CXCR4 gene humanized non-human animal and construction method and application thereof
阅读说明：本技术 Cxcr4基因人源化的非人动物及其构建方法和应用 (CXCR4 gene humanized non-human animal and construction method and application thereof ) 是由 李惠琳 张赞 赵磊 于 2021-02-05 设计创作，主要内容包括：本发明提供了一种CXCR4基因人源化的非人动物及其构建方法和应用。本发明还提供了包含CXCR4基因修饰的多基因修饰的非人动物的构建方法,以及制备获得的CXCR4基因人源化的非人动物或多基因修饰的非人动物在靶向人CXCR4信号通路药物的筛选、药效评价中的应用。(The invention provides a CXCR4 gene humanized non-human animal and a construction method and application thereof. The invention also provides a construction method of the polygene modified non-human animal containing CXCR4 gene modification, and application of the prepared CXCR4 gene humanized non-human animal or polygene modified non-human animal in screening and drug effect evaluation of a medicine targeting a human CXCR4 signal pathway.)
1. A method of constructing a non-human animal humanized with a CXCR4 gene, said method comprising administering to a human animal a peptide comprising a sequence encoding SEQ ID NO: 2 to the non-human animal CXCR4 locus.
2. The method of claim 1, comprising administering a peptide comprising SEQ ID NO: 5 to the non-human animal CXCR4 locus.
3. The method for constructing a recombinant vector according to claim 1 or 2, wherein the non-human animal CXCR4 locus is a non-human animal having a nucleotide sequence identical to the sequence of 128516580-128519946 of NCBI accession No. NC-000067.7, and the non-human animal is a mouse or a rat.
4. The method of claim 1 or 2, wherein the non-human animal body expresses human CXCR4 protein with reduced or absent endogenous CXCR4 protein expression, wherein the human CXCR4 protein comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
5. The method of claim 1 or 2, wherein the genome of said non-human animal comprises a humanized CXCR4 gene, and wherein the mRNA transcribed from said humanized CXCR4 gene comprises the amino acid sequence of SEQ ID NO: 8.
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 a5 ' 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 the CXCR4 gene, wherein said targeting vector comprises a nucleic acid sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5, and the targeting vector further comprises a5 ' 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 CXCR4 gene, wherein said humanized CXCR4 gene comprises a nucleotide sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5.
9. The humanized CXCR4 gene of claim 8 wherein said humanized CXCR4 gene transcribes mRNA as set forth in SEQ ID NO: shown in fig. 8.
10. Use of a non-human animal obtained by the construction method of any one of claims 1 to 6 or of the humanized CXCR4 gene of any one of claims 8 to 9 for non-disease diagnostic and non-disease therapeutic purposes, said use comprising:
A) use in the development of products involving CXCR 4-related immune processes of human cells;
B) use in model systems related to CXCR4 as pharmacological, immunological, microbiological and medical research;
C) to the production and use of animal experimental disease models for the research of the etiology associated with CXCR4 and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
D) the application of the CXCR4 signal channel regulator in screening, detecting drug effect, evaluating curative effect, verifying or evaluating in vivo; alternatively, the first and second electrodes may be,
E) the research on the function of CXCR4 gene, the research on the medicine and the drug effect aiming at the target site of human CXCR4, and the research on the application of the medicine for treating the immune related diseases related to CXCR4 and the anti-tumor medicine.
Technical Field
The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a CXCR4 gene humanized non-human animal, a construction method thereof and application thereof in the field of biomedicine.
Background
CXCR4 (C-C chemokine receptor type 4) is a member of the chemokine receptor family, GPCRs (G protein coupled receptors) consisting of 352 amino acids, with 7 transmembrane structures, stromal cell derived factor-1 (SDF-1/CXCL 12) being the only known ligand for CXCR4, also known as the T cell co-receptor for HIV. CXCR4 is expressed primarily in lymphocytes (T/B), monocytes, macrophages, Natural Killer (NK) cells and dendritic cells, with small amounts expressed in endothelial and epithelial cells. CXCL12/CXCR4 up-regulates VEGF by activating PI3K/Akt signaling pathway, and tumor cells make the tumor cells highly express CXCL12 lymphocytes or tissues to metastasize by increasing the expression of CXCR 4; CXCL12/CXCR4 promotes the increase of vascular endothelial growth factor VEGF, thereby leading to the generation of tumor blood vessels and promoting the proliferation of tumor cells. The monoclonal antibody medicine targeting CXCR4 is mainly used for treating malignant tumors such as acute lymphocytic leukemia, multiple myeloma, metastatic breast cancer, metastatic pancreatic cancer and the like.
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, that is, the gene editing technology is used to replace the homologous gene in animal genome with human normal or mutant gene, so as to establish normal or mutant gene animal model with physiological or disease characteristics similar to that of human. 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 medicine only capable of identifying the amino acid sequence of the human protein, thereby providing possibility for screening anti-human antibodies and other medicines at the animal level. However, due to differences in physiology and pathology between animals and humans, coupled with the complexity of genes (i.e., genetic factors), how to construct "efficient" humanized animal models for new Drug development remains the greatest challenge (Scheer N, Snaith M, Wolf CR, Seibler J. Generation and compliance of genetic humanized models, Drug Discov Today; 18(23-24):1200-11, 2013).
In view of the wide involvement of CXCR4 in the development of various diseases such as tumors and the great value of targeting this signaling pathway, there is still a great need in the art to develop non-human animal models related to the humanized CXCR4 signaling pathway in order to make preclinical trials more effective and minimize development failures.
Disclosure of Invention
In a first aspect of the invention, a construction method of a non-human animal humanized by a CXCR4 gene is provided, wherein the genome of the non-human animal comprises all or part of a human CXCR4 gene. Preferably, exon 1 and/or exon 2 of the human CXCR4 gene are included. Further preferably, the recombinant vector further comprises an intron 1-2.
In one embodiment of the invention, the genome of the non-human animal comprises a portion of human exon 1 and a portion of exon 2, wherein the portion of exon 1 comprises at least the nucleotide sequence of exon 1 encoding CXCR4 protein and the portion of exon 2 comprises at least the nucleotide sequence of exon 1 encoding CXCR4 protein.
In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding SEQ ID NO: 2.
In one embodiment of the invention, the genome of the non-human animal comprises SEQ ID NO: 5.
Preferably, the non-human animal body expresses a human or humanized CXCR4 protein. Further preferably, said humanized CXCR4 protein comprises all or part of a human CXCR4 protein. Still further preferably, said humanized CXCR4 protein comprises an amino acid sequence encoded by exon 1 and/or exon 2 of the human CXCR4 gene.
In one embodiment of the invention, said human CXCR4 protein comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
Preferably, said non-human animal has reduced or absent expression of endogenous CXCR4 protein.
Preferably, the genome of the non-human animal comprises a humanized CXCR4 gene. Further preferably, said humanized CXCR4 gene comprises all or part of a human CXCR4 gene. Even more preferably, exon 1 and/or exon 2 of the human CXCR4 gene is included. Still further preferred, comprises a nucleic acid sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5.
In one embodiment of the invention, the mRNA transcribed from the humanized CXCR4 gene comprises SEQ ID NO: 8, or a nucleotide sequence identical to SEQ ID NO: 8, or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 8 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 set forth in SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
The construction method of the invention comprises inserting or replacing a number 1 and/or number 2 exon containing the human CXCR4 gene into the CXCR4 locus of a non-human animal. Preferably, the inserted or substituted sequence further comprises introns 1-2. Preferably, the replacement is a replacement of a corresponding position. The corresponding position is the amino acid or nucleotide sequence which has the same function compared with the human and the mouse.
The construction method of the invention comprises inserting or replacing a nucleotide sequence encoding human or humanized CXCR4 protein into a non-human animal CXCR4 locus.
The construction method of the invention comprises inserting or replacing a nucleotide sequence containing human CXCR4 gene into a non-human animal CXCR4 locus.
In one embodiment of the invention, the method comprises administering to a subject a polypeptide comprising a nucleotide sequence encoding SEQ ID NO: 2 to the non-human animal CXCR4 locus.
In one embodiment of the invention, the method of construction comprises the step of using a nucleic acid comprising SEQ ID NO: 5 to the non-human animal CXCR4 locus.
Preferably, the non-human animal CXCR4 locus is a substitution in the non-human animal CXCR4 gene encoding SEQ ID NO: 1.
Preferably, the non-human animal CXCR4 locus is a nucleotide sequence which replaces the same nucleotide sequence as the sequence shown in NCBI accession No. NC-000067.7 128516580-128519946 in a non-human animal.
Preferably, the non-human animal is a mouse or a rat.
Preferably, the insertion site is located after the endogenous regulatory elements of the CXCR4 gene.
Preferably, the human CXCR4 gene, humanized CXCR4 gene, humanized CXCR4 protein or a nucleotide sequence encoding human CXCR4 protein are regulated by CXCR4 endogenous regulatory elements.
The construction of CXCR4 gene humanized non-human animals is performed using gene editing techniques including gene targeting techniques using embryonic stem cells, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases, or other molecular biology techniques.
In one embodiment of the invention, the construction of the non-human animal is performed using a targeting vector.
The targeting vector comprises all or part of a human CXCR4 gene. Preferably comprises all or part of exon 1 and/or exon 2 of human CXCR4 gene, and further preferably comprises intron 1-2.
The targeting vector comprises a nucleotide sequence encoding a human CXCR4 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.
The targeting vector further comprises a5 '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 _ 000067.7. More preferably, the length is 3000-4000 bp.
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 _ 000067.7. Further preferably, it is 5000-.
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 1 to exon 2 of the CXCR4 gene in a non-human animal.
In a specific embodiment of the invention, the construction method comprises introducing the targeting vector into cells of a non-human animal, culturing the cells (preferably embryonic stem cells), transplanting the cultured cells into oviducts of a female non-human animal, allowing the cells to develop, and identifying and screening the non-human animal humanized with the CXCR4 gene.
Preferably, to improve recombination efficiency, sgRNA targeting CXCR4 gene can also be used to construct non-human animals along with the targeting vectors described above. Wherein the sgRNA targets a non-human animal CXCR4 gene while the sequence of the sgRNA is on a target sequence on the CXCR4 gene to be altered. Preferably, the target site of the sgRNA is located on exon 1 to exon 2 of the CXCR4 gene.
In a second aspect of the invention, there is provided a non-human animal humanized with CXCR4 gene obtained by the above construction method.
In a third aspect of the invention, a construction method of a CXCR4 gene-deleted non-human animal is provided, wherein the construction method comprises knocking out exons 1 and/or 2 of an endogenous CXCR4 gene.
In a fourth aspect of the invention, a non-human animal with a deleted CXCR4 gene obtained by the above construction method is provided.
In the fifth aspect of the invention, a CXCR4 gene-deleted cell, tissue or organ is provided, wherein the cell, tissue or organ is obtained by the construction method of the CXCR45 gene-deleted non-human animal or is derived from the constructed CXCR4 gene-deleted non-human animal.
In a sixth aspect of the invention, there is provided a targeting vector for the CXCR4 gene, said targeting vector comprising all or part of the human CXCR4 gene. Preferably comprises all or part of exon 1 and/or exon 2 of human CXCR4 gene, and further preferably comprises intron 1-2.
The targeting vector comprises a nucleotide sequence encoding a human CXCR4 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.
The targeting vector further comprises a5 '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 _ 000067.7. More preferably, the length is 3000-4000 bp.
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 _ 000067.7. Further preferably, it is 5000-.
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 1 to exon 2 of the CXCR4 gene in a non-human animal.
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 seventh aspect of the invention, there is provided a cell comprising the targeting vector described above.
In an eighth aspect of the invention there is provided the use of a targeting vector as described above or a cell as described above in the modification of the CXCR4 gene.
In the ninth aspect of the invention, a humanized CXCR4 gene is provided, wherein the humanized CXCR4 gene comprises all or part of a human CXCR4 gene. Further preferred, exon 1 and/or exon 2 of the human CXCR4 gene is included. Still further preferred, comprises a nucleic acid sequence encoding SEQ ID NO: 2 or a nucleotide sequence comprising SEQ ID NO: 5.
In one embodiment of the invention, the mRNA transcribed from the humanized CXCR4 gene comprises SEQ ID NO: 8, or a nucleotide sequence identical to SEQ ID NO: 8, or a nucleotide sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 8 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 set forth in SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
In a tenth aspect of the invention there is provided a humanized CXCR4 protein, said humanized CXCR4 protein comprising all or part of a human CXCR4 protein. Further preferably, said humanized CXCR4 protein comprises an amino acid sequence encoded by exon 1 and/or exon 2 of human CXCR4 gene.
In one embodiment of the invention, said human CXCR4 protein comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
In one embodiment of the invention, the humanized CXCR4 protein is encoded by the humanized CXCR4 gene described above.
In an eleventh aspect of the invention there is provided a construct comprising a humanized CXCR4 gene as described above. Preferably, said construct expresses the above-described humanized CXCR4 protein.
In a twelfth aspect of the invention, there is provided a cell comprising the above construct.
In a thirteenth aspect of the invention, there is provided a tissue comprising the above-described cells.
In a fourteenth aspect of the present invention, there is provided a method for constructing a polygene-modified non-human animal, said 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 CXCR4, inseminating in vitro, or directly performing gene editing, and screening to obtain a polygenetically modified non-human animal.
Preferably, the polygenetically modified non-human animal is a double genetically modified non-human animal, a triple genetically modified non-human animal, a quadruple genetically modified non-human animal, a quintuple genetically modified non-human animal, a hexa genetically modified non-human animal, a hepta genetically modified non-human animal, an octa genetically modified non-human animal or a nona genetically modified non-human animal.
Preferably, the animals modified by other genes except CXCR4 are selected from one or more than two of animals modified by genes such as PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3 or CD 73.
In a fifteenth aspect of the present invention, there is provided a tumor-bearing animal model, wherein the method for preparing the animal model comprises the step of preparing a non-human animal by the above-mentioned 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 a sixteenth aspect of the present invention, there is provided a CXCR4 gene modified cell, tissue or organ obtained by the above method for constructing a CXCR4 gene humanized non-human animal or a method for constructing a polygenic modified non-human animal, or derived from the above constructed CXCR4 gene humanized non-human animal or the above constructed polygenic modified non-human animal, or the above tumor-bearing animal model.
In a seventeenth aspect of the present invention, there is provided an application of the above-mentioned non-human animal obtained by the above-mentioned construction method or the above-mentioned humanized CXCR4 gene, said application being for non-disease diagnosis, non-disease treatment purposes, said application comprising:
A) use in the development of products involving CXCR 4-related immune processes of human cells;
B) use in model systems related to CXCR4 as pharmacological, immunological, microbiological and medical research;
C) to the production and use of animal experimental disease models for the research of the etiology associated with CXCR4 and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
D) the application of the CXCR4 signal channel regulator in screening, detecting drug effect, evaluating curative effect, verifying or evaluating in vivo; alternatively, the first and second electrodes may be,
E) the research on the function of CXCR4 gene, the research on the medicine and the drug effect aiming at the target site of human CXCR4, and the research on the application of the medicine for treating the immune related diseases related to CXCR4 and the anti-tumor medicine.
The eighteenth aspect of the invention provides a non-human animal obtained by humanizing the CXCR4 gene obtained by the above-mentioned construction method, a non-human animal obtained by deleting the CXCR4 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 preparing an animal model.
In a nineteenth aspect, the invention provides a non-human animal obtained by humanizing the CXCR4 gene obtained by the above-mentioned construction method, a non-human animal obtained by deleting the CXCR4 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 tumor, immune-related diseases, cardiovascular and cerebrovascular diseases, nervous system diseases or inflammation.
In a twentieth aspect of the invention, there is provided a method of screening for a modulator specific for human CXCR4, said method comprising administering the modulator to an individual, detecting the effect of the modulation; wherein the individual is selected from the group consisting of the above non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, and the above-mentioned disease model.
Preferably, the modulator is selected from CAR-T, a drug; preferably, the drug is an antibody. Preferably, the modulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.
Preferably, the screening method further comprises the step of implanting a tumor into the individual.
Preferably, the detection comprises determining the size and/or proliferation rate of the tumor cells.
Preferably, the detection method comprises vernier caliper measurement, flow cytometry detection and/or animal in vivo imaging detection.
Preferably, the detecting comprises assessing the weight, fat mass, activation pathways, neuroprotective activity or metabolic changes in the individual, including changes in food consumption or water consumption.
Preferably, the tumor cell is derived from a human or non-human animal.
Preferably, the screening method further comprises the step of implanting a tumor into the individual.
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 acute lymphocytic leukemia, multiple myeloma, metastatic breast cancer, metastatic pancreatic cancer.
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 term "inflammation" as used herein includes, but is not limited to, acute inflammation, and also includes chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).
The cardiovascular and cerebrovascular diseases comprise, but are not limited to hypertension, hyperlipidemia, cerebral apoplexy, myocarditis, arrhythmia, myocardial infarction, heart failure, arteriosclerosis, coronary heart disease, angina pectoris or congenital heart disease.
The "nervous system diseases" described in the present invention include, but are not limited to, cerebrovascular diseases, nervous system degenerative diseases, central nervous system infections, central nervous system demyelinating diseases, dyskinetic diseases, epilepsy, spinal cord diseases, peripheral neuropathy, autonomic nervous system diseases, neuromuscular junction and muscle diseases, nervous system genetic diseases, nervous system dysplasia diseases, and other diseases with sleep disorders such as insomnia, narcolepsy, restless leg syndrome, headache, 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, natural killer cells (NK), dendritic cells or tumor cells.
The "humanized CXCR4 protein" of the invention comprises a part derived from a human CXCR4 protein and a part of a non-human animal CXCR4 protein.
The "humanized CXCR4 protein" of the invention comprises a part derived from a human CXCR4 gene and a part of a non-human animal CXCR4 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 "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 "CXCR 4 locus" refers to a DNA fragment of an optional stretch of the CXCR4 gene. In one embodiment of the invention, the CXCR4 locus that is replaced can be a DNA fragment from exon 1 to exon 2 of the CXCR4 gene, optionally.
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. inchief, Academic Press, Inc., New York), specific, Vols, 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 structural comparison (not to scale) of human and mouse CXCR4 genes;
FIG. 2: humanization of the mouse CXCR4 gene is shown schematically (not to scale), where the UTR region is from murine;
FIG. 3: CXCR4 gene targeting strategies and targeting vector design schematic (not to scale);
FIG. 4: southern blot results of ES cells after recombination, in which WT was the wild type control;
FIG. 5: schematic representation (not to scale) of mouse FRT recombination process for humanization of CXCR4 gene;
FIG. 6: f1 mouse tail PCR identification result, wherein WT is wild type, H2O is water control, and PC is positive control;
FIG. 7: expression of C57BL/6 wild type mouse (WT) and CXCR4 gene humanized homozygote mouse (H/H) in CD4+ T cells, wherein Iso is isotype control, mCXCR4 is murine CXCR4 protein, hCXCR4 is human CXCR4 protein.
FIG. 8A: expression of C57BL/6 wild type mouse (WT) and CXCR4 gene humanized homozygote mouse (H/H) in B cell, wherein Iso is isotype control, mXCR 4 is murine CXCR4 protein, and hXCR 4 is human CXCR4 protein.
FIG. 8B: expression of C57BL/6 wild type mouse (WT) and CXCR4 gene humanized homozygote mouse (H/H) in T cell, wherein Iso is isotype control, mXCR 4 is murine CXCR4 protein, and hXCR 4 is human CXCR4 protein.
FIG. 9: RT-PCR (reverse transcription-polymerase chain reaction) is used for detecting the expression condition of CXCR4 protein in a CXCR4 gene humanized mouse, wherein +/-is wild type, H/H is a CXCR4 gene humanized homozygote, and H2O is water control and GAPDH is internal control.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In each of the following examples, the equipment and materials were obtained from several companies as indicated below:
c57BL/6 mice and Flp tool mice were purchased from the national rodent laboratory animal seed center of the Chinese food and drug assay institute;
brilliant Violet 510 anti-mouse CD45 Antibody, from Biolegend, cat # 103138;
PerCP/Cy5.5 anti-mouse TCR β chain Antibody from Biolegend, cat # 109228;
brilliant Violet 421 anti-mouse CD4 Antibody, from Biolegend, cat # 100438;
brilliant Violet 711 Antibody-mouse CD8a Antibody from Biolegend, cat # 100759;
PE anti-mouse CD184 (CXCR4) Antibody, available from Biolegend under cat number 146505;
PE Rat IgG2b, k isotype Ctrl Antibody, from Biolegend, cat # 400608;
zombie NIR ™ Fixable visualization Kit, available from Biolegend, cat # 423106.
Example 1 preparation of CXCR4 Gene humanized mice
This example modifies a non-human animal (e.g., a mouse) to include a nucleotide sequence encoding a humanized CXCR4 protein in the non-human animal, resulting in a genetically modified non-human animal that expresses a human or humanized CXCR4 protein. Mouse CXCR4 Gene (NCBI Gene ID: 12767, Primary source: MGI: 109563, Unit Probe ID: G3XA59, located at positions 128515936 to 128520036 of chromosome 1 NC-000067.7, based on transcript NM-009911.3 and its encoded protein NP-034041.2 (SEQ ID NO: 1)) and human CXCR4 Gene (NCBI Gene ID: 7852, Primary source: HGNC: 2561, Unit Probe ID: P61073, located at positions 136114349 and 136118149 of chromosome 2 NC-000002.12, based on transcript NM-003467.3 and its encoded protein NP-003458.1 (SEQ ID NO: 2)). The comparative schematic is shown in fig. 1.
To achieve the object of the present invention, a gene sequence encoding a human CXCR4 protein may be introduced at the endogenous CXCR4 locus of a mouse, such that the mouse expresses a human or humanized CXCR4 protein. In particular, humanized modification of the mouse CXCR4 gene can be achieved by replacing the mouse corresponding sequence with a nucleotide sequence (e.g., genomic DNA sequence, cDNA sequence, CDS sequence, etc.) of the human CXCR4 gene at the mouse endogenous CXCR4 locus, such as replacing the sequence comprising at least the start codon ATG to the stop codon TGA of the mouse CXCR4 gene with the corresponding human genomic DNA sequence, resulting in a humanized CXCR4 locus, in some embodiments, from the start codon of exon 1 to the stop codon of exon 2 with the corresponding human genomic DNA sequence, as shown in fig. 2.
Further engineering the targeting strategy as shown in figure 3, a homologous arm sequence containing both upstream and downstream of the mouse CXCR4 gene on the targeting vector is shown in figure 3, along with an a fragment containing the human CXCR4 sequence. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from 128519947 to 128523767 of NCBI accession No. NC-000067.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is the same as the nucleotide sequence from 128510022 to 128515618 of NCBI accession No. NC-000067.7; the sequence of human CXCR4 (SEQ ID NO: 5) is identical to the nucleotide sequence at positions 136114869 to 136118060 of NCBI accession No. NC-000002.12.
The targeting vector also comprises a resistance gene used for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein, the connection design of the 5 'end of the Neo box and the mouse is 5' -CTTCCTCGCCAGAGCTGAGTGAGATT AAGATCTGAATTCCGAAGTTCCTATTCTCTAG-3' (SEQ ID NO: 6), wherein the sequence "GATTA"the last" A "of" is the last nucleotide, sequence, of mouse "AGATC"the first" A "of is the first nucleotide of the Neo cassette; the connection of the 3 'end of the Neo-box to the mouse is designed to be 5' -AGGAACTTCATCAGTCAGGTACATAATGGTGGATCCAGTACTGATATCAAGCATATACCACGATGCCCAGCTTTTTATGAGCAT-3' (SEQ ID NO: 7), wherein the sequence "GATATC"the last" C "of a" is the last nucleotide, sequence of the Neo cassette "AAGCAT"the first" A "of" is the first nucleotide of the mouse. The 5 'homology arm to human connection design 5' -CGTTTGGTGCTCCGGTAACCACCACGGCTGTAGAGCGAGTGTTGCCatggaggggatcagtgtaagtccagtttcaacctgctttgtca-3' (SEQ ID NO: 36), wherein "TGTTGCCThe "last" C "of a" is the last nucleotide of the 5' homology arm,“atggag"the first" A "of" is the first nucleotide of a human; the connection of the 3 'end of human CXCR4 to mouse was designed to be 5' -tttccactgagtctgagtcttcaagttttcactccagctaaCACTTATGCAAAGACATATATAATATATATATATATATATATGATAAAGAACTTTTTTA-3' (SEQ ID NO: 37), wherein "agctaaThe last of "A" is the last nucleotide of a human, ".CACTTAThe "first" C "of" is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CXCR4 after being modified is shown as SEQ ID NO: 8, the expressed protein sequence is shown as SEQ ID NO: 2, respectively.
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 targeting vector with correct sequencing verification is transfected into embryonic stem cells of a C57BL/6 mouse by electroporation, the obtained cells are screened by using a positive clone screening marker gene, PCR (PCR primers are detailed in table 1) and Southern Blot technology are used for detecting and confirming the integration condition of an exogenous gene, the correct positive clone cells are screened, the clone which is identified as positive by PCR is detected by Southern Blot (cell DNA is digested by BglII or ScaI or EcoRV respectively and is hybridized by using 3 probes, the lengths of enzyme, the probes and a target fragment are shown in table 2), and the detection result of the Southern Blot is shown in figure 4, which shows that 1 embryonic stem cell (ES-01) which is verified as positive by PCR is a positive clone without random insertion.
TABLE 1 PCR detection primer sequences and target fragment lengths
TABLE 2 Southern Blot enzyme and Probe Table
The Southern Blot detection comprises the following probe primers:
5 'Probe (5' Probe):
5’Probe-F：5’- CAGGGTGACTCTAAACCCTTTCTGT -3’（SEQ ID NO：13），
5’Probe-R：5’-AGGGCTATCAAATGTCAAGTAGGGA-3’ （SEQ ID NO：14）；
3 'Probe (3' Probe):
3’Probe-F：5’- GGGAAGCTGGTCATGCCTTGAAGAT -3’ （SEQ ID NO：15），
3’Probe-R：5’- ACAGCAGAGGTTAGCCAACAGCATT -3’ （SEQ ID NO：16）；
neo Probe (Neo Probe):
Neo Probe-F：5’- GGATCGGCCATTGAACAAGAT -3’ （SEQ ID NO：17），
Neo Probe-R：5’- CAGAAGAACTCGTCAAGAAGGC -3’ （SEQ ID NO：18）。
the selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (the process is schematically shown in figure 5), and then the humanized CXCR4 gene homozygote mice can be obtained by mating with each other. The results of the identification of the exemplary F1 mouse generations are shown in FIG. 6, in which the mice numbered F1-01 to F1-07 are all positive heterozygous mice and the PCR assay primers are shown in Table 3.
TABLE 3 PCR detection primer sequences and target fragment lengths
The expression of CXCR4 protein in a humanized mouse of the CXCR4 gene is further detected by flow cytometry. 1 Mouse of 9 weeks old wild-type C57BL/6 and humanized homozygous Mouse of CXCR4 gene were selected, and given i.p. to mice Anti-mCD3 (7.5. mu.g/Mouse), 24 hours later, spleen cells were harvested, and Anti-Mouse CD45 Antibody Brilliant Violet 510 Anti-Mouse CD45, murine T-cell surface Antibody PerCP/Cy5.5 Anti-Mouse TCR. beta. chain, Anti-Mouse CD4 Antibody Brilliant Viole 421 Anti-Mouse CD4, Anti-Mouse CD8a Antibody Brilliant Viole Anti-Mouse CD8a, Mouse Antibody Anti-Mouse CD184 (CXCR4) Antibody, PE, biolegged homo-type (mCR 4-PE) (Anti-PE b K-Mouse Antibody IgG 184, Anti-Mouse IgG 184 (CXCR4) Antibody), Anti-Mouse IgG 857 Antibody IgG 184 (CtR) Antibody, Anti-Mouse IgG 857 Antibody IgG 21, Anti-Mouse IgG (Anti-Mouse IgG) Antibody hCR b, Anti-Mouse IgG 184 (Anti-Mouse IgG) Antibody), kappa Isotype Ctrl Antibody, APC, Biolegend @) and then flow detection, the detection results are shown in FIGS. 7, 8A and 8B, CD4+ T cells, T cells and B cells of the spleen of C57BL/6 mouse only express mouse CXCR4 protein, and CD4+ T cells, B cells and T cells of the spleen of humanized homozygote mouse CXCR4 gene only express human CXCR4 protein. The results show that this example successfully constructs a CXCR4 gene humanized mouse capable of expressing human CXCR4 protein.
And further detecting the expression condition of the CXCR4 protein in a humanized mouse of the CXCR4 gene by adopting RT-PCR. Total RNA from spleen cells of wild type C57BL/6 mice and humanized CXCR4 homozygote mice was extracted and reverse transcribed into cDNA using a reverse transcription kit, and the primer sequences are shown in Table 4.
TABLE 4 RT-PCR detection primer sequences and target fragment lengths
The experimental results show (see fig. 9) that mRNA expression of murine CXCR4 can be detected in wild-type C57BL/6 mouse cells and human CXCR4 can be detected in humanized CXCR4 homozygous mouse cells.
Example 2 in vivo efficacy verification
The CXCR4 humanized mouse prepared by the method can be used for evaluating targetingThe potency of modulators of human CXCR 4. For example, humanized mouse homozygotes of CXCR4 are inoculated subcutaneously with mouse colon cancer cells MC38 until the tumor volume grows to about 100mm3Then dividing the tumor into a control group or a treatment group according to the tumor volume, randomly selecting a medicine targeting human CXCR4 and the like in the treatment group, and injecting equal volume of physiological saline into the control group. The tumor volume is measured regularly and the weight of the mouse is weighed, and the in vivo safety and the in vivo efficacy of the compound can be effectively evaluated by comparing the change of the weight of the mouse and the size of the tumor.
EXAMPLE 3 two-Gene or Multi-Gene humanized mice
The mouse with the CXCR4 gene humanized prepared by the method can also be used for preparing a mouse model with double gene modification or multiple gene modification. As in the foregoing example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as PD-1, PD-L1, CTLA4, OX40, LAG3, TIM3, CD73, etc., or can be obtained from humanized CXCR4 mice by using isolated mouse ES embryonic stem cells and gene recombination targeting techniques to obtain a mouse model of double or multiple gene modifications of CXCR4 and other genes. The homozygote or heterozygote of the CXCR4 mouse obtained by the method can be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the heterozygote of the CXCR4 gene and double gene or multiple gene modification heterozygote of other gene modification can be obtained with a certain probability according to Mendel genetic rule, the heterozygote is mated with each other to obtain the homozygote of double gene or multiple gene modification, and the in vivo efficacy verification of the targeted human CXCR4 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
<110> Baiosai Diagram (Beijing) pharmaceutical science and technology Co., Ltd
<120> CXCR4 gene humanized non-human animal and construction method and application thereof
<130> 1
<160> 37
<170> SIPOSequenceListing 1.0
<210> 1
<211> 359
<212> PRT
<213> Mouse (Mouse)
<400> 1
Met Glu Pro Ile Ser Val Ser Ile Tyr Thr Ser Asp Asn Tyr Ser Glu
1 5 10 15
Glu Val Gly Ser Gly Asp Tyr Asp Ser Asn Lys Glu Pro Cys Phe Arg
20 25 30
Asp Glu Asn Val His Phe Asn Arg Ile Phe Leu Pro Thr Ile Tyr Phe
35 40 45
Ile Ile Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile Leu Val
50 55 60
Met Gly Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu
65 70 75 80
His Leu Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp
85 90 95
Ala Val Asp Ala Met Ala Asp Trp Tyr Phe Gly Lys Phe Leu Cys Lys
100 105 110
Ala Val His Ile Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile
115 120 125
Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr
130 135 140
Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Ala Val Tyr Val
145 150 155 160
Gly Val Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile Phe
165 170 175
Ala Asp Val Ser Gln Gly Asp Ile Ser Gln Gly Asp Asp Arg Tyr Ile
180 185 190
Cys Asp Arg Leu Tyr Pro Asp Ser Leu Trp Met Val Val Phe Gln Phe
195 200 205
Gln His Ile Met Val Gly Leu Val Leu Pro Gly Ile Val Ile Leu Ser
210 215 220
Cys Tyr Cys Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His Gln
225 230 235 240
Lys Arg Lys Ala Leu Lys Thr Thr Val Ile Leu Ile Leu Ala Phe Phe
245 250 255
Ala Cys Trp Leu Pro Tyr Tyr Val Gly Ile Ser Ile Asp Ser Phe Ile
260 265 270
Leu Leu Gly Val Ile Lys Gln Gly Cys Asp Phe Glu Ser Ile Val His
275 280 285
Lys Trp Ile Ser Ile Thr Glu Ala Leu Ala Phe Phe His Cys Cys Leu
290 295 300
Asn Pro Ile Leu Tyr Ala Phe Leu Gly Ala Lys Phe Lys Ser Ser Ala
305 310 315 320
Gln His Ala Leu Asn Ser Met Ser Arg Gly Ser Ser Leu Lys Ile Leu
325 330 335
Ser Lys Gly Lys Arg Gly Gly His Ser Ser Val Ser Thr Glu Ser Glu
340 345 350
Ser Ser Ser Phe His Ser Ser
355
<210> 2
<211> 352
<212> PRT
<213> human (human)
<400> 2
Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp Asn Tyr Thr Glu Glu Met
1 5 10 15
Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pro Cys Phe Arg Glu Glu
20 25 30
Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Thr Ile Tyr Ser Ile Ile
35 40 45
Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile Leu Val Met Gly
50 55 60
Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu His Leu
65 70 75 80
Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp Ala Val
85 90 95
Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Phe Leu Cys Lys Ala Val
100 105 110
His Val Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile Leu Ala
115 120 125
Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr Asn Ser
130 135 140
Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Val Val Tyr Val Gly Val
145 150 155 160
Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile Phe Ala Asn
165 170 175
Val Ser Glu Ala Asp Asp Arg Tyr Ile Cys Asp Arg Phe Tyr Pro Asn
180 185 190
Asp Leu Trp Val Val Val Phe Gln Phe Gln His Ile Met Val Gly Leu
195 200 205
Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys Ile Ile Ile Ser
210 215 220
Lys Leu Ser His Ser Lys Gly His Gln Lys Arg Lys Ala Leu Lys Thr
225 230 235 240
Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp Leu Pro Tyr Tyr
245 250 255
Ile Gly Ile Ser Ile Asp Ser Phe Ile Leu Leu Glu Ile Ile Lys Gln
260 265 270
Gly Cys Glu Phe Glu Asn Thr Val His Lys Trp Ile Ser Ile Thr Glu
275 280 285
Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile Leu Tyr Ala Phe
290 295 300
Leu Gly Ala Lys Phe Lys Thr Ser Ala Gln His Ala Leu Thr Ser Val
305 310 315 320
Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys Gly Lys Arg Gly Gly
325 330 335
His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser Phe His Ser Ser
340 345 350
<210> 3
<211> 3821
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcatagattc tgggagaccg agttctacaa agcaagcact aactaagtca tctctgaacc 60
cttcattaaa tcggccttta atgccgatgc ctcctgccat gtaccatcaa gataaaggac 120
tgtgcataag ctttctcccc gactaacttg gagcctgttt tggacagagc agagctttct 180
atcgcatacc cagggacggg cgaattaacc agctgcagtg taagcaacag gaggatgctt 240
gcccctgtgc cactggcttt ggaagtatcc cccacaaact gtggccaaaa ctagttattc 300
cctgggctaa agccatcctt agaacttggg gacactgcca aagccctgac agcccctgag 360
cctggccttt gttgccagag ccccagggga aagctgttaa ttctttaaat gaaggttcag 420
taaacaacca aggagcttgg ttcgctcagc ggtgggctag ataggaaaac tgctatctgt 480
gcactgtctt ccaggagctt ggactgtttg ggagagagga gtaaggcaca tttgctttaa 540
aaaaaaaaaa aagttgaatc acgagagatg tcactcaaag cagtatgtaa gtaaatgtgg 600
aggaagaagc actcagaggg ttcattgcaa ggatcagaaa aagacagtca tggtaatgga 660
gtggagagag cctgggctct ggagccaggc agactaggtg ggcacagatc cttaagtgaa 720
tacaaggagg ggagctagct cctccccctc agcaaacttc agcttccttg tcctgaaaaa 780
cagacctgcc tagtgagggg cttatcccag gttggatgca tgcgtggaac aagctttcag 840
gagacgtttg caatgttttt tctgatgtgc atcacaaagc tggggctgca actaagtagg 900
cagaccgccc atctcatgtg tacataaccc tgagttcaat cctagcacca cataaactgg 960
gcacggtgtt ggagacagga gcaccagaga ttcaaaaatg aactcagctc cacggggaac 1020
ttgaggccag accgggatac aggaaaccca ctgccagtgg agtaaagcaa gagcttccag 1080
caagctgggc aagcactctt accaacggag ctacgctact agtcctcggc atatccaggc 1140
acgactgaga tcatagacta acacagtttt cattcccacc ctgtgcagtt tctttcctaa 1200
gtatttcccc ttagtaataa caactcttca caaacactta ccacatccat attcactccg 1260
ttcgcagatg tactaaatca tctcaactag tctgctcatg gcggacactt agggctactc 1320
agctattact aactattcta ttattaacac tattgtcagc agcagccccg tgaagagctt 1380
tgtagcatga ctgcagtttc ctggggctac atcatgaaag gtagaattac taatctcaaa 1440
aggttctggc tgcatacaaa atacttttca ctgaagctgt tcaaacacca cacacacaca 1500
cacacacaca cacacacaca cacacactcc tgctactgac aagcgagaga cagctggtct 1560
tttccaatcc agaagccaaa tattatcttt gaaaggggga aaaaatccct ctcttttttt 1620
ttctctaatt ctatagtcac caaatagcat gtcattgttg ttttatctgc tttcctccag 1680
caaagagact tcacaggagc taaagagacc tgtgcagaag ctttcttttg tccaccaata 1740
atcaacaaat attaattatg caaatcattt tactcagatt gtactctctg tcgttcaggg 1800
acaggttcgc aagattccct ttctcccatt ttagcctttg ggaatgcagg aatttggagg 1860
ctagacacag ctcaccatcc tgctgctacc gatcttgaac accagcgctc ttttaatggg 1920
tctgatattc cggtccacgg gctcttctgt ctgtcctaac tagtttatgg aaatatgagc 1980
ctatgtttta gtaatgattg gatataatta aaagtgcttg tggaagttct tggcttttaa 2040
cttagcagtt tttgagggtc gcacttccta atttcatcct tagcttttct ggatcacata 2100
agtgcagggc agaagaggac tttgacagca cattttgaag gagagtgccc aggccataca 2160
ggaagatgga gagagacgag ttcccaggct gtaaaacagc tccccagcac ttccaaccga 2220
aagccttcct tagctaaaga aggaggtgca aatgatagta cagttagtag actgcttgcc 2280
tagcctacac aaagctctga tttaagccct atcagcacga aaattgggcg tggtggcaca 2340
caactgtaat cccagtacta ggcaggtgac aaacgggatc atcagaaatt catggtcatc 2400
cttaactaca tacggagcct gagtccagcc tgtactacat aaaactgcct caaacaaaac 2460
aaacaaacaa acaaacaaac aaacaaaaaa ggaacgggag aagtggggcg gggatggagt 2520
tgagggcgca gtctgggcct gcctatcctt tggtccattc caggaggggg tgggggcggt 2580
atctgggttg catagcaacc caggctcctg ctgccggatg tctcaccacc cccagtgccc 2640
ccggcattgg atcccgcggt ctctggctgg aggtctggtt ggttctgacc ccaccctttt 2700
ctccctcccc gcctagccca ggctcggcgc cggtcggatc ccgcgcgagc ctgggaagca 2760
gtctgggcgc ccactgcaac cttagcctgt ctgagattcc ctagccatcc acgcccccaa 2820
cctgacctaa ctagctcttc actaagactt cccatcttag tgttctgcgt gggagacgcg 2880
agataggcat gaagcgaaag ctctgggctc aggccaggct ttgcagtccc aggctgctcc 2940
atttggtctc ttggagctcg tggagcgccc actgactgcg catgaatcgc ggctagcttc 3000
ccagctggga ctctgtaatt atctgttaac tagatgcatc cccctatgtg cttatgaaat 3060
gaaaatgaac cagtgtttct gtgcggattt cttaggatgt gattacacaa agtcctcgcc 3120
gctcaattct ttataataaa gcacatttca atcattttac atacacacac acccaaatct 3180
cttcacgaaa ttgttgccac tattgtgact ttcttcctgg gagtatatct gcttctggat 3240
gggtccgcaa gtgctttctg ccacacctga ccctaatgtc attatatgaa tatgtagtaa 3300
cacacacttg cttggaagag ttttggtagt aatcactcct gacagctcag agtatagaac 3360
cctggcggag gttccaacat tgccgcctac tggttaggca ggctccaagt cacacacccc 3420
catccccagt cttgtctcaa gttctccacc cgtaaactcc aggtctaaaa ttctatccac 3480
gtgggtaagg atggatgcgc gacccgcaga aacaccctgg ctggcatcct gctttcccca 3540
actctgagaa cattatccgg ccaggctctg gacccatact gacttaaaac acaacagcag 3600
tggctcttgg acttctgttt gcccgcgccc tgcgccccgc ccccgccagt ctcctcccgc 3660
cccgtccgag ctgcgcatgc gccgccccgg gagcgtgttt ttataaaagt ccggttcggg 3720
ccagaaactt caattttgtt gcctggtgca gcaggtagca gtgaaacctc tgaggcgttt 3780
ggtgctccgg taaccaccac ggctgtagag cgagtgttgc c 3821
<210> 4
<211> 5597
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aagcatatac cacgatgccc agctttttat gagcattgtg gaagtcagac ctaagttttc 60
acgcttacaa cagcaagcac tcaaagaacc aagccatctc cccagcccct gtcttaagtc 120
ctgtgtattt acttaagctt taccttaaga ggcagacttt ctgcttctac cactaaggga 180
gcccaggagc agaaagttta agaaactgca ttaaagatga gggacagcaa cagccagtat 240
ttcaaagcct gtgtggagag aagttgaagc ccttacactt tcaactcctc gtggaaggtg 300
aggaaagcag gatacacatt cgcatttcac tgtagtttcc cacctgtccc agtcaggcta 360
ggcgctgtgt gtcttctggt gtagatggca atcccacccc cacccccgtc cccttttgac 420
aaaggtggga cctgaggctc ataaacctgt ggtaactttt attttcagct tacacaaccc 480
aaacttaaca ctgttgatcc atcttaccca gtattgccac cccaaaacac ctggtcagat 540
gccggctgag ttgttactga acatgaggcc aaaagtgaat gttttcttca taaaattagc 600
catgcccaaa ataccctcct tgcaatatca cccatgcaaa tctcagcacc tttaccctga 660
ctcttattac aacatctagt gtctcctgaa gggctcaact aaatctgtta aagattaatg 720
ctggtgtttt ttggttggtt ggggtttttt tgtttgtttg ttttttgttt tgttttgttt 780
tttaagagtt gaaagctcta ctgaagcaag ctcttcattt tgcacaagat ttgagagcct 840
tatgtaataa cccagagcta tttacactaa cattgtgcaa gccgcaaact cttgagtaaa 900
ttgcaggtcc ggtctcctgg agaccggaga agataacttc tcatccatac tggtttgccc 960
cttggaacat atacagggtg ctgtaggcac acacccaggc agtaagtgat atataattaa 1020
ggcaatattt ggtcttttag taattgtttc tggcacagaa aatccgtttg ggaaggaaaa 1080
attgcaaggc cttatctttc ttaggcaaat cacatttgtt caagacaaat tatagatcct 1140
gtgaagggaa ataacttaat tacttaaaat aaaatctaat ttagctgtag gtttttgcag 1200
cagcctatgg atctggggta cttcatgccg gtgttcctat tccacttgag gacacaatta 1260
gattttcagt aggaaattat cttgagattt cttgccttcc tctggggagt cttaattgga 1320
tcacgcttag acatactttt cgtagttttc gatgtgcaca ctggaaggct tggtctcgaa 1380
gctgtggcca ttgtggcagg cttcactcgg aatctgctct ataaatattt attcgcaggg 1440
agacaatagg agaaaacctt gggaggaatt ctttctcttt tctcatcttg gcttgaaacc 1500
tcctcacccc agattcctct cctttgcctt ggacccccaa caaaaggaac ttagtctaaa 1560
ccaaattccc agcactgtca gtctccatgg caattgcagc ctccctggga acaaaggcag 1620
atagctcctt ctctaaaaaa gaaaagaatt tgtaaaaaaa aaaaaaaaaa aaaaaaagtc 1680
tttactccct ccctcctcca aaaggagact ccccagagtc attctctgtc ccaactctct 1740
catgacaaac tcctccaaag ccttccctgt gtcctggttg gtctggcaat gtgaggaaac 1800
ccagtctgct tgccagccct cacatgaagc aactgattgt tccttgggct cctcccccat 1860
gaatggggct cttgaaggtc agtaatgtag ctacaatcgt ataatggaag ctaaaatatt 1920
taaattgtat gcatgctgcc aataatggcg tgaacatgac atctgactta caagtgatgt 1980
caggtctcta actgggcagg aacttgaaat gccactaggt gatctatctt tcaggcccat 2040
gggcttccca gcctcctttc atggttatat ttgtatgaac acatccattc cagggtgctc 2100
ttttagaaga aaatgggctt tgttttagta attgaaccaa gtatctatag tttttctccc 2160
atctgatatt cagatgctaa ataaaagctc cacaaacagt tgggcattat tgttccgtgt 2220
gatgctcatt taggagaacc aaggacccct ggagacaaaa ttcaaattac tacagaaagc 2280
agcccagcat ccccacaatg ctatgcttag aaatgtgctc ttacaaccta gcatcataag 2340
agtgcacaca actatcatgg gtagcctctt ttgttagtca ctgctaaaga acttccctag 2400
aagccataac aacattccca agagtacaac ctcagaggaa cagtagtgtt ctggggccaa 2460
aagtaaagaa aatgaattct ttaaatgaaa tggtcctgga gccatattgc cctccaggtc 2520
acatactgat agcttctgga catcttctga gtgatcctat tgaagagagc aggaaactgg 2580
agctagaggc cagacacatt acttcatatc ctataatgtt cacaaaacta tctgcctcaa 2640
agaactacct gacccagaat atcaattgta ttccaacttc agaaaagcct ttttaagggc 2700
agtaactcta tcagtaaatc actcacaggg gagggggtgg agacagactg aaccacagca 2760
cttgatgggc agatgtcttg gttagggttt tactgctgtg aacagacacc atgaccaagg 2820
cagcatttat aaggacaaca tttaattggg gctggcttac aggttcagag gttcagtcta 2880
ttatcatcaa ggtgagaaca tggcagcgtc caggcagtca tggtacagga ggagttgaga 2940
gttctacatc ttcatactga cttctagaca ggtaggatga gggtcttaag gtccacaccc 3000
acaatgacac acctactcca acaaggccac acctcctaat agtgccactc cctgggacaa 3060
gcatatataa accagcatag cagccatttc ctacttggtg ggttcaggac taggagagac 3120
cctttctcaa aataaacaac aaaaaaatgt agatggcatc tgaagaatga cagccatggt 3180
tgtcttctgt cttacacaca cacacacaca cacacacaca cacacgtgca cacatgaata 3240
cacatacaga cacctgcaca gtcatgcaca aatgctcaca cacaaacaca tgcatgcaca 3300
cacacactca cataaagaat cccttttaga gttatggtgc ttatgaaaat aaagatatat 3360
ataaccagta taatctgtgt ttatataaat aagtttattc tagtgtgtgt gtgtgtgtgt 3420
gagagagaga gagagagaga gagagagaga gagagagaga gagagagtac catcgctcga 3480
atgtggaatt cagaagacaa cttaaaggag ttgattgtct cctttcaaca tgtgggcccc 3540
tgggatgaaa ctcaagcttg ccagctaggc tacaagtgcc tttacccact cagccatgtt 3600
gtcagcccca aaccagaata attccacaaa ataacatgaa tccatgaagg ctcttgaatg 3660
atttgctcaa ccaaaatcca gaatttgaaa tcctaccacc ttaaggggat ggctttgaag 3720
tgagacctct gggaagaggc aggtcataga attcctgatc cagagcagga gcggtgccct 3780
aggaacctag ctagcctcgt agatcacaga ggactcagct caaagcccat catctgtgaa 3840
ccacaacgca ggcccttgcc acataccgaa tctgctagtg ccttaatctc cactttccca 3900
gcctcctgag aaagacgttt ctgttgttgc taaggaatcc aacctagtat acatgagagt 3960
ttctgttatg gattccaact tcttcctcta gccttggcta ttgggtttca ttccagggca 4020
ctgtgctccc tccctggcat tcccttctca cacaggccta gctgtccatc ctaacacttt 4080
gcaacacgtc ctggttttct tcttttaaga ctagctggga atgatgaggg gccagcccat 4140
tcttccacca actgccatcc atcgattcag agtcttttaa accaaccctg ttaagtcaga 4200
caattaaaat cttgtaaacc ttccagaggg gaggggtggg gacttcaact gtaaatgcct 4260
gctagctaca gactaacttg agcgatctgc tctgccaaga ggccacataa ggggaaagaa 4320
ttttttaaaa ccaaacaaac aaaaggtgtt caaccaaact aggccaggtg ctcagtgttt 4380
atctgcgtga ttaagttgat ttacatactg cagttcaact aaccagaacc cagctcatca 4440
acactcttca ttaaagaaaa ttacttcctc atatgcgact ctgtcaagaa gaaagtcaca 4500
catgctgaca gccactgaat cctaggcact cttactcagg atacaagagt cccaccagat 4560
agatgtccct atctctgtct tcaggagagg acactgcaaa cgaaattaaa caacaaatga 4620
aaggccattg ctttcccact ccatacagta aagatggcat tgaaactgaa cctctaagtt 4680
ctgagacata gtctcaagtt cttataattg tgtgtgtatg tgtgtgtgtg tgtgtgtgtg 4740
tgtgtgtgtg tgtgtgatat gttctgtgtc tctgtttgtc tctatttgtc tgtctgtctg 4800
tctgtctctc tgtctctgtc tctgtctctg tctctgtctc tctctctctc tctctctctc 4860
tctcacacac acacacacac acacacacac acacacacac ttgtatgttt tgagataggt 4920
tctcatgtag cccagtctag cctcaagcag ccaaggctaa gctctccctc ttaaataatg 4980
gaattgtatc tttgaaccat aatgcccctt gtaatgtgtg ctgggaacca tacccaggct 5040
tttgtgcata ctaggcaagc actgtcctga ctgaatcaca ccctagcctt attaatccaa 5100
gttaatttac catttctttc cagttctagt tgatggcatt aatggttacc accacttacc 5160
agtgacatac accaacgact ttgattattt catctttata gatgaaatcc ctccacacag 5220
taggtattat taccgttgta tagacagaga agcagaatca tagaacgctg tctaaagctg 5280
acccaaagtc acatacttaa tacatgctaa aacctggagt tagacaaggc agtctaacct 5340
gtagcaagcc tctcttcact ttggggtctg gttaattcct ttgtgtcatt cagtattcaa 5400
agaactggcc atgaggtcaa ataggtattt tgtatgactg gggggtgtgg tgttgagagg 5460
ggcaaaagga ttgggtgttc gagaccagtc tgtactacac aagattactg cctcaaaata 5520
ataaaaacac ccaaaaggtc atccattctc ttttttattt ttcaggttta aacaatagac 5580
atttacctgg tcctatt 5597
<210> 5
<211> 3801
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agtttgttgg ctgcggcagc aggtagcaaa gtgacgccga gggcctgagt gctccagtag 60
ccaccgcatc tggagaacca gcggttacca tggaggggat cagtgtaagt ccagtttcaa 120
cctgctttgt cataaatgta caaacgtttg aacttagagc gcagcccctc tccgagcggg 180
cagaagcggc caggacattg gaggtacccg tactccaaaa aagggtcacc gaaaggagtt 240
ttcttgacca tgcctatata gtgcgggtgg gtgggggggg agcaggattg gaatcttttt 300
ctctgtgagt cgaggagaaa cgactggaaa gagcgttcca gtggctgcat gtgtctcccc 360
cttgagtccc gccgcgcgcg gcggcttgca cgctgtttgc aaacgtaaga acattctgtg 420
cacaagtgca gagaaggcgt gcgcgctgcc tcgggactca gaccaccggt ctcttccttg 480
gggaagcggg gatgtcttgg agcgagttac attgtctgaa tttagaggcg gagggcggcg 540
tgcctgggct gagttcccag gaggagattg cgcccgcttt aacttcgggg ttaagcgcct 600
ggtgactgtt cttgacactg ggtgcgtgtt tgttaaactc tgtgcggccg acggagctgt 660
gccagtctcc cagcacagta ggcagagggc gggagaggcg ggtggaccca ccgcgccgat 720
cctctgaggg gatcgagtgg tggcagcagc taggagttga tccgcccgcg cgctttgggt 780
ttgaggggga aaaccttccc gccgtccgaa gcgcgcctct tccccacggc cgcgagtggg 840
tcctgcagtt cgagagtttg gggtcgtgca gaggtcagcg gagtggtttg acctcccctt 900
tgacaccgcg cagctgccag ccctgagatt tgcgctccgg ggataggagc gggtacgggg 960
tgaggggcgg gggcggttaa gaccgcacct gggctgccag gtcgccgccg cgaagactgg 1020
caggtgcaag tggggaaacc gtttggctct ctccgagtcc agttgtgatg tttaaccgtc 1080
ggtggtttcc agaaaccttt tgaaaccctc ttgctaggga gtttttggtt tcctgcagcg 1140
gcgcgcaatt caaagacgct cgcggcggag ccgcccagtc gctccccagc accctgtggg 1200
acagagcctg gcgtgtcgcc cagcggagcc cctgcagcgc tgcttgcggg cggttggcgt 1260
gggtgtagtg ggcagccgcg gcggcccggg gctggacgac ccggcccccc gcgtgcccac 1320
cgcctggagg cttccagctg cccacctccg gccgggttaa ctggatcagt ggcggggtaa 1380
tgggaagcca cccgggagag tgaggaaatg aaacttgggg cgaggaccac gggtgcagac 1440
cccgttacct tctccaccca ggaaaatgcc ccgctcccta acgtcccaaa cgcgccaagt 1500
gataaacacg aggatggcaa gagacccaca caccggagga gcgcccgctt gggggaggag 1560
gtgccgtttg ttcattttct gacactcccg cccaatatac cccaagcacc gaagggcctt 1620
cgttttaaga ccgcattctc tttacccact acaagttgct tgaagcccag aatggtttgt 1680
atttaggcag gcgtgggaaa attaagtttt tgcgctttag gagaatgagt ctttgcaacg 1740
cccccgccct ccccccgtga tcctcccttc tcccctcttc cctccctggg cgaaaaactt 1800
cttacaaaaa gttaatcact gcccctccta gcagcaccca ccccaccccc cacgccgcct 1860
gggagtggcc tctttgtgtg tatttttttt ttcctcctaa ggaaggtttt ttttcttccc 1920
tctagtgggc ggggcagagg agttagccaa gatgtgactt tgaaaccctc agcgtctcag 1980
tgcccttttg ttctaaacaa agaattttgt aattggttct accaaagaag gatataatga 2040
agtcactatg ggaaaagatg gggaggagag ttgtaggatt ctacattaat tctcttgtgc 2100
ccttagccca ctacttcaga atttcctgaa gaaagcaagc ctgaattggt tttttaaatt 2160
gctttaaaaa ttttttttaa ctgggttaat gcttgctgaa ttggaagtga atgtccattc 2220
ctttgcctct tttgcagata tacacttcag ataactacac cgaggaaatg ggctcagggg 2280
actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc aataaaatct 2340
tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat ggattggtca 2400
tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac aggctgcacc 2460
tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt gatgccgtgg 2520
caaactggta ctttgggaac ttcctatgca aggcagtcca tgtcatctac acagtcaacc 2580
tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg gccatcgtcc 2640
acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc tatgttggcg 2700
tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac gtcagtgagg 2760
cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg gttgtgttcc 2820
agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg tcctgctatt 2880
gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag gccctcaaga 2940
ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac attgggatca 3000
gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt gagaacactg 3060
tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt ctgaacccca 3120
tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca ctcacctctg 3180
tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga cattcatctg 3240
tttccactga gtctgagtct tcaagttttc actccagcta acacagatgt aaaagacttt 3300
tttttatacg ataaataact tttttttaag ttacacattt ttcagatata aaagactgac 3360
caatattgta cagtttttat tgcttgttgg atttttgtct tgtgtttctt tagtttttgt 3420
gaagtttaat tgacttattt atataaattt tttttgtttc atattgatgt gtgtctaggc 3480
aggacctgtg gccaagttct tagttgctgt atgtctcgtg gtaggactgt agaaaaggga 3540
actgaacatt ccagagcgtg tagtgaatca cgtaaagcta gaaatgatcc ccagctgttt 3600
atgcatagat aatctctcca ttcccgtgga acgtttttcc tgttcttaag acgtgatttt 3660
gctgtagaag atggcactta taaccaaagc ccaaagtggt atagaaatgc tggtttttca 3720
gttttcagga gtgggttgat ttcagcacct acagtgtaca gtcttgtatt aagttgttaa 3780
taaaagtaca tgttaaactt a 3801
<210> 6
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cttcctcgcc agagctgagt gagattaaga tctgaattcc gaagttccta ttctctag 58
<210> 7
<211> 84
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aggaacttca tcagtcaggt acataatggt ggatccagta ctgatatcaa gcatatacca 60
cgatgcccag ctttttatga gcat 84
<210> 8
<211> 1800
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aattttgttg cctggtgcag caggtagcag tgaaacctct gaggcgtttg gtgctccggt 60
aaccaccacg gctgtagagc gagtgttgcc atggagggga tcagtatata cacttcagat 120
aactacaccg aggaaatggg ctcaggggac tatgactcca tgaaggaacc ctgtttccgt 180
gaagaaaatg ctaatttcaa taaaatcttc ctgcccacca tctactccat catcttctta 240
actggcattg tgggcaatgg attggtcatc ctggtcatgg gttaccagaa gaaactgaga 300
agcatgacgg acaagtacag gctgcacctg tcagtggccg acctcctctt tgtcatcacg 360
cttcccttct gggcagttga tgccgtggca aactggtact ttgggaactt cctatgcaag 420
gcagtccatg tcatctacac agtcaacctc tacagcagtg tcctcatcct ggccttcatc 480
agtctggacc gctacctggc catcgtccac gccaccaaca gtcagaggcc aaggaagctg 540
ttggctgaaa aggtggtcta tgttggcgtc tggatccctg ccctcctgct gactattccc 600
gacttcatct ttgccaacgt cagtgaggca gatgacagat atatctgtga ccgcttctac 660
cccaatgact tgtgggtggt tgtgttccag tttcagcaca tcatggttgg ccttatcctg 720
cctggtattg tcatcctgtc ctgctattgc attatcatct ccaagctgtc acactccaag 780
ggccaccaga agcgcaaggc cctcaagacc acagtcatcc tcatcctggc tttcttcgcc 840
tgttggctgc cttactacat tgggatcagc atcgactcct tcatcctcct ggaaatcatc 900
aagcaagggt gtgagtttga gaacactgtg cacaagtgga tttccatcac cgaggcccta 960
gctttcttcc actgttgtct gaaccccatc ctctatgctt tccttggagc caaatttaaa 1020
acctctgccc agcacgcact cacctctgtg agcagagggt ccagcctcaa gatcctctcc 1080
aaaggaaagc gaggtggaca ttcatctgtt tccactgagt ctgagtcttc aagttttcac 1140
tccagctaac acttatgcaa agacatatat aatatatata tatatatata tgataaagaa 1200
cttttttatg ttacacattt tccagatata agagactgac cagtcttgta cagttttttt 1260
tttttattga ctgttgggag tttatgttcc tctagttttt gtgaggtttg acttaattta 1320
tataaatact tttttttgtt tgtttgtttc atgtgaatga gtgtctaggc aggacctgtg 1380
gccaagttct tagtagctgt ttatctgtgt gtaggactgt agaactgtag aggaagaaac 1440
tgaacattcc agaatgtgtg gtaaattgaa taaagctagc cgtgatcctc agctgttgct 1500
gcataatctc ttcattccga ggagcacccc acccccaccc ccacccccac cccattctta 1560
aattgtttgg ttatgctgtg tgatggtttg tttggttttt ttttttgttg tttttgtttt 1620
tgtttttttt ctgtaaaaga tggcacttaa aaccaaagcc tgaaatggtg gtagaaatgc 1680
tgggggtttt tttgtttgtt tgttttttca gttttcaaga gtagattgat ttcactccct 1740
acaaatgtac agtcttgtat tacattgtta ataaaagtca atgataaact taaaaaaaaa 1800
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggacatag cgttggctac 20
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tctctaaacg ttctgctttg ttgct 25
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctctccagtg gtggcattgc aagta 25
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggcggatcaa ctcctagctg c 21
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cagggtgact ctaaaccctt tctgt 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agggctatca aatgtcaagt aggga 25
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gggaagctgg tcatgccttg aagat 25
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acagcagagg ttagccaaca gcatt 25
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ggatcggcca ttgaacaaga t 21
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cagaagaact cgtcaagaag gc 22
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cggttcgggc cagaaacttc aa 22
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
actcctgttt tcaggaggac cag 23
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gaaggccatg ccagtgagct t 21
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ccgcactata taggcatggt caag 24
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cccaatgatc tagctgtctt cctc 24
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gaaagtctgc ctcttaaggt aaagc 25
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gacaagcgtt agtaggcaca tatac 25
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gctccaattt cccacaacat tagt 24
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
taggatcttc ctgcccacca 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agggcctctg tgatggagat 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cttgactggc atagtcggca 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aacagtggaa gaaggcgagg 20
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cttcctatgc aaggcagtcc a 21
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agatgaatgt ccacctcgct 20
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
aggaaccctg tttccgtgaa g 21
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gaaagctagg gcctcggtg 19
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tcaccatctt ccaggagcga ga 22
<210> 36
<211> 89
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cgtttggtgc tccggtaacc accacggctg tagagcgagt gttgccatgg aggggatcag 60
tgtaagtcca gtttcaacct gctttgtca 89
<210> 37
<211> 100
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tttccactga gtctgagtct tcaagttttc actccagcta acacttatgc aaagacatat 60
ataatatata tatatatata tatgataaag aactttttta 100