CN112458117A

CN112458117A - Construction method and application of CD155 gene humanized non-human animal

Info

Publication number: CN112458117A
Application number: CN202110141140.0A
Authority: CN
Inventors: 张淑金
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd; Beijing Biocytogen Co Ltd
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-03-09
Anticipated expiration: 2041-02-02
Also published as: CN112458117B

Abstract

The invention provides a construction method of a CD155 gene humanized non-human animal, which is characterized in that a nucleotide sequence for coding human CD155 protein is introduced into the genome of the non-human animal in a homologous recombination mode, the humanized CD155 protein can be normally expressed in the animal body, and the animal body can be used as an animal model for human CD155 signal mechanism research and tumor and immune disease drug screening and has important application value for the research and development of new drugs of immune targets. The invention also provides a humanized CD155 protein, a humanized CD155 gene, a targeting vector of the CD155 gene, a non-human animal obtained by the construction method and application thereof in the field of biomedicine.

Description

Construction method and application of CD155 gene humanized non-human animal

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a CD155 gene modified non-human animal model and application thereof in the field of biomedicine.

Background

CD155, also known as PVS, HVED, NECL5, TAGE4 or NECL-5, is a Nectin-likeI type transmembrane glycoprotein belonging to the PVR-related (prr) family of the immunoglobulin superfamily, an adhesion protein that promotes cell binding, binds to Poliovirus (PV) and allows it to enter susceptible cells, and is more commonly referred to as PVR (Poliovirus Receptor). CD155 is detected in monocytes, macrophages, endothelial cells, epithelial cells, CD34+ thymocytes and neurons, but is highly expressed in most tumor cells, including lung cancer, breast cancer, ovarian cancer, melanoma, and the like. Studies have shown that CD155 plays an important role in mediating metastasis of malignant tumors. In addition to acting as receptors for PV and cytomegalovirus, CD155 is involved in cell-to-cell, cell-to-matrix adhesion by interacting with CD96, Nectin 1-3 (CD 111, CD112, CD 113), CD226, and the like. Some adhesion proteins which can be combined with Nectin and Nectin-like family are expressed on NK cells and play an important role in regulating the functions of the NK cells. The receptor includes TIGIT, CD226 and CD96, CD155 is the common ligand of TIGIT, CD226 and CD96, CD226 is the CD155 co-stimulation receptor, TIGIT and CD96 are the co-inhibition receptor of CD155, and the activation or inhibition of NK cells and T cells is realized through a complex receptor/ligand and receptor/receptor balance regulation mechanism.

The experimental animal disease model is an indispensable research tool for researching etiology and pathogenesis of human diseases, developing prevention and treatment technologies and developing medicines. However, due to the differences between the physiological structures and metabolic systems of animals and humans, the traditional animal models cannot reflect the real conditions of human bodies well, and the establishment of disease models closer to the physiological characteristics of human bodies in animal bodies is an urgent need of the biomedical industry.

With the continuous development and maturation of genetic engineering technology, the replacement or substitution of animal homologous genes with human genes has been realized, and the development of humanized experimental animal models in this way is the future development direction of animal models. The gene humanized animal model is one animal model with normal or mutant gene replaced with homologous gene in animal genome and similar physiological or disease characteristics. The gene humanized animal not only has important application value, for example, the humanized animal model of cell or tissue transplantation can be improved and promoted by gene humanization, but also more importantly, the human protein can be expressed or partially expressed in the animal body due to the insertion of the human gene segment, and the gene humanized animal can be used as a target of a drug which can only recognize the human protein sequence, thereby providing possibility for screening anti-human antibodies and other drugs at the animal level. However, the production of humanized animal models can be successfully achieved without any gene or by replacing any region of any gene. In this field, it is also most important and most challenging to obtain a humanized animal model that can express a humanized protein and has functions such as antibody screening by inserting or replacing a specific sequence of a specific non-human animal gene and a specific region of a corresponding human gene according to actual needs.

Thus, since there are physiological and pathological differences between animals and humans, plus the complexity of the CD155 gene, e.g., only 59% identity between human and mouse CD155 proteins, and mouse CD155 protein does not bind human CD226, whereas human CD155 protein can bind mouse CD226, plus the complex ligand-receptor and receptor-receptor regulation mechanisms between CD155 and CD96, Nectin 1-3 (CD 111, CD112, CD 113), CD226, etc., the present application provides a method for constructing a humanized non-human animal of the CD155 gene, selecting a partial region of a specific human CD155 gene to replace a partial region of a specific animal CD155 gene.

Disclosure of Invention

In view of the complex mechanism of action of CD155 and the huge application value in the field of tumor therapy, there is an urgent need in the art to develop a non-human animal model of CD 155-associated signaling pathway in order to further explore its relevant biological properties, improve the effectiveness of preclinical drug efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research. In particular, the method comprises the following steps of,

in a first aspect of the present invention, there is provided a non-human animal humanized with a CD155 gene, whose genome includes exons 2 to 6 of the human CD155 gene, or a method for constructing the same.

Preferably, the genome of the non-human animal further comprises exon 1, exon 7 and/or exon 8 of the human CD155 gene.

In one embodiment of the present invention, the genome of the non-human animal comprises any one or a combination of two or more of exon 1 to exon 8 of the human CD155 gene.

Preferably, the genome of the non-human animal includes any one or a combination of two or more of intron 1-2 to intron 7-8 of the human CD155 gene.

In a specific embodiment of the present invention, the genome of the non-human animal comprises a portion of exon 2, all of exons 3 to 5, and a portion of exon 6 of the human CD155 gene. Preferably, it further comprises all of the introns 2 to 3 and/or all of the introns 5 to 6. Wherein, the part of No. 2 exon at least comprises the nucleotide sequence with the length of 338bp, 339bp, 340bp, 341bp, 342bp, 343bp, 344bp, 345bp, 346bp or 347bp from the 3 '-5' of No. 2 exon, and the part of No. 6 exon at least comprises the nucleotide sequence with the length of 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 15bp, 20bp, 25bp, 30bp, 35bp or 38bp from the 5 '-3' of No. 6 exon.

Preferably, the construction method comprises inserting or replacing any one or more than two exons from exon 1 to exon 8 of human CD155 gene into the CD155 locus of a non-human animal. Further preferred, comprises insertion or substitution of exon 2 to exon 6 of the human CD155 gene into the non-human animal CD155 locus. Even more preferred, this involves insertion or substitution into the non-human animal CD155 locus with a part comprising exon 2, all of exons 3 to 5 and part of exon 6 of the human CD155 gene, preferably a substitution at the corresponding position.

In a second aspect of the invention, there is provided a non-human animal humanized with a CD155 gene, which expresses a human or humanized CD155 protein, or a method of constructing the same.

Preferably, the human or humanized CD155 protein comprises an extracellular region of a human CD155 protein. Further preferably, the peptide further comprises a signal peptide, a transmembrane region and/or a cytoplasmic region.

In one embodiment of the present invention, the human or humanized CD155 protein comprises the extracellular domain of human CD155 protein from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus to 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the C-terminus.

Preferably, the method of construction comprises insertion or substitution into the non-human animal CD155 locus with a nucleotide sequence comprising an extracellular region encoding a human CD155 protein. Further preferably, the method comprises inserting or replacing the nucleotide sequence from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus to 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the C-terminus of the extracellular region encoding the human CD155 protein into the CD155 locus of the non-human animal, preferably replacing the corresponding position.

In a third aspect of the invention, there is provided a method of constructing a non-human animal humanized with a CD155 gene, said method comprising administering to the animal a peptide comprising a sequence encoding SEQ ID NO: 2, from position 29 to 332, into or in place of the non-human animal CD155 locus.

Preferably, the method of construction comprises the use of a polynucleotide comprising SEQ ID NO: 5 into or into the non-human animal CD155 locus.

Wherein said replacement to a non-human animal CD155 locus is a replacement of the nucleotide sequence of the non-human animal CD155 gene encoding SEQ ID NO: 1 from 30 to 336. Preferably, the nucleotide sequence is a nucleotide sequence that replaces a non-human animal with the sequence shown at positions 19643191 to 19652829 of NCBI accession No. NC-000073.7.

Preferably, the non-human animal is a mouse or a rat.

Preferably, the non-human animal body expresses a human or humanized CD155 protein.

Preferably, the expression of the endogenous CD155 protein in the non-human animal is reduced or absent.

Preferably, the humanized CD155 protein comprises SEQ ID NO: 2 from position 29 to 332.

In one embodiment of the invention, the humanized CD155 protein comprises SEQ ID NO: 10, or comprises an amino acid sequence identical to SEQ ID NO: 10, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 10 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 amino acid difference, or, alternatively, comprises a sequence having the amino acid sequence of SEQ ID NO: 10 comprising substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the genome of the non-human animal comprises a humanized CD155 gene, and the humanized CD155 gene comprises SEQ ID NO: 5.

In one embodiment of the invention, the mRNA transcribed from the humanized CD155 gene comprises SEQ ID NO: 9, or a nucleotide sequence identical to SEQ ID NO: 9, or a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 9 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 nucleotide, or alternatively, a nucleic acid sequence comprising a nucleotide sequence having the sequence of SEQ ID NO: 9, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the method of construction comprises inserting or replacing the nucleotide sequence of the humanized CD155 gene or the nucleotide sequence encoding the humanized CD155 protein into the non-human animal CD155 locus.

Preferably, the insertion site follows the endogenous regulatory elements of the CD155 gene.

Preferably, the human CD155 gene, the humanized CD155 protein or the nucleotide sequence encoding the human CD155 protein is regulated by CD155 endogenous regulatory elements.

The invention uses gene editing technology to construct CD155 gene humanized non-human animals, wherein the gene editing technology comprises gene targeting technology using embryonic stem cells, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

In one embodiment of the invention, the construction of the non-human animal is performed using a targeting vector.

Wherein the targeting vector comprises exons 2 to 6 of human CD155 gene. Preferably comprising part of exon 2, all of exons 3 to 5, and part of exon 6 of the human CD155 gene. Further preferably comprises a nucleotide sequence encoding the extracellular domain of human CD 155. More preferably, the polypeptide comprises a nucleotide sequence from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus to 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the C-terminus of the extracellular region encoding the human CD155 protein.

In one embodiment of the invention, the targeting vector comprises a nucleic acid sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5.

Preferably, the targeting vector further comprises a 5 'arm and/or a 3' arm.

Wherein the 5 'arm is a DNA fragment homologous to the 5' end of the transition region to be altered. Preferably, it is selected from nucleotides having at least 90% homology with NCBI accession No. NC _ 000073.7. Further preferably, the length is 4000-.

In one embodiment of the invention, the nucleotide sequence of the 5' arm is as set forth in SEQ ID NO: 3, respectively.

The 3 'arm is a DNA fragment homologous to the 3' end of the transition region to be altered. Preferably, it is selected from nucleotides having at least 90% homology with NCBI accession No. NC _ 000073.7. More preferably, the length is 2500-.

In one embodiment of the invention, the nucleotide sequence of the 3' arm is as set forth in SEQ ID NO: 4, respectively.

Preferably, the transition region to be altered is located in exon 2 to exon 6 of the non-human animal CD155 gene.

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector into a cell of a non-human animal, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal humanized with the CD155 gene.

Preferably, to improve recombination efficiency, a non-human animal can also be constructed using sgRNA targeting the CD155 gene together with the above-described targeting vector. Wherein the sgRNA targets the non-human animal CD155 gene while the sequence of the sgRNA is on the target sequence on the CD155 gene to be altered. Preferably, the target site of the sgRNA is located on exon 2 and/or exon 6 of the CD155 gene.

In a fourth aspect of the present invention, there is provided a non-human animal humanized with the CD155 gene obtained by the above construction method.

In the fifth aspect of the invention, a method for constructing a CD155 gene-deleted non-human animal is provided, wherein the method comprises knocking out any one or a combination of more than two exons 1 to 8 of an endogenous CD155 gene. Preferably, exons 2 to 6 of the endogenous CD155 gene are knocked out.

In a sixth aspect of the present invention, there is provided a non-human animal with a deletion of the CD155 gene obtained by the above-mentioned construction method.

In a seventh aspect of the present invention, there is provided a cell, tissue or organ with a CD155 gene deletion, wherein the cell, tissue or organ is obtained by the method for constructing a non-human animal with a CD155 gene deletion or is derived from a non-human animal with a CD155 gene deletion constructed as described above.

In the eighth aspect of the invention, a targeting vector of the CD155 gene is provided, wherein the targeting vector comprises exons 2 to 6 of the human CD155 gene. Preferably comprising part of exon 2, all of exons 3 to 5, and part of exon 6 of the human CD155 gene. Further preferably comprises a nucleotide sequence encoding the extracellular domain of human CD 155. More preferably, the polypeptide comprises a nucleotide sequence from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus to 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the C-terminus of the extracellular region encoding the human CD155 protein.

Preferably, the targeting vector further comprises a 5 'arm and/or a 3' arm.

Preferably, the targeting vector further comprises a selectable gene marker.

Preferably, the marker gene is a gene encoding a negative selection marker. Further preferably, the gene encoding the negative selection marker is a gene encoding diphtheria toxin subunit a (DTA).

Preferably, the targeting vector further comprises a resistance gene for positive clone selection. Further preferably, the resistance gene selected by the positive clone is neomycin phosphotransferase coding sequence Neo.

Preferably, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (a conventional LoxP recombination system can also be selected). The number of the specific recombination systems is 2, and the specific recombination systems are respectively arranged at two sides of the resistance genes.

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

In the tenth aspect of the invention, the invention provides an application of the targeting vector or the cell containing the targeting vector in CD155 gene modification.

In the eleventh aspect of the invention, a humanized CD155 protein is provided, wherein the humanized CD155 protein comprises an extracellular region of a human CD155 protein. Preferably, the polypeptide comprising SEQ ID NO: 2 from position 29 to 332.

In one embodiment of the present invention, the amino acid sequence of the humanized CD155 protein is as shown in SEQ ID NO: 10, or, alternatively, a variant of SEQ ID NO: 10, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 10 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 amino acid difference, or an amino acid sequence having the amino acid sequence shown in SEQ ID NO: 10 comprising substitution, deletion and/or insertion of one or more amino acid residues.

In the twelfth aspect of the present invention, there is provided a humanized CD155 gene encoding the above humanized CD155 protein, wherein the humanized CD155 gene comprises exons 2 to 6 of a human CD155 gene.

Preferably, the humanized CD155 gene comprises SEQ ID NO: 5.

In one embodiment of the invention, the mRNA transcribed from the humanized CD155 gene comprises SEQ ID NO: 9, or a nucleotide sequence identical to SEQ ID NO: 9, or a nucleotide sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 9 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 nucleotide, or alternatively, a nucleic acid sequence comprising a nucleotide sequence having the sequence of SEQ ID NO: shown at 9.

In a thirteenth aspect of the invention, there is provided a construct expressing the humanized CD155 protein described above. Preferably, the construct comprises the humanized CD155 gene described above.

In a fourteenth aspect of the invention, there is provided a cell comprising the construct described above.

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

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

(a) preparing and obtaining the non-human animal by applying the construction method;

(b) mating the non-human animal obtained in step (a) with a genetically modified animal other than CD155, inseminating in vitro or directly performing gene editing, and screening to obtain a polygenic humanized modified non-human animal.

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

Preferably, the animal which is genetically modified except for CD155 is one or the combination of more than two of animals which are genetically modified such as PD-1, PD-L1, TIGIT or CD 226.

In the seventeenth aspect of the invention, a tumor-bearing animal model is provided, and the preparation method of the animal model comprises the step of preparing an animal by the construction method.

Preferably, the method for preparing the tumor-bearing animal model further comprises the step of implanting tumor cells into the non-human animal prepared by the above method or its offspring.

In an eighteenth aspect of the present invention, there is provided a cell, tissue or organ modified with a CD155 gene, which is obtained by the above-mentioned method for constructing a non-human animal humanized with a CD155 gene or a method for constructing a multi-gene-modified non-human animal, or which is derived from the above-mentioned non-human animal humanized with a CD155 gene or the above-mentioned multi-gene-modified non-human animal, or the above-mentioned tumor-bearing animal model.

In a nineteenth aspect of the present invention, there is provided a non-human animal humanized with CD155 gene obtained by the above-mentioned construction method, a non-human animal deficient with CD155 gene obtained by the above-mentioned construction method, a multi-gene modified non-human animal obtained by the above-mentioned construction method, the above-mentioned tumor-bearing animal model, the above-mentioned humanized CD155 protein or the application of the above-mentioned humanized CD155 gene, said application being for non-disease diagnosis, non-disease treatment purposes, said application comprising:

A) use in the development of products involving CD 155-related immune processes in human cells; preferably, human antibodies can be produced or screened.

B) Use as a model system in association with CD155 for pharmacological, immunological, microbiological and medical research;

C) to the production and use of animal experimental disease models for the study of CD 155-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) the application of the human CD155 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo; alternatively, the first and second electrodes may be,

E) the application of the CD155 gene function, the medicine and the drug effect aiming at the target site of the human CD155, the immune-related disease medicine related to the CD155 and the anti-tumor medicine are researched. The drug directed to the target site of human CD155 may be a human CD155 antibody.

The twentieth aspect of the present invention provides a humanized non-human animal of the CD155 gene obtained by the above-mentioned construction method, a non-human animal deficient in the CD155 gene obtained by the above-mentioned construction method, a multi-gene modified non-human animal obtained by the above-mentioned construction method, and an application of the above-mentioned tumor-bearing animal model in the preparation of a medicament for treating or preventing tumors.

The "tumor" according to the present invention includes, but is not limited to, lymphoma, brain cancer, non-small cell lung cancer, cervical cancer, esophageal cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

In one embodiment of the invention, the tumor is selected from lung cancer, breast cancer, ovarian cancer or melanoma.

The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, dermatitis, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, etc.

The "cell" of the present invention may be any cell of animal or human origin, including but not limited to lymphocytes, monocytes, macrophages, endothelial cells, epithelial cells, CD34+ thymocytes, neurons or tumor cells.

The "humanized CD155 protein" of the present invention comprises a portion derived from a human CD155 protein and a portion of a non-human animal CD155 protein.

The "humanized CD155 protein" of the present invention comprises a portion derived from the human CD155 gene and a portion of the non-human animal CD155 gene.

The term "comprising" or "comprises" as used herein is open-ended, and when used in this application to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be comprised of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described herein.

The "exon" from xx to xx or the "exon from xx to xx" in the present invention includes exons and introns therebetween. For example, "exon nos. 2 to 6" comprises the nucleotide sequence of exon No. 2, intron nos. 2 to 3, exon No. 3, intron nos. 3 to 4, exon No.4, intron nos. 4 to 5, exon No. 5, intron nos. 5 to 6 and exon No. 6.

The term "intron" used herein means an intron from exon x to exon xx. For example, "intron 2-3" means an intron between exon 2 and exon 3.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "CD 155 locus" refers to a DNA fragment of an optional stretch of the CD155 gene. In one embodiment of the invention, the CD155 locus to be replaced may be a DNA fragment of any one of exons 2 to 6 of the CD155 gene.

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

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

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

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

In one aspect, the non-human animal is a mammal, preferably any non-human mammal that can be genetically engineered to make a gene humanized, such as a rodent, pig, rabbit, monkey. In one aspect, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal to which the gene is humanized is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from the family of cricotidae (e.g., mouse-like hamsters), cricotidae (e.g., hamsters, new world rats and mice, voles), muridae (true mice and rats, gerbils, spiny mice, crow rats), marmoraceae (mountaineers, rock mice, tailed rats, madagaska rats and mice), spiny muridae (e.g., spiny mice), and spale (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In a particular embodiment, the non-human animal is a rodent, a strain of C57BL, C58, a/Br, CBA/Ca, CBA/J, CBA/CBA/mouse selected from BALB/C, a/He, a/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10 sn, C57BL/10Cr and C57 BL/Ola.

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

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

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

FIG. 1: schematic comparison of mouse CD155 gene and human CD155 locus (not to scale).

FIG. 2: schematic representation of humanization of mouse CD155 gene (not to scale).

FIG. 3: CD155 gene targeting strategies and targeting vector design schemes (not to scale).

FIG. 4: southern blot results of cells after CD155 recombination, in which WT was the wild type control.

FIG. 5: FRT recombination process schematic (not to scale) for humanized mouse CD155 gene.

FIG. 6: CD155 gene humanized mouse F1 mouse tail PCR identification result, wherein WT is wild type, H₂O is water control and PC is positive control.

FIG. 7: the results of flow assay of CD155 protein on spleen T cells of C57BL/6 wild type mice (WT) and CD155 gene humanized heterozygote mice (CD 155 (+/-)), wherein ISO is isotype control, mCD155 represents murine CD155 protein, and hCD155 represents humanized CD155 protein.

FIG. 8: the results of flow assay of CD155 protein on splenic mononuclear cells of C57BL/6 wild type mice (WT) and CD155 humanized heterozygote mice (CD 155 (+/-)), wherein ISO is isotype control, mCD155 represents murine CD155 protein, and hCD155 represents humanized CD155 protein.

FIG. 9: the results of flow assay of CD155 protein on splenic macrophages of C57BL/6 wild type mice (WT) and CD155 humanized heterozygote mice (CD 155 (+/-)), wherein ISO is isotype control, mCD155 represents murine CD155 protein, and hCD155 represents humanized CD155 protein.

FIG. 10: the flow detection result of CD155 protein on abdominal cavity macrophage of C57BL/6 wild type mouse (WT) and CD155 gene humanized heterozygote mouse (CD 155 (+/-)), wherein ISO is isotype control, mCD155 represents murine CD155 protein, and hCD155 represents humanized CD155 protein.

Detailed Description

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

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

EcoNI, AseI, SpeI enzymes were purchased from NEB under the respective cat numbers R0521S, R0526M, R0133M;

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

brilliant Violet 510 anti-mouse CD45 was purchased from Biolegend, cat # 103138;

brilliant Violet 711 Antibody-mouse TCR β chain Antibody was purchased from Biolegend under accession number 109243;

PE anti-mouse CD155 (PVR) Antibody, available from Biolegend under cat number 132205;

v450 Rat Anti-mouse CD11b was purchased from Biolegend, cat # 560455;

PE Rat IgG2a, kappa Isotype Ctrl Antibody from Biolegend, cat # 400507;

APC anti-human CD155 (PVR) Antibody, available from Biolegend under Cat 337617;

APC Mouse IgG1, kappa Isotype Ctrl (FC) Antibody from Biolegend, cat # 400121;

zombie NIR [ Fixable visual Kit ] from Biolegend, cat # 423106;

FITC anti-mouse F4/80 from Biolegend, cat # 123108;

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, available from Biolegend under cat # 108426;

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

EXAMPLE 1 humanized mouse with CD155 Gene

A comparison scheme between the mouse CD155 Gene (NCBI Gene ID: 52118, Primary source: MGI: 107741, UniProt: Q8K094, located at positions 19637503 to 19655068 of chromosome 7 NC-000073.7, based on transcript NM-027514.2 and its encoded protein NP-081790.1 (SEQ ID NO: 1)) and the human CD155 Gene (NCBI Gene ID: 5817, Primary source: HGNC:9705, UniProt ID: A0A0C4DG49, located at positions 44643910 to 44666162 of chromosome 19 NC-000019.10, based on transcript NM-006505.5 and its encoded protein NP-006496.4 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purposes of the present invention, a nucleotide sequence encoding a human CD155 protein may be introduced at the endogenous CD155 locus of a mouse, such that the mouse expresses a human or humanized CD155 protein. Specifically, the gene editing technology is used to replace part of the sequence from exon 2 to exon 6 of mouse with part of the sequence from exon 2 to exon 6 of mouse containing the human CD155 gene under the control of mouse CD155 gene regulatory elements to obtain a humanized CD155 locus diagram as shown in FIG. 2, so as to realize the humanized modification of mouse CD155 gene.

The targeting strategy was designed as shown in FIG. 3, with the homology arm sequences on the targeting vector containing the upstream and downstream of the mouse CD155 gene, and an A fragment containing the human CD155 sequence shown in FIG. 3. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from position 19652827 to 19657369 of NCBI accession No. NC-000073.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence from position 19638888 to 19641899 of NCBI accession No. NC-000073.7. The nucleotide sequence of human CD155 on fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence 44647228 to 44658746 of NCBI accession No. NC-000019.10; the ligation downstream of the human CD155 sequence to the mouse was designed to be 5' -ttccttacctaaatacctgtttccttctctttcagagggacctgagaatatgcagcaaaatacaagattacacctaggct-3' (SEQ ID NO: 6), wherein the sequence "agggaThe last "a" in "is the last nucleotide, sequence, of a human"cctgaThe first "c" of "is the first nucleotide of the mouse sequence.

The targeting vector also comprises a positive cloneThe selected resistance gene, namely neomycin phosphotransferase coding sequence Neo, is provided with two site-specific recombination system Frt recombination sites which are arranged in the same direction on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the connection between the 5 'end of the Neo box and the mouse gene is designed to be 5' -atccatgccctgagcccctcactttccatagaccacactcGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTT-3' (SEQ ID NO: 7), wherein the sequence "acactc"last of" c "is the last nucleotide, sequence" of the mouse "GAATT"G" of "is the first nucleotide of the Neo cassette; the connection between the 3 'end of the Neo box and the mouse gene is designed to be 5' -TCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGGTGGATCCttgtctaagcactagctctgaatgccactgtagtctgcacaggggcctat-3' (SEQ ID NO: 8), wherein the sequence "GATCC"the last" C "of a" is the last nucleotide, sequence of the Neo cassette "ttgtcThe "first" t "of a" is the first nucleotide of a mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CD155 after being transformed is shown as SEQ ID NO: 9, the expressed protein sequence is shown as SEQ ID NO: shown at 10.

Given that human CD155 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The method comprises the steps of performing electroporation transfection on a targeting vector which is verified to be correct by sequencing into embryonic stem cells of a C57BL/6 mouse, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technologies, screening correct positive clone cells, detecting clones which are verified to be positive by PCR by using Southern Blot (cell DNA is digested by EcoNI or AseI or SpeI respectively and hybridized by using 3 probes, the lengths of the probes and target fragments are shown in Table 1), and detecting the result shown in figure 4, wherein the detection result shows that 12 clones which are verified to be positive by PCR, and the detection result shows that the remaining 9 clones are positive and have no random insertion except 1-E03, 2-E01 and 3-C05, and are further verified by sequencing, and are 1-D04, 1-E02, 1-H07, 2-E03, 2-E05 and random insertion is not shown, 2-G01, 2-G04, 3-G02, 3-G05 and 3-H01.

Table 1: specific probes and target fragment lengths

Wherein the PCR assay comprises the following primers:

F1：5’-catgatggaggagccctgagaatgg-3’（SEQ ID NO：11），

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

F2：5’-GCTCGACTAGAGCTTGCGGA-3’（SEQ ID NO：13），

R2：5’-TGTTTGTGTATGGACAGGGCTTTGC-3’（SEQ ID NO：14）；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

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

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

3 'Probe (3' Probe):

3’Probe-F：5’-TTTAACCATCACCCAACCCCTGCTC-3’（SEQ ID NO：17），

3’Probe-R：5’-CCTATGTGGGGCTACTCACAAGTGC-3’（SEQ ID NO：18）；

neo Probe (Neo Probe):

Neo Probe-F：5’-GGATCGGCCATTGAACAAGAT-3’（SEQ ID NO：19），

Neo Probe-R：5’-CAGAAGAACTCGTCAAGAAGGC-3’（SEQ ID NO：20）。

the selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 5 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous CD155 gene mice. The somatic genotypes of the progeny mice were identified by PCR (primers shown in Table 2), and the results of identification of exemplary F1 generation mice (with the Neo marker gene removed) are shown in FIG. 6, in which all 5 mice numbered F1-01, F1-02, F1-03, F1-04, and F1-05 were positive heterozygous mice.

Table 2: primer name and specific sequence

This indicates that using this method, humanized mice of the CD155 gene can be constructed that can be stably passaged without random insertions.

The expression of the humanized CD155 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. Specifically, 1 mouse of a 9-week-old female C57BL/6 wild-type mouse and 1 mouse of a 18-week-old humanized heterozygote of male CD155 gene were each taken, spleen tissues were taken after cervical euthanization, and a granulocyte-specific recognition Antibody, PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, an anti-mouse CD155 Antibody, PE anti-mouse CD155 (PVR) anti-human APC, anti-human CD155 Antibody, anti-human CD155 (PVR) Antibody, and flow-type detection were carried out using anti-mouse CD45 Antibody, Brilliant Violet 510 anti-mouse CD45, mouse T cell-specific recognition Antibody, Brilliant Ville TCR β chain Antibody, macrophage-specific recognition Antibody, FITC-mouse F4/80, granulocyte-specific recognition Antibody, PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, anti-mouse CD155 Antibody, PE anti-mouse CD155 Antibody CD155 (PVR) anti-human body APC, anti-human CD155 Antibody, and macrophage expression results on T cells, CD, As shown in fig. 8 and 9.

As can be seen in fig. 7, expression of murine CD155 protein was detected on C57BL/6 wild type mouse T cells (fig. 7B), and no humanized CD155 protein was detected (fig. 7E); both murine CD155 protein (fig. 7C) and humanized CD155 protein expression were detected on CD155 gene humanized heterozygote mouse T cells (fig. 7F).

As can be seen in fig. 8, expression of murine CD155 protein was detected on C57BL/6 wild type mouse monocytes (fig. 8B), and no humanized CD155 protein was detected (fig. 8E); both murine CD155 protein (fig. 8C) and humanized CD155 protein expression were detected on CD155 gene humanized heterozygote mouse monocytes (fig. 8F).

As can be seen in fig. 9, expression of murine CD155 protein was detected on C57BL/6 wild-type mouse macrophages (fig. 9B), and no humanized CD155 protein was detected (fig. 9E); both murine CD155 protein (fig. 9C) and humanized CD155 protein expression (fig. 9F) were detected on CD155 gene humanized hybrid mouse macrophages.

Further, flow cytometry was used to detect the expression of CD155 on the macrophages in the abdominal cavity of both the C57BL/6 wild-type mice and the CD155 humanized heterozygote mice, and the detection results are shown in fig. 10. As can be seen, expression of murine CD155 protein was detected on C57BL/6 wild-type mouse peritoneal macrophages (fig. 10B), and no humanized CD155 protein was detected (fig. 10E); both murine CD155 protein (fig. 10C) and the expression of humanized CD155 protein was detected on CD155 gene humanized heterozygote mouse peritoneal macrophages (fig. 10F).

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

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared CD155 mouse. As described above, in example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other gene modifications such as PD-1, PD-L1, TIGIT, and CD226, or may be humanized CD155 mice and then subjected to a two-gene or multi-gene modified mouse model of CD155 and other gene modifications using isolated mouse ES embryonic stem cells and gene recombination targeting techniques. The homozygote or heterozygote of the CD155 mouse obtained by the method can also be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of the humanized CD155 and double gene or multiple gene modification heterozygote of other gene modification can be obtained with a certain probability according to Mendel genetic rules, then the heterozygote is mated with each other to obtain double gene or multiple gene modification homozygote, and the in vivo efficacy verification of the targeted human CD155 and other gene regulators can be carried out by utilizing the double gene or multiple gene modification mice.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

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

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

Sequence listing

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gaagtggcat ttggcacctc agacagtttc atgttttgtg gtgacattaa tttgatttag 60

aagtacttga ggagaacgag aaaattaaaa ccactgtggc tgtgttcccc tctgcagaac 120

acagttagaa ttgccagtca gttttactta ttcatttttg tgctatctgc tctgtacttg 180

tcctcctgca tgcttctccc acccctaaca ccgccagctt ggttggccaa acctacctag 240

gcctaggcag caactgaaga gtcgccattc actggaagag tgtctaaaga aggaaatgtt 300

gggattcctc atttagaaag taacactgtg aattaggaaa gcaggggctt caccgcgcct 360

caggcatttt gcttacaggc ttcataactt gagacagatt ctcagagccc acattgtagc 420

ggcaagaatc gactcctgag gattgtcccc tgaccttcac acaggtatca aggcatgcat 480

aggcatcatg tgcacactca cactacacac gtgcatgcat tcatacacag aaacttcatt 540

cacacacaca tacacactgc acacatacac atcaggcaaa cactacatac ataggcacac 600

tcatgcaccc tcacatacac ttcatataca ctacacgtgc atacactcat gcatactaca 660

tacatacata catacataca tacatacaca cacagtacaa acacatatat tcaagcacac 720

acacttcata tatgtgcaca ctcatataca cactacatac ataagcacac tcacacacat 780

actacataca cacacacatg cacacttata ctagaaacac acactacata aatacactaa 840

ctcatacata cacatccagg catacacata catacataca cacatacata catagcagtt 900

tagaatctat caaaattgaa aacagacatt aaagaacagc aaaaaaaaaa aaaagttaat 960

aagaaaaatg taaaggttgg tgtcagcttc cccaggaatc cgacttttcc accgtttgtt 1020

ttctttcact tcaggaagct aacaagctct gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 1080

gtgtgtgtgt gtgtgtgtgt tatgggaaag gactagagac gctgtattca cagggcagaa 1140

acagggaggg agagagaagg gcacagaact ttttcccttt tcgggtgtgt gtgggaggtg 1200

gtttagtgac ctgccagttt ataccacagc aatggggaga tctgggagaa ggggaagccc 1260

acagtcatga cagcatggtc agagaaggaa cagaatggtt acaggctctc agtgtggaaa 1320

tgccacagga ctcaagaaac tccagagatt tccagaagac ttgcccctca gattcccatt 1380

cagaattaac cgagagcagg aactgggggg ggaggaggtg aaggttgggg agcacaggcc 1440

cggcattgct gcattccaag gtgttgggag gaagatcagg agttcaaggg tagtctagaa 1500

caatcgttct caacctgcga gcttcaaccc ctttggggaa ccatatcaga tatcccacat 1560

gtcatattta cattatgata tttacatgtc ccatattcac attatgattc ataacagtag 1620

caaaattaca gttataagta gcaatgaaaa tgattttatg attaggggtc accacagcat 1680

gaagaactgt attaaagaat ccgggcatgg tggcacacgc ctttaatccc agcacttggg 1740

aggcagaggc aggctgattt ctgagttcga ggccagcctt gtctacaaag tgagttccag 1800

gacagtaaaa ccctgtctcg aaaacaaaca cacagaaaca aaaacaaaca aagaaaaaaa 1860

gtatcgcgca ctgggaagat tgagaaccac tgacctagaa tgtgcttcat gagaccttat 1920

ttcttttttt aaacagtttc actgtgtaac tcagacgagg ctggatttgt ctacggtgga 1980

cccggctgac acagaactca cagatctgcc tgccttccaa agtgctgtga ttaaagacat 2040

ttgaagccta gccccctgcc ccccattatt tttaattaaa aaaaaaatcc gggatgatct 2100

cacgtggccc aggctggcct taaacccgct ttcaattctg atgatcctct tgcctccatc 2160

ctccccaccc gactcaggtg ctgggatgac aggtttacag aacactaatc gtcagggtcg 2220

ttcactcatc aaccagcccg gcagacgtgc ttggctgtgt gcgcctgccc agcaggggac 2280

cgccctcaat gggccgtccc taggcggcac ccgcttagct gagattccag cacttgactt 2340

cagggtttcg gagagataag gcgcttggcc gttactaact ggactacaaa gagctggatc 2400

ggaccggaac cacatggctc aactcgcccg agccacccgc tccccgctgt catggctgct 2460

gctgctgttc tgctatgcac tccggaaagc gggtaagtag ccccgcgccc cgcgccccgc 2520

gccccgcgcc ccgcgccccg gcctgtgcgc acctcgagag gccctcccag gccacccgat 2580

cccttcttcc cggcccaaag tcctaggtct ggattctgga gcagagattc cagaaaatag 2640

ggcgtggtga ctcacaccgg ccaccggagt acaggggagg atgaggtagg agggttgcct 2700

tgagttccaa cgtagcctag actcgggaat gagaaccgga tttgctccac ttcgcgcacg 2760

cttgcgcacg cgcacacaca cacacacaca cgcgcgcgcg cgcgcacaca cacgcacgca 2820

cgcgcgcaaa agatgtttta ttattgtaaa ataaatgact cgtacactga gaaaaagtct 2880

gggtgactca tgccaggaac cttaacccta gtggccacac tctctccagc aagttaatcg 2940

tgactgactc agagttgatt tattagttca ttttcctttc gctgagttgg agactgaagc 3000

ctcactgggg aattgtagca caggagtcta cccctgagca cacagggtgc tgaggataga 3060

cccttggtcc tcacacaacc taattcggag ccctgtgcta ttcccattgt ttcagtggtt 3120

gaccagcagc aagtcaggct gggcgctcag agtgagctca gagtgactgg aaccaaaaaa 3180

ccaaaccaaa ccaaaccaaa acagggaggt ggtggctcat gcctttaacc ccaatagggg 3240

aggcagagac aggtggagcc ccgtacaggg gtaccctagg gtcaccatga cgggtcctgg 3300

gttcccagaa cttcactctg aagtcattat cagacttctc aagtattcag aaatgcctga 3360

tactgtattc cctaatgttg agggcctact gtgtattggg ctaaaggaga gagtctcaca 3420

tagggcctag cagagtgact attgaatggc tcggattcaa accccaccgt atcttgtcgt 3480

cttatgcctg cgtgatcccc ctagcaaatt cttctatgcc tccatttctg catatgtaag 3540

tggtacctga agcccagtgt gatggtgctc acccttaatc gcagaattta ggaggcagag 3600

gcaagcagat ctctgagttc cagaccagcc tggtgaatgt actgaggtga tgtttagtgt 3660

ctctactact actaataata ataataataa caataataat aataatgaat tataaatact 3720

atatgattat tacaaggcaa aattatcatt gtgcattata atagattttg taatataaac 3780

atttaattac attaataata tttaaatata gtatgctgaa ttatattata atgtataaat 3840

atttagatat aacgataaca ttgttattat taaagtaaga taaggggctg gagagagcac 3900

tggctactct tgtagaagac ctgggtttgg ttctcagcac ccacatggtg attcacagtc 3960

ctctgtatct caaattcaga ggaatttcat ccctcccttt tggcttctat gggcaccgta 4020

gtgcaaacac ctatagagaa aaatctctta taaacatcac ctgtcaataa aaaaaccagt 4080

ggccaatgag ctggggcagg aaataggggg tgggacactg acaggacgag aggattctgg 4140

gaaatagtaa gagataaagg agaaatggag gggggtggga tgcagatgta aaacatcagg 4200

gagacactga ggagactctg aggaagaagc aggccagcac tgaaggagag caacctacca 4260

catggtagac atagaatagt ctaagtgggt taaataaatt acgagctact cagggaatga 4320

gccaaagctt atgacctggg tatttaataa taaataacta gtctcagagt tgttattgat 4380

gggaaggggc cgaaaggaaa aatggattta aacttatttt aatacataca tccatacata 4440

catgctagca aacactaaca cacataaact aaaaattaat acatctttaa aataaattaa 4500

tttaaatgga ttaattaaca cctctggttt cccaggtggg gat 4543

<210> 4

<211> 3012

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttgtctaagc actagctctg aatgccactg tagtctgcac aggggcctat gctgtgtgga 60

ggaagggctg tgtgatttaa ctgaccaacc tgcctgctgc agccacacag gtacaggaat 120

gactgacagg tcgtagaagg ccatcatttg tgactgacat ttttctttaa acatgaaata 180

tacctgcccc caccctatcc ccagccacct gggttctgcc tcctcctctg cagaatcctc 240

tctgatttct cacagaacag gtcaagacct accttcagtc tctcccagac cagccttgct 300

cagtcctggt ggtcattgtg tggggacatg tgtgtctctt ccggtggact gtgggcacag 360

agggttggca ggaactgtca gggtcaaggg tatcctcagc tacataggtc atcttgggct 420

gcatgagacc ttgcctcaaa aaaaccaaac atgcgaggca atggtggtgc acacctttaa 480

tctcaaccct tgcgggaggc agagacagta ggatctctgt gagcccaaga ctagcctggt 540

ctacagagtg acttctagga cagccaaaat ctacacagca aaaccctgtc atgaagagaa 600

aaataaataa ataaataaat aaataaataa ataaataaat agtggaaaga tgaggaaaga 660

gtgaatatat cagggtccca ataattcctg caaggtcaca cacacacaca cacacacaca 720

cacacacaca ccagtgacca cttcgccctg ccaggctctg cctcttacag ttgaatggcg 780

tcctgacaac ttgcattgaa tcttgacctc taacacatga cacataagag ccatattctg 840

gggcatggta atgcagcctg tcatcccagc gcttgggagg cagagacagg aagattgtga 900

gttagaagcc agccctgaca acttgcattg aatcttgacc tctaacacat gacacataag 960

agccatattc tagggcatgg taatgcagcc tgtcatccca gcgcttggga ggcagagaca 1020

ggaagattgt gagttagaag ccagcctggg ctacatgtca gggccttttc ctccaaaata 1080

aagctaaatt atagcccccg ttttagtggt tatatccctg ggggaagatt gctgcagggg 1140

aagccacatg ctggggatac gctagctacg atcattattg actttaatta aatgattatt 1200

gtcactatta aagctccaga atggttgttc tcagctgtaa ccccaatgtg ttcaattcct 1260

ttgtcacaaa agaggcatct tccatttagc ccccactgga tagttggatg tgggtactgg 1320

gaactgaatc caggtcctct ataagaatgg tcagtgctct taaccactct tcccccaacc 1380

ccaccccacc ccaccccacc cccttttttt tttttttttt tttttttttt gagactgagt 1440

ctcatgaagt ctaggctggc ctcaaattct atatgtagcc agagatgacc ttaaagcact 1500

ggtctacttc tagaatgtgt gcaccaccat gcttgggtct tctctgtcct gtgtcaatac 1560

aaaggccata gaagcccaga cacccccaga gacaacctct tcctataatt ccagggtctc 1620

aggcatcctc ttcccataat tccagagtct cgggcatcct cttcccatga ttccagggcc 1680

tcgggcatcc tcttcccatg attccagggc ctcgggcatc ctcttcccat aattccagag 1740

tctcgggcat cctcttccca tgattccagg gcctcgggca tcctcttccc atgattccag 1800

ggcctcgggc atcctcttcc catgattcca gggcctcggg catcctcttc ccttgattcc 1860

agggcctcgg gcatcctctt cccatgattc cagggcctcg ggcatcctct tcccatgatt 1920

ccagggcctc gggcatcctc ttcccatgat tccagggcct cgggcatcct cttcccatga 1980

ttccagggcc tcgggcatcc tcttcccatg attccgggtc tcgggcatcc tcttcccatg 2040

attccagggc ctcgggcatc ctcttcccat gattccaggg cctcgggcat cctcttccca 2100

tgattccggg tctcgggcat cctcttccca tgattcgtga cgagttttct tgtttctcca 2160

ggtcgtgctc tgcagtccaa tccctcagag agggaggtga gtacagaggc accagggagg 2220

agaacatagg ggtctgagtg gaccacagac ctattctgga actttctcaa gggggcaggg 2280

cagagagctg cagggtggca ttgggagccg ggattttcta ggtccccagc ttgtgcacca 2340

ctaggaggga atggaaactt ttggagatgg gacctagtga gagaaggcta gatcattgct 2400

ccaagtgtgt ttttagaggg aattttgaaa gcattcatct gatttttgag agcagactat 2460

cctttgaact cactgtctag ccaaggttgg cccacctgcc tctatctccc cagtgatggg 2520

attacaggcc tgtgccattg catccagctc tctgtttttt ggaggacatc tccctatgta 2580

gcctagaata gattctgatg atcctcctac ctctgacacc agagtgccaa ggttccaaga 2640

tatgcctacc tcctgatcct tgcctatggc cactgcttag gggcctagtg ggccgcctcc 2700

ttgagcgtct cacttctccg ggaagaccgt ttacttgatg gaggccctga ctgaagtgtg 2760

atgtctgttg tcccccatcc agaacgtcca gtattcatct gtgaacggcg actgtagact 2820

gaacatggag ccaaacagca caaggtgacg gtgctgggta gacagaacta aggaacttga 2880

aggcatagca actggaaccc tactctcata aatgaagaag cctccagaga gactggctgc 2940

tcagtgtgat gagcatagca agtttggggg gtctcccagg atgctgccga attccacgtt 3000

gtcaaaagga cc 3012

<210> 5

<211> 11519

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcgtcgtgc aggcgcccac ccaggtgccc ggcttcttgg gcgactccgt gacgctgccc 60

tgctacctac aggtgcccaa catggaggtg acgcatgtgt cacagctgac ttgggcgcgg 120

catggtgaat ctggcagcat ggccgtcttc caccaaacgc agggccccag ctattcggag 180

tccaaacggc tggaattcgt ggcagccaga ctgggcgcgg agctgcggaa tgcctcgctg 240

aggatgttcg ggttgcgcgt agaggatgaa ggcaactaca cctgcctgtt cgtcacgttc 300

ccgcagggca gcaggagcgt ggatatctgg ctccgagtgc ttggtgagca gggggttttg 360

gggaggctga atgaaaggca gagacttggt gggaggatca gggaagttgg caaagagcgg 420

ggaggcctgg gagggaggga gattccctcc acagcagatc ccctggggac aaaaggaggg 480

ggcagcgcaa tgatgtgggg tggggtgggg ggaggtgagt ggggggaggg gctgcagggc 540

aggaagaaag cagagatctc agggagtaag gacccccaag cctgggatca gaaagccctg 600

agggaggagg aggggtatat tggaaaggga gctgggaggg gactgggaag gaagggagga 660

ggcctgggtg gcctcttctg ggtccctcct ttcctcatct gtaaaagggt tggtgacaac 720

agcatcaccc ttggaaagct gtcctgagtg tcatcactgc ctaggaaaga gtctgtctgc 780

ccccttggac tgtgtaccca tgagggcaga accggggcta ttttgatcac tgctgggccc 840

cagcactttc caccaaacaa tatttattga gcacttatta tgtgccattc taagagcttt 900

acctgcatat tctaattgcc tgtgattcca tcattatcac ctgttgacag acaaggaaac 960

tgaggcacag acttgcccaa gaccaaagct gcagccttag ggcagcggcg cttcaccgct 1020

ggcccatgtg agccccttag agctgtcggt tactgttact ttatctggtg aagaaggaga 1080

aagttgaaac agacagacag gaaagcaagg atcctgatac ccaaaagagg aggggatcat 1140

gctttcattt tcatgagctc catgaagcct tcctcttgga acttgagcaa gaagtctctg 1200

tgcctcagtt tcctcatctg tgggataggg atcaccatga ggattcaaaa aaactgaccc 1260

tttttttttt tttgagatag tttagttctg tcgcccaggc tggagtgcag tggtgtgatc 1320

atagtaggtc ctcactgcag cctggaactc ctgggctcaa gggatgttcc taccctccct 1380

gtagctggaa ttacaggaat gtgccaccat gcccaacttg ttttctgttt tgttttgttt 1440

tgttttgttt tcttttcttt ttaagcttat gtgccaagaa ctattccaag cctgtgcatg 1500

agaaactcat gaaatgtcaa gggcttggaa ctgtgcctag cacacaacac ccaaccaaag 1560

gcagctgttg tcactccagc aagcctggga ttctgggtat catggtttag gatgagggta 1620

aaacagaaac agtgagtcaa gttaaggaga aatcttcggt aaataatata ggtaatgatg 1680

gggaaggaag ttccaggttt gggaatcacc aggatgatga ttaagagatg gtcaaggtgg 1740

ttttggagtc agacagactt tatgtgaaag cccagctctg ctgcttacag ctgtgtgacc 1800

atgggcaagt cacttccctt ctcaacttca gggctcaggc ttcctctcct ccactgagcc 1860

ccagaccagg ataaccccct ttgggtttcc cctttccagc cctgccctct ccgagtcatc 1920

actgcctagg aaagagtccg tctgccccct tggactgtgt acccatgagg gcagaaccag 1980

ggctattttg gtcactgctg ggctccagca ctttccacca aacaatattt attgagcgct 2040

tattatatac cattctaaga gctttacctg catattctca ttgcctgtga tcccatcact 2100

atcccctgtc aacagacaag gaaactgagg cacagacttg cccaagacca cacagctgct 2160

cagggacaca gctgggattt aaaccatgcc attctgtatc tttttttttt tttttttttt 2220

gagacagaat ctcactctgt cgcccaggct ggagagcagt gacacaatct cagctcactg 2280

caacctccgc ctcccgagtt caagcaattc tctggtctca gcctcccaag taactgggat 2340

tactggcgtg tgccaccata ccctggctaa tttttaatag agacagtttc tccatgttgg 2400

ccaggctggt ctcgaactcc tgaccccaag tgatccaccc acctcagccc cccaaagttc 2460

tgggattata ggcgtgagcc accccgccca gcaaccatgc catcctgtac ccttaatgaa 2520

tgcccccttc tgccacggag gggttcattg aatgacttgt tgcttttgtt cctcttccca 2580

gccaagcccc agaacacagc tgaggttcag aaggtccagc tcactggaga gccagtgccc 2640

atggcccgct gcgtctccac agggggtcgc ccgccagccc aaatcacctg gcactcagac 2700

ctgggcggga tgcccaatac gagccaggtg ccagggttcc tgtctggcac agtcactgtc 2760

accagcctct ggatattggt gccctcaagc caggtggacg gcaagaatgt gacctgcaag 2820

gtggagcacg agagctttga gaagcctcag ctgctgactg tgaacctcac cgtgtactgt 2880

gagtgtgccc aagtcagcga tggcaagaac ccctgccggg ctgcccccac cactgtctac 2940

actgactccc caaggcactg taggcattgc ttccatcatc tgcaccggct cccctgactc 3000

ctgtctacac gacccacccc cattgtctgc accagctccc ctgggccgta aacaacactg 3060

tccctattgt ctacaccagt ttacttgggc catagtcaat gctggtaccc agactgtctg 3120

ccctggctcc tctagcggct gtctgcacgg atacctgaca ctctgttgac tcccttgagc 3180

acagtctaca ctgcccctaa actgcctaca cccatttccc caggcaagta tccacaatga 3240

ccctccccac attgctacac tggttcttct ggccacgatc aacactgcct cccttattgt 3300

ctacacccac tcccttgtcc ttttctttct ttcctttttt tttttttttt tttgaaatag 3360

agtctcactc tgttacccag gctgcagtac agtggcatgt tctcggctca ctgtaacctc 3420

tgcctcccaa attcaggtgg ttctcctacc tcagcctccc aaatagctgg gattaaaggt 3480

gcacaccacc acacctggct aatttttgta tttttagtag agacagggtt tcatcatgtt 3540

ggccaggctg gtttcgaact cctgacctca ggtgatcggc ctgcctcagc ctcccaaagt 3600

atgctggaat tataggcatg agccactgtg cccggctttt tgttttgttt tgtttttcct 3660

ttggacaggg tctcgctctg tcacccaggc tggagtgtag gggcatgatc atggttccta 3720

cagcctcacc cttccaggct caagcaatcc ttctccctca gcctcccaag taactgggac 3780

tacaggtgca gccaccatgc ccggctaatt ttacagtttt tttctagaga gagttttgct 3840

gtgttgccca ggctggtctc aaactcctag gcttaagtga tcctcccgcc tcagtctccc 3900

gaagtgctga gattacaggc gtgagccact gcacctggcc ccttgtccat tttctacact 3960

gccttaccat tgtctacact ggctctcctg ggccacagac agcactaacc ccattgtcta 4020

caccacctat tttggccacc agctgtgggg tgttttcact gtctcccata gtgtctacac 4080

tggctcctat gaccactgtc tatgtaaaca ccccactttt tctacactgg ttctcctagg 4140

cactctctaa agtgccccct gacactgtct acactgactc cctggagaac tgtctgcatt 4200

cacctcccac attgtatcga ccagttcccc tgagccacga tcaactgtct ccgccgggtg 4260

ctagttgcat tgatcttctc acattgtttg tgctggctcc cctaagcagt gtctgcagag 4320

tcccccagtg tagtctccat gttcctctgc ccaatattgt ctacactggc tcatctacag 4380

gctatccagt ctcaccccac atcaccacac tggcctcctt gtgttctgtc tgcacagccc 4440

cctgacagca tctgtcctga ttctcctgca cagcgttacc tccaccagct ccctcatggc 4500

atcatcgaca ttgacaccca cacatttatc tccactgaca gccccaggac tctctaacca 4560

gtgctgtcta acagaaatag catgcaagct gcatatagga ttttaaattc tccagcggcc 4620

accccattaa aaaattaaaa gaaagaggcc aggcacggtg gctcacgcct gtaatcccag 4680

cactttggga ggccgaggca ggtggatcac ctgaggtcag gcattcaaga ccagcctgat 4740

caatgtggtg aaactctgtc ttcactaaaa aatacaaaaa ttagccaggc acggtggcgg 4800

gtgcctgtaa tcccagctac tcgggagact gaggcaggag aatcacttga acctgggagg 4860

cggagattgc aatgagcgag attacgccat tgcactccag cctgggcagc agagcaaaac 4920

tccatctcaa aaacaagaaa agaaagagat gaaatttatt tgtttattta tttatctatt 4980

tatttatttg agacggggtc ttactctgtc acccagactg gagtgcagtg gcacagctcc 5040

ctgcagcctc aacctcctgg gcttaagtga tcctcccacc tcagcctcag tctcctgagt 5100

agctgggact acaggcgtgt gacaccatgc ctagctaatt tttgtttttg tttttgtttt 5160

tgtttttttg ttttgtgatg aagtcttgct ctgtcaccca ggctagagtg cagtggcacg 5220

atctcaactc actgcagcct ccgcctctcg ggttcaagtg attctcctgc ctcagcctcc 5280

cgagtagctg ggattacaag ccctggccac agtgcccagc taatttttgt atttgtggta 5340

gagacgggat ttcaccatgt tggccaggct ggtctcaaac tcctgacctc gtgatccacc 5400

caccttggcc tcccaaattg ctgggattac agacgtgagc caccgcaccc ggcctatttt 5460

tattttttct ggagacagga tctccctatg ttgcccaggc tggtctcaaa ctcctgggct 5520

caagtgatcc tcctgcctca acctcccaag gtactgggat tacaggcatg agccactgtg 5580

cctggccaaa attaattttt aataattgcc tttacttaac ccaatatacc taaaatatta 5640

tttcaacttg taattgtttt tttaaaatta ttcttgagat ctttcccatt tattttgtcc 5700

tagtaaagcc tttgaaatct ggtgtgtatt tcggcctggc tatatttcga gtgctcagta 5760

gtcacatgtg gccagtaggt gccatattgg acagcacaga tctagactaa gctcccacat 5820

catctacact ggctcatcca aatactgtct acacacatac acagagcctg tcttaatttc 5880

gtcagctccc ggggacacac tgtatatgtg ggggtgatct cacccccacc accacatttt 5940

caacactggc ccgacaatat agcatctgct gtaatattcc aacaccctgt ctacactgaa 6000

cccatcagta atatccacac aagctcttct agacatcatt catactgttc ctcccaccat 6060

tgtctatact gcttactccc cgccaggcaa ttggtacaca gattccctca ggtactgtct 6120

atactggagt ccctcaacac tgtcactggg tctgagcgag gaggagatct acactgactc 6180

ctacccctcg cccccgaata aaaccctgtc ccattggcct gcccccaagg acattgaaag 6240

gtcttcgctg tcaatcacgt atgggatggc tgagcagcag cctagagcag ggttatctgg 6300

gactctttcc tgcaggactt gtcagaacct tgaatacact gctgtgtgtt gtgagtggtc 6360

attgcctatg agtgagaaaa ggaggaggca ttttcctgca ggacacccaa gacatgcctg 6420

gtccaggcta cccaggaaag ctagtggaga aaagcagctc ctcagaagcc ttctccatgt 6480

ccccactgac caaggactcc taggcttctg ctctctgtgg caccccatct acctagactc 6540

ctcctaggcc tctccagctc ccgctgtttt ccaaatatcc ccgggccttt tcctgcagtg 6600

tcgtgaatcc cgcgtagccc caggcccccc aaagcctccc agtctctgaa cctctgtatc 6660

catttcctgc agacccccca gaggtatcca tctctggcta tgataacaac tggtaccttg 6720

gccagaatga ggccaccctg acctgcgatg ctcgcagcaa cccagagccc acaggctata 6780

attggagcac gtgagtcctg ggtctcaggg aggaggggct gggggtctgg atttctagca 6840

ctgagggagg aggggctggg ggcctggact cctgggactg agggagaagg ggctgggggc 6900

ccggacccct gggtctgagg gacgagaggc tgggagcccg gactcctggg tctgagggaa 6960

gaggggctgg gggtctggac ccctgggact gagagaggag gggctgggcc tggacccctg 7020

ggtctgaggg aggaggggct ggggggtctg gacccctggg tctgagggag gagaagctgg 7080

gagcctggac ccctgggtct gaggcaggac agactggggc ctggactcct ggtctaagag 7140

aggagggaga ttcagaaaag agaaaaagag gaggttgact tcgggccccc agtggggggc 7200

atctctgttt tgacatctct gttttgcggt tgaggcgttt agcaggtggt tgaatcctgg 7260

ctggtggatg cacctgcttt ttggggtgtg cctgctctgt gcccggtact tcccccgaat 7320

cccatgtgac cccatgctgt gcacagtcag cagctgttgc cctgcccagg ttaaagacca 7380

gccacgagag ggcagaagcg gcccttgaat cagcgctcgg ctccaaagct ggtcctccgc 7440

ctcttggcca agcttgtcta acctgcaggc cacatcattt gtaaactttc ttaaaacatt 7500

atgagacttt ttttttgcga atttttttag ctcattagct attgttagtg ttagtgtatt 7560

ttatgtatgg cccaggacag ttcttcttct tccaatgtgg cccggggaag ccaaaagatt 7620

ggacaccccc actctagact gtaaccacct caccgtcacc ttgtgtaacc tacttggtta 7680

tctctggatg gagcaagtac tatatatccc cattgtctag atgagaaagt tgaggctcac 7740

agcaataaga tttccccaga ttcacactga ggctgagatc aagagcaagg taagaaaaag 7800

gcaaagagat aatacataat tttataatac ataattttac ataaattaca taattttact 7860

gaaccgccct ttttttttaa agaaggggct ctgttgccga ggctggagtg cagtggcgtg 7920

atcatagctc actgcagcct ggacctccct gggctcaggt gatcctccca cctcagcctc 7980

ctgagtagct gggactacag gcacacgcca ccacactcag ctaattttgt tgtatttttt 8040

ttgcagagac agggttttgc cacattgccc aggctggtct gcaactcctg ggctcaagca 8100

atcaacccac ctcagcctcc taaagtgctg ggattacagg cgtgagccac ctcgcccagc 8160

cttaggtctt tttccttatt acaaaaataa cttgtgtcca ctgtagaaat atcaaacaat 8220

ttagttatga gcaaaaagaa gaaagtagtg cctatagttc aagtgtaccc agccacctcc 8280

agaaataccc agtgacccct tattatacat cctgtctttt ttctctctgt ttctaaacaa 8340

agactggaat tatgctgtac atataatttt gaaccttttc gtctgtcttg tgaatacctt 8400

tccatgtcaa agcacataga tccacaactc cattttcccc cacgttgtat taggaagact 8460

gtcaaacata cagaaaaatc ataggaattt tccagtgaac acttaagatt ccaccacgtg 8520

gattcccctg ctaacatttt actgtgcttg ctttatcacg tttctgtgat tccttcagtc 8580

cctctattca tccatcaatc atattatgca tttcagagca cccccagctc tcctgctttt 8640

tttttttttt tttttttttt gaggcagggc ctcactctgt tgctcaggct gtagtgccgt 8700

ggcacaatca cagctcactg cagccttgaa ttcctgggct caagcgatcc tccagcttca 8760

gcctcccaga tagctgggac tacaggcaca caccaccacg cctggctaac ctggcccctt 8820

ttttagtgtt gctgggattc caccacttta aaaaaaaaat tcaaagaata gagacaggat 8880

ctcactatgt tgcccaggct ggtcttgaac tcctgtactc aagcagtcat ctcacctggg 8940

cctcccaaag ggcagggaag ggattccaac atatgtggat gtcatcagtt atttaaccag 9000

tccctagtag aggacacaga tgtcattttc cattttcaac tctgcaaatg ccatggtggt 9060

gtccatcccc actggtcact tgtcagcagt tgtccgtgga aggaattgcc ggtcaaaggt 9120

tttcaactgg tgtgatggtg cctttactgt cctgcctcaa agcaccgcaa ttcaggctct 9180

tgagccagat agctgcgttt gcatcccagc tctgcggagc acccaaagcc tgatgcttcc 9240

atctcctcca cctctgttca ttaaaccagt gttccttgac tacttactgt gtgccgggtg 9300

caattaacca acatacgttg gtcgataaaa cagaccaaat ttcctcccca tatagagctt 9360

ccattctagt ggtgaaagaa acaagaagca agataaagtg tgtagtgtgt gtgcaatggt 9420

gataaatacc atggagaaga caaagcagag aagggaatgg gactgggcgc catgactcat 9480

acctgtaatc ccagcacttt gggaggcaga gatgggagga tcacttgagg ctaggagttc 9540

aagaccagcc tggccaacat ggcgaaaccc catctctacc aaaaaagata caaaaattgg 9600

ccgggtgtgg tggcatgcac ctgtagtccc agctactcag gaggctgagg tgggaagatt 9660

ccatgcaccc aggaggtgga ggttgcagtg agtcaagatg gtgccactgc actccagcct 9720

gggtgacaga gtgagacgct gtctcaaaaa aataataatt aaataaatta aagagaaggg 9780

gatgggaagg gtgatcatca cagttgaaat agggtggcca agtgaggtgt tactaaggtg 9840

acatcccagc agaggcctga aggagatgag ggtgagctct gcgggtgcct agggggaaag 9900

cattccagca gagggaacag catgggcaaa cccatttcta tccatcagtc cctgcataag 9960

caaacaccct ggacacctaa ctggagagtt tgaggaccaa caaagaggcc actgtggatg 10020

cagaggcggg agcaaagggg agagtaatgg aaaacgtggg cagaggtagc aggggccaga 10080

tcacgtgggg cctgtgacac cagaagactt cggtttaact ctgagccagg ctaggccccc 10140

agagggtact gagctgaggt atgggaactg acttagggtt cacaggcgac ctctggtggc 10200

catgtgggga acagacgagg gggcgagggc agaaacggag cccagcgtgg aggcggctgc 10260

tgggggccgg gatgcacagc gccagagcag ggaggtaccg tggaagccct gaggagaggt 10320

gggattctgg atggatctta aggcggggcc gacatgattg gctaatgggt tgggtgtggg 10380

catgagagga agggaagagt tggggggcct ccagagttgc tctccgctta tgctatagag 10440

ggcgtgaggg tttctgggca cgtagtgcgt gctcaatcac tgtcactccg gacctgcagc 10500

agaggccacc tcctcacctt tctgtctctc ccaggaccat gggtcccctg ccaccctttg 10560

ctgtggccca gggcgcccag ctcctgatcc gtcctgtgga caaaccaatc aacacaactt 10620

taatctgcaa cgtcaccaat gccctaggag ctcgccaggc agaactgacc gtccaggtca 10680

aaggtgagga actccctggg tgggaagaac agggaccaaa ggaagaggtc gaggcagggt 10740

tccctgtcta atgctctctg gctcccatcc ttctcctaag ccttctcaca aaaggaccag 10800

tggggccagc tttactcccc tgttaactcc atacctatat cactggtacc gttgttcagc 10860

tgcctggata cactccctcc acacacacag gttgggatag atgtggaatc cctctctctc 10920

agggactcag gcctttccaa atatattagg cagacctcta tcaattgcaa ttgacagaaa 10980

cacaaagtag tctaagcaaa aaaaaccaaa caaaacagga ctttattgaa aattcacctt 11040

caggtatggt tagatccagg ggctccaacc ttgtaaccca ggaactttta ttttatacat 11100

ctcttgtctc tgcttttcgt tatactggct ttattctcag gcaggctctg aagcaagatg 11160

gccctcggca gggacaggct cacattctac cagaatggca acagcaggag aaaaaacata 11220

ccactttcag taactgtagc catagtccca gggctggctc tcattggccc agcttaggcc 11280

acagcccacc tatgaaccaa tcatagcagc cagggagaat ggaatattct gattggccag 11340

gcctgtgtca aagtcccatt ctgggcacca gagggtggga tccattccac ctgaattaaa 11400

tagactgagg cagtggagtg gtggctccct aaggaaattc aagatgccat ttcaagaatg 11460

agggataaac cccaagagtt tccttaccta aatacctgtt tccttctctt tcagaggga 11519

<210> 6

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttccttacct aaatacctgt ttccttctct ttcagaggga cctgagaata tgcagcaaaa 60

tacaagatta cacctaggct 80

<210> 7

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atccatgccc tgagcccctc actttccata gaccacactc gaattccgaa gttcctattc 60

tctagaaagt ataggaactt 80

<210> 8

<211> 100

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tctctagaaa gtataggaac ttcatcagtc aggtacataa tggtggatcc ttgtctaagc 60

actagctctg aatgccactg tagtctgcac aggggcctat 100

<210> 9

<211> 2893

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aggcggcacc cgcttagctg agattccagc acttgacttc agggtttcgg agagataagg 60

cgcttggccg ttactaactg gactacaaag agctggatcg gaccggaacc acatggctca 120

actcgcccga gccacccgct ccccgctgtc atggctgctg ctgctgttct gctatgcact 180

ccggaaagcg ggtggggatg tcgtcgtgca ggcgcccacc caggtgcccg gcttcttggg 240

cgactccgtg acgctgccct gctacctaca ggtgcccaac atggaggtga cgcatgtgtc 300

acagctgact tgggcgcggc atggtgaatc tggcagcatg gccgtcttcc accaaacgca 360

gggccccagc tattcggagt ccaaacggct ggaattcgtg gcagccagac tgggcgcgga 420

gctgcggaat gcctcgctga ggatgttcgg gttgcgcgta gaggatgaag gcaactacac 480

ctgcctgttc gtcacgttcc cgcagggcag caggagcgtg gatatctggc tccgagtgct 540

tgccaagccc cagaacacag ctgaggttca gaaggtccag ctcactggag agccagtgcc 600

catggcccgc tgcgtctcca cagggggtcg cccgccagcc caaatcacct ggcactcaga 660

cctgggcggg atgcccaata cgagccaggt gccagggttc ctgtctggca cagtcactgt 720

caccagcctc tggatattgg tgccctcaag ccaggtggac ggcaagaatg tgacctgcaa 780

ggtggagcac gagagctttg agaagcctca gctgctgact gtgaacctca ccgtgtacta 840

ccccccagag gtatccatct ctggctatga taacaactgg taccttggcc agaatgaggc 900

caccctgacc tgcgatgctc gcagcaaccc agagcccaca ggctataatt ggagcacgac 960

catgggtccc ctgccaccct ttgctgtggc ccagggcgcc cagctcctga tccgtcctgt 1020

ggacaaacca atcaacacaa ctttaatctg caacgtcacc aatgccctag gagctcgcca 1080

ggcagaactg accgtccagg tcaaagaggg acctgagaat atgcagcaaa atacaagatt 1140

acacctaggc tacatctttc ttatcgtctt tgtcctcgct gtagtcatca tcatcgcagc 1200

actatacact atacgaagat gcaggcatgg tcgtgctctg cagtccaatc cctcagagag 1260

ggagaacgtc cagtattcat ctgtgaacgg cgactgtaga ctgaacatgg agccaaacag 1320

cacaaggtga cggtgctggg tagacagaac taaggaactt gaaggcatag caactggaac 1380

cctactctca taaatgaaga agcctccaga gagactggct gctcagtgtg atgagcatag 1440

caagtttggg gggtctccca ggatgctgcc gaattccacg ttgtcaaaag gacccatgga 1500

ggccagtgtg ttggctcact cttgacatct cagcaagctg gggggggggg ggggagcata 1560

aagcaaggtt gagtctagct tgggctatag agcaaagccc tgtccataca caaacaagct 1620

aaggggcttt gagacggtca gaaactgaag tcttgctttg ggtaaggtaa atcctctacc 1680

gcatgtatgt gctagacttg aaagacttcc acacagacct ctttataagt tgactccatt 1740

ggggctatcc cctcctctct ggacaaggtc tctgtatgta gccaaggcta ggctcaaact 1800

cacagagata tgtctgcttc tacctcccca gtgctagagt tgaaagtatt tgtgccactg 1860

cacttttcta ggtcttcttt taatgaagta aagtatatat ttataaaaag ctatttagtt 1920

atatatatat atatttttga gactatttca tagagcccaa gctaacctca aacttactat 1980

gtagccaaga gtgatggtaa actaatttat tttaatttat ttgtcttcaa ttttaaccat 2040

cacccaaccc ctgctccctt ccatatcttc tttcaatcca tttcattgtc tttttcttcc 2100

cagacactat tctgacttac gtctccatta caaacatttt attgaactac ataaaaatgt 2160

gtgaaccaca aaaaaaaaat gtatttgtca aaattgtagt tgtctttctg aggctgacct 2220

gagttctctg ataccattct ctccagttgt atccagtttc ctgtaaacaa tgtgactttg 2280

tttttctcag tagctaaaac atcccaatta tgtgagtgta cactttcttt actcattcct 2340

ctgtgggcca ccagctgggt tggttccata tctgagctat tgtgcatgga attgtctctg 2400

tggtgggttt agtaaactcc caggaatgcc tgtacatgtt tgtagaggcc agaagaaggc 2460

acaaaatctt gagccaggct tacatgcact tgtgagtagc cccacatagg tgctaagaac 2520

ccagttcagg tcctctgctg tgggatggtg ggctgtgcac agaaagcctg gtcccggtct 2580

agcaaaggtc tggaactccg gagccggtgg gctgtgattt acaccagcat gggatggaag 2640

gagttggacc tcgcctcctg ggcacctggc tcctgtcaca tagctacagc ctcccacagc 2700

ccccctatag ggaggtatgc agcatcaatc acatagtagc tgcactaagc cctcccacat 2760

gcaaataagg tttccccaaa ctctcagtcc aagccaatga aaagtacctg ctgtcaaacc 2820

ctaaatcatc cccaaaactc tgtaagtcct atcagggaat aaaatgtgtg tgaaaactaa 2880

aaaaaaaaaa aaa 2893

<210> 10

<211> 405

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Ala Gln Leu Ala Arg Ala Thr Arg Ser Pro Leu Ser Trp Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Pro Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln

50 55 60

Leu Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His

65 70 75 80

Gln Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val

85 90 95

Ala Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe

100 105 110

Gly Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr

115 120 125

Phe Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala

130 135 140

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

145 150 155 160

Pro Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala

165 170 175

Gln Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln

180 185 190

Val Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile

195 200 205

Leu Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val

210 215 220

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

225 230 235 240

Val Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp

245 250 255

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

260 265 270

Pro Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Met Gln Gln Asn Thr Arg Leu His Leu Gly Tyr Ile Phe Leu Ile Val

340 345 350

Phe Val Leu Ala Val Val Ile Ile Ile Ala Ala Leu Tyr Thr Ile Arg

355 360 365

Arg Cys Arg His Gly Arg Ala Leu Gln Ser Asn Pro Ser Glu Arg Glu

370 375 380

Asn Val Gln Tyr Ser Ser Val Asn Gly Asp Cys Arg Leu Asn Met Glu

385 390 395 400

Pro Asn Ser Thr Arg

405

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

catgatggag gagccctgag aatgg 25

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gggatctgct gtggagggaa tctc 24

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gctcgactag agcttgcgga 20

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgtttgtgta tggacagggc tttgc 25

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

accaccacaa ggccggaaac cat 23

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agaatagcca aacaaccagc ttagag 26

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttaaccatc acccaacccc tgctc 25

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cctatgtggg gctactcaca agtgc 25

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggatcggcca ttgaacaaga t 21

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagaagaact cgtcaagaag gc 22

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttgttattg atgggaaggg gccga 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gcttctactg cctctgggga atgtg 25

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

catgctgcca gattcaccat gcc 23

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atccatgccc tgagcccctc actttc 26

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ggtctgggag agactgaagg tagg 24

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gacaagcgtt agtaggcaca tatac 25

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gctccaattt cccacaacat tagt 24

Claims

1. A method of constructing a non-human animal humanized with a CD155 gene comprising the step of expressing a polypeptide comprising the amino acid sequence encoding SEQ ID NO: 2 from position 29 to 332 to the non-human animal CD155 locus.

2. The method of claim 1, comprising administering a peptide comprising SEQ ID NO: 5 to the non-human animal CD155 locus.

3. The construct of claim 1 or 2, wherein the replacement to the non-human animal CD155 locus is a replacement of the nucleotide sequence of the non-human animal CD155 gene encoding SEQ ID NO: 1 from 30 to 336 or a nucleotide sequence which replaces the same sequence as shown in positions 19643191 to 19652829 of NCBI accession No. NC _000073.7 in a non-human animal which is a mouse or a rat.

4. The method of claim 1 or 2, wherein the non-human animal expresses a humanized CD155 protein comprising the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

5. The method of claim 1 or 2, wherein the genome of the non-human animal comprises a humanized CD155 gene, and the mRNA transcribed from the humanized CD155 gene comprises SEQ ID NO: 9, or a nucleotide sequence shown in the specification.

6. The method of claim 1 or 2, wherein the non-human animal is constructed using a targeting vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5, and the targeting vector further comprises a 5 ' arm and/or a 3 ' arm, and the nucleotide sequence of the 5 ' arm is shown as SEQ ID NO: 3, and the nucleotide sequence of the 3' arm is shown as SEQ ID NO: 4, respectively.

7. A targeting vector for a CD155 gene, said targeting vector comprising a nucleic acid sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5, and the targeting vector further comprises a 5 ' arm and/or a 3 ' arm, and the nucleotide sequence of the 5 ' arm is shown as SEQ ID NO: 3, and the nucleotide sequence of the 3' arm is shown as SEQ ID NO: 4, respectively.

8. A humanized CD155 protein, wherein the amino acid sequence of the humanized CD155 protein is as shown in SEQ ID NO: shown at 10.

9. A humanized CD155 gene encoding the humanized CD155 protein of claim 8, wherein the humanized CD155 gene comprises the amino acid sequence of SEQ ID NO: 5.

10. Use of a non-human animal humanized with a CD155 gene obtained by the construction method according to any of claims 1 to 6, a humanized CD155 protein according to claim 8 or a humanized CD155 gene according to claim 9 for non-disease diagnostic, non-disease therapeutic purposes, the use comprising:

A) use in the development of products involving CD 155-related immune processes in human cells;

E) the application of the CD155 gene function, the medicine and the drug effect aiming at the target site of the human CD155, the immune-related disease medicine related to the CD155 and the anti-tumor medicine are researched.