CN112501206A - Construction method and application of PSMA (PSMA) gene humanized non-human animal - Google Patents

Construction method and application of PSMA (PSMA) gene humanized non-human animal Download PDF

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CN112501206A
CN112501206A CN202110173706.8A CN202110173706A CN112501206A CN 112501206 A CN112501206 A CN 112501206A CN 202110173706 A CN202110173706 A CN 202110173706A CN 112501206 A CN112501206 A CN 112501206A
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CN112501206B (en
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李志霞
李霞
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Baccetus Beijing Pharmaceutical Technology Co ltd
Beijing Biocytogen Co Ltd
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Abstract

The application provides a construction method of a PSMA gene humanized non-human animal, the non-human animal obtained by adopting the construction method and application thereof in the field of biomedicine. The application also provides a targeting vector and sgRNA for constructing the PSMA gene for non-human animals. The application further provides humanized PSMA proteins and chimeric PSMA genes. The non-human animal obtained by the construction method can normally express humanized PSMA protein, can be used as an animal model for human PSMA signal mechanism research and drug screening of tumor and immune related diseases, and has important application value for research and development of new drugs of immune targets.

Description

Construction method and application of PSMA (PSMA) gene humanized non-human animal
Technical Field
The application relates to a method for establishing a humanized gene modified animal model and application thereof, in particular to a method for establishing a PSMA gene humanized modified animal model and application thereof in biomedicine.
Background
Prostate cancer is one of the most common malignant tumors in men, and the incidence rate is the first year in European and American countries. Although the incidence rate of Chinese prostate cancer is lower than that of Europe and America, the incidence population of Chinese prostate cancer is greatly increased in recent years along with the coming of the aging society of China and the change of westernization of living habits. Meanwhile, the number of middle and high risk patients and advanced stage patients in the prostate cancer population in China is more, and the proportion is obviously higher than that in Europe and America. The curative effect of the tumor is closely related to the disease stage, so that the death rate of the prostate in China is still at a global high level at present.
PSMA (FOLH 1, GCP2, NAALAD 1), a type II transmembrane glycoprotein, belongs to the zinc-dependent exopeptidase superfamily, has folate hydrolase and neuropolycarboxypeptidase activity; expression in different tissues, such as prostate, kidney, brain, salivary gland, liver, ganglia, small intestine, but mainly in prostate. PSMA is upregulated in prostate cancer and increases as the disease progresses, with the highest expression in metastatic, castration-resistant prostate cancer. Are considered to be ideal targets for diagnosis and treatment of prostate cancer.
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, it is not intended that any gene or any region into which any gene is introduced be used to produce a humanized animal model. In this field, it is more important to select a specific region of a specific gene and introduce a human gene, thereby obtaining a humanized animal model which can express a humanized protein and has a function such as antibody screening.
Therefore, because of the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, it remains the greatest challenge to construct "efficient" humanized animal models for new drug development.
Disclosure of Invention
In view of the complicated mechanism of action of PSMA 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 PSMA-related 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 a first aspect of the present invention, a method for constructing a PSMA gene-humanized non-human animal whose genome includes all or part of a human PSMA gene is provided.
Preferably, the human PSMA gene comprises any one of exons 1 to 19 or a combination of two or more of them.
Preferably comprising a nucleotide sequence encoding a human PSMA protein. Further preferably, the human PSMA protein comprises all or part of a nucleotide sequence encoding an extracellular domain of human PSMA protein.
In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding SEQ ID NO:2 or a nucleotide sequence comprising SEQ ID NO: 5. Preferably, it further comprises a stop codon.
Preferably, the genome of the non-human animal comprises a chimeric PSMA gene, and the chimeric PSMA gene comprises human PSMA, non-human animal PSMA and STOP sequences from 5 'to 3'.
Preferably, the non-human animal expresses a human or humanized PSMA protein. The humanized PSMA protein comprises a part of human PSMA protein. Preferably, it comprises all or part of the extracellular domain of human PSMA protein.
In one embodiment of the invention, the humanized PSMA protein comprises SEQ ID NO:2, or, comprises an amino acid sequence corresponding to SEQ ID NO:2, position 51-750, 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:2, position 51-750, by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by NO more than 1 amino acid, or, alternatively, comprises a polypeptide having the amino acid sequence of SEQ ID NO:2, position 51-750, comprising substitution, deletion and/or insertion of one or more amino acid residues.
In one embodiment of the invention, the humanized PSMA protein comprises SEQ ID NO: 7 or, comprises an amino acid sequence identical to SEQ ID NO: 7, 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: 7 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: 7 comprising substitution, deletion and/or insertion of one or more amino acid residues.
Preferably, the expression of endogenous PSMA protein in the non-human animal is reduced or absent.
Preferably, the construction method comprises inserting or replacing any one or a combination of two or more exons 1 to 19 containing human PSMA gene into the PSMA locus of a non-human animal.
Preferably, the method of construction comprises inserting or replacing a nucleotide sequence comprising a sequence encoding a human PSMA protein into the PSMA locus of a non-human animal. Further preferably, the nucleotide sequence comprising the extracellular region encoding human PSMA protein is inserted or substituted into the PSMA locus of a non-human animal
In one embodiment of the invention, the method of construction comprises contacting a nucleic acid comprising a nucleotide sequence encoding SEQ ID NO:2 or a nucleotide sequence comprising SEQ ID NO: 5 into or into the PSMA locus of a non-human animal.
Preferably, the inserted sequence further comprises non-human animal PSMA.
Preferably, the inserted sequence further comprises a helper sequence.
In one embodiment of the present invention, the helper sequence is a STOP sequence.
In one embodiment of the invention, the method of construction comprises inserting or replacing a nucleotide sequence comprising a humanized PSMA protein into the non-human animal PSMA locus.
In one embodiment of the invention, the method of construction comprises inserting or replacing a nucleotide sequence comprising a chimeric PSMA gene into the PSMA locus of a non-human animal. Namely, the inserted sequence is a chimeric PSMA gene, and the chimeric PSMA gene sequentially comprises a human PSMA, a non-human animal PSMA and a STOP sequence from 5 '-3', wherein the nucleotide sequence of the human PSMA is shown as SEQ ID NO: 5, the nucleotide sequence of the non-human animal PSMA is shown as SEQ ID NO: 8, the STOP sequence is shown as SEQ ID NO: shown at 9.
The non-human animal is a mouse or a rat.
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.
Preferably, said insertion into the PSMA locus of the non-human animal is after insertion of an endogenous regulatory element of the non-human animal.
Preferably, said insertion into the PSMA locus of the non-human animal is after insertion into the nucleotide sequence of the transmembrane region of the non-human animal encoding PSMA.
Preferably, the insertion into the PSMA locus of the non-human animal is into exon 2 of the PSMA gene of the non-human animal.
In one embodiment of the present invention, the insertion into the non-human animal PSMA locus is between the nucleotide sequence encoding the 51 st amino acid and the nucleotide sequence encoding the 52 th amino acid of non-human animal PSMA, or between the 86422483 th position and the 86422484 th position of NC _000073.7 of the non-human animal PSMA gene.
Preferably, the human PSMA gene, the nucleotide sequence encoding the human PSMA protein or the chimeric PSMA gene is regulated by a non-human animal endogenous PSMA regulatory element. Among them, the regulatory element is preferably PSMA promoter.
The invention uses gene editing technology to construct PSMA 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.
The targeting vector comprises all or part of exons 1 to 19 of the human PSMA gene. Preferably, the human PSMA gene comprises any one of exons 1 to 19 or a combination of two or more of them. Preferably comprising a nucleotide sequence encoding a human PSMA protein. Further preferably, the human PSMA protein comprises all or part of the extracellular domain encoding human PSMA protein.
Preferably it may be a genomic DNA, cDNA or CDS sequence.
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.
In one embodiment of the invention, the targeting vector comprises a nucleotide sequence encoding a humanized PSMA protein.
In one embodiment of the invention, the targeting vector comprises the nucleotide sequence of a chimeric PSMA gene.
The targeting vector further comprises a 5 'arm and/or a 3' arm.
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 1000-2000 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 _ 000073.7. Further preferably, the length is 1000-2000 bp.
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 exons 1 to 19 of the PSMA gene of a non-human animal.
In one embodiment of the invention, the transition region to be altered is located in exon 2 of the PSMA gene of a non-human animal.
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 PSMA gene.
In another embodiment of the invention, the sgRNA is used for the construction of a non-human animal.
The sgRNA targets the exon 2 of the PSMA gene of the non-human animal.
The target site sequence of the sgRNA on the PSMA gene is shown as SEQ ID NO: 10-17.
In a specific embodiment of the invention, the construction of the non-human animal is performed using a targeting vector and sgrnas.
In a second aspect of the present invention, there is provided a PSMA gene-humanized non-human animal obtained by the above-described construction method.
In a third aspect of the present invention, there is provided a method for constructing a PSMA gene-deleted non-human animal, which is deleted for all or part of the PSMA gene. Preferably, exons 1 to 19 of the PSMA gene are deleted.
In a fourth aspect of the present invention, there is provided a PSMA gene-deleted non-human animal obtained by the above-described construction method.
In a fifth aspect of the present invention, there is provided a PSMA gene-deleted cell, tissue or organ obtained by the above-described method for constructing a PSMA gene-deleted non-human animal, or the above-described cell, tissue or organ is derived from the above-described constructed PSMA gene-deleted non-human animal.
In a sixth aspect of the present invention, there is provided a targeting vector for PSMA gene, the targeting vector comprising all or part of exons 1 to 19 of human PSMA gene. Preferably, the human PSMA gene comprises any one of exons 1 to 19 or a combination of two or more of them. Preferably comprising a nucleotide sequence encoding a human PSMA protein. Further preferably, the human PSMA protein comprises all or part of the extracellular domain encoding human PSMA protein.
Preferably it may be a genomic DNA, cDNA or CDS sequence.
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.
In one embodiment of the invention, the targeting vector comprises a nucleotide sequence encoding a humanized PSMA protein.
In one embodiment of the invention, the targeting vector comprises the nucleotide sequence of a chimeric PSMA gene.
The targeting vector further comprises a 5 'arm and/or a 3' arm.
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 1000-2000 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 _ 000073.7. Further preferably, the length is 1000-2000 bp.
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 exons 1 to 19 of the PSMA gene of a non-human animal.
In one embodiment of the invention, the transition region to be altered is located in exon 2 of the PSMA gene of a non-human animal.
Preferably, the targeting vector further comprises a selectable gene marker.
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, a sgRNA is provided that targets exon 2 of a PSMA gene in a non-human animal.
In a specific embodiment of the invention, the sequence of the target site of the sgRNA on the PSMA gene is as shown in SEQ ID NO: 10-17. Preferably SEQ ID NO: 13.
in an eighth aspect of the invention, a DNA molecule encoding the sgRNA is provided. Preferably, the double strands of the DNA molecules are the upstream and downstream sequences of the sgRNA, or the forward and reverse oligonucleotide sequences after the addition of the enzyme cleavage site.
In one embodiment of the invention, the DNA molecule duplexes are SEQ ID NO 13 and 19, and SEQ ID NO 18 and 20, respectively.
In a ninth aspect of the invention, a sgRNA vector is provided, which includes the sgRNA described above.
In a tenth aspect of the present invention, there is provided a cell comprising the targeting vector, the sgRNA, the DNA molecule, or the sgRNA vector.
In an eleventh aspect of the present invention, there is provided a use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector, or the cell for PSMA gene editing. Preferably, said use should include the use in knock-out, insertion or substitution of the PSMA gene.
In a twelfth aspect of the present invention, there is provided a method for constructing a polygene-modified non-human animal, comprising the steps of:
I) providing the PSMA gene humanized non-human animal or PSMA gene deleted non-human animal, or the PSMA gene humanized non-human animal obtained by the construction method;
II) mating the non-human animal provided in step I) with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a polygenetically modified non-human animal.
Preferably, the other genetically modified non-human animal includes, but is not limited to, genetically modified non-human animals of PD-1, PD-L1, TIGIT or CD 226.
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, each of the plurality of genes modified in the genome of the polygenic modified non-human animal may be homozygous or heterozygous.
In a thirteenth aspect of the present invention, there is provided a polygene-modified non-human animal obtained by the above-mentioned construction method.
In a fourteenth aspect of the present invention, there is provided an animal disease model, wherein the animal disease model is derived from the above non-human animal or the non-human animal obtained by the above method.
In the fifteenth aspect of the invention, a PSMA gene humanized non-human animal obtained by the above construction method, a PSMA gene deleted non-human animal obtained by the above construction method, or an application of the above non-human animal in preparing a disease model of an animal are provided. Preferably, the disease model may be a tumor, inflammation or immune-related disease. The tumor is preferably prostate cancer.
In a sixteenth aspect of the present invention, there is provided a PSMA gene-humanized non-human animal obtained by the above-mentioned construction method, a PSMA gene-deleted non-human animal obtained by the above-mentioned construction method, an application of the above-mentioned non-human animal or a disease model of the above-mentioned animal in the preparation of a medicament for treating tumor, inflammation or immune-related diseases.
In a seventeenth aspect of the present invention, there is provided a cell or cell line or primary cell culture derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or progeny thereof, or the above-mentioned disease model.
In an eighteenth aspect of the present invention, there is provided a tissue or organ or a culture thereof derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned disease model.
In a nineteenth aspect of the present invention, there is provided a tumor tissue after tumor loading, wherein the tumor tissue is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned disease model.
In a twentieth aspect of the present invention, there is provided a humanized PSMA protein comprising a portion of a human PSMA protein. Preferably, it comprises all or part of the extracellular domain of human PSMA protein.
In one embodiment of the invention, the humanized PSMA protein comprises SEQ ID NO:2, or, comprises an amino acid sequence corresponding to SEQ ID NO:2, position 51-750, 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:2, position 51-750, by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by NO more than 1 amino acid, or, alternatively, comprises a polypeptide having the amino acid sequence of SEQ ID NO:2, position 51-750, comprising substitution, deletion and/or insertion of one or more amino acid residues.
In one embodiment of the invention, the humanized PSMA protein comprises SEQ ID NO: 7 or, comprises an amino acid sequence identical to SEQ ID NO: 7, 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: 7 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: 7 comprising substitution, deletion and/or insertion of one or more amino acid residues.
In one embodiment of the present invention, the humanized PSMA protein has an amino acid sequence as set forth in SEQ ID NO: shown at 7.
In a twenty-first aspect of the present invention, there is provided a chimeric PSMA gene comprising, in order from 5 'to 3', a human PSMA, a non-human animal PSMA, and a STOP sequence. Wherein, the nucleotide sequence of the human PSMA is shown as SEQ ID NO: 5, the nucleotide sequence of the non-human animal PSMA is shown as SEQ ID NO: 8, the STOP sequence is shown as SEQ ID NO: shown at 9.
In one embodiment of the invention, the mRNA transcribed from said chimeric PSMA gene comprises SEQ ID NO: 6, or a nucleotide sequence identical to SEQ ID NO: 6, 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: 6by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 nucleotide, or alternatively, a nucleic acid molecule comprising a nucleotide sequence having the sequence set forth in SEQ ID NO: 6, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.
Preferably, the chimeric PSMA gene encodes the humanized PSMA protein.
In a twenty-second aspect of the present invention, there is provided a construct comprising the chimeric PSMA gene described above. Preferably, the construct expresses the humanized PSMA protein described above.
In a twenty-third aspect of the invention, there is provided a cell comprising the above construct.
In a twenty-fourth aspect of the invention, there is provided a tissue comprising the above-described cells. Preferably, the tissue includes, but is not limited to, prostate, kidney, brain, salivary gland, liver, ganglia or small intestine.
In a twenty-fifth aspect of the present invention, there is provided a cell humanized with the PSMA gene described above, said cell comprising all or part of the human PSMA gene. Preferably, the human PSMA gene comprises one or a combination of two or more of exons 1 to 19. Preferably comprising a nucleotide sequence encoding a human PSMA protein. Further preferably, the human PSMA protein comprises all or part of the extracellular domain encoding human PSMA protein.
In one embodiment of the invention, the cell comprises the chimeric PSMA gene described above.
Preferably, the cell expresses the human or humanized PSMA protein described above.
Preferably, the cell has reduced or absent expression of endogenous PSMA protein.
The twenty-sixth aspect of the present invention provides a PSMA gene humanized non-human animal obtained by the above construction method, the above humanized PSMA protein, or the above chimeric PSMA gene, for the purposes of non-disease diagnosis and non-disease treatment, and the applications include:
A) use in the development of products involving PSMA-related immune processes in human cells;
B) use in model systems related to PSMA as pharmacological, immunological, microbiological and medical research;
C) to the production and use of animal experimental disease models for the study of PSMA-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
D) the application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human PSMA signal channel modulators is studied in vivo; alternatively, the first and second electrodes may be,
E) research on the function of PSMA gene, research on the medicine and drug effect of human PSMA target site, and research on the application of PSMA-related immune-related disease medicine and antitumor medicine.
"tumors" as referred to herein include, but are not limited to, lymphomas, B cell tumors, T cell tumors, myeloid/monocytic tumors, non-small cell lung cancer, leukemias, 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 sarcomas. 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.
The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, etc.
The term "inflammation" as used herein includes acute inflammation as well as 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 humanized PSMA protein comprises a part derived from human PSMA protein and a part of non-human PSMA protein.
The chimeric PSMA gene of the invention comprises a part derived from a human PSMA gene and a part of a non-human PSMA 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, the "exon 1 to 19" includes exon 1, intron 1-2, exon 2, intron 2-3, exon 3, intron 3-4, exon 4, intron 4-5, exon 5, intron 5-6, exon 6, intron 6-7, exon 7, intron 7-8, exon 8, intron 8-9, exon 9, intron 9-10, exon 10, intron 10-11, exon 11, intron 11-12, exon 12, intron 12-13, exon 13, intron 13-14, exon 14, intron 14-15, exon 15-16, exon 3-3, intron 16, Nucleotide sequences of intron 16-17, exon 17, intron 17-18, exon 18, intron 18-19 and exon 19.
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 "PSMA locus" refers to a DNA fragment of an optional segment of the PSMA 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
FIG. 1: a schematic comparison of the murine PSMA gene and the human PSMA gene (not to scale).
FIG. 2: PSMA gene targeting strategies and targeting vector design schemes, involving only the altered portion of the PSMA locus (not to scale).
FIG. 3: PSMA gene humanization mouse gene map, only involving the altered portion of the PSMA locus (not to scale).
FIG. 4: and detecting the result of sgRNA, wherein Con is a negative control, and PC is a positive control.
FIG. 5: mouse tail PCR identification (F0), M is Maker, WT is C57BL/6 mouse, H2O is water control, and the detection results of the PCR-1F/PCR-1R primers are shown in the figure A and the figure B; FIG. C and FIG. D show the results of PCR-2F/PCR-2R primer detection.
FIG. 6: mouse tail PCR identification (F1), wherein M is Marker, WT is C57BL/6 mouse, H2O is water control; FIG. A and FIG. B show the results of PCR-1F/PCR-1R primer detection; FIG. C and FIG. D show the results of PCR-2F/PCR-2R primer detection.
FIG. 7: and F1 mouse Southern blot result, wherein WT is C57BL/6 mouse.
FIG. 8: the immunohistochemistry results of the formalin and paraffin-embedded PSMA humanized mouse are shown in the figure (A) as the expression result of the C57BL/6 mouse kidney humanized PSMA protein, and the figure (B) as the expression result of the PSMA humanized mouse kidney humanized PSMA 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:
c57BL/6 mice were purchased from the national rodent laboratory animal seed center of the Chinese food and drug testing institute;
reverse transcription kit source Takara, cat # 6110A;
the AIO kit is from Beijing Baiosaixi map gene biotechnology limited company with the cargo number BCG-DX-004;
TOP10 competent cells were purchased from Tiangen, Inc. under the accession number CB 104-02;
BglII and AseI enzymes were purchased from NEB under the respective accession numbers; R0144S, R0526S;
Anti-PSMA antibodies were purchased from Abcam, cat # cat: ab 133579;
anti-mouse IgG (H + L), made in goat purchased from Vectorlab, cat # cat: ab205718, respectively.
Example 1 PSMA Gene-humanized mouse
A comparison scheme of the mouse PSMA Gene (NCBI Gene ID: 53320, Primary source: MGI: 1858193, Unit Probe ID: O35409, from position 86365733 to 86425171 on chromosome 7 NC-000073.7, based on NM-016770.3 and its encoded protein NP-058050.3 (SEQ ID NO: 1)) and human (NCBI Gene ID: 2346, Primary source: HGNC:3788, Unit Probe ID: Q04609-1, from position 49145092 to 49208642 on chromosome 11 NC-000011.10, based on NM-004476.3 and its encoded protein NP-004467.1 (SEQ ID NO: 2)) is shown in FIG. 1.
To achieve the object of the present invention, a nucleotide sequence encoding a human PSMA protein can be introduced at the endogenous mouse PSMA locus, so that the mouse expresses the human or humanized PSMA protein. For example, by using gene editing technology, under the control of endogenous mouse PSMA gene regulatory elements, a CDS partial sequence containing human PSMA is inserted into mouse exon 2, and a coding frame of the mouse PSMA gene is simultaneously destroyed to obtain a humanized PSMA locus schematic diagram as shown in FIG. 2, so that humanized transformation of the mouse PSMA gene is realized.
Gene editing using CRISPR/Cas system further designs targeting strategy schematic as shown in figure 3, which shows homology arm sequences containing the upstream and downstream of mouse PSMA on the targeting vector, and a fragment comprising human PSMA sequence in figure 3. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from position 86422483 to 86424000 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 86420962 to 86422482 of NCBI accession No. NC-000073.7; the nucleotide sequence of human PSMA on fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence at position 344-2446 of the sequence having NCBI accession No. NM-004476.3. The sequence of murine PSMA on fragment a is shown in SEQ ID NO: 8, the STOP sequence is shown as SEQ ID NO: shown at 9. The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, direct synthesis 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 vector plasmid with the correct sequencing verification was used for subsequent experiments.
The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for constructing sgRNA expression vectors. A total of 8 sgRNA sequences of sgRNA1-sgRNA8 were designed and synthesized. The target site sequence of each sgRNA on PSMA is as follows:
sgRNA-1 target site sequence (SEQ ID NO: 10): 5'-CAACTGGTCTTTCCCCCTACAGG-3'
sgRNA-2 target site sequence (SEQ ID NO: 11): 5'-ATTTGTTGGCTTGCAACAACTGG-3'
sgRNA-3 target site sequence (SEQ ID NO: 12): 5'-AACATTACCAGTAGCTTCATTGG-3'
sgRNA-4 target site sequence (SEQ ID NO: 13): 5'-AAACCTTCCAATGAAGCTACTGG-3'
sgRNA-5 target site sequence (SEQ ID NO: 14): 5'-CCAGAAAACATTTCCTGTAGGGG-3'
sgRNA-6 target site sequence (SEQ ID NO: 15): 5'-TTACCAGTAGCTTCATTGGAAGG-3'
sgRNA-7 target site sequence (SEQ ID NO: 16): 5'-ACTGGTAATGTTTCCCATTCTGG-3'
sgRNA-8 target site sequence (SEQ ID NO: 17): 5'-CTCCTTCTTCATGCCAGAATGGG-3'
The activity of multiple sgrnas is detected by using a UCA kit, and the results show that the sgrnas have different activities, and the detection results are shown in fig. 4. From which sgRNA-4 was randomly selected for subsequent experiments. Respectively adding enzyme cutting sites on the 5' end and the complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide (the sequences are shown in a table 1), respectively connecting the annealed products to pT7-sgRNA plasmids (the plasmids are firstly linearized by BbsI) after annealing, and obtaining an expression vector pT 7-PSMA-4.
TABLE 1 sequence List of sgRNA-4
Figure DEST_PATH_IMAGE001
Randomly selecting clones, sending the clones to a sequencing company for sequencing verification, and selecting the expression vector pT7-PSMA-4 with correct connection for subsequent experiments.
pT7-sgRNA plasmid sources:
the plasmid backbone of the pT7-sgRNA vector was derived from Takara, cat # 3299. A fragment DNA containing a T7 promoter and sgRNA scaffold is synthesized by a plasmid synthesis company, is sequentially connected to a skeleton vector through enzyme digestion (EcoRI and BamHI), and is verified by sequencing of a professional sequencing company, so that a target plasmid is obtained. Fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 21):
gaattctaatacgactcactatagggggtcttcgagaagacctgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaggatcc
taking a C57BL/6 mouse prokaryotic stage fertilized egg, and injecting a premixed pT7-PSMA-4 plasmid in-vitro transcription product (transcribed by using an Ambion in-vitro transcription kit according to the method of the instruction) and Cas9 mRNA and a targeting vector plasmid into the cytoplasm or nucleus of the mouse fertilized egg by using a microinjection instrument. Microinjection of embryos is performed according to the method in the manual for mouse embryo manipulation experiments (third edition), fertilized eggs after injection are transferred to a culture solution for short-term culture, and then are transplanted to the oviduct of a recipient mother mouse to produce a genetically modified humanized mouse, so that a founder mouse (founder mouse, i.e., F0 generation) is obtained. The mRNA sequence of the humanized mouse PSMA after being transformed is shown as SEQ ID NO: 6, the expressed protein sequence is shown as SEQ ID NO: shown at 7.
The somatic cell genotype of F0 generation mice can be identified by conventional detection methods (e.g., PCR analysis), and the results of some F0 generation mice are shown in FIG. 5. As seen by combining the detection results of the 5 '-end primer and the 3' -end primer, the mice numbered F0-1 and F0-2 in FIG. 5 were positive mice. The PCR analysis included the following primers:
PCR-1F（SEQ ID NO：22）：5’- TCAGCACTGCCTGAACACATCCTTA -3’；
PCR-1R（SEQ ID NO：23）：5’- AGTCCTGGAGTCTCTCACTGAACTTG -3’
PCR-2F（SEQ ID NO：24）：5’- GTTTGAGCTAGCCAATTCCATAGTG -3’；
PCR-2R（SEQ ID NO：25）：5’- TGACATTGATAAAGAGCTTTCAAAGGGA -3’
PSMA humanized mice identified as positive for F0 were mated with C57BL/6 mice to give F1 generation mice. The same PCR method was used to genotype the F1 mice, and the results are shown in FIG. 6, which shows that 7F 1 mice were positive and numbered: f1-1, F1-2, F1-3, F1-4, F1-5, F1-6 and F1-7.
Mice identified as positive by F1 PCR were subjected to Southern blot analysis to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting genome with BglII enzyme or AseI enzyme, transferring membrane and hybridizing. Probes P1, P2 were located outside the 5' homology arm and on the human fragment, respectively.
Primer synthesis for P1 Probe (P1 Probe):
P1-F（SEQ ID NO：26）：5’-ATTAGGTCTGACCCATTGGGACCCT-3’
P1-R（SEQ ID NO：27）：5’-GCAGATGGTTTCTTACAGAGTTGGGC-3’
primer synthesis for P2Probe (P2 Probe):
P2-F（SEQ ID NO：28）：5’-AACTGATGAATGGGAGCAGTGGT-3’
P2-R（SEQ ID NO：29）：5’-GCAGACACTCTATGCCTGTGTGG-3’
the Southern blot assay results are shown in FIG. 7. The results of the P1 and P2 probes were combined to show that 2 of 7 mice had no random insertions, and the numbers of the two mice are F1-1 and F1-2. These 7 mice were confirmed as positive heterozygous mice and no random insertions were present. This shows that the PSMA humanized gene engineering mouse which can be stably passaged and has no random insertion can be constructed by using the method.
The expression of the humanized PSMA protein in a PSMA humanized homozygous mouse is detected by IHC, and the kidney of the PSMA humanized mouse fixed by formalin and the kidney tissue of C57BL/6 mouse are subjected to paraffin embedding, and then the tissue is dewaxed and sealed. Adding Anti-PSMA antibodyy, Anti-Rabbit IgG HRP, and made in goat for color development, and finally adding hematoxylin for staining, wherein the detailed microscopic examination is shown in figure 8. The results showed that no staining was seen in the kidney of C57BL/6 mice (FIG. 8A), no humanized PSMA protein was expressed in the kidney of C57BL/6 mice, and tubular epithelial cells were deeply stained with the cytoplasmic membrane and the cytoplasmic yellow brown color in PSMA humanized mice (FIG. 8B). Thus, the PSMA mouse has the expression of the humanized PSMA protein.
Example 2 double-Gene or Multi-Gene humanized mice containing human PSMA
The PSMA mouse prepared by the method can also be used for preparing a double-humanized or multi-humanized mouse model. For example, in example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other genetic modifications such as PD-1, PD-L1, TIGIT, CD226, etc., or may be humanized PSMA mice and then isolated mouse ES embryonic stem cells and gene recombination targeting techniques may be used to obtain a two-or multi-gene modified mouse model of PSMA with other genetic modifications. The PSMA mouse homozygote or heterozygote obtained by the method can also be mated with other gene modified homozygote or heterozygote mice, the offspring of the PSMA mouse is screened, the humanized PSMA and other gene modified double-gene or multi-gene modified heterozygote mice can be obtained with certain probability according to Mendel genetic rule, then the heterozygote is mated with each other to obtain double-gene or multi-gene modified homozygote, and the double-gene or multi-gene modified mice can be used for in vivo efficacy verification of targeted human PSMA and other gene regulators, and the like.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features of the present invention will not be described separately, and the same shall be considered as the disclosure of the present invention.
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Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val Arg
385 390 395 400
Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile
405 410 415
Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr
420 425 430
Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala
435 440 445
Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val
450 455 460
Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu
465 470 475 480
Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser
485 490 495
Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile
500 505 510
Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg Leu
515 520 525
Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn
530 535 540
Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu
545 550 555 560
Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val
565 570 575
Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val
580 585 590
Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala
595 600 605
Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr
610 615 620
Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr
625 630 635 640
Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys Ser
645 650 655
Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu
660 665 670
Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg
675 680 685
His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser
690 695 700
Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp
705 710 715 720
Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala Ala
725 730 735
Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala
740 745 750
<210> 3
<211> 1518
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tttgatgagg gatctgtttc ctactggact ttagtacaca ggccagtcta acttgtcatc 60
aagtcatccg tctttgctct gcattttgaa ttacttctat gtcattggta aagacaggct 120
ctgaaccaat tgtgtagcct ttggccagag acttcaatca aactaaacac aaagatatca 180
gtaattacct atgattgctg tgagccccaa tgagattgaa ttaaattgag tttctgttat 240
cagcttgaaa tgagaatcaa gttttggaac ggttagcatg ggattcaaaa gcctacatat 300
cctaaaagca agtcatttct actttgttac acccactatc ccttggtttc actgttcaat 360
aacccagttt gttagtttct tagtattctg tctctaccac aggcctctat ctttcttact 420
tgtgagttat ttcaggccta gggtgttctt tcctaatctc acagtgaaca ctagggcctt 480
tagttaagcc ttttgctcag ccatatgtct caatgtgcat atttgtgtgc atatatgtct 540
tcaaatatat tatcaatagt ggtaaagcac catgaggttt ggttcagtaa ctctctttat 600
tctcaggtaa ggtactctgt aaactctata aggatgttct ctccctctca gattaaaatc 660
aatttatgag gccaggggtc aaggcacagg ttataacctg tctgtggctt tctgtgcatt 720
ggatgtcttg cagagctacg aagtatattg cagtttacta tagttctcag aacttcagtt 780
tctcaagcca attttaaagg agaaacaaaa atatccctgc attttgcctt agtgtccaat 840
tcaaagcaaa ttgaagtctg ttttttaaat gttttgagat aatttatata tatgaaatat 900
attcataatt aagatcctct ttagttttca ataacttcat atgttatatc tttgagaacg 960
tatgtatgta gatgtgagag gagatgtcca tgggcttgtg ttgggaaatg tgtgctaagg 1020
atcgaagtca ggcccctttg caagaatatt acattcttct cttaattgct tgagcaatct 1080
ctccagtcac tgtactcata aataattcat tgtgagcttc tgtttagctc tacagagaag 1140
caagaaatta ttaagagaag caagtgtaga gaaactacat tttcatagca ttttatctct 1200
aataattgaa acaacactac atacttgaat tttcacattc aacttttctt agaaataaga 1260
gcaacacata taactgaaat taggtaaaat atgttgtact tgctttgcaa ttaaaagcaa 1320
tacagatatg ctcttttctt tgtagaaaaa aaacaagggg aaaacatgag aagcacagat 1380
catataagaa ataatttatt aactgtgtcc ctcataaaaa aaaaaaaaac caaaagatcc 1440
tgacatgagc tcgtatatca ttcatacttt tgcttgtatt tttcagggtg gtttataaaa 1500
ccttccaatg aagctact 1518
<210> 4
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggtaatgttt cccattctgg catgaagaag gagtttttgc atgaattgaa ggctgagaac 60
atcaaaaaat ttttatagta agaatatact tgtattacat tccatatggt gattatacct 120
tcagctttaa cttcatatct tcacaaagct ttctgagtag tacccagaaa acatttcctg 180
tagggggaaa gaccagttgt tgcaagccaa caaatatctg ccataactac ttaactttag 240
ctccataaga gcaaaagaag ccataggaag tatctgacaa atgcaggttc tgataaaatt 300
gcattcagga gcagagaaat ttgattatta tttgttgatg cattacaaaa tcccatctta 360
tttcctcacc tacttaaatt ataaagctgg tctttgcctg tggagaatac aaaaacaaat 420
tatggccaga tttggactga ggaacgtact ttggtgactt atcctttaag gagagataat 480
aggttttatt taagaaaaag ctgatttaaa aatcgcacga tgtttagttt gagctccatc 540
tctgctcatt atgagggaac taactttgag tgactaatga aactgaccaa gcttctattc 600
tcatctatga gataaggata atgatactgc agtgataagg gtgaacctgg gaattagatg 660
ttctgaaaac actaaaaact aattctattc ttttcaaaag agtgtaaagc aaaagagata 720
aatgaatata ttgtaaaatt attggccctt gcacatgcta gacctaaact atattcctga 780
atatgtacaa ggcactagtt tgattctcat tagtacaaga acataaaacg taatcataga 840
cacaaagtta gatttatatt tagttaaaat acaatatgtg gatgtatata ggattagtat 900
tatctaatca cctagaacac attataggtt ttcaaaaaat caggttaata tatatgtcaa 960
aagtctttaa aaaattcctc tactgatagg atttcctaat gaacacagag tgatttgata 1020
taaaagacta ttaaatattc acatatggtt aaaagtttca tatctgtagg aatatatata 1080
tatatatata tatatatata tatatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 1140
tgtgtgtgtg tgtgtgtacg ttaattacaa tagatttaaa atctatgaga gctattgctt 1200
ttaggagtgc cccattgagt aagaggcatt aggcttaaac ttcttcatga aaagtcccca 1260
catcttatat ttacttcatt tattatatat tataggcatg aaaaatgtag ggtttttggg 1320
gtgcctattg aaaacgcaga cagactaaaa gtaatgttta aacattttca attttccttc 1380
accagcaatt tcacacggac accacacttg gcaggaacac aaaataattt tgagcttgca 1440
aagcaaattc atgaccagtg gaaagaattt ggcctggatt tggttgagtt atcccattac 1500
gatgtcttgc tgtcctatcc a 1521
<210> 5
<211> 2103
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aacattactc caaagcataa tatgaaagca tttttggatg aattgaaagc tgagaacatc 60
aagaagttct tatataattt tacacagata ccacatttag caggaacaga acaaaacttt 120
cagcttgcaa agcaaattca atcccagtgg aaagaatttg gcctggattc tgttgagcta 180
gcacattatg atgtcctgtt gtcctaccca aataagactc atcccaacta catctcaata 240
attaatgaag atggaaatga gattttcaac acatcattat ttgaaccacc tcctccagga 300
tatgaaaatg tttcggatat tgtaccacct ttcagtgctt tctctcctca aggaatgcca 360
gagggcgatc tagtgtatgt taactatgca cgaactgaag acttctttaa attggaacgg 420
gacatgaaaa tcaattgctc tgggaaaatt gtaattgcca gatatgggaa agttttcaga 480
ggaaataagg ttaaaaatgc ccagctggca ggggccaaag gagtcattct ctactccgac 540
cctgctgact actttgctcc tggggtgaag tcctatccag atggttggaa tcttcctgga 600
ggtggtgtcc agcgtggaaa tatcctaaat ctgaatggtg caggagaccc tctcacacca 660
ggttacccag caaatgaata tgcttatagg cgtggaattg cagaggctgt tggtcttcca 720
agtattcctg ttcatccaat tggatactat gatgcacaga agctcctaga aaaaatgggt 780
ggctcagcac caccagatag cagctggaga ggaagtctca aagtgcccta caatgttgga 840
cctggcttta ctggaaactt ttctacacaa aaagtcaaga tgcacatcca ctctaccaat 900
gaagtgacaa gaatttacaa tgtgataggt actctcagag gagcagtgga accagacaga 960
tatgtcattc tgggaggtca ccgggactca tgggtgtttg gtggtattga ccctcagagt 1020
ggagcagctg ttgttcatga aattgtgagg agctttggaa cactgaaaaa ggaagggtgg 1080
agacctagaa gaacaatttt gtttgcaagc tgggatgcag aagaatttgg tcttcttggt 1140
tctactgagt gggcagagga gaattcaaga ctccttcaag agcgtggcgt ggcttatatt 1200
aatgctgact catctataga aggaaactac actctgagag ttgattgtac accgctgatg 1260
tacagcttgg tacacaacct aacaaaagag ctgaaaagcc ctgatgaagg ctttgaaggc 1320
aaatctcttt atgaaagttg gactaaaaaa agtccttccc cagagttcag tggcatgccc 1380
aggataagca aattgggatc tggaaatgat tttgaggtgt tcttccaacg acttggaatt 1440
gcttcaggca gagcacggta tactaaaaat tgggaaacaa acaaattcag cggctatcca 1500
ctgtatcaca gtgtctatga aacatatgag ttggtggaaa agttttatga tccaatgttt 1560
aaatatcacc tcactgtggc ccaggttcga ggagggatgg tgtttgagct agccaattcc 1620
atagtgctcc cttttgattg tcgagattat gctgtagttt taagaaagta tgctgacaaa 1680
atctacagta tttctatgaa acatccacag gaaatgaaga catacagtgt atcatttgat 1740
tcactttttt ctgcagtaaa gaattttaca gaaattgctt ccaagttcag tgagagactc 1800
caggactttg acaaaagcaa cccaatagta ttaagaatga tgaatgatca actcatgttt 1860
ctggaaagag catttattga tccattaggg ttaccagaca ggccttttta taggcatgtc 1920
atctatgctc caagcagcca caacaagtat gcaggggagt cattcccagg aatttatgat 1980
gctctgtttg atattgaaag caaagtggac ccttccaagg cctggggaga agtgaagaga 2040
cagatttatg ttgcagcctt cacagtgcag gcagctgcag agactttgag tgaagtagcc 2100
taa 2103
<210> 6
<211> 3044
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gacttctatt tcccagggga atagcagtgg gaggaaattt taacccgcag aatctctgca 60
gtggatgtag aagagaactg ctgaggaaaa ctaaagttgc cagagatgtg gaacgcactg 120
caggacagag actccgcgga ggtcctggga caccgccagc gctggctccg tgttgggaca 180
ctggtgctgg ctttaaccgg aaccttcctc attggcttcc tctttgggtg gtttataaaa 240
ccttccaatg aagctactaa cattactcca aagcataata tgaaagcatt tttggatgaa 300
ttgaaagctg agaacatcaa gaagttctta tataatttta cacagatacc acatttagca 360
ggaacagaac aaaactttca gcttgcaaag caaattcaat cccagtggaa agaatttggc 420
ctggattctg ttgagctagc acattatgat gtcctgttgt cctacccaaa taagactcat 480
cccaactaca tctcaataat taatgaagat ggaaatgaga ttttcaacac atcattattt 540
gaaccacctc ctccaggata tgaaaatgtt tcggatattg taccaccttt cagtgctttc 600
tctcctcaag gaatgccaga gggcgatcta gtgtatgtta actatgcacg aactgaagac 660
ttctttaaat tggaacggga catgaaaatc aattgctctg ggaaaattgt aattgccaga 720
tatgggaaag ttttcagagg aaataaggtt aaaaatgccc agctggcagg ggccaaagga 780
gtcattctct actccgaccc tgctgactac tttgctcctg gggtgaagtc ctatccagat 840
ggttggaatc ttcctggagg tggtgtccag cgtggaaata tcctaaatct gaatggtgca 900
ggagaccctc tcacaccagg ttacccagca aatgaatatg cttataggcg tggaattgca 960
gaggctgttg gtcttccaag tattcctgtt catccaattg gatactatga tgcacagaag 1020
ctcctagaaa aaatgggtgg ctcagcacca ccagatagca gctggagagg aagtctcaaa 1080
gtgccctaca atgttggacc tggctttact ggaaactttt ctacacaaaa agtcaagatg 1140
cacatccact ctaccaatga agtgacaaga atttacaatg tgataggtac tctcagagga 1200
gcagtggaac cagacagata tgtcattctg ggaggtcacc gggactcatg ggtgtttggt 1260
ggtattgacc ctcagagtgg agcagctgtt gttcatgaaa ttgtgaggag ctttggaaca 1320
ctgaaaaagg aagggtggag acctagaaga acaattttgt ttgcaagctg ggatgcagaa 1380
gaatttggtc ttcttggttc tactgagtgg gcagaggaga attcaagact ccttcaagag 1440
cgtggcgtgg cttatattaa tgctgactca tctatagaag gaaactacac tctgagagtt 1500
gattgtacac cgctgatgta cagcttggta cacaacctaa caaaagagct gaaaagccct 1560
gatgaaggct ttgaaggcaa atctctttat gaaagttgga ctaaaaaaag tccttcccca 1620
gagttcagtg gcatgcccag gataagcaaa ttgggatctg gaaatgattt tgaggtgttc 1680
ttccaacgac ttggaattgc ttcaggcaga gcacggtata ctaaaaattg ggaaacaaac 1740
aaattcagcg gctatccact gtatcacagt gtctatgaaa catatgagtt ggtggaaaag 1800
ttttatgatc caatgtttaa atatcacctc actgtggccc aggttcgagg agggatggtg 1860
tttgagctag ccaattccat agtgctccct tttgattgtc gagattatgc tgtagtttta 1920
agaaagtatg ctgacaaaat ctacagtatt tctatgaaac atccacagga aatgaagaca 1980
tacagtgtat catttgattc acttttttct gcagtaaaga attttacaga aattgcttcc 2040
aagttcagtg agagactcca ggactttgac aaaagcaacc caatagtatt aagaatgatg 2100
aatgatcaac tcatgtttct ggaaagagca tttattgatc cattagggtt accagacagg 2160
cctttttata ggcatgtcat ctatgctcca agcagccaca acaagtatgc aggggagtca 2220
ttcccaggaa tttatgatgc tctgtttgat attgaaagca aagtggaccc ttccaaggcc 2280
tggggagaag tgaagagaca gatttatgtt gcagccttca cagtgcaggc agctgcagag 2340
actttgagtg aagtagccta aacacattct acagaatcac aaaatcccta tggaactatg 2400
gagcttatat atatatatat acacatgcat acatatattc ttattttttt catatgcatc 2460
aaaaaatcca aaagtggata atttattttt taaatttcaa catgtattca taaaatttaa 2520
aattcaaata agtattaaaa ctaaggtgaa cagtctaata aagtgataca tttgacagta 2580
tcagacaatt atcagaaata taatacagcc ttcccagatc tactctgtca tttcaataaa 2640
aacaagtatc aattccttat ggaacacagc tggaatagtt cacaagtctg cttcatctgg 2700
gggactagat acactctggc cacgaaaggc tgggaagatc atattagcca caatagctga 2760
caagttataa tctattctca tatcttgtaa aactcagcac tctgaattat aacgtttaat 2820
tatttagact taggaaaata tcattttatg aaaatgtata aaatataaat gcacaaccaa 2880
taattaattt gtagtcccaa taaccatgta cttctaaaca attgcatcat ttatatcaaa 2940
tataatcctg gtcttaaatt ttatgtttat gatttccttg tttatataga agtcatcaaa 3000
catggctgta ttcattacat atcttgaata tatacataat tttg 3044
<210> 7
<211> 751
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Trp Asn Ala Leu Gln Asp Arg Asp Ser Ala Glu Val Leu Gly His
1 5 10 15
Arg Gln Arg Trp Leu Arg Val Gly Thr Leu Val Leu Ala Leu Thr Gly
20 25 30
Thr Phe Leu Ile Gly Phe Leu Phe Gly Trp Phe Ile Lys Pro Ser Asn
35 40 45
Glu Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp
50 55 60
Glu Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln
65 70 75 80
Ile Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln
85 90 95
Ile Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala
100 105 110
His Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr
115 120 125
Ile Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu
130 135 140
Phe Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro
145 150 155 160
Pro Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val
165 170 175
Tyr Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp
180 185 190
Met Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys
195 200 205
Val Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys
210 215 220
Gly Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val
225 230 235 240
Lys Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg
245 250 255
Gly Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly
260 265 270
Tyr Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val
275 280 285
Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln
290 295 300
Lys Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp
305 310 315 320
Arg Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly
325 330 335
Asn Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu
340 345 350
Val Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu
355 360 365
Pro Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe
370 375 380
Gly Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val
385 390 395 400
Arg Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr
405 410 415
Ile Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser
420 425 430
Thr Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val
435 440 445
Ala Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg
450 455 460
Val Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys
465 470 475 480
Glu Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu
485 490 495
Ser Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg
500 505 510
Ile Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg
515 520 525
Leu Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr
530 535 540
Asn Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr
545 550 555 560
Glu Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr
565 570 575
Val Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile
580 585 590
Val Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr
595 600 605
Ala Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys
610 615 620
Thr Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe
625 630 635 640
Thr Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys
645 650 655
Ser Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu
660 665 670
Glu Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr
675 680 685
Arg His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu
690 695 700
Ser Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val
705 710 715 720
Asp Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala
725 730 735
Ala Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala
740 745 750
<210> 8
<211> 983
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acacattcta cagaatcaca aaatccctat ggaactatgg agcttatata tatatatata 60
cacatgcata catatattct tatttttttc atatgcatca aaaaatccaa aagtggataa 120
tttatttttt aaatttcaac atgtattcat aaaatttaaa attcaaataa gtattaaaac 180
taaggtgaac agtctaataa agtgatacat ttgacagtat cagacaatta tcagaaatat 240
aatacagcct tcccagatct actctgtcat ttcaataaaa acaagtatca attccttatg 300
gaacacagct ggaatagttc acaagtctgc ttcatctggg ggactagata cactctggcc 360
acgaaaggct gggaagatca tattagccac aatagctgac aagttataat ctattctcat 420
atcttgtaaa actcagcact ctgaattata acgtttaatt atttagactt aggaaaatat 480
cattttatga aaatgtataa aatataaatg cacaaccaat aattaatttg tagtcccaat 540
aaccatgtac ttctaaacaa ttgcatcatt tatatcaaat ataatcctgg tcttaaattt 600
tatgtttatg atttccttgt ttatatagaa gtcatcaaac atggctgtat tcattacata 660
tcttgaatat atacataatt ttgtattttt cagtgtcatc ttttctattt gcctttgagt 720
tactcttgta gtttaccaga ttttttgtga taccacattg agatgagtat atgtcacttt 780
tactcagagt tgtacaaaga gttcacatag tcacatagag agatttcaga aagaatgaca 840
agttatatca gtattaatta tttgaattgt tgcaatcaga aaaggaaaaa aaaattatat 900
atagaaggta taaggagtgg tcactatgct gacagtttat atcctcttgt agtcctgtca 960
ccttctaact tatccaactt tat 983
<210> 9
<211> 1349
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
attaagggtt ccggatcctc ggggacacca aatatggcga tctcggcctt ttcgtttctt 60
ggagctggga catgtttgcc atcgatccat ctaccaccag aacggccgtt agatctgctg 120
ccaccgttgt ttccaccgaa gaaaccaccg ttgccgtaac caccacgacg gttgttgcta 180
aagaagctgc caccgccacg gccaccgttg tagccgccgt tgttgttatt gtagttgctc 240
atgttatttc tggcacttct tggttttcct cttaagtgag gaggaacata accattctcg 300
ttgttgtcgt tgatgcttaa attttgcact tgttcgctca gttcagccat aatatgaaat 360
gcttttcttg ttgttcttac ggaataccac ttgccaccta tcaccacaac taactttttc 420
ccgttcctcc atctctttta tatttttttt ctcgagggat ctttgtgaag gaaccttact 480
tctgtggtgt gacataattg gacaaactac ctacagagat ttaaagctct aaggtaaata 540
taaaattttt aagtgtataa tgtgttaaac tactgattct aattgtttgt gtattttaga 600
ttccaaccta tggaactgat gaatgggagc agtggtggaa tgcctttaat gaggaaaacc 660
tgttttgctc agaagaaatg ccatctagtg atgatgaggc tactgctgac tctcaacatt 720
ctactcctcc aaaaaagaag agaaaggtag aagaccccaa ggactttcct tcagaattgc 780
taagtttttt gagtcatgct gtgtttagta atagaactct tgcttgcttt gctatttaca 840
ccacaaagga aaaagctgca ctgctataca agaaaattat ggaaaaatat tctgtaacct 900
ttataagtag gcataacagt tataatcata acatactgtt ttttcttact ccacacaggc 960
atagagtgtc tgctattaat aactatgctc aaaaattgtg tacctttagc tttttaattt 1020
gtaaaggggt taataaggaa tatttgatgt atagtgcctt gactagagat cataatcagc 1080
cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 1140
ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 1200
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 1260
agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg acatggtaag 1320
taagcttggg ctgcaggtcg agggaccta 1349
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caactggtct ttccccctac agg 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atttgttggc ttgcaacaac tgg 23
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aacattacca gtagcttcat tgg 23
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaaccttcca atgaagctac tgg 23
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccagaaaaca tttcctgtag ggg 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttaccagtag cttcattgga agg 23
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
actggtaatg tttcccattc tgg 23
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctccttcttc atgccagaat ggg 23
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
taggaaacct tccaatgaag ctactgg 27
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ccagtagctt cattggaagg ttt 23
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aaacccagta gcttcattgg aaggttt 27
<210> 21
<211> 132
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60
agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120
tttaaaggat cc 132
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tcagcactgc ctgaacacat cctta 25
<210> 23
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agtcctggag tctctcactg aacttg 26
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtttgagcta gccaattcca tagtg 25
<210> 25
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgacattgat aaagagcttt caaaggga 28
<210> 26
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
attaggtctg acccattggg accct 25
<210> 27
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gcagatggtt tcttacagag ttgggc 26
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aactgatgaa tgggagcagt ggt 23
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gcagacactc tatgcctgtg tgg 23

Claims (10)

1. A method for constructing a PSMA gene-humanized non-human animal, comprising administering a nucleic acid sequence comprising a nucleotide sequence encoding SEQ ID NO:2 or a nucleotide sequence comprising SEQ ID NO: 5 into the PSMA locus of a non-human animal.
2. The construction method according to claim 1, wherein the inserted sequence is a chimeric PSMA gene, and the chimeric PSMA gene comprises a human PSMA, a non-human animal PSMA and a STOP sequence from 5 'to 3' in sequence, wherein the nucleotide sequence of the human PSMA is as shown in SEQ ID NO: 5, the nucleotide sequence of the non-human animal PSMA is shown as SEQ ID NO: 8, the STOP sequence is shown as SEQ ID NO: 9, the non-human animal is a mouse or a rat.
3. The construct of claim 1 or 2, wherein the insertion into the non-human animal PSMA locus is between the nucleotide sequence encoding the 51 st amino acid and the nucleotide sequence encoding the 52 th amino acid of the non-human animal PSMA, or between the 86422483 th position and the 86422484 th position of NC _000073.7 of the non-human animal PSMA gene.
4. The construct of claim 1 or 2, wherein the non-human animal expresses a humanized PSMA protein comprising the amino acid sequence of SEQ ID NO: 7.
5. The method of construction according to claim 1 or 2, characterized in that the construction of a non-human animal is carried out using targeting vectors and/or sgRNAs,
the targeting vector comprises a nucleotide 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 is shown in the specification;
the target site sequence of the sgRNA on the PSMA gene is shown as SEQ ID NO: 10-17.
6. A targeting vector of PSMA gene, which is characterized in that the targeting vector comprises a nucleotide 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.
7. The sgRNA has a target site sequence on PSMA gene as shown in SEQ ID NO: 10-17.
8. A humanized PSMA protein comprising the amino acid sequence of SEQ ID NO: 7.
9. The chimeric PSMA gene is characterized by comprising a human PSMA, a non-human animal PSMA and a STOP sequence from 5 'to 3', wherein the nucleotide sequence of the human PSMA is shown as SEQ ID NO: 5, the nucleotide sequence of the non-human animal PSMA is shown as SEQ ID NO: 8, the STOP sequence is shown as SEQ ID NO: shown at 9.
10. The non-human animal humanized with PSMA gene obtained by the construction method according to any of claims 1 to 5, the humanized PSMA protein according to claim 8 or the chimeric PSMA gene according to claim 9, for non-disease diagnosis, non-disease treatment purposes, the use comprising:
A) use in the development of products involving PSMA-related immune processes in human cells;
B) use in model systems related to PSMA as pharmacological, immunological, microbiological and medical research;
C) to the production and use of animal experimental disease models for the study of PSMA-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;
D) the application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human PSMA signal channel modulators is studied in vivo; alternatively, the first and second electrodes may be,
E) research on the function of PSMA gene, research on the medicine and drug effect of human PSMA target site, and research on the application of PSMA-related immune-related disease medicine and antitumor medicine.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113447654A (en) * 2021-07-15 2021-09-28 广州济士源生物技术有限公司 Application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108239659A (en) * 2016-12-23 2018-07-03 北京百奥赛图基因生物技术有限公司 The preparation method and application of humanization genetic modification animal model
CN111172190A (en) * 2018-12-25 2020-05-19 百奥赛图江苏基因生物技术有限公司 Construction method and application of humanized cell factor CSF2 gene modified non-human animal
CN111304248A (en) * 2018-12-25 2020-06-19 百奥赛图江苏基因生物技术有限公司 Construction method and application of humanized cytokine IL15 gene modified non-human animal
CN111793647A (en) * 2020-09-08 2020-10-20 北京百奥赛图基因生物技术有限公司 Construction method and application of CD226 gene humanized non-human animal

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108239659A (en) * 2016-12-23 2018-07-03 北京百奥赛图基因生物技术有限公司 The preparation method and application of humanization genetic modification animal model
CN111172190A (en) * 2018-12-25 2020-05-19 百奥赛图江苏基因生物技术有限公司 Construction method and application of humanized cell factor CSF2 gene modified non-human animal
CN111304248A (en) * 2018-12-25 2020-06-19 百奥赛图江苏基因生物技术有限公司 Construction method and application of humanized cytokine IL15 gene modified non-human animal
CN111793647A (en) * 2020-09-08 2020-10-20 北京百奥赛图基因生物技术有限公司 Construction method and application of CD226 gene humanized non-human animal

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ZINK CF ET AL: "glutamate carboxypeptidase 2 isoform 1[Homo sapiens]", 《GENBANK登录号：NP_004467.1》 *
ZINK CF ET AL: "Homo sapiens folate hydrolase 1 (FOLH1),transcript variant 1, mRNA", 《GENBANK登录号：NM_004476.3》 *
张玲玲 等: "过表达前列腺特异性膜抗原和荧光素酶的PC3稳定细胞系的建立及成瘤性鉴定", 《细胞与分子免疫学杂志》 *
王子龙 等: "前列腺特异性膜抗原在泌尿系统肿瘤中的诊疗进展", 《肿瘤综合治疗电子杂志》 *
顾正勤: "抗人前列腺干细胞抗原抗体的制备及其在前列腺癌诊断治疗中的应用研究", 《中国博士学位论文全文数据库 医药卫生科技辑 E072-147》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113447654A (en) * 2021-07-15 2021-09-28 广州济士源生物技术有限公司 Application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer

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