctgtga acgcttgcct tatgatgcca 2760

gcaagtggga attccccagg gaccggctga aactaggaaa acctcttggc cgcggtgcct 2820

tcggccaagt gattgaggca gacgcttttg gaattgacaa gacagcgact tgcaaaacag 2880

tagccgtcaa gatgttgaaa gaaggagcaa cacacagcga gcatcgagcc ctcatgtctg 2940

aactcaagat cctcatccac attggtcacc atctcaatgt ggtgaacctc ctaggcgcct 3000

gcaccaagcc gggagggcct ctcatggtga ttgtggaatt ctgcaagttt ggaaacctat 3060

caacttactt acggggcaag agaaatgaat ttgttcccta taagagcaaa ggggcacgct 3120

tccgccaggg caaggactac gttggggagc tctccgtgga tctgaaaaga cgcttggaca 3180

gcatcaccag cagccagagc tctgccagct caggctttgt tgaggagaaa tcgctcagtg 3240

atgtagagga agaagaagct tctgaagaac tgtacaagga cttcctgacc ttggagcatc 3300

tcatctgtta cagcttccaa gtggctaagg gcatggagtt cttggcatca aggaagtgta 3360

tccacaggga cctggcagca cgaaacattc tcctatcgga gaagaatgtg gttaagatct 3420

gtgacttcgg cttggcccgg gacatttata aagacccgga ttatgtcaga aaaggagatg 3480

cccgactccc tttgaagtgg atggccccgg aaaccatttt tgacagagta tacacaattc 3540

agagcgatgt gtggtctttc ggtgtgttgc tctgggaaat attttcctta ggtgcctccc 3600

cataccctgg ggtcaagatt gatgaagaat tttgtaggag attgaaagaa ggaactagaa 3660

tgcgggctcc tgactacact accccagaaa tgtaccagac catgctggac tgctggcatg 3720

aggaccccaa ccagagaccc tcgttttcag agttggtgga gcatttggga aacctcctgc 3780

aagcaaatgc gcagcaggat ggcaaagact atattgttct tccaatgtca gagacactga 3840

gcatggaaga ggattctgga ctctccctgc ctacctcacc tgtttcctgt atggaggaag 3900

aggaagtgtg cgaccccaaa ttccattatg acaacacagc aggaatcagt cattatctcc 3960

agaacagtaa gcgaaagagc cggccagtga gtgtaaaaac atttgaagat atcccattgg 4020

aggaaccaga agtaaaagtg atcccagatg acagccagac agacagtggg atggtccttg 4080

catcagaaga gctgaaaact ctggaagaca ggaacaaatt atctccatct tttggtggaa 4140

tgatgcccag taaaagcagg gagtctgtgg cctcggaagg ctccaaccag accagtggct 4200

accagtctgg gtatcactca gatgacacag acaccaccgt gtactccagc gacgaggcag 4260

gacttttaaa gatggtggat gctgcagttc acgctgactc agggaccaca ctgcgctcac 4320

ctcctgttta aatggaagtg gtcctgtccc ggctccgccc ccaactcctg gaaatcacga 4380

gagaggtgct gcttagattt tcaagtgttg ttctttccac cacccggaag tagccacatt 4440

tgattttcat ttttggagga gggacctcag actgcaagga gcttgtcctc agggcatttc 4500

cagagaagat gcccatgacc caagaatgtg ttgactctac tctcttttcc attcatttaa 4560

aagtcctata taatgtgccc tgctgtggtc tcactaccag ttaaagcaaa agactttcaa 4620

acagtggctc tgtcctccaa gaagtggcaa cggcacctct gtgaaactgg atcgaatggg 4680

caatgctttg tgtgttgagg atgggtgaga tgtcccaggg ccgagtctgt ctaccttgga 4740

ggctttgtgg aggatgcggg ctatgagcca agtgttaagt gtgggatgtg gactgggagg 4800

aaggaaggcg caagctcgct cggagagcgg ttggagcctg cagatgcatt gtgctggctc 4860

tggtggaggt gggcttgtgg cctgtcagga aacgcaaagg cggccggcag ggtttggttt 4920

tggaaggttt gcgtgctctt cacagtcggg ttacaggcga gttccctgtg gcgtttccta 4980

ctcctaatga gagttccttc cggactctta cgtgtctcct ggcctggccc caggaaggaa 5040

atgatgcagc ttgctccttc ctcatctctc aggctgtgcc ttaattcaga acaccaaaag 5100

agaggaacgt cggcagaggc tcctgacggg gccgaagaat tgtgagaaca gaacagaaac 5160

tcagggtttc tgctgggtgg agacccacgt ggctgccctg gtggcagtgt ctgagggttc 5220

tctgtcaagt ggcggtaaag gctcaggctg gtgttcttcc tctatctcca ctcctgtcag 5280

gcccccaagt cctcagtatt ttagctttgt ggcttcctga tggcagaaaa atcttaattg 5340

gttggtttgc tctccagata atcactagcc agatttcgaa attacttttt agccgaggtt 5400

atgataacat ctactgtatc ctttagaatt ttaacctata aaactatgtc tactggtttc 5460

tgcctgtgtg cttatgttaa aaaaaaaaag aaagaaagaa actgttcttt tcatttggta 5520

ccatagtgtg aagagctggg agcaatgact gttaaacatg ctatggcaca tctatttata 5580

gtctgttatg tagaacaaat gtaatatatt aaaacgttat attatatata atgaactttg 5640

tactacccac cttttgtatc agtattatgt accactagag agattacaag gctttcagca 5700

gccgctgttg ttttgttaaa gactttgaga aactcgaagg aatcctttca tggaatatgc 5760

agctatatac cctaccgtct ctctcatctc aaacggagga ggaggaggag gagtcaggta 5820

taatgtgagt gtgttctacg tgtccttgtt ctctgttctt aggaggaatg atttcatcaa 5880

atgtttatat gctttataaa ccaataaacg tattctgagt aaagaga 5927

<210> 13

<211> 1347

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Glu Ser Lys Ala Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu

1 5 10 15

Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro

20 25 30

Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr

35 40 45

Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro

50 55 60

Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser

65 70 75 80

Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn

85 90 95

Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser

100 105 110

Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser

115 120 125

Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys

130 135 140

Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser

145 150 155 160

Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg

165 170 175

Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile

180 185 190

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

195 200 205

Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr

210 215 220

Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu

225 230 235 240

Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile

245 250 255

Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu

260 265 270

Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe

275 280 285

Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu

290 295 300

Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr

305 310 315 320

Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met

325 330 335

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

340 345 350

Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly

355 360 365

Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr

370 375 380

Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu

385 390 395 400

Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val

405 410 415

Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val

420 425 430

Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr

435 440 445

Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu

450 455 460

Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr

465 470 475 480

Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys

485 490 495

Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys

500 505 510

Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr

515 520 525

Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser

530 535 540

Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln

545 550 555 560

Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser

565 570 575

Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro

580 585 590

Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr

595 600 605

Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile

610 615 620

Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr

625 630 635 640

Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val

645 650 655

Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn

660 665 670

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

675 680 685

Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn

690 695 700

Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg

705 710 715 720

Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr

725 730 735

Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe

740 745 750

Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu Val Ile Ile Leu

755 760 765

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

770 775 780

Val Leu Arg Thr Val Lys Arg Ala Asn Glu Gly Glu Leu Lys Thr Gly

785 790 795 800

Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg

805 810 815

Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp

820 825 830

Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val

835 840 845

Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr

850 855 860

Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg

865 870 875 880

Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu

885 890 895

Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu

900 905 910

Met Val Ile Val Glu Phe Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu

915 920 925

Arg Gly Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg

930 935 940

Phe Arg Gln Gly Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys

945 950 955 960

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

965 970 975

Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Ser

980 985 990

Glu Glu Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr

995 1000 1005

Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys

1010 1015 1020

Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn

1025 1030 1035 1040

Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp

1045 1050 1055

Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met

1060 1065 1070

Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile Gln Ser Asp Val

1075 1080 1085

Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser

1090 1095 1100

Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys

1105 1110 1115 1120

Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr

1125 1130 1135

Gln Thr Met Leu Asp Cys Trp His Glu Asp Pro Asn Gln Arg Pro Ser

1140 1145 1150

Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala

1155 1160 1165

Gln Gln Asp Gly Lys Asp Tyr Ile Val Leu Pro Met Ser Glu Thr Leu

1170 1175 1180

Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser

1185 1190 1195 1200

Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn

1205 1210 1215

Thr Ala Gly Ile Ser His Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg

1220 1225 1230

Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu

1235 1240 1245

Val Lys Val Ile Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu

1250 1255 1260

Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro

1265 1270 1275 1280

Ser Phe Gly Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala Ser

1285 1290 1295

Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp

1300 1305 1310

Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu Leu Lys

1315 1320 1325

Met Val Asp Ala Ala Val His Ala Asp Ser Gly Thr Thr Leu Arg Ser

1330 1335 1340

Pro Pro Val

1345

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttgctctcag atgcgacttg cc 22

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtgtatgcat gttggcagag aatag 25

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gctcgactag agcttgcgga 20

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttcactgata gcaaacgcct ctcc 24

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cactctagca gccactggag aagga 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgtagccagt aatgggctct gagac 25

<210> 20

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gacttacagc tctctgttag catctg 26

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcagaattcc acaatcacca tgaga 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> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gctataaagc agccacctca catt 24

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gatcgaattg ccagagacca tct 23

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

aggagattag tcagcagtag atccc 25

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gtctgtgagg tctgagttac gg 22

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

caggggaaac gaagtttcct ttcat 25

<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

acatgcacag tctacgccaa 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gccatgcgct ctaggatgat 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ccctgcgaag taccttggtt 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

tctcccgact ttgttgaccg 20

<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

<210> 37

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

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

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 38

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Gly Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A humanized VEGFR2 protein, wherein the humanized VEGFR2 protein comprises all or a portion of a human VEGFR2 protein.

2. The humanized VEGFR2 protein of claim 1, wherein the humanized VEGFR2 protein comprises at least 200 consecutive amino acids of the extracellular region of human VEGFR2 protein; preferably, the humanized VEGFR2 protein comprises a sequence identical to SEQ ID NO: 4, amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 or comprising the amino acid sequence of SEQ ID NO: 4, amino acid sequence shown in positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751; and/or the presence of a gas in the gas,

the humanized VEGFR2 protein comprises an amino acid sequence encoded by a part of exon 2, all of exons 3 to 14 and a part of exon 15 of human VEGFR2 gene; preferably, the polypeptide comprising SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

3. The humanized VEGFR2 protein of claim 1 or 2, wherein the amino acid sequence of the humanized VEGFR2 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 VEGFR2 gene, wherein the humanized VEGFR2 gene comprises a portion of the human VEGFR2 gene; preferably, the humanized VEGFR2 gene encodes the humanized VEGFR2 protein of any one of claims 1-3.

5. The humanized VEGFR2 gene of claim 4, wherein the humanized VEGFR2 gene comprises part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, and preferably further comprises intron 2-3 and/or intron 14-15; preferably, the humanized VEGFR2 gene comprises SEQ ID NO: 7.

6. The humanized VEGFR2 gene of claim 4 or 5, wherein the nucleotide sequence of the humanized VEGFR2 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 VEGFR2 gene, preferably comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of the human VEGFR2 gene, preferably further comprising intron 2-3 and/or intron 14-15; further preferably, said humanized VEGFR2 gene comprises SEQ ID NO: 7;

C) a nucleotide sequence encoding the humanized VEGFR2 protein of any of claims 1-3; or the like, or, alternatively,

D) the humanized VEGFR2 gene of any one of claims 4-6;

preferably, the targeting vector further comprises a 5 ' arm and/or a 3 ' arm, said 5 ' arm having at least 90% homology to NCBI accession No. NC _ 000071.6; 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-000071.6; preferably, the 3' arm is identical to SEQ ID NO: 6 or as shown in SEQ ID NO: and 6.

8. A method for constructing a non-human animal with a humanized VEGFR2 gene, wherein the non-human animal expresses a human or humanized VEGFR2 protein and/or the genome of the non-human animal comprises a portion of the human VEGFR2 gene; preferably, the non-human animal expresses a humanized VEGFR2 protein of any of claims 1-3.

9. The method of claim 8, wherein the genome of the non-human animal comprises the humanized VEGFR2 gene of any one of claims 4-6, preferably the genome of the non-human animal comprises part of exon 2, all of exons 3 to 14, and part of exon 15 of human VEGFR2 gene, preferably further comprises intron 2-3 and/or intron 14-15; more preferably, the genome of said non-human animal comprises SEQ ID NO: 7.

10. The method of construction of claim 8 or 9, comprising introducing into the non-human animal VEGFR2 locus a nucleotide sequence comprising any one of:

A) a portion of the human VEGFR2 gene, preferably comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of the human VEGFR2 gene, preferably further comprising intron 2-3 and/or intron 14-15; further preferred, comprises SEQ ID NO: 7; or the like, or, alternatively,

preferably, said introducing is an insertion or a substitution, and more preferably, said introducing is at the VEGFR2 locus of a non-human animal to replace the corresponding region of the non-human animal; it is still further preferred that all or part of exons 2 to 15 of non-human animal VEGFR2 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 of any one of claims 8-11, further comprising mating the VEGFR2 gene-humanized non-human animal with another genetically modified non-human animal, in vitro fertilization, or direct gene editing, and screening to obtain a polygenetically modified non-human animal;

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

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 that expresses the humanized VEGFR2 protein of any of claims 1-3, or that comprises the humanized VEGFR2 gene of any of claims 4-6 in its genome, or that is derived from a non-human animal obtained by the construction method of any of claims 8-13.

15. Use of a protein derived from the humanized VEGFR2 of any one of claims 1-3, the humanized VEGFR2 gene of any one of claims 4-6, the non-human animal obtained by the method of construction of any one of claims 8-13, or the cell, tissue or organ of claim 14, said use comprising:

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

D) screening, validating, evaluating or studying VEGFR2 pathway function; or the like, or, alternatively,

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