CN111793647B - Construction method and application of CD226 gene humanized non-human animal - Google Patents

Abstract

The invention provides a construction method of a CD226 gene humanized modified non-human animal, which is characterized in that a nucleotide sequence for coding human CD226 protein is introduced into the genome of the non-human animal in a homologous recombination mode, the humanized CD226 protein can be normally expressed in the animal body, and the animal body can be used as an animal model for human CD226 signal mechanism research and tumor and immune disease drug screening and has important application value for the research and development of new drugs of immune targets. The invention also provides a CD226 chimeric protein, a CD226 chimeric gene, a targeting vector of the CD226 gene, a non-human animal constructed by the construction method and application thereof in the field of biomedicine.

Description

Construction method and application of CD226 gene humanized non-human animal

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a CD226 gene modified non-human animal model and application thereof in the field of biomedicine.

Background

CD226 is a glycoprotein belonging to the Ig superfamily and is expressed mainly on the surface of NK and T cells, as well as on platelets and monocytes. Current studies indicate that the ligands for CD226 are CD112 and CD155, both of which are expressed in many malignant and virally infected cells, and that overexpression is associated with cancer invasion and metastasis. Thus, CD226 plays a crucial role in fighting tumor immune responses, controlling viral infections in vivo. The research finds that the change of the Gly307Ser amino acid of the CD226 is also related to autoimmune diseases, such as type I diabetes, rheumatoid arthritis, systemic lupus erythematosus and the like. anti-CD 226 monoclonal antibodies can inhibit T cell and NK cell mediated cytotoxicity against certain tumor cells, while having no effect on the cytolysis of other cells, suggesting that CD 226-induced cytotoxicity depends on specific ligands from tumor cells. The interaction of CD226 with the ligands CD155 and CD112 triggers NK or T cell mediated cytotoxicity with an increase in cytokine production. Further in vitro studies indicate that tumor cells with higher CD155 expression are more susceptible to CD 226-induced killing. In addition, CD226 has been found to mediate NK cell inhibition of CD155 positive tumor metastasis.

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, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes, how to construct an "efficient" humanized animal model for new drug development remains the greatest challenge.

In view of the great application value of CD226 in the field of treatment of tumors, autoimmune diseases and the like, there is an urgent need in the art to develop a non-human animal model of CD 226-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.

Disclosure of Invention

In the first aspect of the invention, a construction method of a non-human animal humanized and transformed by a CD226 gene is provided, and the genome of the non-human animal comprises a human or humanized CD226 nucleotide sequence.

Preferably, the human or humanized CD226 nucleotide sequence comprises all or part of exon 2 to exon 7 of the human CD226 nucleotide sequence. Preferably a portion comprising exon 2, all of exons 3 to 5, and a portion of exon 6; wherein the part of exon 2 of the nucleotide sequence of human CD226 comprises at least the nucleotide sequence from the nucleotide sequence encoding the signal peptide to the last 1 nucleotide sequence of exon 2, and the part of exon 6 comprises at least the nucleotide sequence from the first nucleotide of exon 6 to the nucleotide sequence encoding 1-10 cytoplasmic domains.

Preferably, the human or humanized gene comprises the CDs sequence of the nucleotide sequence of human CD226 or the full-length nucleotide sequence of human CD 226.

More preferably, the human or humanized CD226 gene comprises a nucleotide sequence encoding amino acids 1 to 278 of SEQ ID No.2 or comprises the nucleotide sequence of SEQ ID No. 5.

Further, the humanized CD226 nucleotide sequence also includes a portion of exon 6 and a portion of exon 7 of the murine CD226 nucleotide sequence.

Preferably, the nucleotide sequence of the murine CD226 comprises the nucleotide sequence encoding the amino acid sequence as shown at position 279-333 of SEQ ID NO.1 or the nucleotide sequence of SEQ ID NO. 6.

In one embodiment of the invention, the humanized CD226 nucleotide sequence comprises the nucleotide sequences of SEQ id No.5 and SEQ id No.6 in order from 5 'to 3'.

More preferably, the LoxP STOP sequence is inserted after the murine CD226 nucleotide sequence.

In one embodiment of the present invention, the humanized CD226 nucleotide sequence comprises one of the following groups:

a) the human CD226 nucleotide sequence in the humanized CD226 nucleotide sequence is a nucleotide sequence which codes the 1 st to 278 th amino acids of SEQ ID NO.2 or a part or all of the nucleotide sequence shown in SEQ ID NO. 5;

b) the degree of identity of the human CD226 nucleotide sequence in the humanized CD226 nucleotide sequence to a part or all of the nucleotide sequence encoding amino acids 1 to 278 of SEQ ID No.2 or the nucleotide sequence set forth in SEQ ID No.5 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) the human CD226 nucleotide sequence in the humanized CD226 nucleotide sequence has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide difference with the nucleotide sequence encoding amino acids 1 to 278 in SEQ ID NO.2 or the nucleotide sequence shown in SEQ ID NO. 5;

d) the nucleotide sequence of the human CD226 in the humanized CD226 nucleotide sequence is shown by the nucleotide sequence which codes the 1 st to 278 th amino acids of SEQ ID NO.2 or the nucleotide sequence shown by SEQ ID NO.5 and comprises one or more nucleotide substitutions, deletions and/or insertions.

Preferably, the construction method comprises insertion, inversion, knockout or substitution. Preferably by insertion.

Preferably, the construction method comprises inserting or replacing the nucleotide sequence comprising the human or humanized CD226 into the CD226 locus of the non-human animal

In a specific embodiment, the method of construction comprises inserting into the CD226 locus of a non-human animal a nucleotide sequence comprising the nucleotide sequence encoding amino acids 1 to 278 of SEQ ID No.2, a nucleotide sequence comprising SEQ ID No.5, or a nucleotide sequence comprising the 5 '-3' sequence comprising, in order, SEQ ID No.5 and SEQ ID No. 6.

Preferably, the insertion or substitution site follows an endogenous regulatory element of the CD226 gene.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD226 gene of the non-human animal and then performing the insertion operation, or the insertion step may be performed by causing a frame shift mutation at the endogenous CD226 gene or by performing the insertion step of the human sequence.

Preferably, the humanized CD226 gene is homozygous or heterozygous in the animal model. Preferably, the genome of the animal model comprises a humanized CD226 gene on at least one chromosome.

Preferably, the CD226 gene humanized and modified animal model is constructed by using gene editing technology, wherein the gene editing technology comprises gene targeting technology by using embryonic stem cells, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

Preferably, the targeting vector is used for constructing the CD226 gene humanized and transformed animal model.

Preferably, the target site sequence targeted using the sgRNA is as shown in any one of SEQ ID No. 8-15; more preferably, the sgRNA target site sequence used is SEQ ID No.8, 10, 11, 13, 14 and/or 15.

Preferably, the transition region to be altered is located at the CD226 locus of a non-human animal. Further preferably, it is located on exon 2 to exon 7 of the non-human animal CD226 gene. More preferably, it is located on the exon No. 2.

In one 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 to obtain an animal model.

Preferably, the animal models express human or humanized CD226 protein in vivo with reduced or absent expression of endogenous CD226 protein.

Preferably, the humanized CD226 protein comprises part or all of the amino acid sequence encoded by exon 2 through exon 7 of the human CD226 gene. More preferably, the humanized CD226 protein comprises a signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of the human CD226 protein.

Preferably, the humanized CD226 protein further comprises a portion of a non-human animal CD226 protein, more preferably a cytoplasmic region of the non-human animal CD226 protein. Further, the part of the non-human animal CD226 protein is an amino acid sequence shown in position 279-333 of SEQ ID NO. 1.

In one embodiment of the present invention, the humanized CD226 protein comprises one of the following groups:

a) part or all of the amino acid sequence shown in positions 1-278 of SEQ ID NO.2 or the amino acid sequence of SEQ ID NO. 32;

b) a degree of sequence identity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% to the amino acids shown in positions 1-278 of SEQ ID No.2 or the amino acid sequence of SEQ ID No. 32;

c) no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference from the amino acids shown in positions 1-278 of SEQ ID No.2 or the amino acid sequence of SEQ ID No. 32;

d) has the amino acid sequence shown by the amino acids from 1 st to 278 th of SEQ ID NO.2 or SEQ ID NO.32 and comprises substitution, deletion and/or insertion of one or more amino acid residues.

In a second aspect of the invention, a CD226 gene humanized and modified animal model is provided, and the non-human animal is obtained by adopting the construction method.

In a third aspect of the invention, there is provided a targeting vector comprising the above human or humanized CD226 nucleotide sequence

Preferably, the targeting vector further comprises a DNA fragment homologous to the 5 'end of the transition region to be altered, i.e.the 5' arm, selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD226 gene; preferably, said 5' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 5' arm sequence has at least 90% homology with SEQ ID NO.3, or is as shown in SEQ ID NO. 3.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 3 'end of the transition region to be altered, i.e., the 3' arm, selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD226 gene; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 3' arm sequence has at least 90% homology with SEQ ID NO.4, or is as shown in SEQ ID NO. 4.

Preferably, the targeting vector further comprises a marker gene, more preferably, the marker gene is a gene encoding a negative selection marker, and even more preferably, the gene encoding the negative selection marker is a gene encoding diphtheria toxin subunit a (DTA).

In a specific embodiment of the present invention, the targeting vector further comprises a resistance gene selected by a positive clone, and further preferably, the resistance gene selected by the positive clone is neomycin phosphotransferase coding sequence Neo.

In a specific embodiment of the present invention, the targeting vector further comprises a specific recombination system, and further preferably, the specific recombination system is a Frt recombination site, and a conventional LoxP recombination system can also be selected, and the specific recombination system has two Frt recombination sites, which are respectively connected to both sides of the resistance gene.

The targeting vector is used in the construction method of the humanized and modified animal model of the CD226 gene.

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

In a fifth aspect of the invention, there is provided the use of the above-described targeting vector, or the above-described cell, in the modification of the CD226 gene, preferably, said use includes, but is not limited to, inversion, knock-out, insertion or substitution.

In a sixth aspect, the invention relates to a humanized CD226 gene-engineered cell, wherein the genome of the humanized CD226 gene-engineered cell comprises the human or humanized CD226 nucleotide sequence as described above. Preferably, it is regulated by endogenous CD226 regulatory elements; the humanized CD226 gene engineered cells express human or humanized CD226 protein in vivo with reduced or absent expression of endogenous CD226 protein.

In a seventh aspect, the invention relates to a cell with a deleted CD226 gene, wherein the CD226 gene deleted cell deletes exons 1 to 6 of an endogenous CD226 gene.

In an eighth aspect, the invention relates to an sgRNA, wherein a target site sequence targeted by the sgRNA is shown in any one of SEQ ID No. 8-15; more preferably, the sgRNA target site sequence is SEQ ID No.8, 10, 11, 13, 14 and/or 15.

In a ninth aspect of the invention, a vector is provided, the vector comprises the sgRNA, and preferably, the vector is shown in SEQ ID No. 20.

The tenth aspect of the present invention provides a method for constructing a polygene-modified non-human animal, comprising the steps of:

(a) preparing an animal model by applying the construction method;

(b) mating the animal model obtained in the step (a) with a humanized animal except CD226, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-gene humanized modified animal.

Preferably, the other genetically modified non-human animals include, but are not limited to, non-human animals humanized with the genes PD-1, PD-L1, TIGIT.

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

Preferably, each of the plurality of genes humanized in the genome of the polygenic modified non-human animal may be homozygous or heterozygous.

Preferably, the other gene is PD-1.

The eleventh aspect of the present invention relates to the polygenic modified non-human animal or its offspring prepared by the above-described method for preparing a polygenic humanized transformed animal.

In a twelfth aspect, the invention relates to a tumor-bearing animal model, and the preparation method of the animal model comprises the step of preparing the animal by the humanized CD226 gene modified animal model or the method for preparing the multi-gene humanized modified animal.

Preferably, the method for preparing the tumor-bearing animal model further comprises the step of implanting tumor cells into the humanized genetically modified animal prepared by the method or the progeny thereof.

In the thirteenth aspect of the invention, the invention provides an application of the animal model obtained by the construction method or the polygene modified non-human animal obtained by the construction method in preparing a tumor-bearing animal model.

In a fourteenth aspect, the present invention relates to a cell or cell line or a primary cell culture derived from the humanized modified animal model of CD226 gene obtained by the above construction method, the humanized modified animal model of CD226 gene, the polygene-modified non-human animal prepared by the above construction method, the progeny thereof or the tumor-bearing animal model thereof.

In a fifteenth aspect, the present invention relates to a tissue or an organ or a culture thereof derived from the humanized modified animal model of CD226 gene obtained by the above-mentioned construction method, the humanized modified animal model of CD226 gene, the polygene-modified non-human animal obtained by the above-mentioned construction method, the offspring thereof or the tumor-bearing animal model thereof.

Preferably, the tissue or organ or culture thereof is spleen, tumor or culture thereof.

In a sixteenth aspect of the invention, there is provided a humanized CD226 protein, wherein the humanized CD226 protein is as defined in the first aspect of the invention.

In a seventeenth aspect of the invention, there is provided a humanized CD226 gene encoding the above humanized CD226 protein, wherein the humanized CD226 gene has a humanized CD226 nucleotide sequence as described in the first aspect of the invention.

In an eighteenth aspect, the present invention relates to a construct expressing the humanized CD226 protein described above.

In a nineteenth aspect, the present invention relates to a cell comprising the above construct.

In a twentieth aspect, the present invention relates to a tissue comprising the above-described cells.

The twenty-first aspect of the present invention relates to an animal model obtained by the above-mentioned construction method, a polygene-modified non-human animal obtained by the above-mentioned construction method, the above-mentioned cell or cell line or primary cell culture, the above-mentioned tissue or organ or culture thereof, the above-mentioned humanized CD226 protein or the above-mentioned humanized CD226 gene for use in the preparation of a medicament for treating or preventing a tumor.

The twenty-second aspect of the present invention relates to an animal model obtained by the above construction method, a polygenic modified non-human animal obtained by the above construction method, the above cell or cell line or primary cell culture, the above tissue or organ or culture thereof, the above humanized CD226 protein or the above humanized CD226 gene, and applications thereof in CD226 gene or protein related research, wherein the applications comprise:

A) product development involving the immunological process of human cells, use in the manufacture or screening of human antibodies;

B) as model systems for pharmacological, immunological, microbiological and medical research;

C) the production of immune processes involving human cells and the use of animal experimental disease models for pathogenic research, for the development of diagnostic strategies or for the development of therapeutic strategies;

D) screening, drug effect detection, efficacy evaluation, validation or evaluation of human CD226 signaling pathway modulators are studied in vivo; or,

E) the application in the aspects of researching the functions of CD226 genes, researching human CD226 antibodies, researching medicaments and drug effects aiming at human CD226 target sites, and researching medicaments for immune-related diseases and antitumor medicaments.

The disease is a disease related to CD226, preferably, the disease is tumor, autoimmune disease and the like.

"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. In one embodiment of the invention, the tumor is selected from the group consisting of a B cell tumor, a T cell tumor, a bone marrow/monocyte tumor. Preferably B-or T-cell Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), non-Hodgkin's lymphoma (NHL) and Multiple Myeloma (MM), nasopharyngeal carcinoma, lung carcinoma.

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, type I diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, etc. In one embodiment of the invention. The immune-related disease is rheumatoid arthritis.

Preferably, the use is not a method of diagnosis or treatment of disease. The cells are also unable to develop into an individual.

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as a muridae or superfamily murinus. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the family. 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 invention has the beneficial effects that: the humanized non-human animal body of the CD226 gene can normally express the human or humanized CD226 protein. Can be used for drug screening, drug effect evaluation, immunity-related diseases and tumor treatment aiming at human CD226 target sites, can accelerate the development process of new drugs, and saves time and cost. Provides effective guarantee for researching CD226 protein function and screening related disease drugs.

Unless otherwise defined herein, scientific and technical terms and their abbreviations used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art to which the invention belongs. Some of the terms and abbreviations used herein are listed below.

The invention relates to a whole or part, wherein the whole is a whole, and the part is a part of the whole or an individual forming the whole.

The "humanized CD226 protein" of the present invention comprises a portion derived from a human CD226 protein and a portion of a non-human CD226 protein. Wherein, the "human CD226 protein" is identical to the whole "human CD226 protein, namely, the amino acid sequence of the" human CD226 protein "is identical to the full-length amino acid sequence of the human CD226 protein. The "part of the human CD226 protein" is a sequence of 5-336 amino acids which are continuous or spaced and are consistent with the amino acid sequence of the human CD226 protein. Preferably consecutive or spaced 10-239, for example consecutive 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 240, 300 amino acid sequences are identical to the amino acid sequence of the human CD226 protein.

The "humanized CD226 gene" of the present invention comprises a portion derived from a human CD226 nucleotide sequence and a portion of a non-human CD226 gene. Wherein, the "human CD226 nucleotide sequence" is identical to "all of the human CD226 nucleotide sequence", namely, the nucleotide sequence is identical to the full-length nucleotide sequence of the human CD226 nucleotide sequence. The "part of the human CD226 nucleotide sequence" is a continuous or spaced nucleotide sequence of 20-1008bp consistent with the human CD226 nucleotide sequence, preferably 20-831, for example, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800 consistent with the human CD226 nucleotide sequence.

The "exon" from xx to xxx or the whole of the "exon from xx to xxx" in the present invention includes nucleotide sequences of exons and introns therebetween, for example, the "exon 1 to 6" includes all nucleotide sequences of 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 and exon 6.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 1-2" means an intron between exon 1 and exon 2.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "CD 226 locus" refers to a DNA fragment of any one of exons 1 to 6 of the CD226 gene. Preferably any one of exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6, or a combination of two or more thereof, or all or part of one or two or more thereof.

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.

"homology" as used herein means that, in the context of using a protein sequence or a nucleotide sequence, one skilled in the art can adjust the sequence as needed to obtain a sequence having (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.

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 is a schematic representation of the comparison of the mouse CD226 gene and the human CD226 gene (not to scale);

FIG. 2: schematic representation of humanization of mouse CD226 gene (not to scale);

FIG. 3 schematic (not to scale) of CD226 gene targeting strategy and targeting vector design, wherein the targeting vector comprises a5 'homology arm, a 3' homology arm, and an A fragment comprising human CD226 and murine CD226 DNA;

FIG. 4: a detection result of sgRNA activity, wherein Con is a blank control, and PC is a positive control;

FIG. 5: genotype identification of F0 mouse, in which WT was a wild-type control, H₂O is water control;

FIG. 6: genotype identification results of F1 mouse, wherein WT is wild type control, PC is positive control, H₂O is water control;

FIG. 7: southern blot assay results, where WT is wild type control;

FIG. 8: and (3) flow cytometry detection results, wherein the WT is a wild type C57BL/6 mouse, and the H/+ is a CD226 gene humanized mouse heterozygote.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, the devices and materials were obtained from several companies indicated below:

BbsI, EcoRI, BamHI, StuI, BglII enzymes were purchased from NEB under the respective accession numbers R0539L, R0101M, R0136M, R0187M, R0144M;

c57BL/6 mice were purchased from the national rodent laboratory animal seed center of the Chinese food and drug testing institute;

ambion in vitro transcription kit purchased from Ambion, cat # AM 1354;

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

UCA kit comes from Baiosai chart company, Cat number BCG-DX-001;

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

PerCP/Cy5.5 anti-mouse TCR β chain (mTCRb-BV 711) purchased from Biolegend, cat # 109228;

APC anti-mouse CD226 (DNAM-1) Antibody (mCD 226-APC) was purchased from Biolegend under Cat 128809;

PE anti-human CD226 (DNAM-1) Antibody (hCD 226-PE) was purchased from Biolegend under cat # 338305.

Example 1 humanized mouse of CD226 Gene

A comparison scheme of the mouse CD226 Gene (NCBI Gene ID: 225825, Primary source: MGI: 3039602, UniProt: Q8K4F0, located at positions 89176954 to 89272232 of chromosome 18 NC-000084.6, based on transcript NM-178687.2 and its encoded protein NP-848802.2 (SEQ ID NO. 1)) and the human CD226 Gene (NCBI Gene ID: 10666, Primary source: HGNC:16961, UniProt ID: Q15762, located at positions 69853274 to 69962086 of chromosome 18 NC-000018.10, based on transcript NM-006566.4 and its encoded protein NP-006557.2 (SEQ ID NO. 2)) is shown in FIG. 1.

For the purposes of the present invention, a nucleotide sequence encoding a human CD226 protein may be introduced at the endogenous CD226 locus of a mouse, such that the mouse expresses the human or humanized CD226 protein. Specifically, by using gene editing technology, under the control of mouse CD226 gene regulatory elements, a sequence comprising part sequence from No.2 exon to No.6 exon of human CD226 gene and part sequence from mouse No.6 exon to No.7 exon is inserted, and a LoxP STOP sequence is inserted after the mouse sequence, so as to obtain a humanized CD226 locus schematic diagram as shown in FIG. 2, thereby realizing humanized transformation of mouse CD38 gene.

The CRISPR/Cas system was introduced for gene editing and the targeting strategy was designed as shown in figure 3, which shows the homology arm sequences containing the upstream and downstream of the mouse CD226 gene on the targeting vector, and an a fragment comprising the human CD226 sequence. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO. 3) is identical to the nucleotide sequence from position 89205265 to 89206595 of NCBI accession No. NC-000084.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO. 4) has 99% homology with the nucleotide sequence from position 89206599 to 89208210 of NCBI accession No. NC-000084.6, except that G is replaced with C at position 89206694; the human CD226, the mouse CD226 and the LoxP STOP sequence on the A fragment are sequentially arranged according to the 5 '-3' sequence, wherein, the nucleotide sequence (SEQ ID NO. 5) of the human CD226 is in phase with the 73 th to 906 th nucleotide sequences with NCBI accession number NM-006566.4The sequence of the mouse CD226 is shown as SEQ ID NO. 6. The ligation of the murine CD226 sequence downstream to the LoxP STOP sequence upstream in fragment A was designed to be 5' -gattgcttgaacatttttacgtctcaatttgtgaactgttatta agggttccggatcctcggggacaccaaatatggcgatc-3' (SEQ ID NO. 7), wherein the sequence "ctgttThe last "t" in "is the last nucleotide, sequence, of the mouse"attaaThe first "a" in "is the first nucleotide of the LoxP STOP sequence. The amino acid sequence expressed by the humanized mouse CD226 gene after being transformed is shown as SEQ ID NO. 32.

Given that human CD226 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, 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 correct targeting vector was verified by sequencing 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. sgRNA sequences recognizing the target sites were designed and synthesized, and the target site sequence of each sgRNA on the CD226 gene was as follows:

sgRNA1 target site sequence (SEQ ID No. 8): 5'-CAGAGATGGCTTATGTTACTTGG-3'

sgRNA2 target site sequence (SEQ ID No. 9): 5'-GTTTTGCTATGGGGAGCACCAGG-3'

sgRNA3 target site sequence (SEQ ID No. 10): 5'-CAAGTAACATAAGCCATCTCTGG-3'

sgRNA4 target site sequence (SEQ ID No. 11): 5'-TTTTGCTATGGGGAGCACCAGGG-3'

sgRNA5 target site sequence (SEQ ID No. 12): 5'-TCCTGAACTGTTTCCAGAGATGG-3'

sgRNA6 target site sequence (SEQ ID No. 13): 5'-GCCATCTCTGGAAACAGTTCAGG-3'

sgRNA7 target site sequence (SEQ ID No. 14): 5'-GCTGATAAGATCTGAGAGGAAGG-3'

sgRNA8 target site sequence (SEQ ID No. 15): 5'-GTTTTGCTATGGGGAGCACCAGG-3'

The activity of a plurality of sgrnas is detected by using a UCA kit, the detection result is shown in fig. 4, although some sgrnas have relatively low activity, which may be caused by the specificity of a target site sequence, according to our experiments, the numerical values of the sgrnas are still significantly higher than the numerical values of a control group, and the sgrnas can still be judged to be active, and the activity meets the requirements of gene targeting experiments. From these sgRNA8 were selected for subsequent experiments. The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide sequence and a reverse oligonucleotide sequence (see table 1), and after annealing, an annealing product is connected to pT7-sgRNA plasmid (the plasmid is firstly linearized by BbsI), so that an expression vector pT7-CD226-8 is obtained.

Table 1 sgRNA8 sequence listing

SEQ ID NO.16	Upstream: 5'-GTTTTGCTATGGGGAGCACC-3'
		SEQ ID NO.17	Upstream: 5'-TAGGTTTTGCTATGGGGAGCACC-3'
SEQ ID NO.18	Downstream: 5 '-GGTGCTCCCCATAGCAAAAC-3'
		SEQ ID NO.19	Downstream: 5 '-AAACGGTGCTCCCCATAGCAAAAC-3'

The pT7-sgRNA vector was synthesized by plasmid synthesis company as a fragment DNA (SEQ ID NO. 20) containing the T7 promoter and sgRNA scaffold, and was linked to a backbone vector (Takara, cat. No. 3299) by digestion with EcoRI and BamHI in sequence, and sequencing by the professional sequencing company was verified, and the result indicated that the objective plasmid was obtained.

Taking mouse prokaryotic fertilized eggs, such as C57BL/6 mice, and injecting in vitro transcription products (transcribed by using an Ambion in vitro transcription kit according to a method of a specification) of pT7-CD226-8 plasmid, a targeting vector and Cas9mRNA into cytoplasm or nucleus of the mouse fertilized eggs after being premixed by using a microinjection instrument. Microinjection of fertilized eggs is carried out according to the method in the experimental manual for mouse embryo manipulation (third edition) (Andrass, chemical industry publishers, 2006), the fertilized eggs after injection are transferred to a culture solution for short-term culture, then the fertilized eggs are transplanted to the oviduct of a recipient mother mouse for development, and the obtained mice (F0 generation) are hybridized and selfed to expand the population number and establish a stable CD226 gene mutation mouse strain.

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. The 5 'end primer detection result and the 3' end primer detection result are combined, and the 6 mice numbered from F0-01 to F0-06 in figure 5 are all positive mice through further verification of sequencing. The PCR analysis included the following primers:

5' end primer:

L-GT-F（SEQ ID NO.21）：5’-TCTATTATGGTCATACATACCTCCTGG-3’

L-GT-R（SEQ ID NO.22）：5’-AGTGATCCCCATAGTTTTGAGGTAACTA-3’

3' end primer:

R-GT-F（SEQ ID NO.23）：5’-TGACTGTAGCCGAGGGTAAAACCGA-3’

R-GT-R（SEQ ID NO.24）：5’-AGGTGCTGAAACTAGTGCCAAATGA-3’

wherein, the position of the primer L-GT-F is positioned at the left side of the 5 'homology arm, the position of the primer R-GT-R is positioned at the right side of the 3' homology arm, and the position of the primer L-GT-F and the position of the primer R-GT-F are both positioned on the A fragment sequence.

The CD226 gene humanized mouse identified as positive for F0 was mated with the wild type mouse to give F1 generation mice. The same PCR method can be used for genotyping F1 generation mice, and exemplary assay results are shown in FIG. 6, which shows 5 mice numbered F1-01 through F1-05 as positive mice.

The PCR detection primer sequence comprises:

L-GT-F（SEQ ID NO.21）：5’-TCTATTATGGTCATACATACCTCCTGG-3’，

L-GT-R（SEQ ID NO.22）：5’-AGTGATCCCCATAGTTTTGAGGTAACTA-3’；

R-GT-F（SEQ ID NO.23）：5’-TGACTGTAGCCGAGGGTAAAACCGA-3’，

R-GT-R（SEQ ID NO.24）：5’-AGGTGCTGAAACTAGTGCCAAATGA-3’；

WT-F（SEQ ID NO.25）：5’-GCAGCTGATAAGATCTGAGAGGAAGG-3’，

WT-R（SEQ ID NO.26）：5’-GATAAGCTCATGGTGAAGACTGACA-3’；

Mut-F（SEQ ID NO.27）：5’-TGGGCTGCAGGTCGAGGGACCTA-3’

the 5 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 StuI enzyme or BglII enzyme, transferring membrane, and hybridizing. The 5 ' probe and the 3 ' probe are respectively positioned outside the 5 ' homologous arm and on the sequence of the A fragment, and the lengths of the specific probe and the target fragment are shown in Table 2. The Southern blot detection result is shown in FIG. 7, and the result of combining the 3 'probe and the 5' probe shows that 5 mice have no random insertion, which confirms that the 5 mice are positive heterozygous mice and have no random insertion. This shows that the method can construct the humanized gene engineering mouse of the CD226 gene which can be stably passaged and has no random insertion.

TABLE 2 Length of the particular probes and target fragments

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				StuI	5 'Probe (5' Probe)	12.8kb	16.7kb
BglII	3 'Probe (3' Probe)	——	3.8 kb

The probe synthesis primers were as follows:

5’ Probe-F（SEQ ID NO.28）：5’-GGACCTGACTTCAATGATCACCTGGC-3’

5’ Probe-R（SEQ ID NO.29）：5’-TGTGTCCACATGGGGCATGCTTAG-3’

3’ Probe-F（SEQ ID NO.30）：5’-AACTGATGAATGGGAGCAGTGGTGG-3’

3’ Probe-R（SEQ ID NO.31）：5’-GCAGACACTCTATGCCTGTGTGGAG-3’

heterozygous mice identified as positive in the F1 generation were mated with each other to obtain humanized homozygote mice of the CD226 gene in the F2 generation.

The expression of the humanized CD226 protein in positive mice can be confirmed by a conventional detection method, for example, using flow cytometry or the like. Specifically, 1 mouse of wild-type C57BL/6 female (7 weeks old) and 1 mouse of humanized heterozygote of CD226 gene (10 weeks old) prepared in this example were selected, spleen cells were harvested after cervical dislocation and euthanization, and flow detection was performed after recognition staining with anti-mouse CD45 Antibody Brilliant Violet 510 anti-mouse CD45, mouse-specific T cell surface Antibody PerCP/Cy5.5 anti-mouse TCR β chain (mTCRb-BV 711), anti-mouse CD226 Antibody APC anti-mouse CD226 (DNAM-1) Antibody (mCD 226-APC) or anti-human CD226 Antibody PE anti-human CD226 (DNAM-1) Antibody (hCD 226-PE), and the detection results are shown in FIG. 8. As can be seen from the figure, only the expression of the murine CD226 protein could be detected in the wild type C57BL/6 mouse (FIG. 8A), and the expression of the humanized CD226 protein could not be detected (FIG. 8C); both murine CD226 protein expression (fig. 8B) and humanized CD226 protein expression (fig. 8D) were detected in CD226 humanized heterozygote mice.

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

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

Sequence listing

<110> Beijing Baiosai map Gene Biotechnology Co., Ltd

Construction method and application of CD226 gene humanized non-human animal

<130> 1

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 333

<212> PRT

<213> mouse (Mus musculus)

<400> 1

Met Ala Tyr Val Thr Trp Leu Leu Ala Ile Leu His Val His Lys Ala

1 5 10 15

Leu Cys Glu Glu Thr Leu Trp Asp Thr Thr Val Arg Leu Ser Glu Thr

20 25 30

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

35 40 45

Glu Trp Thr Lys Asn Thr Gly Thr Lys Thr Val Ser Ile Ala Val Tyr

50 55 60

Asn Pro Asn His Asn Met His Ile Glu Ser Asn Tyr Leu His Arg Val

65 70 75 80

His Phe Leu Asn Ser Thr Val Gly Phe Arg Asn Met Ser Leu Ser Phe

85 90 95

Tyr Asn Ala Ser Glu Ala Asp Ile Gly Ile Tyr Ser Cys Leu Phe His

100 105 110

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

115 120 125

Asp Ser Phe Glu Ile Ala Ala Pro Ser Asp Ser Tyr Leu Ser Ala Glu

130 135 140

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

145 150 155 160

Val Gln Gln Val Ile Trp Glu Lys Val Gln Pro His Gln Val Asp Ile

165 170 175

Leu Ala Ser Cys Asn Leu Ser Gln Glu Thr Arg Tyr Thr Ser Lys Tyr

180 185 190

Leu Arg Gln Thr Arg Ser Asn Cys Ser Gln Gly Ser Met Lys Ser Ile

195 200 205

Leu Ile Ile Pro Asn Ala Met Ala Ala Asp Ser Gly Leu Tyr Arg Cys

210 215 220

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

225 230 235 240

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

245 250 255

Gly Gly Leu Val Ser Leu Leu Leu Val Ile Leu Ile Ile Ile Ile Phe

260 265 270

Ile Leu Tyr Asn Arg Lys Arg Arg Arg Gln Val Arg Ile Pro Leu Lys

275 280 285

Glu Pro Arg Asp Lys Gln Ser Lys Val Ala Thr Asn Cys Arg Ser Pro

290 295 300

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

305 310 315 320

Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Pro Arg Leu

325 330

<210> 2

<211> 336

<212> PRT

<213> human (Homo sapiens)

<400> 2

Met Asp Tyr Pro Thr Leu Leu Leu Ala Leu Leu His Val Tyr Arg Ala

1 5 10 15

Leu Cys Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn

20 25 30

Met Ser Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val

35 40 45

Glu Trp Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser

50 55 60

Pro Thr His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr

65 70 75 80

Phe Leu Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg

85 90 95

Asn Ala Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr

100 105 110

Tyr Pro Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp

115 120 125

Ser Phe Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro

130 135 140

Gly Lys Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val

145 150 155 160

Gln Ala Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu

165 170 175

Thr Tyr Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro

180 185 190

Arg Gln Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val

195 200 205

Ile Pro Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu

210 215 220

Gln Ala Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val

225 230 235 240

Ala Glu Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Gly Gly

245 250 255

Thr Val Leu Leu Leu Leu Phe Val Ile Ser Ile Thr Thr Ile Ile Val

260 265 270

Ile Phe Leu Asn Arg Arg Arg Arg Arg Glu Arg Arg Asp Leu Phe Thr

275 280 285

Glu Ser Trp Asp Thr Gln Lys Ala Pro Asn Asn Tyr Arg Ser Pro Ile

290 295 300

Ser Thr Ser Gln Pro Thr Asn Gln Ser Met Asp Asp Thr Arg Glu Asp

305 310 315 320

Ile Tyr Val Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Thr Arg Val

325 330 335

<210> 3

<211> 1331

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cttgaacttc atatgtgatg agtccattta aaagattatc agtgtgaatc aattattttg 60

tgtatcagca tgagtataaa acagggagat gtttttagag aacatgaaat ttcagctgag 120

aataagaaat aacctcattt ataatgtgca cattgtatca tggttctgct aatggggcca 180

aatgtaatat aattcttctt tttataaaga taaaagtttg agttttgatt gtcttgttga 240

aatattcttc acaaattttt aagaattaga attaatattt tattatatat tagttattca 300

tattataatt gtgtgtgttt gtttggtatg tgtggtttca tgtgccaccg tgtacatgtg 360

ctagttcaag cataactttg tgaagttagt tctatcctct tacctttatg taggttctgg 420

agactcaggt catcaggctt ttttaataag cattgttact ggctaaccca tcttgtcaca 480

accctaagtt gtttttataa ttgaagagag aaatcttaga aatgaatctc actttgatag 540

tgtatcacag ttttagatat ataaggtgta acctctaagt tgtaatatct agttttttcg 600

tagtactcta tgcttatttt aaaattaaaa agtggaaaaa aaatcatcag atataatggt 660

atattacatg tgttttggat cgtaatattt tgtagttgga ggtccattca gttgaatatc 720

tttgttcaaa tctaacccag aggtgtggtg cagctggaag gccacacctg agtggttgtc 780

tgatatgcag gctcctccca tcagctccag tcttcacccc atttcacttt ctcagtgagc 840

agttcctgga cttgcctata tgaaaagatc ttgactgtcc tgctttgctt cacttcttct 900

caggtaccaa ccaccttcag cacactatat ctagtacacc tttagcattg tatcaacctc 960

ttcctaacaa aatggcatgg gattttattt ttaagatgca tttttgtata ttcttgttgc 1020

acagagctaa gtctggcaca gaggacacac tcagtaaaca tttgtcaaat gaaagaatct 1080

ttctcgaaaa ttatatcttt taaagtaaac tgattatctg acagagaaca ataactgcag 1140

cctatccatc cctgtacgat agcaaagctt tctttgaagc tgaaacttct ttcctgtgct 1200

catacttcag agaaaaagat gcagctgata agatctgaga ggaaggaaac agtacttcat 1260

gctgagctgt gaatataaca tcatacacat ttttttttta tacagtccaa cctcctgaac 1320

tgtttccaga g 1331

<210> 4

<211> 1612

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcttatgtta cttggctttt ggctattctt catgtgcaca aaggtaaagc cagtcctatg 60

tattcattta aaggttttgc tatggggagc accagcgtca agaagtggga gtagataggt 120

aggggagggt ggaggagggc atagggaact ttcgggatag catttgaaat gtaaatgaag 180

aaaatacata ataaaaaaat tggaaagaaa aaaaagtttt gctttttttt ttgtgacatg 240

tgcaggatct cttaggcagt ttctcacttg tttagaaata ctaaaattta caagaggcaa 300

ttttgtagtt gaaattgtca gtcttcacca tgagcttatc atgctatttg ttaagtctta 360

taagttgtgt ttatttggct tcgtcatcat tggatctaat caatttatgt gattgatata 420

cattttagca ctgtgtgaag agacattgtg ggacacaaca gttcggcttt ctgagactat 480

gactctggaa tgtgtatatc cattgacgca taacttaacc caggtggagt ggaccaagaa 540

cactggcaca aagacagtga gcatagcagt ttacaaccct aaccataata tgcatataga 600

atctaactac ctccatagag tacacttcct aaactcaaca gtggggttcc gcaacatgag 660

cctttccttt tacaatgcct cagaagcaga cattggcatc tactcctgct tgtttcatgc 720

tttcccaaat ggaccttggg aaaagaagat aaaagtagtc tggtcaggta aggaaaaact 780

ttattttctt tttgtctact gacaatgttt aattctaaga aacccaacca aaagcttaaa 840

atgttattgt attcttttcc caattccatt gctagatttt tgatctaatt tttcctcaac 900

gaataatgtc gtaaaccatc tcattttttt cttaattgct gtcatttctg agtgcaagtg 960

tctcccaggt ttcagtgatg tgggaggaaa tgcagattta catagttttt atgatgacat 1020

aaggttagat tgaaataata ttgtcaacat gtagattcag agcaatccct tctttttttt 1080

cattttattt atttattttt taattaggta ttttcttcat ttacatttca aatgctatcc 1140

caaaagtccc tcataccccc ccccactccc ctacccacct actcccaatt cttggtcctg 1200

gcgttcccct gtactgaggc atataaagtt tgcaagacca aggggcctct cttcccaatg 1260

atggccaact aggccatctt ctgatacata tgcagctaga gacacgagct ccgggggtaa 1320

tggttagttc atattgttgt tccacctata aggttgcaga cccctttagc tccttgagta 1380

ctttctctag ctcctccatt ggcagccctg tgttccatcc aatagctgac tgtgagcatc 1440

cacttctgtg tttgccaggc cctggcatag cctcacaaga gacagcaata tcagggtcct 1500

ttcagcaaaa tctttctggt gcatgcaatg gtgtcagcat ttggaggctg attatgggat 1560

ggatccccgg gtatgccagt ctctagatgg tccatccttt actggtaaga ag 1612

<210> 5

<211> 834

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggattatc ctactttact tttggctctt cttcatgtat acagagctct atgtgaagag 60

gtgctttggc atacatcagt tccctttgcc gagaacatgt ctctagaatg tgtgtatcca 120

tcaatgggca tcttaacaca ggtggagtgg ttcaagatcg ggacccagca ggattccata 180

gccattttca gccctactca tggcatggtc ataaggaagc cctatgctga gagggtttac 240

tttttgaatt caacgatggc ttccaataac atgactcttt tctttcggaa tgcctctgaa 300

gatgatgttg gctactattc ctgctctctt tacacttacc cacagggaac ttggcagaag 360

gtgatacagg tggttcagtc agatagtttt gaggcagctg tgccatcaaa tagccacatt 420

gtttcggaac ctggaaagaa tgtcacactc acttgtcagc ctcagatgac gtggcctgtg 480

caggcagtga ggtgggaaaa gatccagccc cgtcagatcg acctcttaac ttactgcaac 540

ttggtccatg gcagaaattt cacctccaag ttcccaagac aaatagtgag caactgcagc 600

cacggaaggt ggagcgtcat cgtcatcccc gatgtcacag tctcagactc ggggctttac 660

cgctgctact tgcaggccag cgcaggagaa aacgaaacct tcgtgatgag attgactgta 720

gccgagggta aaaccgataa ccaatatacc ctctttgtgg ctggagggac agttttattg 780

ttgttgtttg ttatctcaat taccaccatc attgtcattt tccttaacag aagg 834

<210> 6

<211> 1714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agacggagac aggtgagaat tccacttaaa gagcccaggg ataaacagag taaggtagcc 60

accaactgca gaagtcctac ttctcccatc cagtctacag atgatgaaaa agaggacatt 120

tatgtaaact atccaacttt ctctcgaaga ccaaaaccaa gactctaagc tgctcttttg 180

gcctgaacac attagtgatg acttctatgg catggaattt tacccatgat ttccttacca 240

ctaggatcta cattgataaa aaaaattgat taaatttatt tcatctcata tatagaagta 300

ctttattacc tggaaacatt cttaatagag attcattaga aaacccaaat ctaatgttca 360

tgtgttcaag gaaccttctt ccattatgta acagaacagt ctagagaaga ttaaggacca 420

catggctttc ttgctctact tgaaattaat tgtgagcata agcttgtttc tggagtcttc 480

ttacattgtt ggttctactt acatactact ggtccaactc tcatgctgtt tctctcagat 540

gttcccatga tggttgccaa ggacacttga tagaaagact actggttaaa cacaataaac 600

aaagttcatt attcacttat tagcaagaag gtagcattat cataaaggat tagatgactt 660

aagttagcta taggttcaag acctggacta aagtattact tggaaattct gagtattgct 720

aaaaaggagg atgaaaggga cctagaagtt gagttattac taaaaacttt gagtgcgaag 780

atattactca ttaaccagat aacaagtgaa tatgctgtag catcaacata attcaaaaga 840

gtaaagaaat ggctaggaat gaggtagttg tgtaattatt tcttctctta ctagtttcaa 900

ataaattcat ctctaattct atagagaatt cttgcctccc attcaggact ggccttctat 960

acagtgagat ggtccagtaa gaaataattt ttattagtgt tttttctatt ttgagaatta 1020

ttttaatata tattttaata tataaacttg tgagttaaat tttttttttg caaaattagc 1080

acatgaaaag agattgatgg ttttaagtag tagaacacag tagtgtagga atctgagagc 1140

agagagtttg ggagggggtg aagagaaaac aacatcacca aatagtgata tataagagaa 1200

aatctgtgct tcagagtttg atcagggcca tctctcccaa ctctgctgga actgagagaa 1260

tgcacctgat gttgtctcca ttttagatag agaaaaaaaa aacccgaata tttataaaac 1320

taaataaaac tatagttacc tcaaaactat ggggatcact ataacataga atagaataga 1380

atagaataga atagaataga atagaataga atagatttag tatctatttt ctaatctcta 1440

aaaaatttta aattatcata atctttgaat gctagtgctg ttacatatat tctaaaatgt 1500

aatccatagt tttaaggtag ctgggggata tttcatattt ttaaccttag atccatgcat 1560

aatgaatagg ccaattattt tgccaattat agaaattcag aatgtaataa ataaaaatga 1620

cttttttctt agttgttctt aaataagaat gattaattaa ctcagatttt gactgattgc 1680

ttgaacattt ttacgtctca atttgtgaac tgtt 1714

<210> 7

<211> 82

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gattgcttga acatttttac gtctcaattt gtgaactgtt attaagggtt ccggatcctc 60

ggggacacca aatatggcga tc 82

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagagatggc ttatgttact tgg 23

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gttttgctat ggggagcacc agg 23

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caagtaacat aagccatctc tgg 23

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttttgctatg gggagcacca ggg 23

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcctgaactg tttccagaga tgg 23

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gccatctctg gaaacagttc agg 23

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctgataaga tctgagagga agg 23

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gttttgctat ggggagcacc agg 23

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gttttgctat ggggagcacc 20

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

taggttttgc tatggggagc acc 23

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggtgctcccc atagcaaaac 20

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aaacggtgct ccccatagca aaac 24

<210> 20

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 21

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tctattatgg tcatacatac ctcctgg 27

<210> 22

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

agtgatcccc atagttttga ggtaacta 28

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tgactgtagc cgagggtaaa accga 25

<210> 24

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aggtgctgaa actagtgcca aatga 25

<210> 25

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gcagctgata agatctgaga ggaagg 26

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gataagctca tggtgaagac tgaca 25

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgggctgcag gtcgagggac cta 23

<210> 28

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggacctgact tcaatgatca cctggc 26

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tgtgtccaca tggggcatgc ttag 24

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aactgatgaa tgggagcagt ggtgg 25

<210> 31

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gcagacactc tatgcctgtg tggag 25

<210> 32

<211> 333

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Met Asp Tyr Pro Thr Leu Leu Leu Ala Leu Leu His Val Tyr Arg Ala

1 5 10 15

Leu Cys Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn

20 25 30

Met Ser Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val

35 40 45

Glu Trp Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser

50 55 60

Pro Thr His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr

65 70 75 80

Phe Leu Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg

85 90 95

Asn Ala Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr

100 105 110

Tyr Pro Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp

115 120 125

Ser Phe Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro

130 135 140

Gly Lys Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val

145 150 155 160

Gln Ala Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu

165 170 175

Thr Tyr Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro

180 185 190

Arg Gln Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val

195 200 205

Ile Pro Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu

210 215 220

Gln Ala Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val

225 230 235 240

Ala Glu Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Gly Gly

245 250 255

Thr Val Leu Leu Leu Leu Phe Val Ile Ser Ile Thr Thr Ile Ile Val

260 265 270

Ile Phe Leu Asn Arg Arg Arg Arg Arg Gln Val Arg Ile Pro Leu Lys

275 280 285

Glu Pro Arg Asp Lys Gln Ser Lys Val Ala Thr Asn Cys Arg Ser Pro

290 295 300

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

305 310 315 320

Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Pro Arg Leu

325 330

Claims (9)

1. A construction method of a CD226 gene humanized and transformed non-human animal is characterized in that a genome of the non-human animal comprises a humanized CD226 nucleotide sequence, the humanized CD226 nucleotide sequence sequentially comprises a human CD226 nucleotide sequence, a mouse CD226 nucleotide sequence and a LoxP STOP sequence from 5 '-3', wherein the human CD226 protein sequence coded by the human CD226 nucleotide sequence is shown as the amino acid sequence from the 1 st position to the 278 th position of SEQ ID NO.2, and the mouse CD226 nucleotide sequence is shown as SEQ ID NO. 6;

the construction method comprises the steps of inserting or replacing the humanized CD226 nucleotide sequence into the CD226 locus of the non-human animal, and constructing the non-human animal by using a targeting vector or sgRNA;

the targeting vector comprises the humanized CD226 nucleotide sequence, and the targeting vector also comprises DNA fragments which are homologous with the 5 'end and the 3' end of the switching region to be changed, namely 5 'and 3' arms, wherein the sequence of the 5 'arm is shown as SEQ ID NO.3, and the sequence of the 3' arm is shown as SEQ ID NO. 4;

the sequence of the target site targeted by the sgRNA is shown in any one of SEQ ID NO. 8-15.

2. The construction method according to claim 1, wherein the nucleotide sequence of human CD226 is the nucleotide sequence shown in SEQ ID No. 5.

3. The construction method according to claim 1, wherein the humanized CD226 protein is shown as amino acid in SEQ ID No. 32.

4. The method of any one of claims 1-3, wherein the non-human animal is a rat or a mouse.

5. A targeting vector is characterized by comprising a humanized CD226 nucleotide sequence, and further comprising DNA fragments which are homologous with the 5 'end and the 3' end of a switching region to be changed, namely 5 'and 3' arms, wherein the 5 'arm sequence is shown as SEQ ID NO.3, the 3' arm sequence is shown as SEQ ID NO.4, the humanized CD226 nucleotide sequence sequentially comprises a human CD226 nucleotide sequence, a murine CD226 nucleotide sequence and a LoxP STOP sequence from 5 'to 3', wherein the human CD226 protein sequence coded by the human CD226 nucleotide sequence is shown as the amino acid sequence from the 1 st to the 278 th positions of SEQ ID NO.2, and the murine CD226 nucleotide sequence is shown as SEQ ID NO. 6.

6. The targeting vector of claim 5, wherein the nucleotide sequence of human CD226 is the nucleotide sequence set forth in SEQ ID No. 5.

7. A humanized CD226 protein, wherein the humanized CD226 protein comprises an extracellular region, a signal peptide, a transmembrane region and a cytoplasmic region of the human CD226 protein, and the humanized CD226 protein has an amino acid sequence as set forth in SEQ ID No. 32.

8. A humanized CD226 gene encoding the humanized CD226 protein of claim 7, wherein said humanized CD226 gene comprises the nucleotide sequence of SEQ ID No. 5.

9. The non-human animal constructed by the construction method according to any one of claims 1 to 4, the humanized CD226 protein according to claim 7, or the use of the humanized CD226 gene according to claim 8 in CD226 gene or protein-related research, which is not a method for diagnosing and treating a disease, the use comprising:

A) product development involving the immunological process of human cells, use in the manufacture or screening of human antibodies;

B) as model systems for pharmacological, immunological, microbiological and medical research;

C) relates to the production of immune processes of human cells and the use of animal experimental disease models for the application in the research of etiology;

D) screening, drug effect detection, efficacy evaluation, validation or evaluation of human CD226 signaling pathway modulators are studied in vivo; or,

E) the application in the aspects of researching CD226 gene function, researching human CD226 antibody, researching medicine and drug effect aiming at human CD226 target site, researching immune-related disease medicine and anti-tumor medicine.

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