Non-human animals with humanized VEGFR2 gene and construction method and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic engineering and gene genetic modification, and specifically relates to a non-human animal with humanized VEGFR2 gene, a construction method thereof, and application in the field of biomedicine. Background Art

[0002] Angiogenesis refers to the process by which pre-existing capillaries sprout to form new blood vessels. Many important physiological and pathological processes in the human body involve the formation of new blood vessels, including embryonic development, wound healing, diabetic retinopathy, tumor infiltration and metastasis, etc. This process is stimulated 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), etc. Among them, VEGF is one of the most important factors regulating the growth and proliferation of vascular endothelial cells. It induces capillary proliferation, promotes endothelial cell proliferation and migration, enhances vascular permeability, and inhibits cell apoptosis by specifically acting on the glycosylated cell mitogen of endothelial cells. Its role depends on binding to the vascular endothelial cell-specific receptor (VEGFR), thereby activating signal transduction in the body to exert biological effects.

[0003] VEGF family members include VEGFA, VEGFB, VEGFC, VEGFD and VEGFE, among which VEGFA is the most important member and one of the most potent vascular permeability factors known so far. The VEGF receptor family has three members: VEGFR1, VEGFR2 and VEGFR3, among which VEGFR2 is one of the most important regulatory factors, mainly distributed in vascular endothelial cells and lymphatic endothelial cells, and plays a leading role in VEGF signal transduction and vascular endothelial formation. VEGFR2 forms dimers by binding to VEGFA, causing autophosphorylation, activating and transmitting cell membrane / cytoplasmic kinase cascade reaction signals to the nucleus, triggering a series of reactions such as endothelial cell proliferation, invasion, and migration, and regulating endothelial cells through anti-apoptosis and other mechanisms, promoting angiogenesis and maintaining its integrity. The VEGFA / VEGFR2 signaling pathway is the most important rate-limiting step in physiological and pathological angiogenesis, and is also an important pathway for tumor angiogenesis, which not only directly leads to the formation of tumor blood vessels, but also indirectly promotes the formation of lymphatic metastatic lesions. At present, high expression of VEGFR2 has been detected in a variety of tumor tissues, such as non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, gastric cancer, brain glioma, etc., and its expression is related to tumor staging, recurrence and prognosis. Therefore, VEGFR2 has been considered an important target for anti-tumor drugs, and can be used as a tumor prognostic indicator to assist clinical medication, providing an important basis for determining the treatment plan, evaluating efficacy and prognosis.

[0004] Experimental animal disease models are indispensable research tools for studying the causes and pathogenesis of human diseases, developing prevention and treatment technologies, and developing drugs. However, due to the differences in the physiological structure and metabolic system between animals and humans, traditional animal models cannot well reflect the real condition of the human body. It is an urgent need for the biopharmaceutical industry to establish disease models in animals that are closer to human physiological characteristics. However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, how to construct an "effective" humanized animal model that is close to human physiological characteristics for new drug development is still the biggest challenge.

[0005] In view of the great application value of the VEGFA / VEGFR2 signaling pathway in the field of tumor immunotherapy, in order to further study the relevant biological characteristics, improve the effectiveness of preclinical efficacy trials, improve the success rate of research and development, make preclinical trials more effective and minimize research and development failures, the field urgently needs to develop non-human animal models involving the VEGFA / VEGFR2 signaling pathway. In addition, the non-human animals obtained by this method can also be mated with other genetically humanized non-human animals to obtain a multi-gene humanized animal model, which is used to screen and evaluate the efficacy of human drugs and combination drugs targeting this signaling pathway. The present invention has broad application prospects in academic and clinical research. Summary of the invention

[0006] The present invention uses gene editing technology to replace homologous genes in non-human animal genomes with normal or mutant human genes, and establish non-human animals with normal or mutant genes that are closer to human physiological or disease characteristics. Gene humanization can improve and enhance cell or tissue transplantation. More importantly, due to the insertion of human gene fragments, human proteins can be expressed or partially expressed in animals, which can be used as targets for drugs that can only recognize human protein sequences, making it possible to screen anti-human antibodies and other drugs at the animal level.

[0007] Specifically, the first aspect of the present invention provides a humanized VEGFR2 protein, wherein the humanized VEGFR2 protein comprises all or part of the human VEGFR2 protein.

[0008] 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 region of the human VEGFR2 protein.

[0009] Preferably, the humanized VEGFR2 protein further comprises a portion of a non-human animal VEGFR2 protein, more preferably all or part of a signal peptide, a transmembrane region and / or a cytoplasmic region of a non-human animal VEGFR2 protein. Further preferably, it may also comprise a portion of an extracellular region of a non-human animal VEGFR2 protein.

[0010] In a specific 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 region comprises all or part of the extracellular region of the human VEGFR2 protein. Preferably, the part of the extracellular region of the human VEGFR2 protein comprises an extracellular region of the 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 the C-terminus. Further preferably, the extracellular region of the human VEGFR2 protein comprising 6 amino acid residues removed from the N-terminus and 13 amino acid residues removed from the C-terminus. The signal peptide, the transmembrane region and the cytoplasmic region are derived from non-human animals.

[0011] 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 in the extracellular region of the human VEGFR2 protein.

[0012] In a specific embodiment of the present invention, the humanized VEGFR2 protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or at least 99% identical to positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4, or comprises the amino acid sequence shown in positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4. 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 region of the human VEGFR2 protein (preferably the extracellular region of the human VEGFR2 protein with 0-20 amino acid residues removed from the N-terminus and / or C-terminus) and a portion of the non-human animal extracellular region (0-20 amino acid residues at the N-terminus and / or C-terminus).

[0013] Preferably, the humanized VEGFR2 protein comprises all or part of the amino acid sequence encoded by exons 1 to 30 of the human VEGFR2 gene. Further preferably, it comprises all or part of the amino acid sequence encoded by any one, two, three or more, or a combination of two or more consecutive exons from exons 1 to 30 of the human VEGFR2 gene. Even more preferably, it comprises all or part of the amino acid sequence encoded by exons 2 to 15 of the human VEGFR2 gene. Still further preferred, the amino acid sequence encoded by a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of the human VEGFR2 gene, wherein the portion of exon 2 of the human VEGFR2 gene comprises the nucleotide sequence of exon 2 encoding 0-3 amino acids at the N-terminus removed, or the portion of exon 2 of the human VEGFR2 gene comprises at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of the nucleotide sequence of exon 2; the portion of exon 15 of the human VEGFR2 gene comprises the nucleotide sequence of exon 15 encoding 0-4 amino acids at the C-terminus removed, or the portion of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 80, 100, 110, 119, 120, 130 or 132 bp of the nucleotide sequence of exon 15. Most preferably, it comprises an amino acid sequence encoded by a nucleotide sequence having at least 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 7 or an amino acid sequence encoded by SEQ ID NO: 7.

[0014] Preferably, the humanized VEGFR2 protein also comprises all or part of the amino acid sequence encoded by the non-human animal VEGFR2 gene, preferably exons 1 and 16-30 of the non-human animal VEGFR2 gene, and preferably also comprises an amino acid sequence encoded by part of exon 2 and / or part of exon 15.

[0015] In a specific embodiment of the present invention, the amino acid sequence of the humanized VEGFR2 protein comprises one of the following groups:

[0016] A) all or part of the amino acid sequence at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO: 4;

[0017] B) is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4;

[0018] C) differs from the amino acid sequence as shown at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, D) the amino acid sequence as shown at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4, including substitution, deletion and / or insertion of one or more amino acid residues.

[0019] In a specific embodiment of the present invention, the amino acid sequence of the humanized VEGFR2 protein comprises one of the following groups:

[0020] I) all or part of the amino acid sequence of SEQ ID NO: 13;

[0021] II) at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 13;

[0022] III) differs from the amino acid sequence of 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

[0023] IV) an amino acid sequence as shown in SEQ ID NO: 13, including substitution, deletion and / or insertion of one or more amino acid residues.

[0024] The second aspect of the present invention provides a humanized VEGFR2 gene, wherein the humanized VEGFR2 gene comprises a portion of the human VEGFR2 gene.

[0025] Preferably, the humanized VEGFR2 gene comprises all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human VEGFR2 protein. Further preferably, it comprises all or part of the nucleotide sequence encoding the extracellular region of the human VEGFR2 protein. Still further preferably, it comprises a nucleotide sequence encoding the extracellular region of the 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-terminus and / or the C-terminus. Still further preferably, it comprises a nucleotide sequence encoding the extracellular region of the 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, it comprises 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 the extracellular region of human VEGFR2 protein.

[0026] Preferably, it further comprises a nucleotide sequence encoding a human VEGFR2 signal peptide. Most preferably, it comprises a nucleotide sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 95% or at least 99% identity with positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO: 4 or a nucleotide sequence consistent with the amino acid sequence shown in positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO: 4.

[0027] Preferably, the humanized VEGFR2 gene further comprises all or part of the nucleotide sequence encoding the non-human animal VEGFR2 protein, such as a nucleotide sequence encoding the cytoplasmic region, signal peptide, transmembrane region and / or extracellular region of the non-human animal VEGFR2 protein.

[0028] Preferably, the humanized VEGFR2 gene encodes the above-mentioned humanized VEGFR2 protein.

[0029] Preferably, the humanized VEGFR2 gene comprises all or part of exons 1 to 30 of the human VEGFR2 gene. Further preferably, it comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons of exons 1 to 30 of the human VEGFR2 gene. Even more preferably, it comprises all or part of exons 2 to 15 of the human VEGFR2 gene. Still further preferred, it comprises a portion of exon 2, all of exons 3 to 14 and a portion of exon 15 of the human VEGFR2 gene, and preferably further comprises introns 2-3 and / or introns 14-15, wherein the portion of exon 2 comprises the nucleotide sequence of exon 2 excluding the nucleotide sequence encoding 0-3 amino acids at the N-terminus, or the portion of exon 2 of the human VEGFR2 gene comprises at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of the nucleotide sequence of exon 2; the portion of exon 15 comprises the nucleotide sequence of exon 15 excluding the nucleotide sequence encoding 0-4 amino acids at the C-terminus, or the portion of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 80, 100, 110, 119, 120, 130 or 132 bp of the nucleotide sequence of exon 15.

[0030] Preferably, the humanized VEGFR2 gene also comprises a portion of a non-human animal VEGFR2 gene; preferably exons 1 and 16-30 of a non-human animal VEGFR2 gene, and preferably further comprises a portion of exon 2 and / or a portion of exon 15.

[0031] Preferably, the humanized VEGFR2 gene comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95% or at least 99% identical to SEQ ID NO: 8 and / or 9, or comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or 9.

[0032] Preferably, the humanized VEGFR2 gene comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95% or at least 99% identical to SEQ ID NO: 10 and / or 11, or comprises the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

[0033] In a specific embodiment of the present invention, the humanized VEGFR2 gene comprises one of the following groups:

[0034] (A) all or part of the nucleotide sequence shown in SEQ ID NO: 7;

[0035] (B) is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:7;

[0036] (C) differs from the nucleotide sequence of SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

[0037] (D) A nucleotide sequence having the nucleotide sequence shown in SEQ ID NO: 7, including a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or inserted.

[0038] In a specific embodiment of the present invention, the nucleotide sequence of the humanized VEGFR2 gene comprises one of the following groups:

[0039] (i) the transcribed mRNA is all or part of the nucleotide sequence shown in SEQ ID NO: 12;

[0040] (ii) the transcribed mRNA is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:12;

[0041] (iii) the transcribed mRNA differs from the nucleotide sequence of SEQ ID NO: 12 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

[0042] (iv) the transcribed mRNA has the nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO: 12, including substitution, deletion and / or insertion of one or more nucleotides.

[0043] Preferably, the humanized VEGFR2 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a conventional inducing or repressing substance.

[0044] In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0045] In a third aspect of the present invention, a targeting vector is provided, wherein the targeting vector comprises any one of the following:

[0046] A) a portion of the human VEGFR2 gene, preferably comprising a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of the human VEGFR2 gene, and preferably further comprising introns 2-3 and / or introns 14-15, wherein the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of exon 2 excluding the nucleotide sequence encoding 0-3 amino acids at the N-terminus, or the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of exon 2; the portion of exon 15 of the human VEGFR2 gene comprises a nucleotide sequence of exon 15 excluding the nucleotide sequence encoding 0-4 amino acids at the C-terminus, or the portion of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 60, 70, 80, 86, 90 or 94 bp of exon 15. 100, 110, 119, 120, 130 or 132 bp nucleotide sequence; further preferably, the humanized VEGFR2 gene comprises the nucleotide sequence shown in SEQ ID NO: 7;

[0047] B) a nucleotide sequence encoding all or part of a human VEGFR2 protein, preferably comprising all or part of an extracellular region of 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 the extracellular region of a human VEGFR2 protein, more preferably comprising a nucleotide sequence encoding the amino acid sequence shown at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751 or 26-751 of SEQ ID NO:4;

[0048] C) a nucleotide sequence encoding the above-mentioned humanized VEGFR2 protein; or,

[0049] D) The humanized VEGFR2 gene described above.

[0050] Preferably, the targeting vector further comprises a 5' arm (5' homology arm) and / or a 3' arm (3' homology arm). The 5' arm is a DNA fragment homologous to the 5' end of the switch region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the VEGFR2 gene of non-human animals. Preferably, the 5' arm has at least 90% homology with the nucleotides with NCBI accession number NC_000071.6. Further preferably, the 5' arm sequence has at least 90% homology with SEQ ID NO: 5, or as shown in SEQ ID NO: 5. The 3' arm comprises a DNA fragment homologous to the 3' end of the switch region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the VEGFR2 gene of non-human animals. Preferably, the 3' arm has at least 90% homology with the nucleotides with NCBI accession number NC_000071.6. Further preferably, the 3' arm sequence has at least 90% homology with SEQ ID NO: 6, or is as shown in SEQ ID NO: 6.

[0051] Preferably, the switch region to be changed of the targeting vector is located on the non-human animal VEGFR2 gene locus. Further preferably, it is located on exons 1 to 30 of the non-human animal VEGFR2 gene. Even more preferably, it is located on exons 2 to 15 of the non-human animal VEGFR2 gene.

[0052] Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Even further preferably, the gene encoding the negative selection marker is a gene encoding a diphtheria toxin A subunit (DTA).

[0053] In a specific embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo.

[0054] In a specific embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is an Frt recombination site (a conventional LoxP recombination system may also be selected). The specific recombination system has two Frt recombination sites, which are respectively connected to both sides of the resistance gene.

[0055] In a specific embodiment of the present invention, the targeting vector further comprises a nucleotide sequence that is at least 85%, 90%, 95% or at least 99% identical to SEQ ID NO: 8 and / or 9, or comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or 9.

[0056] In a specific embodiment of the present invention, the targeting vector further comprises a nucleotide sequence that is at least 85%, 90%, 95% or at least 99% identical to SEQ ID NO: 10 and / or 11, or comprises the nucleotide sequence shown in SEQ ID NO: 10 and / or 11.

[0057] The fourth aspect of the present invention provides a cell comprising the above-mentioned targeting vector.

[0058] The fifth aspect of the present invention provides the use of the above-mentioned targeting vector or a cell comprising the targeting vector in VEGFR2 gene modification. Preferably, the application includes but is not limited to knockout, insertion or replacement.

[0059] In a sixth aspect, the present invention provides a non-human animal with humanized VEGFR2 gene, wherein the non-human animal expresses human or humanized VEGFR2 protein in vivo; or, the genome of the non-human animal contains part of the human VEGFR2 gene.

[0060] Preferably, the expression of endogenous VEGFR2 protein in the non-human animal is reduced or absent.

[0061] Preferably, the genome of the non-human animal contains a humanized VEGFR2 gene.

[0062] Preferably, a portion of the human VEGFR2 gene or a nucleotide sequence of a humanized VEGFR2 gene is operably linked to an endogenous regulatory element of a non-human animal.

[0063] The seventh aspect of the present invention provides a method for constructing a non-human animal with a humanized VEGFR2 gene, wherein the non-human animal expresses the above-mentioned human or humanized VEGFR2 protein in vivo; or, the genome of the non-human animal contains a portion of the human VEGFR2 gene.

[0064] Preferably, the genome of the non-human animal comprises a nucleotide sequence encoding a human VEGFR2 protein. It is further preferred to comprise all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of the human VEGFR2 protein. It is further preferred to comprise all or part of the nucleotide sequence encoding the extracellular region of the human VEGFR2 protein. It is further preferred to comprise a nucleotide sequence encoding the extracellular region of the 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 the C-terminus. In a specific embodiment of the present invention, it comprises a nucleotide sequence encoding the extracellular region of the 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, it comprises 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 the extracellular region of human VEGFR2 protein.

[0065] In a specific embodiment of the present invention, the genome of the non-human animal contains a nucleotide sequence encoding an amino acid sequence that is at least 80%, 85%, 90%, 95% or at least 99% identical to positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4, or a nucleotide sequence that is consistent with the amino acid sequence shown in positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO:4.

[0066] In a specific embodiment of the present invention, the genome of the non-human animal contains a nucleotide sequence encoding the amino acid shown in SEQ ID NO:13.

[0067] Preferably, the genome of the non-human animal comprises all or part of exons 1 to 30 of the human VEGFR2 gene. Further preferably, the genome comprises all or part of any one, two, three or more, or a combination of two or more consecutive exons of exons 1 to 30 of the human VEGFR2 gene. Even more preferably, the genome comprises all or part of exons 2 to 15 of the human VEGFR2 gene. Still further preferred, it comprises a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of the human VEGFR2 gene, and preferably further comprises introns 2-3 and / or introns 14-15, wherein the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of exon 2 encoding 0-3 amino acids at the N-terminal end, or the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of exon 2; the portion of exon 15 of the human VEGFR2 gene comprises a nucleotide sequence of exon 15 encoding 0-4 amino acids at the C-terminal end, or the portion of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 201, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 510, 520, 530, 540, 550, 560, 570, 580, 590 In one embodiment of the present invention, the genome of the non-human animal comprises the nucleotide sequence shown in SEQ ID NO:7.

[0068] In a specific embodiment of the present invention, the genome of the non-human animal comprises the above-mentioned human or humanized VEGFR2 gene.

[0069] Preferably, the construction method comprises introducing a nucleotide sequence comprising any of the following into the VEGFR2 locus of a non-human animal:

[0070] A) a portion of the human VEGFR2 gene, preferably comprising a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of the human VEGFR2 gene, and preferably further comprising introns 2-3 and / or introns 14-15, wherein the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of exon 2 excluding the nucleotide sequence encoding 0-3 amino acids at the N-terminus, or the portion of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of exon 2; the portion of exon 15 of the human VEGFR2 gene comprises a nucleotide sequence of exon 15 excluding the nucleotide sequence encoding 0-4 amino acids at the C-terminus, or the portion of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 60, 70, 80, 86, 90 or 94 bp of exon 15. 100, 110, 119, 120, 130 or 132 bp nucleotide sequence; further preferably, comprising the nucleotide sequence shown in SEQ ID NO: 7;

[0071] B) a nucleotide sequence encoding all or part of a human VEGFR2 protein, preferably comprising all or part of an extracellular region of a human VEGFR2 protein, preferably comprising a nucleotide sequence encoding the amino acid sequence shown at positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751 or 26-751 of SEQ ID NO: 4;

[0072] C) a nucleotide sequence encoding the above-mentioned humanized VEGFR2 protein; or,

[0073] D) The humanized VEGFR2 gene described above.

[0074] The introduction is insertion or replacement. Preferably, the insertion or replacement site is after the endogenous regulatory element of the VEGFR2 gene.

[0075] Wherein, the insertion is to place the target fragment directly between two adjacent bases without deleting nucleotides. Wherein, the target fragment is, for example, human VEGFR2 gene, humanized VEGFR2 gene, nucleotide sequence encoding human or humanized VEGFR2 protein, nucleotide sequence obtained by splicing human VEGFR2 and non-human VEGFR2 genes. Of course, it can also be a partial nucleotide sequence of the human VEGFR2 gene, preferably, exon x+1 to exon 30 of the human VEGFR2 gene is inserted adjacent to exon x of the non-human animal VEGFR2 gene; for example, exon 2 to exon 30 of the human VEGFR2 gene is inserted adjacent to exon 1 of the non-human animal VEGFR2 gene, exon 3 to exon 30 of the human VEGFR2 gene is inserted adjacent to exon 2 of the non-human animal VEGFR2 gene, exon 4 to exon 30 of the human VEGFR2 gene is inserted adjacent to exon 3 of the non-human animal, exon 4 of the non-human animal is inserted adjacent to exon 30 of the human VEGFR2 gene, and so on.

[0076] Preferably, according to the needs of the specific implementation scheme, the insertion may also include destroying the coding frame of the endogenous VEGFR2 gene of the non-human animal or destroying the coding frame of the endogenous VEGFR2 gene after the insertion sequence, and then performing the insertion operation. Alternatively, the insertion step can cause a frameshift mutation in the endogenous VEGFR2 gene and can also achieve the step of inserting the human sequence.

[0077] Further preferably, according to the needs of the specific embodiment, the insertion may also add an auxiliary sequence (such as a stop codon or a sequence containing a termination function, etc.) or other methods (such as flipping the sequence, or knocking out the sequence) after the inserted target fragment so that the endogenous VEGFR2 protein of the non-human animal after the insertion site cannot be expressed normally.

[0078] Wherein, the replacement includes replacement of corresponding positions or replacement of non-corresponding positions. The replacement of corresponding positions does not only mechanically represent the replacement of the base sites of human and non-human animal VEGFR2 genes directly corresponding to each other, but also includes the replacement of corresponding functional regions, such as replacing the nucleotide sequence encoding the extracellular region of non-human animal VEGFR2 protein with the nucleotide sequence encoding the extracellular region of human VEGFR2 protein, replacing the nucleotide sequence encoding the signal peptide of non-human animal VEGFR2 protein with the nucleotide sequence encoding the signal peptide of human VEGFR2 protein, replacing the nucleotide sequence encoding the transmembrane region of non-human animal VEGFR2 protein with the nucleotide sequence encoding the transmembrane region of human VEGFR2 protein, and replacing the nucleotide sequence encoding the cytoplasmic region of non-human animal VEGFR2 with the nucleotide sequence encoding the cytoplasmic region of human VEGFR2 protein. The nucleotide sequence of the cytoplasmic region of the protein is replaced by the nucleotide sequence encoding the signal peptide and extracellular region of the non-human animal VEGFR2 protein with the nucleotide sequence encoding the signal peptide and extracellular region of the human VEGFR2 protein, the nucleotide sequence encoding the signal peptide, extracellular region and cytoplasmic region of the non-human animal VEGFR2 protein is replaced by the nucleotide sequence encoding the signal peptide, extracellular region and cytoplasmic region of the human VEGFR2 protein, the nucleotide sequence encoding the signal peptide, extracellular region, cytoplasmic region and transmembrane region of the non-human animal VEGFR2 protein is replaced by the nucleotide sequence encoding the signal peptide, extracellular region, cytoplasmic region and transmembrane region of the human VEGFR2 protein, and the nucleotide sequence encoding the extracellular region and transmembrane region of the non-human animal VEGFR2 protein is replaced by the nucleotide sequence encoding the extracellular region and cytoplasmic region of the human VEGFR2 protein.

[0079] Preferably, the introduction into the non-human animal VEGFR2 gene locus is to replace the corresponding region of the non-human animal; more preferably, all or part of exons 2 to 15 of the non-human animal VEGFR2 gene are replaced.

[0080] Preferably, the construction method comprises inserting or replacing a nucleotide sequence encoding a human VEGFR2 protein into a non-human animal VEGFR2 locus. Further preferably, inserting or replacing a nucleotide sequence encoding a human VEGFR2 protein extracellular region into a non-human animal VEGFR2 locus. Still further preferably, inserting or replacing a nucleotide sequence encoding a human VEGFR2 protein extracellular region comprising 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 from the N-terminal and / or C-terminal regions into a non-human animal VEGFR2 locus. Still further preferably, inserting or replacing a nucleotide sequence encoding a human VEGFR2 protein extracellular region comprising 6 amino acid residues from the N-terminal and 13 amino acid residues from the C-terminal regions into a non-human animal VEGFR2 locus. Most preferably, a nucleotide sequence encoding an amino acid sequence that is at least 80%, 85%, 90%, 95% or at least 99% identical to positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO: 4, or an amino acid sequence that is consistent with positions 1-764, 14-764, 20-764, 26-764, 23-760, 20-751, 26-751 of SEQ ID NO: 4 is inserted or replaced into the VEGFR2 locus of a non-human animal.

[0081] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence encoding amino acids 1-762, 14-762, 20-762, 26-762, 23-758, 20-749 or 26-749 of the VEGFR2 gene of a non-human animal with a nucleotide sequence encoding amino acids 1-764, 14-764, 20-764, 26-764, 23-760, 20-751 or 26-751 of SEQ ID NO: 4.

[0082] Preferably, it includes inserting or replacing a partial nucleotide sequence comprising a human VEGFR2 gene into a non-human animal VEGFR2 locus. Further preferably, all or part of a nucleotide sequence comprising exons 1 to 30 of the human VEGFR2 gene is inserted or replaced into a non-human animal VEGFR2 locus. Still further preferably, all or part of a nucleotide sequence comprising any one, two, three or more, or a combination of two or more consecutive exons of exons 1 to 30 of the human VEGFR2 gene is inserted or replaced into a non-human animal VEGFR2 locus. Still further preferably, all or part of a nucleotide sequence comprising exons 2 to 15 of the human VEGFR2 gene is inserted or replaced into a non-human animal VEGFR2 locus. More preferably, a nucleotide sequence comprising part of exon 2, all of exons 3 to 14, and part of exon 15 of the human VEGFR2 gene is inserted or replaced into the non-human animal VEGFR2 locus, preferably further comprising introns 2-3 and / or introns 14-15, wherein the part of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of exon 2 encoding 0-3 amino acids at the N-terminus removed, or the part of exon 2 of the human VEGFR2 gene comprises a nucleotide sequence of at least 20, 30, 40, 50, 60, 70, 80, 86, 90 or 94 bp of exon 2, the part of exon 15 of the human VEGFR2 gene comprises a nucleotide sequence of exon 15 encoding 0-4 amino acids at the C-terminus removed, or the part of exon 15 of the human VEGFR2 gene comprises at least 20, 50, 60, 70, 86, 90 or 94 bp of exon 15. Most preferably, the nucleotide sequence comprising a nucleotide sequence having at least 80%, 85%, 90%, 95% or at least 99% identity with SEQ ID NO: 7 or the nucleotide sequence of SEQ ID NO: 7 is inserted or replaced into the VEGFR2 gene locus of a non-human animal.

[0083] In a specific embodiment of the present invention, the construction method comprises replacing all or part of exons 2 to 15 of a non-human animal VEGFR2 gene with all or part of exons 2 to 15 of a human VEGFR2 gene.

[0084] In a specific embodiment of the present invention, the construction method comprises replacing a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of a non-human animal VEGFR2 gene with a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of a human VEGFR2 gene.

[0085] In a specific embodiment of the present invention, the construction method comprises replacing a portion of exon 2, all of introns 2-3, all of introns 3 to 14, all of introns 14-15, and a portion of exon 15 of a non-human animal VEGFR2 gene with a portion of exon 2, all of introns 2-3, all of exons 3 to 14, all of introns 14-15, and a portion of exon 15 of a human VEGFR2 gene.

[0086] In a specific embodiment of the present invention, the construction method comprises replacing all or part of exons 2 to 15 of the VEGFR2 gene of a non-human animal with a nucleotide sequence shown in SEQ ID NO:7.

[0087] In a specific embodiment of the present invention, the construction method comprises replacing the nucleotide sequence encoding amino acids 26-749 of SEQ ID NO: 2 in the VEGFR2 gene of a non-human animal with the nucleotide sequence shown in SEQ ID NO: 7.

[0088] In a specific embodiment of the present invention, a cDNA sequence encoding human VEGFR2 protein is inserted into or replaced into the VEGFR2 gene locus of a non-human animal.

[0089] In a specific embodiment of the present invention, a nucleotide sequence encoding a humanized VEGFR2 protein is inserted into or replaced into the VEGFR2 gene locus of a non-human animal.

[0090] In a specific embodiment of the present invention, a nucleotide sequence comprising a humanized VEGFR2 gene is inserted into or replaced into the VEGFR2 gene locus of a non-human animal.

[0091] Preferably, the human or humanized VEGFR2 gene is regulated in the non-human animal by endogenous regulatory elements.

[0092] Preferably, the non-human animal is homozygous or heterozygous.

[0093] Preferably, at least one chromosome in the genome of the non-human animal contains a humanized VEGFR2 gene.

[0094] Preferably, at least one cell in the non-human animal expresses human or humanized VEGFR2 protein.

[0095] Preferably, non-human animals are constructed using gene editing technology, including gene targeting technology using embryonic stem cells, clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, homing endonuclease (megabase wide range ribozyme) or other molecular biology technology.

[0096] Preferably, the above-mentioned targeting vector is used for construction of non-human animals.

[0097] In a specific embodiment of the present invention, the construction method comprises introducing the above-mentioned targeting vector into non-human animal cells, culturing the cells (preferably embryonic stem cells), and then transplanting the cultured cells into the fallopian tubes of female non-human animals, allowing them to develop, and identifying and screening to obtain non-human animals with humanized VEGFR2 genes.

[0098] The construction method also includes mating, in vitro fertilization or direct gene editing of non-human animals with humanized VEGFR2 genes with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals. Preferably, the other genes include one or more of CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR or TIGIT.

[0099] The non-human animal described in any of the above aspects can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, pigs, rabbits, monkeys, etc.

[0100] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0101] 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. Even more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - (NRG) mouse, Rag 2 - / - -IL2rg - / - (RG) mice, NOD / SCID mice, or nude mice.

[0102] The eighth aspect of the present invention provides a method for constructing a multi-gene modified non-human animal, comprising the following steps:

[0103] (1) Providing the above-mentioned non-human animals or the non-human animals obtained by the above-mentioned construction methods;

[0104] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0105] Preferably, the other genetically modified non-human animals include but are not limited to non-human animals modified with one or a combination of two or more of the genes CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR or TIGIT.

[0106] Preferably, the multi-gene 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.

[0107] Preferably, each of the multiple humanized genes in the genome of the multi-gene modified non-human animal may be homozygous or heterozygous.

[0108] The ninth aspect of the present invention provides a non-human animal or its offspring obtained by the above-mentioned construction method.

[0109] The tenth aspect of the present invention provides an animal tumor-bearing or inflammation model, 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 offspring.

[0110] The eleventh aspect of the present invention provides a method for constructing a tumor-bearing or inflammation model in an animal, including the above-mentioned method for constructing a non-human animal, a non-human animal or its offspring, or a multi-gene modified non-human animal.

[0111] The twelfth aspect of the present invention provides the use of the non-human animals derived from the above-mentioned non-human animals, the non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animals or their offspring, or the multi-gene modified non-human animals constructed as above in preparing tumor-bearing or inflammatory models of animals.

[0112] In the thirteenth aspect of the present invention, a cell, tissue or organ is provided, wherein the cell, tissue or organ expresses the humanized VEGFR2 protein, or the genome of the cell, tissue or organ contains the humanized VEGFR2 gene, or the cell, tissue or organ is derived from the non-human animal, the non-human animal obtained by the construction method, the non-human animal or its offspring, or the tumor-bearing or inflammatory model. Preferably, the cell, tissue or organ cannot develop into an individual.

[0113] The fourteenth aspect of the present invention provides a tumor tissue after tumor-bearing, wherein the tumor tissue 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 its offspring, or the above-mentioned tumor-bearing or inflammation model.

[0114] In a fifteenth aspect of the present invention, a VEGFR2 gene humanized cell is provided, wherein the cell expresses human or humanized VEGFR2 protein; or, the genome of the cell comprises a portion of the human VEGFR2 gene. Preferably, the cell expresses the humanized VEGFR2 protein.

[0115] Preferably, the cell comprises the above-mentioned humanized VEGFR2 gene.

[0116] In a sixteenth aspect, the present invention provides uses of the humanized VEGFR2 protein, the humanized VEGFR2 gene, the non-human animal, the non-human animal obtained by the construction method, the non-human animal or its progeny, the tumor-bearing or inflammatory model, the cells, tissues or organs, the tumor tissue after tumor-bearing, and the cells, wherein the uses include A) use in the development of products involving the immune process of human cells; the products are preferably antibodies;

[0117] B) Use as a model system in pharmacological, immunological, microbiological or medical research;

[0118] C) Applications involving the production and use of animal experimental disease models for etiological studies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0119] D) screening, verification, evaluation or research of VEGFR2 pathway function; preferably human VEGFR2 pathway signaling mechanism; or, E) screening and evaluation of human medication and drug efficacy research. The drug is preferably an antibody or immune-related drug.

[0120] Preferably, the application is an application related to VEGFR2 gene or protein.

[0121] Preferably, the application is a non-disease diagnosis and treatment method.

[0122] In the seventeenth aspect of the present invention, a method for screening human VEGFR2-specific regulators is provided, the screening method comprising applying a regulator to an individual implanted with tumor cells and detecting tumor inhibitory activity; wherein the individual is selected from the above-mentioned non-human animals, the non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animals or their offspring, or the above-mentioned tumor-bearing or inflammatory models.

[0123] Preferably, the regulator is selected from CAR-T and drugs. Further preferably, the drug is an antibody.

[0124] Preferably, the regulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0125] Preferably, said detecting comprises measuring the size and / or proliferation rate of tumor cells.

[0126] Preferably, the detection method includes vernier caliper measurement, flow cytometry detection and / or in vivo animal imaging detection.

[0127] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0128] Preferably, the tumor cells are derived from humans or non-human animals.

[0129] Preferably, the method for screening human VEGFR2 specific modulators is not a treatment method. The method is used to screen or evaluate drugs, detect and compare the efficacy of candidate drugs to determine which candidate drugs can be used as drugs and which cannot be used as drugs, or compare the sensitivity of the efficacy of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0130] In the eighteenth aspect of the present invention, a method for evaluating an intervention regimen is provided, the evaluation method comprising implanting tumor cells into an individual, applying the intervention regimen to the individual implanted with tumor cells, and detecting and evaluating the tumor inhibition effect on the individual after the application of the intervention regimen; wherein the individual is selected from the above-mentioned non-human animals, the non-human animals obtained by the above-mentioned construction method, the above-mentioned non-human animals or their offspring, or the above-mentioned tumor-bearing or inflammatory models.

[0131] Preferably, the intervention regimen is selected from CAR-T and drug therapy. Further preferably, the drug is an antigen binding protein. The antibody binding protein is an antibody.

[0132] Preferably, the tumor cells are derived from humans or non-human animals.

[0133] Preferably, the evaluation method of the intervention plan is not a treatment method. The evaluation method detects and evaluates the effect of the intervention plan to determine whether the intervention plan has a therapeutic effect, that is, the therapeutic effect is not inevitable, but only a possibility.

[0134] The nineteenth aspect of the present invention provides 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 offspring, and the above-mentioned tumor-bearing or inflammation model in the preparation of a human VEGFR2-specific regulator.

[0135] The twentieth aspect of the present invention provides 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 offspring, the above-mentioned tumor-bearing or inflammation model in the preparation of drugs for treating tumors, angiogenesis-related diseases, embryonic development or immune-related diseases.

[0136] The non-human animal with humanized VEGFR2 gene described in the present invention can normally express human or humanized VEGFR2 protein in its body, and can be used for drug screening, drug efficacy evaluation, immune-related diseases and tumor treatment for human VEGFR2 pathway target sites, which can accelerate the new drug development process and save time and cost.

[0137] The "angiogenesis-related diseases" mentioned in the present invention include but are not limited to wound healing, diabetes, retinopathy, tumor infiltration and metastasis, etc.

[0138] The "immune-related diseases" described in the present invention include, but are not limited to, allergies, 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 disorders, etc.

[0139] The "tumor" described in 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, described leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myeloid leukemia; described lymphoma is selected from Hodgkin lymphoma and non-Hodgkin 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 macroglobulinemia; described sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In a specific embodiment of the present invention, described tumor is non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal carcinoma, gastric cancer, brain glioma.

[0140] The “all or part” mentioned in the present invention, “all” refers to the whole, and “part” refers to a part of the whole, or an individual that makes up the whole.

[0141] The "humanized VEGFR2 protein" of the present invention includes a portion derived from a human VEGFR2 protein and a portion derived from a non-human VEGFR2 protein.

[0142] 400, 450, 500, 550, 600, 650, 700, 726, 750, 800, 900, 1000, 1100, 1200, 1300 or 1356 amino acid sequences are consistent with the amino acid sequence of human VEGFR2 protein.

[0143] 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.

[0144] wherein the “humanized VEGFR2 gene” comprises a continuous or intermittent 20 bp-47000 bp nucleotide sequence that is consistent with the nucleotide sequence of the human VEGFR2 gene, preferably 20-19273 continuous or intermittent nucleotides, 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, The nucleotide sequences of 9000, 9500, 10000, 19273, 20000, 30000, 40000 and 47000 bp are consistent with the nucleotide sequences of human VEGFR2 gene.

[0145] The "exons xx to xxx" or "all of exons xx to xxx" described in the present invention include the nucleotide sequences of exons and introns therebetween, for example, the "exons 2 to 15" described include exon 2, introns 2-3, exon 3, introns 3-4, exon 4, introns 4-5, exon 5, introns 5-6, exon 6, introns 6-7, exon 7, introns 7-8, exon 8, introns 8-9, exon 9, introns 9-10, exon 10, introns 10-11, exon 11, introns 11-12, exon 12, introns 12-13, exon 13, exon 13-14 The complete nucleotide sequences of intron 1 and exon 14, introns 14-15 and exon 15.

[0146] The "intron x-xx" of the present invention refers to the intron between exon x and exon xx. For example, "intron 2-3" refers to the intron between exon 2 and exon 3.

[0147] The "locus" described in the present invention represents the position of a gene on a chromosome in a broad sense, and represents a DNA fragment on a certain gene in a narrow sense, that is, it can be a gene or a part of a gene. For example, the "VEGFR2 locus" described in the present invention represents an arbitrary DNA fragment on exons 1 to 30 of the VEGFR2 gene. In a specific embodiment of the present invention, the replaced VEGFR2 locus can be an arbitrary DNA fragment on exons 1 to 30 of the VEGFR2 gene. In a specific embodiment of the present invention, the replaced VEGFR2 locus can be an arbitrary DNA fragment on exons 2 to 15 of the VEGFR2 gene.

[0148] The "nucleotide sequence" of the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences, preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

[0149] The “more than three” mentioned in the present invention includes but is not limited to three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty etc.

[0150] The "three or more consecutive" mentioned in the present invention includes but is not limited to three consecutive, four consecutive, five consecutive, six consecutive, seven consecutive, eight consecutive, nine consecutive, ten consecutive, eleven consecutive, twelve consecutive, thirteen consecutive, fourteen consecutive, fifteen consecutive, sixteen consecutive, seventeen consecutive, eighteen consecutive, nineteen consecutive, twenty consecutive, twenty-one consecutive, twenty-two consecutive, twenty-three consecutive, twenty-four consecutive, twenty-five consecutive, twenty-six consecutive, twenty-seven consecutive, twenty-eight consecutive, twenty-nine consecutive or thirty consecutive, etc. Among them, "three or more consecutive exons from exons 2 to 15" includes three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, etc. consecutive exons, and also includes the intron nucleotide sequences in between.

[0151] The term "treating" (or "treat" or "treatment") as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treating" and the like refers to therapeutic interventions that improve the signs, symptoms, and the like of a disease or pathological state after the disease has begun to develop.

[0152] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the question. For example, "A and / or B" includes "A", "A and B" and "B". For 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".

[0153] The “comprising” or “including” described in the present invention is an open-ended writing method. When used to describe the 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 have the activity described in the present invention. In addition, those skilled in the art are aware that the methionine encoded by the start codon at the N-terminus of the polypeptide will be retained in certain practical situations (for example, when expressed in a specific expression system), but will not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0154] The term "homology" as used in the present invention refers to that, in terms of the use of amino acid sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs, while ensuring that the structures or functions are similar to those of known sequences, so that the used sequences 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.

[0155] One skilled in the art is able to determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

[0156] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as the family of Jerboas. In one embodiment, the genetically humanized non-human animal is a rodent. In one embodiment, the rodent is selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family of Muridae. In one embodiment, the genetically modified animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malvinas (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the family of Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from the group consisting of a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0157] In a specific embodiment, the non-human animal is a rodent, which is a mouse of the C57BL, C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, CBA / H strains selected from BALB / c, A, A / He, A / J, A / WySN, AKR, AKR / A, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr and C57BL / Ola.

[0158] Unless otherwise indicated, the practice of the present invention will employ 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 literature. For example: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DNGlovered., 1985); Oligonucleotide Synthesis (MJGaited., 1984); Mullisetal. US Pat. No. 4, 683, 195; Nucleic Acid Hybridization (BDHames&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RIFreshney, 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), specifically, Vols.154and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (DMWeir and CCBlackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0159] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0160] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention.

[0161] The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0163] Figure 1 : Schematic diagram of the comparison between the mouse VEGFR2 gene and the human VEGFR2 locus (not to scale);

[0164] Figure 2 : Schematic diagram of the humanization of mouse VEGFR2 gene (not to scale);

[0165] Figure 3 : Schematic diagram of VEGFR2 gene targeting strategy and targeting vector design (not to scale);

[0166] Figure 4 : Southern blot results of cells after VEGFR2 recombinant expression, where WT is the 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, and 3-F07 are clone numbers;

[0167] Figure 5 : Schematic diagram of the FRT recombination process of humanized VEGFR2 mice (not to scale);

[0168] Figure 6: Genotype identification results of F1 generation of VEGFR2 humanized mice, where WT is wild type, H2O is water control, and PC is positive control;

[0169] Figure 7 : Comparison results of human and mouse VEGFR2 protein sequences (partial), where Query is the amino acid sequence of human VEGFR2 protein, and Sbjct is the amino acid sequence of mouse VEGFR2 protein;

[0170] Figure 8 : Results of VEGFR2 mRNA detection in the embryonic lungs of C57BL / 6 wild-type mice (+ / +) and VEGFR2 gene homozygous mice (H / H). DETAILED DESCRIPTION

[0171] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0172] In each of the following examples, equipment and materials were obtained from the following companies:

[0173] XmnI, ScaI, and HindIII enzymes were purchased from NEB with catalog numbers R0194, R0122, and R0104, respectively;

[0174] Heraeus TM Fresco TM 21 Microcentrifuge was purchased from Thermo Fisher Scientific, model Fresco 21;

[0175] Mouse VEGFR2 / Flk-1 Quantikine ELISA Kit was purchased from R&D, catalog number MVR200B;

[0176] Human VEGF Receptor 2 ELISA kit was purchased from Abcam, catalog number ab213476;

[0177] C57BL / 6 mice and Flp tool mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Inspection Institutes;

[0178] BV480 Rat Anti-Mouse CD31 was purchased from BD Horizon TM , item number 565629;

[0179] PE / Cyanine7 anti-mouse CD34 Antibody was purchased from Biolegend, catalog number 128617;

[0180] APC anti-mouse CD309 (VEGFR2, Flk-1) Antibody was purchased from Biolegend, catalog number 136405;

[0181] Human VEGFR2 / KDR / Flk-1 PE-conjugated Antibody was purchased from R&D, catalog number FAB357P-025;

[0182] Zombie NIR TM Fixable Viability Kit was purchased from Biolegend, catalog number 423105;

[0183] Purified anti-mouse CD16 / 32Antibody was purchased from Biolegend, catalog number 101302;

[0184] The Trizol kit was purchased from TakaRa, catalog number 6110A.

[0185] Example 1 VEGFR2 gene humanized mice

[0186] In this embodiment, a non-human animal (such as a mouse) is modified so that the non-human animal contains a nucleotide sequence encoding a humanized VEGFR2 protein, thereby obtaining a genetically modified non-human animal that can express a humanized VEGFR2 protein. A schematic diagram of the comparison 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. Figure 1 shown.

[0187] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a portion of a human VEGFR2 protein can be introduced into the mouse endogenous VEGFR2 locus, so that the mouse expresses a humanized VEGFR2 protein. Specifically, the 18321 bp sequence from the partial sequence of exon 2 to the partial sequence of exon 15 of the mouse VEGFR2 gene can be replaced with a corresponding human DNA sequence by gene editing technology to obtain a humanized VEGFR2 gene sequence (schematic diagram as shown in Figure 2 As shown), the humanized transformation of the mouse VEGFR2 gene was achieved.

[0188] like Figure 3 The schematic diagram of the targeting strategy shown shows that the targeting vector contains homology arm sequences upstream and downstream of the mouse VEGFR2 gene, and an A fragment containing a human VEGFR2 sequence. Among them, the upstream homology arm sequence (5' homology arm, SEQ ID NO: 5) is identical to the nucleotide sequence of positions 75974488 to 75978469 of NCBI accession number NC_000071.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is identical to the nucleotide sequence of positions 75951590 to 75955601 of NCBI accession number NC_000071.6; the A fragment contains a human VEGFR2 sequence (SEQ ID NO: 7), the human VEGFR2 sequence is identical to the nucleotide sequence of positions 55101910 to 55121182 of NCBI accession number NC_000004.12; the connection between the upstream of the human VEGFR2 sequence in fragment A and the mouse VEGFR2 gene is designed to be 5'-ttggatccaactcttttctaatttgtttctaggtt tgcct agtgtttctcttgatctgcccaggctcagcatacaaaaag-3' (SEQ ID NO: 8), wherein the sequence " tgcct The last "t" in the sequence "agtgt" is the last nucleotide of the mouse, and the "a" in the sequence "agtgt" is the first nucleotide of the human; the connection between the downstream of the human VEGFR2 sequence and the mouse VEGFR2 gene is designed as

[0189] 5'-ggcatgcagtgttcttggctgtgcaaaagtggaggcattt ttcat aatagaaggtcagtgtgatgtcataggctcatcag-3' (SEQ ID NO: 9), wherein the sequence The last "t" in the sequence is the last nucleotide in the human genome. ttcatThe first "t" in " is the first nucleotide in mouse.

[0190] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the mouse gene is designed as follows:

[0191] 5'-ctggggaaaaatagatttggtgttgggcattgcta ttcaa TGATATCGAATTCCGAAGTTCCTATTCTCTAGAAA -3' (SEQ ID NO: 10), wherein the sequence " ttcaa The last "a" in the sequence is the last nucleotide in the mouse. The first "A" in the sequence is the first nucleotide of the Neo box; 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 The "C" in the sequence is the last nucleotide of the Neo box. ctagt "c" in " is the first nucleotide of mouse. In addition, a coding gene with a negative selection marker (coding gene of diphtheria toxin A subunit (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, and the expressed protein sequence is shown in SEQ ID NO: 13.

[0192] In addition, based on the amino acid sequence structure of human VEGFR2 protein and mouse VEGFR2 protein (such as Figure 7 The humanized mouse VEGFR2 sequence may include, in addition to positions 26-751 of SEQ ID NO: 4, the same amino acid residues may also come from the extracellular region and / or signal peptide, for example, the extracellular region including positions 20-764, 26-764, 23-760, 20-751 or 26-751 of SEQ ID NO: 4, or the signal peptide and extracellular region at positions 1-764, 14-764. The mRNA sequence of the modified humanized mouse VEGFR2 is shown in SEQ ID NO: 12, and the expressed protein sequence is shown in SEQ ID NO: 13.

[0193] Conventional methods can be used to construct the targeting vector, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene, and PCR and Southern Blot techniques are used to detect and confirm the integration of the exogenous gene, and the correct positive clone cells are screened. The clones identified as positive by PCR are then tested by Southern Blot (cell DNA is digested with XmnI or ScaI or HindIII and hybridized with 3 probes, and the probes and target fragment lengths are shown in Table 1). The results are as follows: Figure 4 As shown, the test results showed that 12 clones were verified to be positive by PCR. Sequencing revealed that except for 3-F07, the other 11 were positive clones without random insertions. The specific numbers were 1-A04, 1-A05, 1-C08, 1-E02, 2-B02, 2-C06, 2-F02, 2-H06, 3-A02, 3-B12, and 3-E05.

[0194] Table 1: Specific probes and target fragment lengths

[0195] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size Xgf 5'Probe 7.0kb 11.6kb ScaI 3'Probe 7.4kb 12.6kb HindIII Neo Probe — 5.9kb

[0196] Among them, the PCR assay includes the following primers:

[0197] F1: 5'-TTGCTCTCAGATGCGACTTGCC-3' (SEQ ID NO: 14),

[0198] R1: 5'-GTGTATGCATGTTGGCAGAGAATAG-3' (SEQ ID NO: 15);

[0199] F2: 5'-GCTCGACTAGAGCTTGCGGA-3' (SEQ ID NO: 16),

[0200] R2: 5'-TTCACTGATAGCAAACGCCTCTCC-3' (SEQ ID NO: 17);

[0201] Southern Blot assay includes the following probe primers:

[0202] 5'Probe:

[0203] 5'Probe-F: 5'-CACTCTAGCAGCCACTGGAGAAGGA-3' (SEQ ID NO: 18),

[0204] 5'Probe-R: 5'-TGTAGCCAGTAATGGGCTCTGAGAC-3' (SEQ ID NO: 19);

[0205] 3'Probe:

[0206] 3'Probe-F: 5'-GACTTACAGCTCTCTGTTAGCATCTG-3' (SEQ ID NO: 20),

[0207] 3'Probe-R: 5'-GCAGAATTCCACAATCACCATGAGA-3' (SEQ ID NO: 21);

[0208] Neo Probe:

[0209] Neo Probe-F: 5'-GGATCGGCATTGAACAAGAT-3' (SEQ ID NO: 22),

[0210] Neo Probe-R: 5'-CAGAAGAACTCGTCAAGAAGGC-3' (SEQ ID NO: 23).

[0211] The correct positive clone cells (black mice) screened out are introduced into the separated blastocysts (white mice) according to the techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium for a short culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), which can produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (see the schematic diagram of this process). Figure 5 ) and then mating with each other to obtain humanized VEGFR2 gene homozygous mice. The genotype of the somatic cells of the offspring mice can be identified by PCR (primers are shown in Table 2). The identification results of the exemplary F1 generation mice (Neo marker gene removed) are shown in Figure 6 Among them, the two mice numbered F1-01 and F1-02 were both positive heterozygous mice. This shows that the method can be used to construct VEGFR2 gene humanized mice that can be stably propagated and have no random insertion.

[0212] Table 2: Primer names and specific sequences

[0213]

[0214] The expression of humanized VEGFR2 protein in VEGFR2 gene humanized mice can be detected by conventional methods, such as ELISA method. According to the operating procedures of Mouse VEGFR2 / Flk-1 Quantikine ELISA Kit and Human VEGFReceptor 2ELISA instructions, the expression of VEGFR2 protein in 11-week-old male C57BL / 6 wild-type mice and VEGFR2 gene humanized homozygous mice was detected. The results showed that the expression of mouse VEGFR2 protein was detected in C57BL / 6 wild-type mice, but the humanized VEGFR2 protein was not detected; the expression of humanized VEGFR2 protein was detected in VEGFR2 gene humanized homozygous mice, but the expression of mouse VEGFR2 was not detected.

[0215] The expression of humanized VEGFR2 protein in VEGFR2 gene humanized mice can also be confirmed by flow cytometry. Specifically, 11-week-old female C57BL / 6 wild-type mouse and 12-14-day-pregnant female VEGFR2 homozygous mouse were taken, and the embryonic lung tissue of the mice was taken and stained with anti-mouse CD31 antibody BV480 Rat Anti-MouseCD31, anti-mouse CD34 antibody PE / Cyanine7 anti-mouse CD34 Antibody, anti-mouse VEGFR2 antibody APCanti-mouse CD309(VEGFR2,Flk-1) Antibody, anti-human VEGFR2 antibody Human VEGFR2 / KDR / Flk-1PE-conjugated Antibody, and Zombie NIR TM After staining with Fixable Viability Kit, Purified anti-mouse CD16 / 32 antibody, etc., flow cytometry was performed to detect the expression of VEGFR2 protein. The test results are shown in Table 3.

[0216] Table 3 Flow cytometry detection results of VEGFR2 protein

[0217]

[0218] The results showed (Table 3) that only mouse VEGFR2 protein was detected in embryonic lung tissues from C57BL / 6 wild-type mice, but no human or humanized VEGFR2 protein was detected. Humanized VEGFR2 protein could only be detected in embryonic lung tissues from homozygous VEGFR2 humanized mice (hVEGFR2). However, mouse VEGFR2 protein was detected in about 15% of endothelial cells (CD31+ cells or CD34+ cells) of hVEGFR2 mice, suggesting that the anti-human VEGFR2 antibody Human VEGFR2 / KDR / Flk-1 PE-conjugated Antibody may be a human-mouse cross antibody.

[0219] In order to further verify the expression of VEGFR2 in VEGFR2 homozygous humanized mice, the expression of human VEGFR2 mRNA in mice can be confirmed by conventional detection methods, such as RT-PCR. Specifically, 11-week-old female C57BL / 6 wild-type mice and 12-14-day-pregnant female VEGFR2 homozygous mice prepared in this example were taken, and after euthanasia by cervical dislocation, the mouse embryonic lung cells were taken, and the cell RNA was extracted according to the instructions of the Trizol kit, and the RT-PCR test was performed after reverse transcription into cDNA (primers are shown in Table 4). The test results are shown in Table 4. Figure 8 As shown: only mouse VEGFR2 mRNA was detected in C57BL / 6 wild-type mice, but no human VEGFR2 mRNA was detected; human VEGFR2 mRNA could only be detected in VEGFR2 humanized homozygous mice. Combined with the results of flow cytometry, it was shown that only humanized VEGFR2 protein could be detected in VEGFR2 humanized homozygous mice.

[0220] Table 4: RT-PCR primer names and specific sequences

[0221]

[0222] Example 2 Efficacy Verification

[0223] (Ramucirumab, the full-length heavy chain (HC) sequence is SEQ ID NO: 37, the full-length light chain (LC) sequence is SEQ ID NO: 38) is a humanized monoclonal antibody that binds to VEGFR2 and was developed by Eli Lilly and Company.

[0224] The tumor model constructed by using the VEGFR2 gene humanized mice prepared by the present invention can be used to test the efficacy of drugs targeting human VEGFR2. Specifically, the VEGFR2 gene humanized homozygous mice prepared in Example 1 were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5×105 When the tumor volume is about 100-150mm 3 Afterwards, the mice were divided into control group or treatment group (n=6 / group) according to the tumor volume. The control group was injected with PBS, and the treatment group was injected with Ramucirumab. Administration method: intraperitoneal injection (ip), administration started on the day of grouping, 2 times a week, and 6 times in total. The tumor volume was measured twice a week. After inoculation, the tumor volume of a single mouse reached 3000mm 3 The specific groups and drug administration are shown in Table 5.

[0225] Table 5: Grouping and drug administration

[0226]

[0227] The results showed that during the experiment in each group, the animals were in good health, and there was no significant difference in the weight of animals in all treatment groups (G2, G3) compared with the control group (G1). Judging from the tumor volume results, the tumors of the mice in the control group continued to grow during the experimental period, while the tumor volumes of all treatment groups showed varying degrees of reduction and / or disappearance compared with the control group, indicating that different concentrations of the anti-human VEGFR2 antibody Ramucirumab have a certain degree of tumor inhibition effect, and do not produce obvious toxic effects on animals, and have good safety. It is proved that VEGFR2 humanized mice can be used to screen drugs (such as antibody drugs) targeting human VEGFR2 and for in vivo efficacy testing. Example 3 Preparation of double humanized or multiple double humanized mice

[0228] The VEGFR2 mouse prepared by the present method can also be used to prepare a double-gene humanized or multi-gene humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice modified with genes such as CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR, TIGIT, etc. Alternatively, on the basis of humanized VEGFR2 mice, a double-gene or multi-gene modified mouse model of VEGFR2 and other genes can be obtained by separating mouse ES embryonic stem cells and gene recombination targeting technology. The homozygous or heterozygous VEGFR2 mice obtained by this method can also be mated with other genetically modified homozygous or heterozygous mice, and their offspring can be screened. According to Mendel's law of inheritance, there is a certain probability of obtaining heterozygous mice with humanized VEGFR2 and other genetically modified double or multiple genes. The heterozygous mice can then be mated with each other to obtain homozygous double or multiple genes. These double or multiple genes modified mice can be used to perform in vivo efficacy verification of targeted human VEGFR2 and other gene regulators.

[0229] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0230] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention. Sequence Listing <110> Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. <120> Non-human animals with humanized VEGFR2 gene 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 tggtaaga ttcaggcatt gtactgagag atgggaaccg gaacctgact atccgcaggg 2460 tgagaagga 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 gaagaagga gcaacaca 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 agatatcccca ttggaggaac 4020 cagaagtaaa agtgatccca gatgacagcc agacagacag tgggatggtc cttgcatcag 4080 aagagctgaa aactctggaa gacaggaaca aattatctcc atctttttggt 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 atttcaagt gttgttcttt ccaccacccg gaagtagcca catttgattt 4440 tcatttttgg aggagggacc tcagactgca aggagcttgt cctcagggca tttccagaga 4500 agatgcccat gacccaagaa tgtgttgact ctactctcttt 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 tatttagct tgtggctc ctgatgggcag aaaatctta attggttggt 5340 ttgctctcca gataatcact agccagatt cgaaattact ttttagccga ggttatgata 5400 acatctactg tatcctttag aatttacc tataaaacta tgtctactgg tttctgcctg 5460 tgtgcttatg ttaaaaaaaa aaagaaagaa agaactgtt ctttcattt ggtaccatag 5520 tgtgaagagc tgggagcaat gactgttaaa catgctatgg cacatctatt tatagtctgt 5580 tatgtagaac aaatgtatata tattaaaacg tattaatgac ttgtactac 5640 ccaccttttg tatcagtatt atgtaccact agagagatta agcagccgct 5700 gttgttttgt taagacttt gagaaactcg aaggaatcct ttcatggaat atgcagctat 5760 ataccctacc gtctctca tctcaacgg aggaggagga ggaggagtca ggtataatgt 5820 gagtgtgttc tacgtgtcct tgttctctgt tcttaggagg aatgatttca tcaatgttt 5880 atatgcttta taaaccata aacgtattct gagtaagag a 5921 <210> 2 <211> 1345 <212> PRT <213> Muscles <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 cattgtcag ggtccatgaa aaaccttttg ttgctttttgg 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 cacccagat tggtgagaaa tctctaatct ctcctgtgga ttcctaccag tacggcacca 1620 ctcaaacgct gacatgtacg gtctatgcca ttcctccccc gcatcacatc cactggtatt 1680 ggcagttgga ggaagagtgc gccaacgagc ccagccaagc tgtctcagtg acaaacccat 1740 acccttgtga agaatggaga agtgtggagg acttccaggg aggaataaa 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 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 agaatttgtgt aggcgattga 3660 aagaaggaac tagaatgagg gcccctgatt atactacacc agaaatgtac cagaccatgc 3720 tggactgctg gcacggggag cccagtcaga gacccacgtt ttcagttg gtggaacatt 3780 tgggaaatct cttgcaagct aatgctcagc aggatggcaa agactacatt gttcttccga 3840 tatcagagac ttgagcatg gagaggatt ctggactctc tctgcctacc tcacctgttt 3900 cctgtatgga ggaggaggaa gtatgtgacc ccaattcca ttatgacaac acagcaggaa 3960 tcagtcagta tctgcagaac agtaagcgaa agagccggcc tgtgagtgta aaaacatttg 4020 aagatatccc gttagagaa ccagaagtaa aagtaatccc agatgacac cagacggaca 4080 gtggtatggt tctgcctca gagagctga aactttgga agacagaacc aaattatctc 4140 catctttgg tgaatggtg cccagcaaaa gcagggagtc tgtggcatct gaggctca 4200 accagacaag cggctaccag tccggatatc actccgatga cacagacacc accgtgtact 4260 ccagtgagga agcagaactt ttaaagctga tagagattgg agtgcaacc ggtagcacag 4320 cccagattct ccagcctgac tcggggacca cactgagctc tcctcctgtt taaaaggaag 4380 catccacacc cccaactcct ggacatcaca tgagaggtgc tgctcagat tcaagtgtt 4440 gttctttcca ccagcaggaa gtagccgcat ttgattttca tttcgacaac agaaaaagga 4500 cctcggactg cagggagcca gtcttctagg catatcctgg aagaggcttg tgacccaaga 4560 atgtgtctgt gtttctccc agtgttgacc tgatcctctt tttcattcat ttaaaaaagca 4620 tttatcatgc cccctgctgc gggtctcacc atgggtttag aacaaagacg ttcaagaaat 4680 ggccccatcc tcaaagagt agcagtacct ggggagctga cacttctgta aaactagaag 4740 ataaaccagg caatgtaagt gttcgaggtg ttgaagatgg gaaggatttg cagggctgag 4800 tctatccaag aggctttgtt taggacgtgg gtcccaagcc aagccttaag tgtggaattc 4860 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 tcaaagttttc 5340 aggaaggatt ttacccttt gttcttcccc ctgtccccaa cccactctca ccccgcaacc 5400 catcagtatt ttagttattt ggcctctact ccagtaaacc tgattgggtt tgttcactct 5460 ctgaatgatt attagccaga cttcaaaatt atttatagc ccaaattata acatctattg 5520 tattatttag acttttaaca tatagagcta tttctactga ttttgccct tgttctgtcc 5580 ttttttcaa aaaagaaaat gtgtttttg tttggtacca tagtgtgaaa tgctgggaac 5640 aatgactata agacatgcta tggcacatat atttatagtc tgtttatgta gaaacaaatg 5700 tatatatta 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 Serves Ile Thr Serves Gln Serves Ala Serves Gly 965,970,975 Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Wing Pro 980,985,990 Glu Asp to Tyr Lys Phe Asp to Thr to Glu His to Cys Tyr 995 1000 1005 Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ser Ser Arg Lys Cys 1010 1015 1020 His Arg Asp Has Only Arg Asn Has Have Has Has 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 Only Pro Glu Thr with Asp Arg Val Tyr Thr with 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 <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 ctttgcggc 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 cttcactta 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 ggaagaaa 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 1920 ttatgataa cctcattgac tggatttgat tgcagacatg tattggtatg gggcatcctt 1980 taagatgag 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 tgattgttg caggttaatg gttctcagtc atgtttagaa 2280 tctacttatc aaaaaattg tttctcatt tcctgctct tctcaacaa agtaagattc 2340 cattattgaa aggcttgttt aagagcattt taactgcttg cctatgttag ggacagtgac 2400 ttatttcata ttgacaata ttatgccgat taattgaata tgaccca gttctatagc 2460 tgtctcaggg cagaccaga gcatctgtga tccagtcact ttaaatgcca tttaaaatgc 2520 ataatttgtt ggtctaggaa taacacact gtaagttta gatcacggc ccaacacaa 2580 gtctttaaca atgccaacta gcttctgaga ttcattaatg tcatttaatt accaatgttt 2640 taaaaatatg tcattaatta ctaatctat agttgtaaca gcacacatg tacatctt 2700 taggttggggt atattcaggg tggcatagct gtagactatt gcacatctgt gtaggtgagc 2760 cagtggacag ctgcctctgg ctgttctcag agggccacag tgtcacggca tggctattt 2820 gccttggctc tttgctaata ctttattgac atggcctcat ctcgttcac 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 <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 ttttgtgat taatggaagc 2820 tgactgggtt tctttcagcg gcttcttccc attgttattg gctcaatggg cacattttta 2880 cctcaataca ataacattct tgtccatttt cttgggtgg actgtgggca ttaattgatg 2940 ggagccacca gaggggtgaa aggtttggac tgtccactgt attaagatt 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 aagttttac ccgctcatct ggctctctgt taagacaatg acagatctgg tctatttaga 3420 tgatgtattc tgattttaa atattaattt tatctcttga ctttgggtaa tcatccattt 3480 agctttctag tagtaaggag cctgtgcaac catccaaagc agggcatttt gaaaagcaaa 3540 tggaaaaacc agaaaaagg aggaaagtgc tctgtgggtc taactgggat gcaccatcct 3600 cttgaccaat tggaatcttc atatgccttt tgacagtgtg aaatcaaag tgtttttgt 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 <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 gaaggcatg 840 gagccttctt aggacctgtg tgtttttggt agagactgtg acaagtggag gtgatgttac 900 cctcctggaa gagtgctggg ggtccacaaa ggaccttggg taggttattg ccattgcttc 960 atacttgttg aatactaagc attaaaccga atgacataca tctatttag actgcagtat 1020 aaagaatacc ctagcccctt accaataccc agcccttggg aaaaaacaca gtagcaggtg 1080 ctgtttctct agctttactt gtttaagaca catttcccat tagatttcc 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 gtagaaact ttgtttttt tttttgagg 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 aaaaatttc atttcccacg tctctttacc agttataaa gataggc 2640 tcaagaga attgagtaca aaagggct tgctctaaag gctgtttgcc agaggaata 2700 cacacaattc ttctctcctg aggctttc tgagaaata gactcattga ttctggagct 2760 tgggccgtgt tacctcttt ttgcccagtt agttttggttc tgatctttgt ttccaggta 2820 aatctgtgtt cactgttggc cattgagact tataaaagt cttcctatgt ttgagagaa 2880 aacctaaaat tcttgaatc gaggagat tggggtga ttatggagaa atttctgtgg 2940 aggataagt tatctacagc agagtaggag atttccca gatgcatag gaagcatttt 3000 3060 taaaaaaaa ttcccatact catgtgcaaa taagacatt gctcagact cttttcagga 3120 caatgtttct ttcctttgct tgtttgtct gagatcttgg atgatatgct gtatctttct 3180 aggatgtgca gtttgggat gatttatga aggctgactt aacatccata tagtataaaa 3240 taaatgtcac acatattctg catttataat gagttatgca ttctttgtg tttcaaaaat 3300 cttacactat cttatctttt ctgtgaaaac ctaacttaac taatgagatc cctatgatat 3420. aaatttaagg aatgtaaggg ctgcatcata gtttggttgg atgtaccaaa tattttctt ttcagtgaag ataaacagac attttatgta tttacgtata tgccttttta catcccagag tatttgagac aggtgaagat gacttagact tttttcccag aagcagcttt tacagggcaa gaatttcatc agctttggga aacacacttg catatctctg cttacatttc agtagtgtaa tatggtcagt gcaatgaaaa agtggagacc acatcaaaat aacctatgcc actggattca caatgtttga gaatatctt tgcccagagt aagcactgtc aaagatagaa ttctgtgccc tcctccttcc ctccacaaga tttgaaagag acaaggctca catcttggag aatttctggc tccttttgac ctggcagtct tgagagatgc agctcggtca gaagattgca aggatttcct 3840 gctttcagcc tgtctgaaaa tctacaaga tgaacatccc ccatatctca ttatttactt cttcctaagt caggaaactt ggagacatgt gaaaattcat ttcatgagtt tcagtaaata ttttattttg agaggctggg tggtggttttg ggttttcttt gttttttcc tttttttgag ataccgaaat agaattgatt tactaaatag gtttagtctt acgtcaaagg gttaatttag 4080 cttccaaagg cttgctctgt aagcaagtta tgtaatattt cataacatgt ggatgaaagg 4140 taggcaatat taagaagtgg caatccctag cactgtttat tggtacactg cctgtctttg 4200 ggtataccat taaattctgc ttcctgtcta agcttaaaagt tctaggagtt gggctgtcca 4260 agattttggc catgaagtta aacaatggga aaggaaacac tgaagtattc tctatggata 4320 ggtgtttaat gtcccctctg gtcgccacct tacttccta gtttctgac cccattctct 4380 tcagcaatgg atggagccag gaagtgagcc ctggcctcat aagataatgg ctatggcatg 4440 tggtgggcta gattggctgc tttctgtgc tttccagctg ggaaggaat 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 gaaatcttt tttttttttt ttttgagac ggagttttgc tcttgttgcc caggctaaag 4800 tgcagtggtg ctatctcagc tcactgcaac ttctgccccc cggttcaaat gattctcctg 4860 cctcagcctc tgaatagct gggactacag gtgcgtgcca tcatgcctgg ctaattttt 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 atttaaata ttagtaaaac ttgtcggtaa agtgtataga tttaacttta aattttaaag 5400 taaattat ccttcatttt gaaaaaatta taatgattaa tcttttaaaa tgtgaaatct 5460 aaaaatat attctgctg tcaataacc tgtgaagg agtcaatctc aattgggagt 5520 ttttttcaa aattttata cacacagata tatacacatg catgtgcatg cacaacaca 5580 cacacacaca tacacaca ccctcatgta gcacagatat ctacagcag ataatctgt 5640 ggatgccttt ggttgtgtga ggtgtccctt ccagtcattc acttgtctgg ttagagttta 5700 ggaacctgaa aaatgaccaa ctttctagt aaatactatt aactcattaa taaaactaaa 5760 tttcttcta gattacagat ctccatttat tgcttctgtt agtgaccaac atggagtcgt 5820 gtacattact gagaacaaaaaaactgt ggtgattcca tgtctcgggt ccattcaaa 5880 tctcaacgtg tcactttgtg cagtaagttg catctcctcc aatcgtct taagtttta 5940 taattttag ctaatattaa gatgggtaac ctgtttata tattcacaat gagttttaag 6000 gatcttttag gaagggtca atgcaatgaa tAaactaat tagtattctt aaaaataga 6060 tgaatttctc agtgatcatt gtacatggct ctcattttg gtactgatt aaatatttga 6120 tatgtctttt tattacccag agatacccag aaagagat tgttcctgat ggtaacagaa 6180 tttcctggga cagcaagaag ggctttacta ttcccagcta catgatcagc tatgctggca tggtcttctg tgaagcaaaa attatgatg aaagttacca gtctattatg tacatagttg tcgttgtagg tag tttcctttcc attack tagatcc ttgtattaag atcttggaga taacaacata gagtgaga ggatattga aagtatagga actcaggata tggtgttggg caattcatct gctcttctct accaaataa cccatgtgca attgaggttg tctcttttct tgccaagatt aaggaaa aagaaaactt tttaaaaaaa ggatgaaagc gaatggtatt actcgagcac attttatgaa gaattcaatg ttcagagcat tgcttgctat caattattc aattatgact attttatgga aacttcagca atttgctaaa gctggcccta ctggcctagg gctactgacc actgaaagtt tactactttt ctgtccactg ggttacaaca tctttgagat ctgtgaaggt agtgctttgt aaacctctgt tggccatttt cctggggagct accaagtatt ggtgaggcct gcagggaaa acaatgtggc atgttttaaa gttgcattac tttaaaaaat aaatctgtgc aaagttatag gcttattgc tctctcatgt tctgtttttt caatttactt gctctagggt ataggattta tgatgtggtt ctgagtccgt ctcatggaat 6960 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 agaagttat gggtaatgtt atatgctgtg ctttgggaagt 7680 tcagtcaact ctttttttca gcatcagcat aagaaacttg taaaccgaga cctaaaaacc 7740 7800 agtgaccaag gattgtacac ctgtgcagca tccagtgggc tgatgaccaa gaagaacagc 7860 acatttgtca gggtccatgg taagctatgg tcttggaaat tattctgtgc cttgacaagt 7920 gagataattt aaaataattt aggtcactta gtgattccta ttttgttcat tcaagagata 7980 gtttctagtt tttcttgtta gggaggccac atgacctaga ggtcaagagc atagctttgt 8040 agtcaggaac ttgggttcaa acctcaactt taagatgag atgtgctgat atacagtaag 8100 agttcattta gtattactta ttatagttat tgctgctatt aggattgtta ctatgataaa 8160 tagtattagc taaggtagtt tttaaatttt cattttattg taaggctgag aggcctactt 8220 gaataagcat gagctttgca aactgggggaa acatttagca atatacagtt gacctgtgag 8280 caactcaggg attgggggaa ctcaggggag ttcccctaac tttccctcct ctgcagtcaa 8340 aaatccatgt ataggccggg cgcggtggct cacgcctgta atcccacac ttggggtc 8400 tgaggtgggt ggatcacctg agatcaggag ttcgaacca gcctggtcaa catggtggaa 8460 cccatctct actaaaaatc caaaaaatta gcctgtgtg gtggtgggag cttgtaatcc 8520 cagctactca ggaggctgag gcaggagaat tgcttgacc caggaggtgg aggttgcagt 8580 gagccaagat cgtgccattg taccccagcc tgggcaacaa gagccaact ccttctcaa 8640 aaaaaaaaa aaaaaaaaat caagtataa ctttgactt ccacaaaca taactaatgg 8700 cctactgttg actggaagcc ctactgataa cataacagt cattaacac atattttata 8760 tgttatatgt attatatact gtattctctcc aaaagcta gagaaagaa atgttatta 8820 agaaaattgt aaggaga aaatatattt actattcatt aagtgtaagt ggatcatcat 8880 aaggtcttc atccttgtct tcacgttgag taggctgagg aaagggggga agaggagggg 8940 gtggttttgc tgtctcaggg gtggcagagg tggaaaaa tctgcttata agtggactca 9000 tgtagttcaa gtttgtgtta tttaagggtc aactgtaatt gaactggaat taaattgaac 9060 tggccttgag aaaatcacct taatttttg tttattctct ttcatttaca taaatgtctg 9120 agtttacatg gtaatttgtg tggcatccta cttataagcc ttggaaagga ttttggagtt 9180 tatattatga gaatgcatca atacagtgaa atttaaaaa 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 ttaaatttatt 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 aagggtcc 9720 gctgatcaat tcattggttg tggagtataa gagaaaaaga gtggaagatg actcgagcgt 9780 tagcatgagc aactgagtaa atgatggtgt tattactga gatggcaag atcgagaagg 9840 cagtgagatt tagggaaca gtgttagata tgtttatctg gagatgcctg ttaaaacatcc 9900 aagtggagat atttacata tcaacccgga acccagagga gtcagggcag agatacac 9960 atttaggagg tacgtgaatg atactttaaa cctgaggcta gaggaggtg taaataaga 10020 ggaggtctga ggactgagtc ctggggcctc atggtggaag aggtgtg aggctgtcat 10080 gggagcagag gagaggagc acccaagcat ccctggggga cttagagaaa gctgcacaga 10140 ggagcaagtg ttgagttga gacttgagca atcactaggc tgtgggt gcactagcgg 10200 ggagagaaaa gcaaatgcaa accaggagg tgtgggagaa acacgggagg tgtgggagaa 10260 gctgaaaagt gacccactga aagatagtac aggaaatctt ggaactgcag ctactcagac 10320 cctcaagtc tttgacgttt cacttgaaat gaaaactaa atcaatgac catttacagt 10380 aagttgacct ttttttttttttctt ccagaaaac cttttgttgc ttttggaagt 10440 ggcatggaat ctctggtgga agccacggtg gggagcgtg tcagaatccc tgcgaagtac 10500 cttggttacc cacccccaga aataaatgg taactactgg aaataatgc aaagcatcat 10560 ttcgtgtgag agcaaatcct ttgactatac taattcctga gaattttttt tcataggtat 10620 aaaaatggaa taccccttga gtccaatcac aaattaaag cggggcatgt actgacgatt 10680 10740 aaggaagc 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 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 aaaaaataaa aaattagcca 11940 ggcatggtgg tgggcacctg taatcccagc tacttgggag gctgaggcag gagaatcact 12000 tgaacccagg aggtggaggt tgcagatagc cgagatcgca ccattgtact ccagcctggt 12060 gacaagagca aaactccgtc tcaaaaaaaa agttaacagg ttccaaaaag gttgtttaga 12120 aggagcatag gtgtagggga ctggggagag gagaaactgg aaagtgtata agtaggatgg 12180 gagggaaa tgaacaggaa ataaaacaa aacacggaca gcaaatagcc catttcatca 12240 gttcatgaag ccactaaata ttttattcac tttagcaaat tctctgctat atgaaataaa 12300 cataaaaaag aagtcaagtc ttcaaagcat aatctgaggc tttaggttga catataag 12360 gaatagttt tgactttgga gtcaaaaaag aaagaaagga aaaagggaga gaaaaagaa 12420 ggaagtgaga gaagggagaa ggagaaagg ggagaagg gaagaggga aagggagtgg agagggaggg 12480 agggagaag 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 agcttcttc tcagctgctg atcatttcct tcacagagac catggtccca 14040 gagaaattac tttaaggagc ccagtggctt ctaagttttcc ttgccttcct ttgaactaaa 14100 ttaacttgaa ttgtcttgtc gatccaattt atgaatgaag gtttattccc agaatagctg 14160 cttccctcct gtatcctgaa tgaatctacc tagaaccttt tccttcattg tcaatgccta 14220 ttttaattg 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 gattaatag agcgtattaa gtcactttga aaataccatt 14640 gacttttagc agtaccatta acttattaat agcttatcag agaaaaataa aaacatctat 14700 gacattaaat ctatgcatct gtgtagggtg attctgattt tataaacatg agaatgaaaa 14760 aatgtgtatc atatdatat 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 gtttgggtt ggatcactca cacagtggt actaagctat gtaattccct 15420 gttgttttg ccattcatgt gagtggcatg gcatttagga agaggactt ggattgatca 15480 ttgatgcttt cattcataaa ttacacttc tcaggtatct cctgggctt tgtgaagtca 15540 gtgcgtctaa ctacactgga gagagaatgg ttcacagat gctttaaacc acagctctg 15600 tgtggtattt acatctcagt cttcagagtc tggcacagtg cctggcttat tgagctcag 15660 tacatattgg tgggtgct gtggacagt tgatgaggt gggcttg gaggcaatca 15720 gaaggacata ggagcagtgc cctcccaatg ctgccgattt tgcctgtgca tcttagtttt 15780 atggataagc tttagctgat tgtgctgaat ggaatattat agccaggct aattcattgg 15840 cataaatgta gcttcatat cattgagtgt tagtgttaat gagacctaa ttttaaaatt 15900 ctgttagaat tagagatttt gctttggatt ttatatat taacattgc gtagagctca 15960 tagtggagat gtggtaaata tctgaggaat tcgtttacat tttcaagtaa tgtgttttggc 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 gaggaaa gaagctaata atatgaat gatatacga aaggaaaaaacc 16740 tgatgatgga gtaattcact agtatatgca agggatatta gcttgaacca gggaaacttc 16800 tgccttatct tgggcatcca tttatttaaa tagacaaata ttgtggaat gcctgctatg 16860 agctaggaga gtgtcagaaa ttcacagtgg taaacatgaa ggaaggagg agaacatagg 16920 caccactgg gaagtcacag cacagtgagg tctctgtgtc catgagaca ggaattgttc 16980 tctgttttgc tccctgctat agctctagtc atagagcata gcagcatata ctaactgctc 17040 aaaggcac ctgctgcatg agagtggga tgatggctg cgtttaagac ctagaagact 17100 ccatgggaag gaagctacat tcactgctg tacctctggg tcatcccaca tgatccagcg 17160 tagcccagg tcaatgggac gatcacttca gtgagcagat agctctgtaa attcctccat 17220 agaggcactg tctacccctt gtctaacctc atgccttgtg caaagctgg gcagccatgg 17280 ctttgtctgt gggaaaatca ggcaatttg gggagcgtct ctttgtgcca cttctctcca 17340 tttctcctc tgtgtgtc cctttccaat tcctaggata tatgtgccct ctgttttttt 17400 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 agaataatc 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 tctttgtga 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 <400> 8 ttggatccaa ctcttttcta atttgtttct aggtttgcct agtgtttctc ttgatctgcc 60 caggctcagc atacaaaaag 80 <210> 9 <211> 80 <212> DNA <213> Artificial Sequence <400> 9 ggcatgcagt gttcttggct gtgcaaaagt ggaggcattt ttcataatag aaggtcagtg 60 tgatgtcata ggctcatcag 80 <210> 10 <211> 80 <212> DNA <213> Artificial Sequence <400> 10 ctggggaaaa atagatttgg tgttgggcat tgctattcaa aagcttgata tcgaattccg 60 aagttcctat tctctagaaa 80 <210> 11 <211> 80 <212> DNA <213> Artificial Sequence <400> 11 gaacttcatc agtcaggtac ataatggtgg atccgatatc ctagtgacaa ggatcaaagg 60 ttcaggattt tccccagagc 80 <210> 12 <211> 5927 <212> DNA <213> 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 1080 cgaagcatca gcatagaaa cttgtaaacc gagacctaaa aacccagtct gggagtgaga 1140 tgaagaaatt tttgagcacc ttaactatagt atggtgtaac ccggagtgac caaggattgt 1200 acacctgtgc agcatccagt gggctgatga ccaagaagaa cagcacattt gtcagggtcc 1260 atgaaaaacc ttttgttgct tttggaagtg gcatggaatc tctggtggaa gccacggtgg 1320 gggagcgtgt cagaatccct gcgaagtacc ttggttaccc acccccagaa ataaaaatggt 1380 ataaaaatgg aatacccctt gagtccaatc acacaattaa agcggggcat gtactgacga 1440 ttatgggaagt gagtgaagaa gacagggaa attacactgt catccttacc aatcccattt 1500 1560 agaaatctct aatctctcct gtggattcct accagtacgg caccactca acgctgacat 1620 gtacggtcta tgccattcct cccccgcatc acatccactg gtattggcag ttggaggag 1680 agtgcgccaa cgagcccagc caagctgtct cagtgacaaa cccataccct tgtgaagaat 1740 ggagaagtgt ggaggacttc caggggaa aaaattga agttaataa atcaatttg 1800 ctctaattga aggaaaaac aaactgtaa gtaccctgt tatccaagcg gcaatgtgt 1860 cagctttgta caatgtgaa gcggtcaca aagtcgggag aggagagg gtgatctccct 1920 tccacgtgac caggggtcct gaaattactt tgcaacctga catgcagccc actgagcagg 1980 agagcgtgtc ttgtggtgc actgcagaca gatctacgtt tgagaacctc acatggtaca 2040 agcttggccc acagccctg ccaatccatg tgggagtt gcccacct gtttgcaaga 2100 acttggatac tcttggaaa ttgaatgcca ccatgttctc taatagcaca atgacattt 2160 tgatcatgga gcttaagaat gcatccttgc aggaccaagg agactatgtc tgccttgctc 2220 aagacaggaa gaccaagaaa agacattgcg tggtcaggca gctcacagtc ctagagcgtg 2280 tggcaccac gatcacagga aacctggaga atcagacgac aagtattggg gaagcatcg 2340 aagtctcatg cacggcatct gggaatcccc ctccacagat catgtggttt aagataatg 2400 agacccttgt agagactca ggcattgtat tgaaggatgg gaaccggaac ctcactatcc 2460 gcagagtgag gaaggaggac gaaggctct acacctgcca ggcatgcagt gttcttggct 2520 gtgcaaagt ggaggcattt ttcaatag aaggtgccca ggaaaagacc aacttggaag 2580 tcattatcct cgtcggcact gcagtgatg ccatgttctt ctggctcctt cttgtcattg 2640 tcctacggac cgttaagcgg gccaatgaag gggaactga gatacctac ttgtctattg 2700 tcatggatcc agatgaattg cccttggatg agcgctgtga acgcttgcct tatgatgcca 2760 gcaagtggga attccccagg gaccggctga aactaggaaa acccttggc cgcggtgcct 2820 tcggccaagt gattgaggca gacgctttg gattgacaa gattgacgact tgcaaacag 2880 tagccgtcaa gatgttgaaa gaaggcaa cacacagcga gcatcgagcc ctcatgtctg 2940 aactcagat cctcatccac attggcacc atctcaatgt ggtgaacctc ctaggcgcct 3000 gcaccaagcc gggagggcct ctcatggtga ttgtggaatt ctgcaagtttt 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 tgatttcat tttggagga gggacctcag actgcagga gcttcctc agggcattc 4500 cagagaagat gcccatgacc cagaatgtg tgacttac tctctttcc attcatttaa 4560 aagtcctata taatgtgccc tgctgtgtc tcactaccag ttaagcaa agactttcaa 4620 acagtggctc tgtcctccaa gaagtggcaa cggcacctct gtgaactgg atcgaatggg 4680 caatgctttg tgtgttgagg atgggtgaga tgtcccaggg ccgagtctgt ctaccttgga 4740 ggctttgtgg aggatgcgggg ctatgagcca agtgttaagt gtgggatgtg gactgggagg 4800 aaggaaggcg caagctcgct cggagagcgg ttggagctg cagatgcatt gtgctggctc 4860 tggtggaggt gggcttgtgg cctgtcagga aacgcaaagg cggccggcag ggttttggttt 4920 tggaaggttt gcgtgctt cacagtcggg ttacaggcga gttccctgtg gcgtttccta 4980 ctcctaatga gagttccttc cggactctta cgtgtctcct ggcctggccc division 5040 atgatgcagc ttgctccttc ctcatctc aggctgtgcc ttaattcaga acaccaaaag 5100 agaggaacgt cggcagaggc tcctgacggg gccgaagaat tgtgagaca gacagaaac 5160 tcagggtttc tgctgggtgg agacccacgt ggctgccctg gtggcagtgt ctgaggttc 5220 tctgtcaagt ggcggtaag gctcaggctg gtgttctcc tctactcca ctcctgtcag 5280 gccccaagt cctcagtatt ttagctttgt ggctcctga tggcagaaaa atctttattg 5340 gttggtttgc tctccagata atcactagcc agatttcga attacttt agccgaggtt 5400 atgataacat ctactgtatc ctttagaatt ttaacctata aaactagtc tactggttttc 5460 tgcctgtgtg cttatgttaa aaaaaaaag aaagaaagaa actgttcttt tcatttgta 5520 ccatagtgtg aagagctggg agcaatgact gttaaacatg ctatggcaca tctatttata 5580 gtctgttatg tagacaat gtatatatatt aaacgttat attatata atgaactttg 5640 tactaccac ctttgtatc agtattagt accactagag agattacaag gctttcagca 5700 gccgctgttg tttgttaaa gacttgaga aactcgaagg aatcctttca tggaatatgc 5760 agctatatac cctaccgtct ctctcatctc aaacggagga ggaggaggag gagtcaggta 5820 taatgtgagt gtgttcacg tgtccttgtt ctctgttctt aggaggaatg atttcatcaa 5880 atgtttatat gctttataaa ccaataaacg tattctgagt aaagaga 5927 <210> 13 <211> 1347 <212> PRT <213> 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 Serves Ile Thr Serves Gln Serves Ala Serves 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 Tyr Lys Asp Phe Thr Glu Glu His Ile Cys Tyr 995 1000 1005 Ser Phe Gln Val Ala Lys Gly Met Glu Phe Leu Ser Ser Arg Lys Cys 1010 1015 1020 His Arg Asp Has Only Arg Asn Has Have Has Has 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 <400> 14 ttgctctcag atgcgacttg cc 22 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 gtgtatgcat gttggcagag aatag 25 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 gctcgactag agcttgcgga 20 <210> 17 <211> twenty four <212> DNA <213> Artificial Sequence <400> 17 ttcactgata gcaaacgcct ctcc 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <400> 18 cactctagca gccactggag aagga 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <400> 19 tgtagccagt aatgggctct gagac 25 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <400> 20 gacttacagc tctctgttag catctg 26 <210> twenty one <211> 25 <212> DNA <213> Artificial Sequence <400> twenty one gcagaattcc acaatcacca tgaga 25 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two ggatcggcca ttgaacaaga t 21 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty three cagaagaact cgtcaagaag gc 22 <210> twenty four <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty four gctataaagc agccacctca catt 24 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <400> 25 gatcgaattg ccagagacca tct 23 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <400> 26 aggagattag tcagcagtag atccc 25 <210> 27 <211> twenty two <212> DNA <213> Artificial Sequence <400> 27 gtctgtgagg tctgagttac gg 22 <210> 28 <211> 25 <212> DNA <213> Artificial Sequence <400> 28 caggggaaac gaagtttcctttcat 25 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <400> 29 gacaagcgtt agtaggcaca tatac 25 <210> 30 <211> twenty four <212> DNA <213> Artificial Sequence <400> 30 gctccaattt cccacaacat tagt 24 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 acatgcacag tctacgccaa 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 gccatgcgct ctaggatgat 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <400> 33 ccctgcgaag taccttggtt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 tctcccgact ttgttgaccg 20 <210> 35 <211> twenty two <212> DNA <213> Artificial Sequence <400> 35 tcaccatctt ccaggagcga ga 22 <210> 36 <211> twenty one <212> DNA <213> Artificial Sequence <400> 36 gaaggccatg ccagtgagct t 21 <210> 37 <211> 446 <212> PRT <213> 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 <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 method for constructing a non-human animal with humanized VEGFR2 gene, characterized in that: The non-human animal expresses humanized VEGFR2 protein in vivo, and the genome of the non-human animal contains a humanized VEGFR2 gene; The non-human animal has reduced or absent endogenous VEGFR2 protein expression; The construction method comprises introducing a nucleotide sequence encoding a portion of the extracellular region of the human VEGFR2 protein into the VEGFR2 gene locus of a non-human animal; the introduction into the VEGFR2 gene locus of the non-human animal is to replace the corresponding region of the non-human animal; The amino acid sequence of the humanized VEGFR2 protein is shown in SEQ ID NO: 13; The mRNA transcribed from the humanized VEGFR2 gene is shown in SEQ ID NO: 12; The non-human animal is a mouse.

2. The construction method according to claim 1, characterized in that: The part of the extracellular region of human VEGFR2 protein comprises the amino acid sequence shown at positions 20-764 of SEQ ID NO:

4.

3. The construction method according to claim 1, characterized in that: The part of the extracellular region of human VEGFR2 protein comprises the amino acid sequence shown at positions 26-764 of SEQ ID NO:

4.

4. The construction method according to claim 1, characterized in that: The part of the extracellular region of human VEGFR2 protein comprises the amino acid sequence shown at positions 23-760 of SEQ ID NO:

4.

5. The construction method according to claim 1, characterized in that: The part of the extracellular region of human VEGFR2 protein comprises the amino acid sequence shown at positions 20-751 of SEQ ID NO:

4.

6. The construction method according to claim 1, characterized in that: The part of the extracellular region of human VEGFR2 protein comprises the amino acid sequence shown at positions 26-751 of SEQ ID NO:

4.

7. The construction method according to claim 5 or 6, characterized in that: The nucleotide sequence encoding the portion of the extracellular region of the human VEGFR2 protein includes a portion of exon 2, all of exons 3 to 14, and a portion of exon 15 of the human VEGFR2 gene.

8. The construction method according to claim 1, characterized in that: The nucleotide sequence encoding a portion of the extracellular region of human VEGFR2 protein comprises SEQ ID NO:

7.

9. The construction method according to claim 1, characterized in that: The humanized VEGFR2 gene comprises a portion of the human VEGFR2 gene, and the portion of the human VEGFR2 gene comprises the nucleotide sequence shown in SEQ ID NO:

7.

10. The construction method according to claim 1, characterized in that: The humanized VEGFR2 gene comprises the nucleotide sequence shown in SEQ ID NO:

8.

11. The construction method according to claim 1, characterized in that: The humanized VEGFR2 gene comprises the nucleotide sequence shown in SEQ ID NO:

9.

12. The construction method according to claim 1, characterized in that: Part of exon 2, all of introns 2-3, all of exons 3 to 14, all of introns 14-15, and part of exon 15 of the non-human animal VEGFR2 gene are replaced.

13. The construction method according to claim 9, characterized in that: The part of the human VEGFR2 gene or the humanized VEGFR2 gene is regulated in non-human animals by endogenous regulatory elements.

14. The construction method according to claim 1, characterized in that: The construction method includes using a targeting vector for construction.

15. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding a portion of the extracellular region of human VEGFR2 protein.

16. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding the amino acid sequence shown at positions 20-764 of SEQ ID NO:

4.

17. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding the amino acid sequence shown at positions 26-764 of SEQ ID NO:

4.

18. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding the amino acid sequence shown at positions 23-760 of SEQ ID NO:

4.

19. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding the amino acid sequence shown at positions 20-751 of SEQ ID NO:

4.

20. The construction method according to claim 14, characterized in that: The targeting vector comprises a nucleotide sequence encoding the amino acid sequence shown at positions 26-751 of SEQ ID NO:

4.

21. The construction method according to claim 14, characterized in that: The targeting vector comprises SEQ ID NO:

7.

22. The construction method according to claim 14, characterized in that: The targeting vector comprises a 5' arm and a 3' arm, wherein the 5' arm comprises a nucleotide sequence as shown in SEQ ID NO:5; and the 3' arm comprises a nucleotide sequence as shown in SEQ ID NO:

6.

23. The construction method according to claim 1, characterized in that: The construction method also includes mating, in vitro fertilization or direct gene editing of non-human animals with humanized VEGFR2 genes and other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

24. The construction method according to claim 23, characterized in that: The other genes include one or more of CD3, PD-L1, PD-1, CD47, CD27, CD28, SIRPA, GITR or TIGIT.

25. A humanized VEGFR2 gene, characterized in that: The mRNA transcribed from the humanized VEGFR2 gene is shown as SEQ ID NO:

12.

26. The humanized VEGFR2 gene according to claim 25, characterized in that The humanized VEGFR2 gene comprises a portion of the human VEGFR2 gene, and the humanized VEGFR2 gene comprises the nucleotide sequence shown in SEQ ID NO:

7.

27. A targeting vector, characterized in that The targeting vector comprises a nucleotide sequence encoding a portion of the extracellular region of human VEGFR2 protein, a 5' arm and a 3' arm; The nucleotide sequence encoding the extracellular region of human VEGFR2 protein is shown in SEQ ID NO: 7; the 5' arm is shown in SEQ ID NO: 5; and the 3' arm is shown in SEQ ID NO:

6.

28. A cell, tissue or organ, characterized in that: The genome of the cell, tissue or organ contains the humanized VEGFR2 gene of claim 25 or 26, or the cell, tissue or organ is derived from a non-human animal obtained by the construction method of any one of claims 1-24.

29. Use of the humanized VEGFR2 gene of claim 25 or 26, the non-human animal obtained by the construction method of any one of claims 1 to 24, or the cell, tissue or organ of claim 28, characterized in that: The application is a non-disease diagnosis and treatment method, and the application includes: A) Applications in product development involving immune processes in human cells; B) Use as a model system in pharmacological, immunological, microbiological or medical research; C) Applications involving the production and use of animal experimental disease models for etiological research; D) Screen, validate, evaluate or study the function of VEGFR2 pathway; or, E) Application in screening and evaluation of human drugs and drug efficacy research.

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