CN114751973B

CN114751973B - Construction method and application of SIGLEC15 gene humanized non-human animal

Info

Publication number: CN114751973B
Application number: CN202210243186.8A
Authority: CN
Inventors: 赵磊; 李惠琳; 赵素曼; 刘帅帅; 田茂鹏
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-03-12
Filing date: 2022-03-11
Publication date: 2024-02-20
Anticipated expiration: 2042-03-11
Also published as: CN114751973A; WO2022188871A1

Abstract

The invention provides a construction method of a non-human animal humanized by a SIGLEC15 gene, a humanized SIGLEC15 protein, a humanized SIGLEC15 gene, a targeting vector of the SIGLEC15 gene and application thereof in the field of biological medicine, and a nucleotide sequence for encoding the human SIGLEC15 protein is introduced into a genome of the non-human animal by utilizing a homologous recombination mode, so that the human or humanized SIGLEC15 protein can be normally expressed in the animal body, and the method can be used as an animal model for researching a human SIGLEC15 signal mechanism and screening medicines for tumors and autoimmune diseases, and has important application value for developing new medicines of immune targets.

Description

Construction method and application of SIGLEC15 gene humanized non-human animal

Technical Field

The invention belongs to the fields of animal genetic engineering and genetic modification, and particularly relates to a construction method of a SIGLEC15 gene humanized non-human animal and application thereof in the field of biological medicine.

Background

SIGLEC15 (Sialic acid-binding immunoglobulin-like lectin 15), which belongs to the SIGLEC family, is a type I transmembrane protein whose mRNA is rarely expressed in most normal human tissues and various immune cell subsets, but expressed in macrophages in relatively high amounts. In 2007, japanese scientist Takashi AngataMayo discovered that the protein can recognize sialic acid for the first time, so that the protein is classified into SIGLEC family, and plays an important regulatory role in autoimmune diseases, inflammatory reactions and tumors of organisms by regulating innate immunity and adaptive immune responses.

SIGLEC15 is a related immune receptor of DAP12 (DNAX activating protein kDa), whereas DAP12 is a adaptor protein based on the tyrosine immune receptor activation motif (ITAM, immunoreceptor tyrosine-based activation motif) signaling pathway, and SIGLEC15, when bound to DAP12, is capable of causing transmission of the ITAM signaling pathway, involved in osteoclast differentiation. The Anti-SIGLEC15 antibody can inhibit the differentiation of osteoclast and down regulate the expression of RANKL (important factor for promoting the differentiation of osteoclast), so as to achieve the effect of treating osteoporosis. Furthermore, professor on display in 2019 indicated that SIGLEC15 screened using the high throughput functional screening system TCAA was able to consistently inhibit T cell activity and was shown to satisfy the main features of normalized cancer immunotherapy. For the vast majority of PD-L1 negative tumor patients, SIGLEC15 and PD-L1 are expressed mutually exclusive, and in particular, the immunosuppression of the protein is independent of PD-1, which provides a new candidate for patients who do not respond to current immunotherapy, especially to anti-PD-1/PD-L1 therapy. TCGA database Meta analysis shows that mRNA of SIGLEC15 is up-regulated in various human cancers, for example, in colon cancer, endometrioid cancer and thyroid cancer, and significantly up-regulated in bladder cancer, kidney cancer, lung cancer and liver cancer, and can be a candidate target for a tumor immunotherapy normalization strategy.

The experimental animal disease model is an indispensable research tool for researching the etiology and pathogenesis of human disease occurrence and developing control technology and medicines. In view of the great application value of SIGLEC15 in the fields of autoimmune diseases and tumor immunotherapy, for further research on related biological characteristics, the effectiveness of preclinical pharmacodynamic tests is improved, the success rate of research and development is improved, preclinical tests are more effective, and research and development failures are minimized, and development of a related non-human animal model related to SIGLEC15 is needed in the art.

Disclosure of Invention

In a first aspect of the invention, there is provided a humanized SIGLEC15 protein, said humanized SIGLEC15 protein comprising all or part of a human SIGLEC15 protein.

Preferably, the humanized SIGLEC15 protein comprises all or part of a signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of a human SIGLEC15 protein.

Further preferred, the humanized SIGLEC15 protein comprises all or part of the extracellular domain of a human SIGLEC15 protein.

In a specific embodiment of the invention, the humanized SIGLEC15 protein comprises a signal peptide, a transmembrane region, a cytoplasmic region and an extracellular region, wherein the extracellular region comprises all or a portion of the extracellular region of a human SIGLEC15 protein, preferably at least 50 consecutive amino acids of the extracellular region of a human SIGLEC15 protein, such as a human SIGLEC15 protein extracellular region comprising at least 50, 70, 90, 100, 150, 197, 200, 210, 211, 212, 213, 214, 215, 216, 223, 217, 218, 219, 220, 230, 233, 240, 244 consecutive amino acids, further preferably 197, 216, 223, or 233 consecutive amino acids; preferably, the human SIGLEC15 protein extracellular region comprising an N-terminal removal of 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and/or a C-terminal removal of 0-50 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 47, 50), further preferably, the human SIGLEC15 protein extracellular region comprising an N-terminal removal of 0, 7, 9 or 11 and a C-terminal removal of 4, 12, 47 or 17 amino acids, more preferably, the amino acid sequence having at least 85%, 90%, 95% or at least 99% identity to positions 31-246 of SEQ ID NO 2 or the amino acid sequence corresponding to positions 31-246 of SEQ ID NO 2.

In some embodiments, the humanized SIGLEC15 protein comprises amino acids 6-15 flanking positions 31-246 of SEQ ID NO. 2, e.g., positions 27-259, 29-251, 20-216 of SEQ ID NO. 2.

Preferably, the humanized SIGLEC15 protein comprises all or part of the amino acid sequence encoded by the human SIGLEC15 gene from exon 1 to exon 6, further preferably comprises all or part of the amino acid sequence encoded by the human SIGLEC15 gene from exon 2 to exon 4, further preferably comprises all or part of the amino acid sequence encoded by any one, two, three, two or three consecutive exons from exon 2 to exon 4, still further preferably comprises all or part of the amino acid sequence encoded by the part of exon 2, all of exon 3 and part of exon 4, wherein the part of exon 2 comprises at least a nucleotide sequence of 5bp, e.g. at least a nucleotide sequence of 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60bp, further preferably a nucleotide sequence of 22 bp; preferably, the portion of exon 2 comprises at least the last nucleotide sequence from nucleotide sequence encoding amino acids 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) from the N-terminus of the extracellular region of human SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably, the last nucleotide sequence from nucleotide sequence encoding amino acids 12 from the N-terminus of the extracellular region of human SIGLEC15 protein to the last nucleotide sequence of exon 2; the part of exon 4 comprises at least a nucleotide sequence of 100bp, for example at least a nucleotide sequence of 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 378bp, further preferred a nucleotide sequence of 242 bp; preferably, the portion of exon 4 comprises at least a nucleotide sequence from nucleotide 4 to the C-terminal 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids encoding the extracellular region of human SIGLEC15 protein, preferably, from nucleotide 4 to the C-terminal 18 amino acids encoding the extracellular region of human SIGLEC15 protein, more preferably, the humanized SIGLEC15 protein comprises an amino acid sequence having at least 85%, 90%, 95% or at least 99% identity to the amino acid sequence encoded by SEQ ID NO:5 or comprises an amino acid sequence encoded by SEQ ID NO: 5.

In a specific embodiment of the present invention, the amino acid sequence of the human SIGLEC15 protein comprised in the humanized SIGLEC15 protein comprises any one of the following groups:

a) All or part of the amino acid sequence comprising positions 31-246, 27-259, 20-263, 20-216 or 29-251 of SEQ ID NO. 2;

b) Comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to amino acid sequence positions 31-246, 27-259, 20-263, 20-216 or 29-251 of SEQ ID NO. 2;

c) Comprises an amino acid sequence which differs by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 amino acid from the amino acid sequence at positions 31-246, 27-259, 20-263, 20-216 or 29-251 of SEQ ID NO. 2; or (b)

D) Comprising the amino acid sequence shown in positions 31-246, 27-259, 20-263, 20-216 or 29-251 of SEQ ID NO. 2, including substitutions, deletions and/or insertions of one or more amino acid residues.

Preferably, the humanized SIGLEC15 protein further comprises a portion of a non-human animal SIGLEC15 protein, preferably a signal peptide, extracellular region, transmembrane region and/or cytoplasmic region of a non-human animal SIGLEC15 protein.

Preferably, up to 30 (e.g., 0, 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) consecutive amino acid sequences of the humanized SIGLEC15 protein are derived from a non-human animal SIGLEC15 protein extracellular domain, wherein the extracellular domain comprises an N-terminal 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid and/or a C-terminal 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acid sequence derived from the non-human animal SIGLEC15 protein extracellular domain, further preferably comprises an N-terminal 3, 5 or 7 amino acids and a C-terminal 17 amino acid sequence derived from the non-human animal SIGLEC15 protein extracellular domain.

Preferably, the humanized SIGLEC15 protein further comprises a portion of an amino acid sequence encoded by a non-human animal endogenous SIGLEC15 gene, further preferably comprises all or a portion of an amino acid sequence encoded by all of exon 1, exon 2, exon 4, exon 5, and exon 6 of a non-human animal endogenous SIGLEC15 gene, wherein the portion of exon 2 of the non-human animal SIGLEC15 gene comprises at least a 20bp nucleotide sequence, such as at least a 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60bp nucleotide sequence, further preferably comprises a 38bp nucleotide sequence; preferably, the portion of exon 2 comprises at least the nucleotide sequence from the first nucleotide to 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids encoding the N-terminus of the extracellular region of SIGLEC15 protein, preferably, the portion of exon 2 comprises the nucleotide sequence from the first nucleotide to 7 amino acids encoding the N-terminus of the extracellular region of SIGLEC15 protein; the non-human animal SIGLEC15 gene exon 4 comprises at least a 100bp nucleotide sequence, for example at least a 100, 110, 120, 230, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 145, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, 370, 375bp nucleotide sequence, more preferably a 136bp nucleotide sequence; preferably, the portion of exon 4 comprises at least from the nucleotide sequence encoding amino acids 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) from the C-terminal end of the extracellular region of SIGLEC15 protein to the last nucleotide sequence of exon 4, preferably from the nucleotide sequence encoding amino acids 17 from the C-terminal end of the extracellular region of SIGLEC15 protein to the last nucleotide sequence of exon 4.

In a specific embodiment of the present invention, the amino acid sequence of the humanized SIGLEC15 protein comprises any one of the following groups:

a) Comprising all or part of the amino acid sequence of SEQ ID NO. 10;

b) Comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of SEQ ID NO 10;

c) Comprises an amino acid sequence which differs from the amino acid sequence shown in SEQ ID NO. 10 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 amino acid; or (b)

d) Comprising the amino acid sequence shown in SEQ ID NO. 10, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the non-human animal is selected from any non-human animal that can be genetically edited to produce a humanized gene, such as rodents, pigs, rabbits, monkeys, etc.

Preferably, the non-human animal is a non-human mammal, more preferably, the non-human mammal is a rodent, even more preferably, the rodent is a rat or a mouse.

The non-human animal is an immunodeficient non-human mammal, preferably an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey, more preferably an immunodeficient rodent is an immunodeficient mouse or rat, even more preferably the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a second aspect of the present invention, there is provided a humanized SIGLEC15 gene, said humanized SIGLEC15 gene comprising a portion of a human SIGLEC15 gene.

Preferably, the humanized SIGLEC15 gene comprises all or part of a nucleotide sequence encoding a signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of a human SIGLEC15 protein; preferably, the humanized SIGLEC15 gene comprises all or part of a nucleotide sequence encoding an extracellular region of a human SIGLEC15 protein, wherein the extracellular region comprises all or part of an extracellular region of a human SIGLEC15 protein, preferably comprises at least 50 consecutive amino acids of an extracellular region of a human SIGLEC15 protein, e.g. comprises at least 50, 70, 90, 100, 150, 197, 200, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 223, 230, 233, 240, 244 consecutive amino acids, which corresponds to an extracellular region of a human SIGLEC15 protein, further preferably comprises 197, 223, 216 or 233 consecutive amino acids; preferably, the human SIGLEC15 protein extracellular domain comprising an N-terminal removal of 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids and/or a C-terminal removal of 0-50 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 47, 50) amino acids, further preferably, the human SIGLEC15 protein extracellular domain comprising an N-terminal removal of 0, 7, 9 or 11 amino acids and a C-terminal removal of 4, 12, 47 or 17 amino acids, further preferably, the extracellular domain comprises an amino acid sequence having at least 85%, 90%, 95% or at least 99% identity to positions 31-246 or 27-259 of SEQ ID NO 2 or comprises an amino acid sequence corresponding to positions 31-246 or 27-259 of SEQ ID NO 2.

Preferably, the humanized SIGLEC15 gene encodes a humanized SIGLEC15 protein of the invention.

Preferably, the humanized SIGLEC15 gene comprises all or part of exons No. 1 to 6, further preferably comprises all or part of exons No. 2 to 4, further preferably comprises all or part of any one, two, three, consecutive two or consecutive three exons of the human SIGLEC15 gene, further preferably comprises all or part of exons No. 2, 3 and part of exons No. 4, further preferably further comprises any one of introns No. 2 to 3 and/or introns No. 3 to 4, further preferably comprises any one of introns between exons No. 2 to 4, wherein the part of exons No. 2 comprises at least a nucleotide sequence of 5bp, such as at least a nucleotide sequence of 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 60, 55, further preferably comprises a nucleotide sequence of at least 5 bp; preferably, the portion of exon 2 comprises at least the nucleotide sequence encoding amino acids 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) from the N-terminus of the extracellular region of human SIGLEC15 to the last nucleotide sequence of exon 2, preferably, from the nucleotide sequence encoding amino acids 12 from the N-terminus of the extracellular region of human SIGLEC15, said portion of exon 4 comprises at least a 100bp nucleotide sequence, e.g., at least a 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 378bp nucleotide sequence, further preferably, 242bp nucleotide sequence; preferably, the portion of exon 4 comprises at least a nucleotide sequence from the first nucleotide of exon 4 to 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids encoding the extracellular region of human SIGLEC15, preferably, a nucleotide sequence from the first nucleotide of exon 4 to 18 amino acids encoding the extracellular region of human SIGLEC15, more preferably, the humanized SIGLEC15 gene comprises a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity to SEQ ID No. 5 or comprises the nucleotide sequence set forth in SEQ ID No. 5.

In a specific embodiment of the present invention, the human SIGLEC15 gene comprised in the humanized SIGLEC15 gene comprises any one of the following groups:

(A) Comprising all or part of the nucleotide sequence set forth in SEQ ID NO. 5;

(B) A nucleotide sequence comprising at least 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. 5;

(C) Comprising a nucleotide sequence which differs from the nucleotide sequence shown in SEQ ID No. 5 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 nucleotide;

(D) A nucleotide sequence having the nucleotide sequence shown in SEQ ID No. 5, comprising a substitution, deletion and/or insertion of one or more nucleotides.

Preferably, the humanized SIGLEC15 gene comprises a nucleotide sequence encoding a signal peptide, a transmembrane region, a cytoplasmic region of a non-human animal, and/or a nucleotide sequence encoding up to 30 (e.g., 0, 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) consecutive amino acids of an extracellular domain of a non-human animal SIGLEC15 protein, wherein the extracellular domain comprises a nucleotide sequence derived from the N-terminus 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and/or the C-terminus 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids of the extracellular domain, and further preferably the extracellular domain comprises an extracellular domain derived from a non-human animal SIGLEC15 protein, and the C-terminus 0-20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 20) amino acids.

Preferably, the humanized SIGLEC15 gene further comprises all of exon 1, part of exon 2, part of exon 4, all of exon 5 and/or all of exon 6 of a non-human animal endogenous SIGLEC15 gene, wherein the non-human animal endogenous SIGLEC15 gene comprises at least a 20bp nucleotide sequence, e.g. at least a 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60bp nucleotide sequence, further preferably a 38bp nucleotide sequence; preferably, the portion of exon 2 comprises at least a nucleotide sequence from the first nucleotide to 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular region of SIGLEC15 protein, preferably, a nucleotide sequence from the first nucleotide to the 2-exon encoding 7 amino acids from the N-terminus of the extracellular region of SIGLEC15 protein, and the portion of exon 4 of the endogenous SIGLEC15 gene of the non-human animal comprises at least a 100bp nucleotide sequence, e.g., at least a nucleotide sequence of 100, 110, 120, 230, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 145, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, 370, 375bp, and more preferably, a nucleotide sequence of 136 bp; preferably, the portion of exon 4 comprises at least from the nucleotide sequence encoding amino acids 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) from the C-terminal end of the extracellular region of SIGLEC15 protein to the last nucleotide sequence of exon 4, preferably from the nucleotide sequence encoding amino acids 17 from the C-terminal end of the extracellular region of SIGLEC15 protein to the last nucleotide sequence of exon 4.

In a specific embodiment of the invention, the humanized SIGLEC15 gene comprises at least the nucleotide sequence set forth in SEQ ID NO. 6, 7, 8, or a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO. 6, 7, 8.

In one embodiment of the invention, the mRNA transcribed from the humanized SIGLEC15 gene comprises any one of the following groups:

(a) Comprises all or part of the nucleotide sequence shown in SEQ ID NO. 9;

(b) A nucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence set forth in SEQ ID No. 9;

(c) A nucleotide sequence comprising NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO 9; or (b)

(d) Comprising the nucleotide sequence set forth in SEQ ID NO. 9, including substitutions, deletions and/or insertions of one or more nucleotides.

Preferably, the humanized SIGLEC15 gene further comprises a specific inducer or repressor, more preferably, the specific inducer or repressor may be a conventionally inducible or repressible substance.

In one embodiment of the invention, the specific inducer is selected from the group consisting of the tetracycline System (Tet-Off System/Tet-On System) and the Tamoxifen System (Tamoxifen System).

Preferably, the non-human animal is an immunodeficient non-human mammal, further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey, more preferably, the immunodeficient rodent is an immunodeficient mouse or rat, still further preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a third aspect of the invention, a targeting vector is provided, said targeting vector comprising a portion of the human SIGLEC15 gene.

Preferably, the part of the human SIGLEC15 gene comprises all or part of exons No. 2 to 4 of the human SIGLEC15 gene, further preferably comprises all or part of any one, two, three, two or three consecutive exons from No. 2 to 4 of the human SIGLEC15 gene, still further preferably comprises all or part of exons No. 2, all of exons No. 3 and part of exons No. 4, further preferably further comprises introns No. 2 to 3 and/or introns No. 3 to 4, still further preferably comprises any one of introns No. 2 to 4, wherein part of exons No. 2 comprises at least a nucleotide sequence of 5bp, such as at least a nucleotide sequence of 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60bp, further preferably comprises a nucleotide sequence of 22 bp; preferably, the portion of exon 2 comprises at least the nucleotide sequence encoding amino acids 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) from the N-terminus of the extracellular region of human SIGLEC15 to the last nucleotide sequence of exon 2, preferably, from the nucleotide sequence encoding amino acids 12 from the N-terminus of the extracellular region of human SIGLEC15, said portion of exon 4 comprises at least a 100bp nucleotide sequence, e.g., at least a 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 378bp nucleotide sequence, further preferably, 242bp nucleotide sequence; preferably, the portion of exon 4 comprises at least a nucleotide sequence from the first nucleotide of exon 4 to 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids encoding the extracellular region of human SIGLEC15 protein, more preferably, the targeting vector comprises a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO:5 or comprises a nucleotide sequence as set forth in SEQ ID NO: 5.

Preferably, the targeting vector comprises the humanized SIGLEC15 gene described above.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 5 'end of the transition region to be altered, i.e. a 5' arm, selected from 100-10000 nucleotides in length of the non-human animal SIGLEC15 genomic DNA; preferably, the 5' arm has at least 90% homology to NCBI accession nc_ 000084.6; further preferred, the 5' arm sequence has at least 90% homology with SEQ ID NO. 3 or 11, or is as shown in SEQ ID NO. 3 or 11.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 3 'end of the switching region to be altered, i.e. a 3' arm, selected from 100-10000 nucleotides in length of the non-human animal SIGLEC15 genomic DNA; preferably, the 3' arm has at least 90% homology to NCBI accession nc_ 000084.6; further preferably, the 3' arm sequence has at least 90% homology with SEQ ID NO. 4 or 12, or is shown as SEQ ID NO. 4 or 12.

Preferably, the targeting vector further comprises the nucleotide sequence shown as SEQ ID NO. 6, 7, 8, or comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO. 6, 7, 8.

Preferably, the transition region to be changed is located at the non-human animal SIGLEC15 locus, and more preferably, the transition region to be changed is located at exons 2 to 4 of the non-human animal SIGLEC15 locus.

Preferably, the targeting vector is used for construction of a non-human animal humanized with the SIGLEC15 gene.

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 marker gene encoding a diphtheria toxin A subunit (DTA).

In a specific embodiment of the present invention, the targeting vector further includes a resistance gene selected from positive clones, and more preferably, the resistance gene selected from positive clones 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 (a conventional LoxP recombination system may be selected), and the specific recombination system has two Frt recombination sites, and preferably, the specific recombination system is connected to both sides of the resistance gene in the same direction.

In a fourth aspect of the invention there is provided an sgRNA which targets the non-human animal SIGLEC15 gene, with the sequence of the sgRNA being on the target sequence on the SIGLEC15 gene to be altered.

Preferably, the target site of the sgRNA is located on the sequence from exon 1 to exon 6 of the SIGLEC15 gene. Further preferably, the 5 '-end target site of the sgRNA is located on the exon 2 sequence of the SIGLEC15 gene, and more preferably, the 3' -end target site of the sgRNA is located on the exon 4 sequence of the SIGLEC15 gene.

Preferably, the 5' -end target site sequence of the sgRNA is shown in any one of SEQ ID NO 13-20.

Preferably, the 3' -end target site sequence of the sgRNA is shown in any one of SEQ ID NOs 21-28.

In a fifth aspect of the invention there is provided a DNA molecule encoding the sgRNA described above. Preferably, the DNA molecule double strand is respectively an upstream sequence and a downstream sequence of the sgRNA, or a forward oligonucleotide sequence and a reverse oligonucleotide sequence after an enzyme cutting site is added.

In one embodiment of the invention, the DNA molecule double strand is SEQ ID NO. 29 and SEQ ID NO. 31, SEQ ID NO. 30 and SEQ ID NO. 32, respectively.

In one embodiment of the invention, the DNA molecule double strand is SEQ ID NO 33 and SEQ ID NO 35, SEQ ID NO 34 and SEQ ID NO 36, respectively.

In a sixth aspect of the invention there is provided an sgRNA vector comprising the above-described sgRNA.

In a seventh aspect of the invention, there is provided a cell comprising the targeting vector described above, the sgRNA described above, the DNA molecule described above or the sgRNA vector described above.

In an eighth aspect of the invention, there is provided the use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector or the cell described above for the modification of the SIGLEC15 gene. Preferably comprises the use in knockout, insertion or replacement of the SIGLEC15 gene.

In a ninth aspect of the present invention, there is provided a non-human animal humanized with a SIGLEC15 gene, said non-human animal being:

(1) Expressing a human or humanized SIGLEC15 protein; and/or the number of the groups of groups,

(2) Comprising the human or humanized SIGLEC15 gene.

Preferably, the non-human animal has reduced or absent expression of endogenous SIGLEC15 protein.

Preferably, the humanized SIGLEC15 protein is expressed in the non-human animal.

Preferably, the non-human animal body comprises a part of the human SIGLEC15 gene, and more preferably, the non-human animal body comprises the humanized SIGLEC15 gene.

Preferably, a portion of the human SIGLEC15 gene or the humanized SIGLEC15 gene is operably linked to an endogenous regulatory element.

According to some embodiments of the invention, the non-human animal further comprises additional genetic modifications selected from at least one of PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40, and IL 10.

According to some embodiments of the invention, the human or humanized SIGLEC15 gene and/or the other gene is homozygous or heterozygous for the endogenous modified locus.

Preferably, the non-human animal is an immunodeficient non-human mammal, further preferably, the immunodeficient non-humanThe mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey, more preferably the immunodeficient rodent is an immunodeficient mouse or rat, even more preferably the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a tenth aspect of the present invention, a method for constructing a non-human animal humanized with SIGLEC15 gene, wherein the non-human animal expresses human or humanized SIGLEC15 protein in vivo.

Preferably, the non-human animal genome comprises all or part of the human or humanized SIGLEC15 gene.

Preferably, the genome of the non-human animal comprises the humanized SIGLEC15 gene.

Preferably, the humanized SIGLEC15 protein is a humanized SIGLEC15 protein as described above.

Preferably, the non-human animal is selected from the non-human animals humanized with the SIGLEC15 gene described above.

The human or humanized SIGLEC15 gene is regulated in a non-human animal by regulatory elements including, but not limited to, promoters. The regulatory element may be endogenous or exogenous. For example, the exogenous regulatory element may be derived from the human SIGLEC15 gene.

Preferably, the humanized SIGLEC15 gene is regulated in a non-human animal by endogenous regulatory elements. Further preferably, the regulatory element comprises a promoter.

Preferably, the construction method comprises introducing all or part of the human SIGLEC15 gene from exon 1 to exon 6 into the non-human animal SIGLEC15 locus; further preferably, the non-human animal SIGLEC15 locus is introduced with all or part of the nucleotide sequence comprising exons No. 2 to 4 of the human SIGLEC15 gene, more preferably with all or part of a combination comprising any one, two, three, consecutive two or consecutive three exons of exons No. 2 to 4, wherein the part of the exons No. 2 comprises at least a nucleotide sequence of 5bp, e.g. at least a nucleotide sequence of 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 40, 45, 60, further preferably a nucleotide sequence of 5bp, 9, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 40, 45, 60, further preferably with all or part of exons No. 2 to 4 of the human SIGLEC15 gene, more preferably also comprising introns No. 2 to 3 and/or No. 3 to 4, even more preferably with any one of the introns between exons No. 2 to 4; preferably, the portion of exon 2 comprises at least the nucleotide sequence encoding amino acids 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) from the N-terminus of the extracellular region of human SIGLEC15 to the last nucleotide sequence of exon 2, preferably, from the nucleotide sequence encoding amino acids 12 from the N-terminus of the extracellular region of human SIGLEC15, said portion of exon 4 comprises at least a 100bp nucleotide sequence, e.g., at least a 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 378bp nucleotide sequence, further preferably, 242bp nucleotide sequence; preferably, the portion of exon 4 comprises at least a nucleotide sequence from nucleotide 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from nucleotide 1 to nucleotide 18 encoding the extracellular domain of human SIGLEC15 protein, more preferably, a nucleotide sequence comprising at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO:5 or a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO:5 is used to introduce into the non-human animal SIGLEC15 locus.

Preferably, the introduction described herein includes, but is not limited to, insertion, substitution or transgene, and the substitution is preferably in situ.

Preferably, the human or humanized SIGLEC15 gene is operably linked to endogenous regulatory elements of the endogenous SIGLEC15 gene on at least one chromosome.

In one embodiment of the invention, the endogenous regulatory element is from a non-human animal SIGLEC15 gene.

Preferably, the site of introduction is located after the endogenous regulatory element of the SIGLEC15 gene.

Preferably, the introducing is a substitution or insertion, and in one embodiment of the present invention, the non-human animal SIGLEC15 locus is a substitution of the corresponding region of the non-human animal, further preferably, all or part of exons No. 2 to No. 4 of the non-human animal SIGLEC15 gene is substituted, still further preferably, all or part of exons No. 2, no. 3 and No. 4 of the non-human animal SIGLEC15 gene are substituted.

Preferably, the sequence encoding SEQ ID NO:1, the nucleotide sequence of the amino acid shown at positions 31-245 of 1 is replaced.

In some embodiments, the nucleic acid encoding SEQ ID NO:1 or 27-257.

Preferably, the construction method comprises introducing into a non-human animal SIGLEC15 locus with all or part of a nucleotide sequence comprising a signal peptide encoding a human SIGLEC15 protein, a transmembrane region, a cytoplasmic region and/or an extracellular region; it is further preferred that all or part of the nucleotide sequence comprising an extracellular region encoding a human SIGLEC15 protein is introduced into a non-human animal SIGLEC15 locus, preferably that a non-human animal SIGLEC15 locus is introduced with at least 50 consecutive amino acids comprising an extracellular region encoding a human SIGLEC15 protein, for example that a human SIGLEC15 protein extracellular region comprising at least 50, 70, 90, 100, 150, 197, 200, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 223, 230, 233, 240, 244 consecutive amino acids is introduced into a non-human animal SIGLEC15 locus, further preferred that a human SIGLEC15 protein extracellular region comprising 197, 223, 216 or 233 consecutive amino acids is introduced; the extracellular region comprises all or a portion of the extracellular region of a human SIGLEC15 protein, preferably, the extracellular region of a human SIGLEC15 protein comprising an N-terminal removal of 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and/or a C-terminal removal of 0-50 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 47, 50), more preferably, the extracellular region of a human SIGLEC15 protein comprising an N-terminal removal of 0, 7, 9 or 11 amino acids and a C-terminal removal of 4, 12, 47 or 17 amino acids, more preferably, an amino acid sequence comprising at least 85%, 90%, 95% or at least 99% identity to positions 31-246 of SEQ ID NO 2 or a non-human SIGLEC15 gene comprising amino acid sequence identical to positions 31-246 of SEQ ID NO 2 is introduced.

Preferably, the construction method comprises introducing a non-human animal SIGLEC15 locus with a nucleotide sequence comprising the humanized SIGLEC15 gene.

Preferably, the construction method comprises introducing a non-human animal SIGLEC15 locus with a nucleic acid sequence comprising a sequence encoding the humanized SIGLEC15 protein.

Preferably, the insertion or substitution site is subsequent to the endogenous regulatory element of the SIGLEC15 gene.

Preferably, the insertion is a first disruption of the coding box of the endogenous SIGLEC15 gene of the non-human animal, followed by an insertion procedure, or the insertion step may be a step of both causing a frame shift mutation in the endogenous SIGLEC15 gene and effecting insertion of a human sequence.

Preferably, the humanized SIGLEC15 gene is contained on at least one chromosome in the genome of said non-human animal.

Preferably, at least one cell in the non-human animal expresses a human SIGLEC15 protein or a humanized SIGLEC15 protein.

Preferably, the construction of the non-human animal is performed using gene editing techniques including gene targeting techniques using embryonic stem cells, CRISPR/Cas9 techniques, zinc finger nuclease techniques, transcription activator-like effector nuclease techniques, homing endonucleases or other molecular biology techniques.

Preferably, the construction of the non-human animal is performed using the targeting vector described above.

Preferably, to increase recombination efficiency, construction of non-human animals can also be performed using sgrnas targeting SIGLEC15 genes along with the targeting vectors described above. Wherein the sgRNA targets the non-human animal SIGLEC15 gene while the sequence of the sgRNA is on the target sequence on the SIGLEC15 gene to be altered.

Preferably, the 5' -end target site of the sgRNA is located on exon 2 of the SIGLEC15 gene.

Preferably, the 3' -end target site of the sgRNA is located on exon 4 of the SIGLEC15 gene.

In one specific embodiment of the present invention, the construction method comprises introducing the targeting vector, sgRNA targeting SIGLEC15 gene and Cas9 into a non-human animal cell, culturing the cell (preferably fertilized egg), transplanting the cultured cell into oviduct of female non-human animal, allowing it to develop, and identifying and screening the non-human animal humanized with the SIGLEC15 gene.

According to some embodiments of the invention, the method of constructing further comprises: mating the SIGLEC15 gene humanized non-human animal with other non-human animals modified by genes, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-gene modified non-human animal.

Preferably, the other gene is a non-human animal genetically modified with at least one of PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40 and IL 10.

Preferably, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40, and IL10 proteins.

Preferably, each of the plurality of genes modified in the genome of the polygenously modified non-human animal is homozygous or heterozygous for the endogenous replaced locus.

In an eleventh aspect of the present invention, there is provided a non-human animal in which the SIGLEC15 gene is knocked out, the non-human animal lacking all or part of the nucleotide sequence of the SIGLEC15 gene.

Preferably, the non-human animal lacks all or part of exons 2 to 4 of the SIGLEC15 gene, further preferably, lacks all or part of the nucleotide sequence of any one, two, three, two consecutive or three consecutive exons of exons 2 to 4, preferably, lacks all or part of exons 2, 3 and part of exons 4, preferably also lacks introns 2 to 3 and/or introns 3 to 4, further preferably, lacks any of the introns 2 to 4, wherein the deleted part of exons 2 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, further preferably, the deleted part of exons 2 comprises a nucleotide sequence of 22 bp; preferably, the deleted portion of exon 2 comprises at least the last nucleotide sequence from the nucleotide sequence encoding 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular region of the non-human animal SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably the last nucleotide sequence from the nucleotide sequence encoding 7 amino acids from the N-terminus of the extracellular region of the non-human animal SIGLEC15 protein to the last nucleotide sequence of exon 2; preferably, the portion of the deleted exon 4 comprises at least a nucleotide sequence of 100bp, e.g., at least a nucleotide sequence of 100, 150, 200, 205, 210, 220, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 250, 300, 310, 320, 330, 340, 350, 360, 370, 375bp, further preferably, a nucleotide sequence of 239 bp; preferably, the portion of exon 4 deleted comprises at least the nucleotide sequence from nucleotide 1 to nucleotide 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) from the first nucleotide of exon 4 to the C-terminus of the extracellular region of the SIGLEC15 protein, preferably, the nucleotide sequence from nucleotide 1 to nucleotide 18 from the C-terminus of the extracellular region of the SIGLEC15 protein.

Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Still more preferably, the rodent is a rat or mouse.

Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferred, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a twelfth aspect of the invention, a method for constructing a non-human animal with SIGLEC15 gene knockout is provided, wherein sgRNA is used for constructing the non-human animal. Wherein the sgRNA targets a non-human animal SIGLEC15 gene, and the sequence of the sgRNA is unique on the target sequence on the SIGLEC15 gene to be altered.

Preferably, the target site of the sgRNA is located on the sequence from exon 1 to exon 6 of the SIGLEC15 gene. Further preferably, the 5 '-end target site of the sgRNA is located on the exon 2 sequence of the SIGLEC10 gene, and more preferably, the 3' -end target site of the sgRNA is located on the exon 4 sequence of the SIGLEC10 gene.

In a thirteenth aspect of the present invention, there is provided a method of constructing a polygenically modified non-human animal comprising the steps of:

i) Providing the non-human animal with the SIGLEC15 gene humanized or the non-human animal with the SIGLEC15 gene knocked out, or the non-human animal with the SIGLEC15 gene humanized obtained by the construction method;

II) mating the non-human animal provided in the step I) with other non-human animals modified by genes, performing in vitro fertilization or directly performing gene editing, and screening to obtain the non-human animal modified by multiple genes.

Preferably, the other genetically modified non-human animals include, but are not limited to, non-human animals modified by the genes PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40, and IL 10.

Preferably, the polygene modified non-human animal is a double-gene humanized non-human animal, a triple-gene humanized non-human animal, a four-gene humanized non-human animal or a five-gene humanized non-human animal.

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

In a fourteenth aspect of the present invention, there is provided a non-human animal or a progeny thereof obtained by the above construction method, wherein the non-human animal or the progeny thereof is selected from the group consisting of a non-human animal humanized with SIGLEC15 gene, a non-human animal knocked out with SIGLEC15 gene, and a non-human animal modified with multiple genes.

In a fifteenth aspect of the present invention, there is provided an animal model of a disease, said animal model being derived from the above-described non-human animal, the above-described non-human animal obtained by the above-described construction method, or, preferably, the above-described non-human animal or progeny thereof, said disease comprising an autoimmune disease, a tumor or an inflammation.

In a sixteenth aspect of the present invention, there is provided a method for preparing an animal model of a disease, the method comprising the steps of humanizing the SIGLEC15 gene, a SIGLEC15 knockout non-human animal, or a polygenic modified non-human animal; preferably, the disease comprises an autoimmune disease, a tumor or an inflammation, further preferably, the method further comprises the step of implanting tumor cells.

In a seventeenth aspect of the present invention, there is provided the use of the above-described SIGLEC15 gene-humanized non-human animal, the above-described SIGLEC15 gene-knocked-out non-human animal, the above-described SIGLEC15 gene-humanized non-human animal obtained by the above-described construction method, the above-described SIGLEC15 gene-knocked-out non-human animal or a polygenic modified non-human animal or a progeny thereof in the preparation of an animal model of a disease, preferably, the disease comprises an autoimmune disease, a tumor or an inflammation.

In an eighteenth aspect, the present invention provides the use of the above-described non-human animal, the above-described non-human animal or progeny thereof, the above-described non-human animal obtained by the above-described construction method or the above-described animal model for the preparation of a medicament for the treatment of autoimmune diseases, tumors and/or inflammations.

In a nineteenth aspect of the present invention, there is provided a cell or cell line or primary cell culture derived from the above-described non-human animal, the non-human animal obtained by the above-described construction method, the above-described non-human animal or progeny thereof, or the above-described animal model. Preferably, the cell or cell line or primary cell culture is incapable of developing into an animal subject.

In a twentieth aspect of the present invention, there is provided a tissue or organ or a culture thereof derived from the above-described non-human animal, the above-described non-human animal obtained by the above-described construction method, the above-described non-human animal or a progeny thereof or an animal model thereof, wherein the tissue is a neoplastic tissue. Preferably, the tissue or organ or culture thereof is incapable of developing into an animal subject.

In a twenty-first aspect of the present invention, there is provided a tumor tissue after tumor-bearing, said tumor tissue comprising the above-described humanized SIGLEC15 protein or the above-described humanized SIGLEC15 gene. Preferably, the tumor tissue after tumor-bearing cannot develop into an animal individual.

Preferably, the tumor tissue is derived from the non-human animal, the non-human animal obtained by the above construction method, the non-human animal or its progeny, or the disease model.

In a twenty-second aspect of the present invention, there is provided a cell humanized with a SIGLEC15 gene, the cell expressing a human SIGLEC15 protein or a humanized SIGLEC15 protein.

Preferably, the humanized SIGLEC15 protein is selected from the humanized SIGLEC15 proteins described above.

Preferably, the expression of the endogenous SIGLEC15 protein is reduced or absent in said cell.

Preferably, the genome of the cell comprises a portion of the human SIGLEC15 gene, and more preferably, the cell comprises the humanized SIGLEC15 gene. Preferably, the cells are unable to develop into an individual animal.

In a twenty-third aspect of the present invention, there is provided a SIGLEC15 gene knockout cell in which all or part of the nucleotide sequence of the SIGLEC15 gene is deleted.

Preferably, the cell lacks all or part of the nucleotide sequence of exons 2 to 4 of the SIGLEC15 gene, further preferably, lacks all or part of the nucleotide sequence of any one, two, three, two consecutive or three consecutive exons of exons 2 to 4, preferably, lacks all or part of exons 2, 3 and 4, preferably also lacks introns 2 to 3 and/or introns 3 to 4, further preferably, lacks any one of the introns 2 to 4, wherein the deleted part of exons 2 comprises at least a nucleotide sequence of 10bp, for example at least a nucleotide sequence of 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, further preferably, the deleted part of exons 2 comprises a nucleotide sequence of 22 bp; preferably, the deleted portion of exon 2 comprises at least a nucleotide sequence of 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular region of the non-human animal SIGLEC15 protein, preferably from the nucleotide sequence of 7 amino acids from the N-terminus of the extracellular region of the non-human animal SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably, the deleted portion of exon 4 comprises at least a nucleotide sequence of 100bp, e.g., at least a nucleotide sequence of 100, 150, 200, 205, 210, 220, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 250, 300, 310, 320, 330, 340, 350, 360, 370, 375bp, further preferably, 239 bp; preferably, the portion of exon 4 deleted comprises at least the nucleotide sequence from nucleotide 1 to nucleotide 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) from the first nucleotide of exon 4 to the C-terminus of the extracellular region of the SIGLEC15 protein, preferably, the nucleotide sequence from nucleotide 1 to nucleotide 18 from the C-terminus of the extracellular region of the SIGLEC15 protein.

Preferably, the SIGLEC15 gene-deleted cells are prepared using the sgrnas described above that target the SIGLEC15 gene. Preferably, the cells are unable to develop into an individual animal.

In a twenty-fourth aspect of the present invention, there is provided a construct expressing the above-described humanized SIGLEC15 protein, preferably, the construct comprising the above-described humanized SIGLEC15 gene.

In a twenty-fifth aspect of the invention, there is provided a cell comprising the construct described above. Preferably, the cells are unable to develop into an individual animal.

In a twenty-sixth aspect of the invention, there is provided a tissue comprising the above-described cells. Preferably, the tissue is unable to develop into an individual animal.

In a twenty-seventh aspect of the present invention, there is provided the use of a humanized SIGLEC15 protein derived from the above, a humanized SIGLEC15 gene as described above, a non-human animal as described above or a progeny thereof, an animal model as described above, a cell or cell line as described above or a primary cell culture, a tissue as described above or organ or a culture thereof, a tumor tissue after tumor bearing as described above, a cell as described above, a construct as described above, a cell as described above or a tissue as described above, which comprises:

The application in the development of products requiring immune processes involving human cells, the manufacture of antibodies, or as model systems for pharmacological, immunological, microbiological, medical research;

the application in the production and utilization of animal experimental disease models for the development of new diagnostic and/or therapeutic strategies;

or,

the application in screening, verifying, evaluating or researching SIGLEC15 function, human SIGLEC15 signaling mechanism, human targeting antibody, human targeting drug, drug effect, immune related disease drug and anti-tumor or anti-inflammatory drug, screening and evaluating human drug and drug effect research. Preferably, the use is not a method of treatment and/or diagnosis of a disease.

In a twenty eighth aspect of the present invention, there is provided use of the above-described non-human animal humanized by SIGLEC15 gene, the above-described non-human animal humanized by SIGLEC15 gene knockout obtained by the above-described construction method, the above-described non-human animal humanized by SIGLEC15 gene knockout or a polygenic modified non-human animal or a progeny thereof in the preparation of a human SIGLEC15 specific modulator or a product for screening a human SIGLEC15 specific modulator. Preferably, the use is not a method of treatment and/or diagnosis of a disease.

In a twenty-ninth aspect of the present invention, there is provided a screening method of a human SIGLEC 15-specific modulator, the screening method comprising applying the modulator to an individual, and detecting the effect of the modulator; wherein the individual is selected from the group consisting of the above-described non-human animal, the above-described non-human animal obtained by the above-described construction method, the above-described non-human animal or progeny thereof, or the above-described animal model.

Preferably, the modulator is selected from CAR-T, a drug, and more preferably, the drug is an antibody.

Preferably, the regulator is monoclonal antibody or bispecific antibody or the combination of two or more drugs.

Preferably, the screening method further comprises the step of implanting a tumor into the individual.

Preferably, the detection comprises determining the size and/or proliferation rate of the tumour cells.

Preferably, the method of detection comprises vernier caliper measurement, flow cytometry detection and/or animal live imaging detection.

Preferably, the detecting comprises assessing the weight, fat mass, activation pathway, neuroprotective activity, or metabolic change in the subject, including a change in food consumption or water consumption.

Preferably, the tumor cells are derived from a human or non-human animal.

Preferably, the method of screening for a human SIGLEC 15-specific modulator is not a therapeutic method. The method is used for screening or evaluating medicines, detecting and comparing the medicine effects of candidate medicines to determine which candidate medicines can be taken as medicines and which can not be taken as medicines, or comparing the medicine effect sensitivity degree of different medicines, namely that the treatment effect is not necessarily the same, but is only one possibility.

In a thirty-first aspect of the present invention, there is provided a method of evaluating an intervention program, the method comprising applying the intervention program to an individual, and detecting and evaluating the effect of modulation on the individual after applying the intervention program; wherein the individual is selected from the non-human animals, the non-human animals obtained by the construction method, the non-human animals or their offspring, or the animal model.

Preferably, the evaluation method further comprises implanting tumor cells into the individual.

Preferably, the intervention regimen is selected from CAR-T, drug therapy, further preferably, the drug is an antigen binding protein and the antibody binding protein is an antibody.

Preferably, the tumor cells are derived from a human or non-human animal.

Preferably, the method of evaluation of the intervention program is not a therapeutic method, and the method of evaluation detects and evaluates the effect of the intervention program to determine whether the intervention program has a therapeutic effect, i.e. the therapeutic effect is not necessarily, but is only one possibility.

In a thirty-first aspect of the present invention, there is provided the use of a non-human animal derived from the above described non-human animal, the above described non-human animal obtained by the above described construction method, the above described non-human animal or a progeny or disease model thereof, in the manufacture of a medicament for the treatment of a tumor, an inflammatory or an autoimmune disease.

"replacement" as used herein refers to the placement of exogenous genetic material at an endogenous locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. In one example, endogenous non-human genes or fragments thereof are replaced with corresponding human genes or fragments thereof. The corresponding human gene or fragment thereof is an ortholog or homolog of the endogenous non-human gene or fragment thereof that is replaced, or is substantially identical or identical in structure and/or function to the endogenous non-human gene or fragment thereof that is replaced. In another embodiment, gene replacement can occur when an endogenous gene is deleted or rendered non-functional (such as by insertion of a missense mutation or a premature stop codon) and the corresponding human gene or fragment thereof is inserted into the germline at a separate location.

The term "transgene" as used herein refers to the insertion of exogenous genetic material into the genome of a cell by manual intervention, such as by microinjection or by direct or indirect introduction into a precursor cell by infection with a recombinant virus, which induces a genetic change in the cell upon incorporation of the exogenous genetic material, wherein the vectors used to stabilize integration during the process include: plasmids, retroviral vectors, and other animal viruses, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and the like.

"tumor" as used herein includes, but is not limited to, lymphoma, B cell tumor, T cell tumor, bone marrow/monocyte tumor, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcomas. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the 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 sarcoma, smooth muscle sarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the invention, the tumor is selected from the group consisting of B cell tumor, T cell tumor, bone marrow/monocyte tumor. Preferably includes B or T cell Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), non-Hodgkin's lymphoma (NHL) and Multiple Myeloma (MM), nasopharyngeal carcinoma, lung cancer.

The "autoimmune diseases" described herein include, but are not limited to, allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, and the like.

"inflammation" as used herein includes acute inflammation as well as chronic inflammation. In particular, including but not limited to, degenerative inflammation, exudative inflammation (serositis, cellulitis, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, jatropha, lymphogranuloma, etc.).

Preferably, the presently claimed subject matter "cell", "cell or cell line or primary cell culture", "tissue or organ or culture thereof" is not capable of developing into an animal, wherein the cell is not a stem cell or fertilized egg cell, the cell may be a somatic cell, a lymphocyte (preferably a T cell or B cell), a tumor cell, or the like, and the tissue may be a spleen, lymph node, bone marrow, tumor or culture thereof, or the like.

The SIGLEC15 gene humanized non-human animal body can normally express human SIGLEC15 protein or humanized SIGLEC15 protein. Can be used for drug screening, drug effect evaluation, cardiovascular and cerebrovascular diseases, neurological diseases, autoimmune diseases and tumor treatment aiming at human SIGLEC15 target sites, and can accelerate the development process of new drugs and save time and cost. Provides effective guarantee for researching SIGLEC15 protein function and screening related disease drugs.

The invention relates to all or part of the whole, the whole is the whole, the part is the part of the whole or the whole individual.

The "humanized SIGLEC15 protein" described herein comprises a portion derived from a human SIGLEC15 protein. Wherein, the human SIGLEC15 protein is identical to the whole human SIGLEC15 protein, i.e. the amino acid sequence of the human SIGLEC15 protein is identical to the full-length amino acid sequence of the human SIGLEC15 protein. The "part of human SIGLEC15 protein" is that 5-328 (preferably 10-216) amino acid sequences which are continuous or spaced are identical with the amino acid sequence of human SIGLEC15 protein or have more than 70% homology with the amino acid sequence of human SIGLEC15 protein.

The "humanized SIGLEC15 gene" of the present invention includes a part derived from a human SIGLEC15 gene and a part derived from a non-human SIGLEC15 gene. Wherein, the human SIGLEC15 gene is identical to the whole human SIGLEC15 gene, i.e. the nucleotide sequence is identical to the full-length nucleotide sequence of the human SIGLEC15 gene. The part of the human SIGLEC15 gene is continuous or interval 20-18420bp (preferably 20-2948bp or 20-1893 bp) nucleotide sequence which is identical with the human SIGLEC15 gene or has more than 70% homology with the human SIGLEC15 gene.

The "xx-to-xxx exons" or the "all of xx-to-xxx exons" described herein include exons and nucleotide sequences of introns therebetween, e.g., the "exon 2-to-3" described herein includes all nucleotide sequences of exon 2, intron 2-3, and exon 3.

The "x-xx number intron" as used herein means an intron between the x-exon and the xx number exon. For example, "intron No. 2-3" means an intron between exon No. 2 and exon No. 3.

"part of an exon" as used herein means that several, tens or hundreds of nucleotide sequences are identical to all exon nucleotide sequences, either consecutively or at intervals. For example, the part of the exon 2 of the human SIGLEC15 gene comprises 5-60bp, preferably 10-22bp, nucleotide sequences that are identical to the nucleotide sequence of the exon 2 of the human SIGLEC15 gene, either consecutively or at intervals. In one embodiment of the present invention, the "portion of exon 2" contained in the "humanized SIGLEC15 gene" includes at least the start codon and the stop codon of the amino acid sequence encoded by exon 2.

The "locus" as used herein refers broadly to the location of a gene on a chromosome, and in a narrow sense to a DNA fragment on a gene, either a gene or a portion of a gene. For example, the "SIGLEC15 locus" means a DNA fragment of an optional stretch on exons 1 to 6 of the SIGLEC15 gene. Preferably any one or a combination of two or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6 or an intron therebetween, or all or part of one or two or more, more preferably on exons 2 to 4 of the SIGLEC15 gene.

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

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

"homology" as used herein means that a person skilled in the art, while using an amino acid sequence or a nucleotide sequence, can adjust the sequence according to actual work requirements on the premise of ensuring a structure or function similar to that of a known sequence, and the sequence used 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%, 96%,97%, 99.99.99.99.99%, 99.7%, 99.99.99% and 99.99.99% as compared with the sequence obtained in the prior art.

In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, e.g. a murine. In one embodiment, the non-human 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 a murine family. In one embodiment, the genetically modified animal is from a family selected from the group consisting of the hamsidae (e.g., hamster-like), hamsidae (e.g., hamster, new world rats and mice, voles), murine superfamily (true mice and rats, gerbils, chinchilla, coronarism), equine island murine (mountain climbing mice, rock mice, tailed rats, motor gas rats and mice), spiny murinaceae (e.g., spiny muzzle) and mole murinaceae (e.g., mole rats, bamboo rats and zokoros). In a particular embodiment, the genetically modified rodent is selected from the group consisting of a true mouse or rat (murine superfamily), a gerbil, a spiny mouse, and a coronal 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, which is a mouse selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strain mice and NOD, NOD/SCID, NOD-Prkcid IL-2 rgb background.

One skilled in the art can determine and compare sequence elements or degrees of identity to distinguish 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,2ndEd., by Sambrook, fritschand Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, volumes I and II (D.N.Glcovered., 1985); oligonucleotide Synthesis (m.j. Gaited., 1984); mullisetal, u.s. Pat. No.4, 683, 195; nucleic Acid Hybridization (B.D.Hames & S.J.Higginseds.1984); transcription And Translation (B.D.Hames & S.J.Higginseds.1984); culture Of Animal Cells (R.I.Freshney, alanR.Liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); perbal, A Practical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m. Simon, eds. Inch, academic Press, inc., new York), special, vols.154and 155 (wuetal. Eds.) and vol.185, "Gene Expression Technology" (d. Goeddel, ed.); gene Transfer Vectors For Mammalian 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 illustrative of some aspects of the present invention and is not, nor should it be construed as limiting the invention in any respect.

All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto 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 present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: schematic of the comparison of human and mouse SIGLEC15 loci (not to scale);

fig. 2: schematic representation (not to scale) of the humanized SIGLEC15 locus;

fig. 3: SIGLEC15 gene targeting strategy schematic one (not to scale);

fig. 4: SIGLEC15 gene targeting strategy schematic two (not to scale);

fig. 5: the sgRNA1-sgRNA16 activity detection result, wherein Con is a negative control, and PC is a positive control;

fig. 6: genotyping results in F0 mice, wherein WT is wild type control, H ₂ O is water contrast, F0-01, F0-02, F0-03, F0-04, F0-05 are mouse numbers;

Fig. 7: genotyping results in F1 mice, wherein WT is wild type control, H ₂ O is water control, PC is positive control, F1-01, F1-02, F1-03, F1-04 are mouse numbers;

fig. 8: the detection result of Southern Blot of F1 generation, wherein WT is wild type control, and F1-01, F1-02, F1-03, F1-04, F1-05, F1-06 and F1-07 are mouse numbers;

fig. 9: detection result of humanized SIGLEC15 mRNA, wherein +/+ is wild type C57BL/6 mice, H/H is SIGLEC15 gene humanized homozygote mice, H ₂ O is water control;

fig. 10: SIGLEC15 gene knockout mouse genotype identification result, wherein WT is wild type control, H ₂ O is water control, KO-01 is mouse number;

fig. 11: SIGLEC15 protein flow detection result, wherein ISO is isotype control, A, B, C is SIGLEC15 gene humanized mouse detection result, D, E, F is SIGLEC15 gene knockout mouse detection result, G, H, I is wild type C57BL/6 mouse detection result;

fig. 12: mice homozygous for the SIGLEC15 gene were subcutaneously inoculated with MC38, which overexpressed the human SIGLEC15 protein, and given anti-human SIGLEC15 antibody AB1 or anti-murine PD-1 antibody AB2, as well as weight measurements of each group of mice during the experimental period;

Fig. 13: mice with humanized homozygotes of the SIGLEC15 gene are subcutaneously inoculated with mouse colon cancer cells MC38 over-expressing human SIGLEC15 protein, and given with anti-human SIGLEC15 antibody AB1 or anti-mouse PD-1 antibody AB2, and the tumor volume of each group of mice is measured during experimental period;

fig. 14: mice with humanized homozygotes of the SIGLEC15 gene are subcutaneously inoculated with MC38, which is a mouse colon cancer cell over-expressing the human SIGLEC15 protein, and given anti-human SIGLEC15 antibody Ab1 or Ab2, and the weight of each group of mice is measured during the experimental period;

fig. 15: the humanized homozygous mice of the SIGLEC15 gene are subcutaneously inoculated with a mouse colon cancer cell MC38 over-expressing the human SIGLEC15 protein, and an anti-human SIGLEC15 antibody Ab1 or Ab2 is given, and the body weight measurement results of the mice in each group in the experimental period are obtained;

fig. 16: flow detection results of leukocyte subfraction ratio in spleen of C57BL/6 wild mice and SIGLEC15 gene humanized homozygote mice;

fig. 17: flow detection results of T cell subfraction ratio in spleen of C57BL/6 wild mice and SIGLEC15 gene humanized homozygote mice;

fig. 18: flow detection results of leukocyte subpopulations in lymph nodes of C57BL/6 wild type mice and SIGLEC15 gene humanized homozygote mice;

Fig. 19: flow detection results of T cell subset ratio in lymph nodes of C57BL/6 wild mice and SIGLEC15 gene humanized homozygote mice;

fig. 20: flow detection results of leukocyte subfraction ratio in blood of C57BL/6 wild mice and SIGLEC15 gene humanized homozygote mice;

fig. 21: flow detection results of T cell sub-population ratio in blood of C57BL/6 wild mice and SIGLEC15 gene humanized homozygote mice;

fig. 22: flow assay strategy diagram, wherein diagram a is a T cell, B cell, NK cell, cd4+ T cell, cd8+ T cell, tregs cell flow assay strategy diagram, diagram B is a DC cell, granulocyte (Granulocyte), monocyte (Monocyte), macrophage (Macrophage) flow assay strategy diagram;

fig. 23: alignment between the mouse SIGLEC15 amino acid sequence (np_ 001094508.1;SEQ ID NO:1) and the human SIGLEC15 amino acid sequence (np_ 998767.1;SEQ ID NO:2).

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

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

BbsI, ecoRI, bamHI, bspHI, ecoNI enzyme was purchased from NEB under the accession numbers R0539S, R0101M, R0136M, R0517L, R0521L, respectively;

c57BL/6 mice and Flp tool mice were purchased from national rodent seed center of China food and drug inspection institute;

ambion in vitro transcription kit was purchased from Ambion under the trade designation AM1354;

cas9mRNA source SIGMA, cat No. Cas9mRNA-1EA;

UCA kit is from Baiocigram company, with the product number BCG-DX-001;

purified anti-mouse CD16/32 is purchased from bioleged under accession number 101302;

fixable Viability Dye eFluorTM506 available from eBioscience under the accession number 65-0866-14;

APC/Cy7 anti-mouse F4/80 was purchased from Biolegend, cat. No. 123118;

v450 Rat Anti-mouse CD11b is available from BD horizons under the accession number 560455;

anti-mouse MHC II (I-A/I-E) Super Bright 600 is available from eBioscience under the trade designation 63-5321-80;

Anti-Mo CD206 (MMR) eBioscience PE/Cyanine 7 available from eBioscience under the trade designation 25-2061-80;

Alexa647-conjugated AffiniPure F (ab') 2Fragment Goat Anti-Human IgG, fcgamma Fragment Specific from Jackson Immuno Research, cat# 109-606-170;

Recombinant Murine M-CSF is available from PeproTech under the accession number 315-02;

attune NxT Flow Cytometer available from Thermo Fisher, model Attune NxT

Brilliant Violet 510TManti-mouse CD45 is purchased from Biolegend under accession number 103138;

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) anti-body available from bioleged under the accession number 108426; brilliant Violet 421TManti-mouse CD4 is purchased from Biolegend under accession number 100438;

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

PE anti-mouse CD8a anti-body is available from Biolegend under accession number 100708;

PE/CyTM7Mouse anti-Mouse NK1.1 is available from BD Pharmingen under the accession number 552878;

APC anti-mouse/rate Foxp3 was purchased from eBioscience, cat# 17-5773-82;

FITC anti-Mouse CD19 from Biolegend, cat# 115506;

PerCP/Cy5.5 anti-mouse TCR beta chain was purchased from Biolegend under the accession number 109228.

EXAMPLE 1 preparation of SIGLEC15 Gene humanized mice

The comparison of the mouse SIGLEC15 Gene (NCBI Gene ID:620235,Primary source:MGI:3646642,UniProt ID:A7E1W8, located at positions 78042493 to 78057441 of chromosome 18 NC-000084.6, based on transcript NM-001101038.2 and its encoded protein NP-001094508.1 (SEQ ID NO: 1)) with the human SIGLEC15 Gene (NCBI Gene ID:284266,Primary source:HGNC:27596,UniProt ID:Q6ZMC9, located at positions 45825675 to 45844094 of chromosome 18 NC-000018.10, based on transcript NM-213602.3 and its encoded protein NP-998767.1 (SEQ ID NO: 2)) is shown in FIG. 1; an alignment of human murine protein sequences is shown schematically in FIG. 23.

For the purposes of the present invention, a nucleotide sequence encoding a human SIGLEC15 protein may be introduced at the endogenous SIGLEC15 locus in a mouse such that the mouse expresses the human or humanized SIGLEC15 protein. Specifically, the humanized modification of the mouse SIGLEC15 gene may be achieved by substituting the DNA sequence of the human SIGLEC15 gene at the mouse endogenous SIGLEC15 locus by gene editing techniques, such as substituting a sequence of about 1.9kb containing at least the mouse SIGLEC15 gene with the corresponding human DNA sequence to obtain a humanized SIGLEC15 locus (schematic diagram is shown in fig. 2).

The schematic diagram of the targeting strategy shown in FIG. 3 is further designed, wherein the targeting vector I contains a 5 'homology arm, a 3' homology arm and an A fragment containing the human SIGLEC15 gene sequence. Wherein, the 5' homology arm (SEQ ID NO: 3) and the N CBI accession number are identical to the 78049252-78053412 th nucleotide sequence of NC_ 000084.6; the 3' homology arm (SEQ ID NO: 4) is identical to the 78042522-78046612 nucleotide sequence of NCBI accession number and NC_ 000084.6; the sequence of the human SIGLEC15 gene (SEQ ID NO: 5) is identical to nucleotide 45837067-45838959 of NCBI accession NC_ 000018.10. The connection of the human SIGLEC15 sequence downstream of the A fragment to the mouse was designed to be 5' -ccgctacacgtgtacggccgccaacagcctgggcc gctccgaggccagcgtctacctgttccgcttccacggcgcccccg-3' (SEQ ID NO: 6) in which the sequence "gctccThe last "c" in "is the last nucleotide in humans, and the first" g "in the sequence" g aggc "is the first nucleotide in mice.

The targeting vector I also comprises a resistance gene for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo box (neocassette). Wherein the connection of the 5' end of the Neo box and the mouse is designed as follows In which the last "t" of the sequence "gtact" is the last nucleotide of the mouse, the sequence +.>Is the first core of the Neo boxA glycoside acid; the connection of the 3' -end of Neo cassette to the mouse was designed as +.> In which the sequence is'GCCGCThe last "C" of the "is the last nucleotide of the Neo cassette, sequence +.>Is the first nucleotide of the mouse. In addition, a gene encoding a negative selection marker (encoding a diphtheria toxin A subunit (DTA)) was also constructed downstream of the 3' homology arm of the recombinant vector. The mRNA sequence of the modified humanized mouse SIGLEC15 is shown as SEQ ID NO:9, the expressed protein sequence is shown as SEQ ID NO: shown at 10.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. And (3) carrying out electroporation transfection on the targeting vector with correct sequencing verification into embryonic stem cells of a wild mouse, screening the obtained cells by utilizing a positive clone screening marker gene, and screening correct positive cloned cells by utilizing PCR to detect the integration condition of exogenous genes. The correctly positive cloned cells (black mice) are introduced into the isolated blasts (white mice) according to the techniques known in the art, and the resulting embedded blasts are transferred to a culture medium for short culture and then transplanted into oviducts of recipient mice (white mice), thereby producing F0-generation chimeric mice (black-white interphase). And backcrossing the F0 generation chimeric mice and the wild mice to obtain F1 generation mice, and then mating the F1 generation heterozygous mice to obtain F2 generation homozygous mice. The SIGLEC15 gene humanized homozygote mice can be obtained by mating positive mice with Flp tool mice to remove positive clone screening marker genes.

In addition, a CRISPR/Cas system can be introduced to carry out gene editing, so that humanized modification of the mouse SIGLEC15 gene can be realized. Designing a targeting strategy shown in FIG. 4, wherein the targeting vector II contains homologous arm sequences of the upstream and downstream of the mouse SIGLEC15 gene and human SIGLEC15 genome DNA sequence, wherein the upstream homologous arm sequence (5 'homologous arm, SEQ ID NO: 11) is identical to 78049252-78050600 th nucleotide sequence of NCBI accession number NC_000084.6, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 12) is identical to 78046002-78047346 th nucleotide sequence of NCBI accession number NC_ 000084.6; the human SIGLEC15 gene sequence is identical to the SIGLEC15 gene sequence of the a fragment in fig. 3. The mRNA sequence and the protein sequence of the modified humanized mouse SIGLEC15 are respectively identical with those of SEQ ID NO:9 and SEQ ID NO:10 are identical.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgrnas and the efficiency of inducing Cas9 cleavage of the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for construction of sgRNA expression vectors. The sgRNA sequence that recognizes the target site is designed and synthesized. The target sites are located on exons 2 and 4 of SIGLEC15 gene, and the target site sequence of each sgRNA on SIGLEC15 is as follows:

sgRNA1 target site sequence (SEQ ID NO: 13): 5'-CCCCTCGACTTCCGGAGGCAGGG-3'

sgRNA2 target site sequence (SEQ ID NO: 14): 5'-GGTAGTGTGGGTCACACGCCAGG-3'

sgRNA3 target site sequence (SEQ ID NO: 15): 5'-TCCCTGCCTCCGGAAGTCGAGGG-3'

sgRNA4 target site sequence (SEQ ID NO: 16): 5'-TGTGCTCCCCCATAACTAAAAGG-3'

sgRNA5 target site sequence (SEQ ID NO: 17): 5'-CCACTCCCCTCGACTTCCGGAGG-3'

sgRNA6 target site sequence (SEQ ID NO: 18): 5'-TTGGACAGTTTGTTTCATCCTGG-3'

sgRNA7 target site sequence (SEQ ID NO: 19): 5'-AAAGAGTTGCTGAGATGCTATGG-3'

sgRNA8 target site sequence (SEQ ID NO: 20): 5'-CCCGTATTACAAGATCCCTAAGG-3'

sgRNA9 target site sequence (SEQ ID NO: 21): 5'-TTGCCCGCGCTGACCCGCGACGG-3'

sgRNA10 target site sequence (SEQ ID NO: 22): 5'-GTGCACGGCGGCCAATAGCCTGG-3'

sgRNA11 target site sequence (SEQ ID NO: 23): 5'-TGGCCGCCGTGCACGTGTAGCGG-3'

sgRNA12 target site sequence (SEQ ID NO: 24): 5'-TGCACGGCGGCCAATAGCCTGGG-3'

sgRNA13 target site sequence (SEQ ID NO: 25): 5'-TCACCTGGTAGCCGTGACCCTGG-3'

sgRNA14 target site sequence (SEQ ID NO: 26): 5'-GCCTGGTCGGGTCCCGCCCCAGG-3'

sgRNA15 target site sequence (SEQ ID NO: 27): 5'-GCCTGGGGCGGGACCCGACCAGG-3'

sgRNA16 target site sequence (SEQ ID NO: 28): 5'-AGGGCAGCGGAGCTGTTGCCTGG-3'

TABLE 1 UCA detection results

/>

The activity of a plurality of sgrnas was detected using UCA kit, and it was found from the results that the sgrnas have different activities, and the detection results are shown in table 1 and fig. 5. In combination with the activity, targeting position and sequence specificity of each sgRNA, the sgrnas 2 and 12 were selected for subsequent experiments. The forward oligonucleotide and the reverse oligonucleotide were obtained by adding cleavage sites to the 5' -end and the complementary strand, respectively (see Table 2), and after annealing, the annealed products were ligated to pT7-sgRNA plasmid (plasmid was linearized with BbsI) to obtain expression vectors pT7-SIGLEC15-2 and pT7-SIGLEC15-12, respectively.

TABLE 2 forward and reverse oligonucleotide sequences of sgRNA2 and sgRNA12

pT7-sgRNA vector A fragment DNA (SEQ ID NO: 37) containing the T7 promoter and sgRNA scaffold was synthesized by plasmid synthesis company and ligated to a backbone vector (source Takara, cat. No. 3299) by cleavage (EcoRI and BamHI) in sequence, and the results were verified by sequencing by a professional sequencing company, and the result showed that the objective plasmid was obtained.

Taking prokaryotic fertilized eggs of C57BL/6 wild mice, and injecting in vitro transcription products of pT7-SIGLEC15-2 and pT7-SIGLEC15-12 plasmids (transcribed by using an Ambion in vitro transcription kit according to the method of the specification) and targeting vectors into cytoplasm or nucleus of the fertilized eggs of the mice after premixing with Cas9 mRNA by using a microinjection instrument. Microinjection of fertilized eggs was performed according to the method of the mouse embryo operation laboratory Manual (third edition) (andela, nagel, chemical industry Press, 2006), the fertilized eggs after injection were transferred into a culture medium for short-term culture, then transplanted into oviducts of recipient mice for development, and the obtained mice (F0 generation) were subjected to hybridization and selfing to expand population numbers and establish stable SIGLEC15 gene mutant mouse strains.

The genotype of somatic cells of F0-generation mice can be identified through PCR primer pairs L-GT-F/L-GT-R and R-GT-F/R-GT-R (primer sequences and target fragment lengths are shown in table 3), the identification result of partial F0-generation mice is shown in fig. 6, 3 mice with the numbers of F0-01, F0-02 and F0-03 are positive mice, and the 3 mice are further verified to be positive mice through sequencing without random insertion.

TABLE 3P CR detection primer sequences and fragment lengths of interest

F1 generation mice were obtained by mating SIGLEC15 gene humanized mice identified as positive for F0 with C57BL/6 wild type mice. The genotype of the F1-generation mice can be identified using PCR primer pairs WT-F/WT-R and WT-F/Mut-R (Table 3), exemplary results are shown in FIG. 7, which shows 4 mice numbered F1-01 to F1-04 as positive mice. Southern blot detection was performed on these 4 mice identified as positive by F1 PCR to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting the genome with BspHI enzyme or EcoNI enzyme, transferring film and hybridizing. The probe 5' probe and 3' probe are respectively positioned on the left side of the 5' homology arm and the human sequence, and the lengths of the specific probe and the target fragment are shown in Table 4.

TABLE 4 Length of specific probes and fragments of interest

Restriction enzyme	Probe with a probe tip	Wild fragment size	Recombinant sequence fragment size
				BspHI	5’Probe	9.8kb	6.6kb
EcoNI	3’Probe	——	2.3kb

The probe synthesis primers were as follows:

5’Probe-F(SEQ ID NO：45)：5’-TGGCCTGAACGCCTAATAACTCTCC-3’

5’Probe-R(SEQ ID NO：46)：5’-CATGGTCGCCAGCCTACTTTCACTT-3’

3’Probe-F(SEQ ID NO：47)：5’-CCACCCTGCTCTGCGACAATAATGG-3’

3’Probe-R(SEQ ID NO：48)：5’-GCACCGAGATGTTGACGATCCGC-3’

the Southern blot detection results are shown in FIG. 8. The results of the 5'probe and the 3' probe were combined and further verified by sequencing that none of the 4 mice numbered F1-01, F1-02, F1-03, F1-04 had random insertions, confirming that these 4 mice were male heterozygous mice and that there was no random insertion. The F1 generation positive heterozygous mice are mutually mated to obtain the F2 generation SIGLEC15 gene humanized homozygous mice. This shows that the SIGLEC15 gene humanized genetically engineered mice which can be stably passaged and have no random insertion can be constructed by using the method.

The expression of the obtained humanized SIGLEC15 mRNA in the positive mice can be confirmed by a conventional detection method, for example, using an RT-PCR method. Selecting female wild type C57BL/6 mice and 1 SIGLEC15 gene humanized homozygous mice prepared in the embodiment, taking ovarian tissues to detect the expression condition of the humanized SIGLEC15 mRNA after neck-removing euthanasia respectively, and displaying the detection result (see figure 9) that only the murine SIGLEC15 mRNA is detected in the wild type C57BL/6 mice (figure 9A); only humanized SIGLEC15 mRNA was detected in the SIGLEC15 gene humanized homozygous mice (FIG. 9B), and no murine SIGLEC15 mRNA was detected.

RT-PCR detection primer sequence:

mSIGLEC15-F(SEQ ID NO：49)：5’-GAGAGTCGCCATGGGGTCCG-3’

mSIGLEC15-R(SEQ ID NO：50)：5’-GCTCGGAGCCTCTGTGAGCAG-3’

hSIGLEC15-F(SEQ ID NO：51)：5’-CGTCCATGACCGCTACGAGA-3’

hSIGLEC15-R(SEQ ID NO：52)：5’-TGACTAGATGGTGATGGCTGAGG-3’

GAPDH-F(SEQ ID NO：53)：5’-TCACCATCTTCCAGGAGCGAGA-3’

GAPDH-R(SEQ ID NO：54)：5’-GAAGGCCATGCCAGTGAGCTT-3’

furthermore, since cleavage of Cas9 causes double strand break of genomic DNA, insertion/deletion mutation is randomly generated by repair means of chromosome homologous recombination, and thus a knockout mouse with loss of function of SIGLEC15 protein may be obtained. For this purpose, a pair of primers was designed for detecting a knockout mouse, the wild type mouse should have no PCR band, the knockout mouse should have 1 PCR band, the product length should be about 569bp, and the result is shown in FIG. 10, wherein the mouse numbered KO-01 was further verified as SIGLEC15 knockout mouse by sequencing. The primers are respectively positioned at the left side of the 5 'end target site and the right side of the 3' end target site, and the sequences are as follows:

SEQ ID NO：55：5’-TTTGCCTCAACATCGCAGTTACTCCA-3’

SEQ ID NO：56：5’-CTCAAAATCCTTTGCAGAGCCACCA-3’

Expression of the humanized SIGLEC15 protein in SIGLEC15 gene humanized mice and SIGLEC15 gene knockout mice can be detected using conventional methods, such as flow cytometry and the like. Specifically, wild-type C57BL/6 mice, SIGLEC15 humanized homozygous mice prepared in this example and SIGLEC15 knockout mice were each 1, bone marrow cells were taken after neck-free euthanasia, red blood cells were lysed and induced to differentiate into macrophages with M-CSF-containing medium, and then stained with blocking antibody Purified Anti-mouse CD16/32, dead living cell identification dye Fixable Viability Dye eFluorTM, macrophage labeled antibody V450 Rat Anti-mouse CD11b and APC/Cy7 Anti-mouse F4/80, M2 type macrophage labeled antibody Anti-Mo CD206 (MMR) ebiscience PE/Cyanine TM7, M1 type macrophage labeled antibody ebiscience Anti-moschus II (I-A/I-E) SuperBright 600, cross-identified Human/murine SIGLEC15 Anti-GLEC 15-1 (conventionally), and secondary Anti-Alex conjugated AffiniPure Goat Anti IgG were measured as shown in FIG. 11. As can be seen from the figures, no expression of SIGLEC15 protein was detected on the surface of macrophages induced to differentiate by SIGLEC15 knockout mice (FIGS. 11D, 11E, 11F), and SIGLEC15 protein was detected on the surface of macrophages induced to differentiate by SIGLEC15 humanized homozygote mice (FIGS. 11A, 11B, 11C) and wild-type C57BL/6 mice (FIGS. 11G, 11H, 11I), and M2 type was higher than M1 type. In combination with the RT-PCR detection results of FIG. 9, only humanized SIGLEC15 protein was detected in mice homozygous for SIGLEC15 gene humanization, whereas only murine SIGLEC15 protein was detected in wild type C57BL/6 mice.

Example 2 preparation of double humanized or multiple double humanized mice

The humanized mice of SIGLEC15 genes prepared by the method can also be used for preparing double-or multi-humanized mouse models. For example, in example 1, embryo stem cells used for blastocyst microinjection may be selected from mice modified with other genes including PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40 and IL 10, or may be obtained from a model of mice modified with two or more genes of SIGLEC15 and other genes by isolating mouse ES embryo stem cells and gene recombination targeting techniques based on SIGLEC15 humanized mice. The homozygote or heterozygote of the SIGLEC15 mouse obtained by the method can be mated with other genetically modified homozygote or heterozygote mice, offspring are screened, according to Mendelian genetic law, the humanized SIGLEC15 and other genetically modified double-gene or polygene modified heterozygote mice can be obtained with a certain probability, and the homozygote of double-gene or polygene modification can be obtained by mating heterozygote with each other, and in vivo efficacy verification of targeting human SIGLEC15 and other gene regulators can be performed by utilizing the double-gene or polygene modified mice.

Example 3 SIGLEC15 Gene humanized mouse model drug efficacy experiment

8-week-old female SIGLEC15 gene humanized homozygote mice prepared in example 1 were selected and subcutaneously inoculated with human SIGLEC15 protein overexpressing mouse colon cancer cell MC38 (5X 10) ⁵ A number) of about 100 mm to treat tumor volume ³ Thereafter, the tumor volume was counted as a control group or a treatment group (n=5/group). The control group was injected with phosphate buffered saline (PB S), and the treatment group used different doses of anti-human SIGLEC15 antibody AB1 or anti-murine PD-1 antibody AB2 (obtained by immunizing mice using conventional methods, see Janeway' S immunology (9 th Edition)). The administration mode is as follows: intraperitoneal injection (i.p.), the group was given on the day, 2 times a week for 6 times. Tumor volume was measured 2 times per week, tumor volume of single mice after inoculationUp to 3000mm ³ Euthanasia is performed at that time. The specific groupings and dosing are shown in Table 5. The body weight and tumor volume measurements of the mice during the experimental period are shown in fig. 12 and 13, respectively.

TABLE 5 grouping and dosing conditions

The main data and analysis results of each experiment are shown in Table 6, and include tumor volume at the time of grouping, 9 days after grouping, 20 days after grouping, survival of mice, tumor-free mice, tumor (volume) inhibition rate (Tumor Growth Inhibition value, TGI) _TV ) And statistical differences in tumor volume (P-value) between treated and control mice.

TABLE 6 tumor volume, survival and tumor inhibition

Overall, the animals in each group had good health status during the experiment, and all the animals in the treatment group (G2, G3 and G4 group) and the control group (G1) had a trend of increasing weight (fig. 12), indicating that the animals had good tolerance to the anti-human SIGLEC15 antibody AB1 and the anti-murine PD-1 antibody AB2, neither AB1 nor AB2 had a significant toxic effect on the animals, and the safety was good. From the tumor volume measurements, the treated group had less tumor volume than the control group at each stage of the experiment (fig. 13); on day 20 post-dose, tumor volumes of mice in treatment groups G2, G3, and G4 were 1643+ -278 mm, respectively ³ 、1398±168mm ³ And 1277 + -274 mm ³ 1735+ -173 mm compared with control group ³ The tumor volume of the anti-mouse PD-1 antibody AB2 treatment group (G4) is smaller than that of the anti-human SIGLEC15 antibody treatment group, and the tumor inhibition effect of different antibodies in the SIGLEC15 gene humanized mice is particularly different. For the anti-human SIGLEC15 antibody AB 1-treated group, the tumor volume of the 30mg/kg dose group (G3) was less than that of the 10mg/kg dose group (G2), indicating that different doses of AB1 were humanized for the SIGLEC15 gene Specific different tumor inhibiting effects in mice. The experimental result proves that the SIGLEC15 gene humanized mice prepared by the method can be used for screening in-vivo efficacy detection of anti-human SIGLEC15 antibodies, can be used as a living body substitution model for in-vivo research, and can be used for screening, evaluating and treating human SIGLEC15 signal path modulators.

Example 4 SIGLEC15 Gene humanized mouse model drug efficacy experiment two

8-week-old female SIGLEC15 gene humanized homozygote mice prepared in example 1 were selected and subcutaneously inoculated with human SIGLEC15 protein overexpressing mouse colon cancer cell MC38 (5X 10) ⁵ A number) of about 100 mm to treat tumor volume ³ Thereafter, the tumor volume was counted as a control group or a treatment group (n=5/group). The control group was injected with phosphate buffered saline (PB S), and the treatment group used different anti-human SIGLEC15 antibodies Ab1 or Ab2 (obtained by immunizing mice using conventional methods, see Janeway' S immunology (9 th Edition)). The administration mode is as follows: dosing was started on the day of grouping, 2 times per week, 6 times total. Tumor volume was measured 2 times per week and the tumor volume of individual mice reached 3000mm after inoculation ³ Euthanasia is performed at that time. The specific groupings and dosing are shown in Table 7. The body weight and tumor volume measurements of the mice during the experimental period are shown in fig. 14 and 15, respectively.

TABLE 7 grouping and dosing conditions

The main data and analysis results of each experiment are shown in Table 8, and include tumor volume at the time of grouping, 14 days after grouping, 25 days after grouping, survival of mice, tumor-free mice, tumor (volume) inhibition (Tumor Growth Inhibition value, TGI) _TV ) And statistical differences in tumor volume (P-value) between treated and control mice.

TABLE 8 tumor volume, survival and tumor inhibition

Overall, the animals in each group had good health status during the experiment, and all animals in the treatment group (G2, G3) and the control group (G1) had a trend of increasing weight (fig. 14), indicating that the animals had good tolerance to human SIGLEC15 antibodies Ab1 and Ab2, neither Ab1 nor Ab2 had significant toxic effects on the animals, and the safety was good. From the tumor volume measurements, the treated group had less tumor volume than the control group at each stage of the experiment (fig. 1 5); tumor volumes of treatment group G2 and G3 mice were 1801+ -181 mm, respectively, on day 25 post-dose ³ Sum 1220.+ -.234 mm ³ Is equal to 2528+ -377 mm of control group ³ The tumor volume of the Ab2 treatment group (G3) is smaller than that of the Ab1 treatment group (G2), which shows that the inhibition effect of Ab2 on tumors is better than that of Ab.

The results prove that the SIGLEC15 gene humanized mice prepared by the method can be used for screening in-vivo drug effects of different anti-human SIGLEC15 antibodies, can be used as a living body substitution model for in-vivo research, and can be used for screening, evaluating and treating human SIGLEC15 signal path modulators.

Example 5 SIGLEC15 Gene human mice immunophenotyping

Flow cytometry was used to detect the in vivo leukocyte immunoassay of SIGLEC15 gene humanized mice. Specifically, 3 wild type C57BL/6 mice and 3 SIGLEC15 gene humanized homozygous mice prepared in example 1 were selected, and spleen, lymph node and blood samples were taken after neck-free euthanasia and single cell suspensions were prepared for flow cytometry detection. A flow analysis strategy diagram is shown in fig. 22. The results of the detection of the leukocyte subtypes and the T cell subtypes in the spleen are shown in FIG. 16 and FIG. 17, respectively, the results of the detection of the leukocyte subtypes and the T cell subtypes in the lymph node are shown in FIG. 18 and FIG. 19, respectively, and the results of the detection of the leukocyte subtypes and the T cell subtypes in the blood are shown in FIG. 20 and FIG. 21, respectively. From the figures, it can be seen that the percentage of T cell, B cell, NK cell, DC cell (fig. 14), granulocyte (Granulocyte), mononuclear cell (Monocyte), macrophage (Macrophage) and other leukocyte subtypes in spleen, lymph node and blood samples of SIGLEC15 gene humanized homozygous mice was consistent with that of C57BL/6 wild type mice (fig. 16, 18, 20), and that the humanized modification of SIGLEC15 gene did not affect the differentiation, development and distribution of leukocytes in mice and lymphoid tissues.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Sequence listing

<110> Baioer chart (Beijing) pharmaceutical technology Co., ltd

Construction method and application of <120> SIGLEC15 gene humanized non-human animal

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gtctacagag aaatagcaag gagatactgt atcggctact gcagcaagga gatactgtat 840

ccgctactgc agcaaggaga tactgtatcc actactgcag ccttcttttt tttttttttt 900

tttttttttt tttttgcagc cttcttaaaa acaaaacaaa aggccttgtc tctggagttt 960

aaactttcta tgcagcttct tttatttttg tttaatcaag tgaacagaag tttgtttttg 1020

gttttttttc cccccatggg gaaacaaaag aatattttgc acttgttctt aagcaactgg 1080

gcacagtggt ggggggcagg gagatggggg tgcagagccc gtcaggggca agctgtggtt 1140

ctgctactgt cagtcaccag ctgactttga gaagtcgctt gggccttcca agccctcagt 1200

ttccccctct ctaaaacggg ccattgacaa catctcctgg ggctgttatg tgttttaagt 1260

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gcatgcgcgc ctttaatccc agcactcggg aggcagaggc aggtggattt ctgagttcaa 1500

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aaataaacaa acaaaaaacc aacctcccga tgtaggtaaa agtggcctga acgcctaata 1680

actctcctcc tgaacattga ggctttctgc acaggctgcc atgtctttca cagtcgtacc 1740

ttacagcttg gttccctgct ccccgtgcac agactgtcaa gcccaggtct ccgtggaatt 1800

cattctctgg actctgcata cacatacctc ctctctgttg ggctctacaa cccctgcctg 1860

ctatttccaa tccctgcaac tctttccacc agccctcatc cttctgaaag cctaaccccc 1920

tttcatccca gggtacaggg gagagatacc agagctaggt tgggtgctca gagactattc 1980

tggcaagggt gattcatcag ataacagagg ttgaatagga gagatctggc aagtgaaagt 2040

aggctggcga ccatgttcag actaaggcaa tattgtctgt atctttcatc agagcacctg 2100

gcagagcaca gcctaattac atgttcactg atctttctcc agctcgatga taggggaccc 2160

tgttcatctt tgtccccaca gctcatatcc cagcacctgg cgcagcgtgc ggaagtatcc 2220

aataaatgtt tgcagaattg atgaatgcac aatagcagac aatgtgtggg caatccgtgc 2280

ctgactccat gagcttgcaa gatgcagaat tcaagcctaa ggtcacagag ggtagggagt 2340

acaggaaaac tgaggtaggg ctgagggtgc acactcatag tcccagctgt taggaagctg 2400

agacaggaag attgcaagtt gaacttgtgg cttgtttgag ctacactgtc agacagacag 2460

acagacagac agacagacag acctaaggaa actttcttca gctagacgtc ctgacaggat 2520

cagcgcaatc atcacacagg tttctcacat ttgaaaggca gccaaacacg ttcttactaa 2580

ggataaacgt ccagcccccg acttgaattc aaacacgtag ttcaacttgt ccagagctaa 2640

aagagattaa gcctaagaac aggccgtgtg aaacaggaac tggggtttaa ctcgatggtc 2700

ttccagctct gtgcccacag caggatcaga ttggacattc agcattccag cccaaggtgc 2760

acaagaactc gctgcactct gcaggtgcag cacactaggg ccacccgggg cgggacttga 2820

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ctccacggta cgtactcact gcccttcccc catctctgcc taccacattg gcccaggcat 540

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cccagctctg gagtgctgtg cctggtgctg gctgggagca cactctttcc tgctgctcaa 1020

gtcttgacag cccgccatat gtgcatctcc tttactgacg tgcaacaaga tcagagtcac 1080

aacactgcct gatgtacact cacctaacaa atatttaccg aatacctacg atctagcagg 1140

caataacatg gatgatagag actggaaatc caagcttact ctccaatgtg cacagttttg 1200

cacaaacata acaagaccat ctgagatctg taaagctagg gataaagcag tgcccagtgg 1260

gtagccaggc ggccatgact gctttagagc tggatacttt ctttgaagac atggccctaa 1320

agacaccaag ctggaggctg gaacctaggg tgaccaaggt gatagggata tcccaagagt 1380

gaaaactgta aaaggatctt ctgttctcct gaagtaactg tcatctctaa ggctcactgc 1440

ctccatctgc taacctaagc ctagttctgg aagcttctag ccttcgtaca atctaatcta 1500

ggcctagaat gttttcaggc tctgagactc actgctgaat aagttcaccc cttctagctc 1560

tttctgagct ctggctggct aattcaactc agctgttctg gctcaaactc ctctcaaagc 1620

tgactgatac agtctggctt ctctcttgga ctctgatatt tttgctgtgc ttggcgtcta 1680

agtctggcaa tctgttctaa ccttctggct ccttctcctt ctctggcttg tcttgtcttc 1740

accagtgtct agcttgcttg ttctctcttc aacctgtgtc tgtacaactc tcccagtaaa 1800

actgcttgct tgcttcctct gtctctgtct ctctcttctt cttcttttct cctcctcctc 1860

ctcctcctcc tcctcctcct tcttcttctt cttcttcttc ttctctctct ctctctctct 1920

ctctctctct ctctctctct ctctctctct gcactgctcc cttaagtagc ttccctttcc 1980

tctctctcct catgagagtt gggcgtatcc tattctgtca aatctttctc cgactcatca 2040

ctttgtctgc cactcaatta gacttaactt tcaagcatgc atgcttcctt ctacaaacta 2100

acttcacctt cattgtttgg gattaaaagt gtgtactaag ggttgagcta taactagaaa 2160

caggcttttt tttttttcag taaataacac aatctctcag ggttcactgt gtgatcaaat 2220

atcctgcaac agaaaacctt aagtagaaag gtcaggtggg atttggaatg gacacagaca 2280

tggggggagc atggaaatgt gggttaagat ggcaaggttg gggcaattct gggtgaggtg 2340

gaaatcagaa tggtttggag tcatacatga gaggttgaat ctgaacagaa gggcccaaga 2400

aatacacccc aaaagcggct gctgtttttt ctgctctggc agcaaccagg ggaagggact 2460

ctctgaggtg aagggaggga agcaggatat ccagagttag agttgagtcc tagtcacaga 2520

gaccaggagg gaatggagta ggggaggggt gtgcgctttg tctcccacgg tgggtgggag 2580

gttggaggta gggatgtctg ggaagcagtc agatctgagg agattgggga catggagctg 2640

gggtctgaaa cctctgacaa agtcactttg taggagatgg taactgatca aggacgctat 2700

caccagcagc atccatcccc ctgtcttatg ttaatcaatt ccccagcctt gctgcttctg 2760

gctccactgc ctcccatgta gtatctttct tgacctgttg ttggggcaca gcttgggaag 2820

tcaaggacct cgccacatat tgggtggtaa gttgattttc tcctgtctac aggtctcagg 2880

ctcaggagtc caattatgaa aatttgagcc agatgagtcc tccaggccac cagctgccac 2940

gtgtttgctg tgaggaactc ctcagccatc accatctagt cattcaccat gagaaataaa 3000

ggacagtacc aggctagacc tggacagctc ataattccca ggttccttgg ccaggcaaca 3060

tccaagggga ccaggaccct gctcacagag gctccgagcc tcacggtatt ataacaacag 3120

tggagccagc aatctgggtg tatatgtatg ccgacatgag cctcagcatg aaacttctgg 3180

aaaaactccc cttgttttat tctttcttag aactacgtag taaagcagac aggaaactag 3240

ccataatgtc tggaggtcca ggctccagtc tgctctgtca tggacttcca gtgcggacca 3300

ggacacttca cctatctggg gctcagctga agaaatagaa gatctgaaag acctgcataa 3360

actccaacgc tagctcaaag gtgggcctct ggattgctga attcttaggt aggtgtggca 3420

ctgctcagcc ctgtggagaa caaagcctga gactggagct gtgcaaactg atcagaggta 3480

cagcaaaccc tgggaatgtt atagcaagaa gtgtggagcc cctgcctacc tgtgatcata 3540

tcagctgacc cacacttcct taatctggga cccaccaaga gaaggagact gaagacccat 3600

agtctcaaag catctgaaag ctgtatgtca aaacctggga gacctgaagg ggctctgggt 3660

cccagtagtt aaggcagctg tggctctgac ccctcctggg gagtgcactc cctcctagga 3720

tgtgaaagtc caggagatct ggcagtgtct tcccacttgc ccaaacccag ccaggctaac 3780

ttcaagagtg gtgctttcct tatttacact tcgggtgcat ttgttggtcc tattaaagga 3840

gagaggtgta cttgggacca agtgagccac aagacaggag gctaaggaga ccatgttttt 3900

ctggggacgg catctctatg tacagggcaa tcggtttcag gcttcccact gcgccagagc 3960

ttcaggcgag gcggagctca gccaagggca atggcagatc atagttattc ccagatccat 4020

cgagagctgt taagaaagca catgggagat ctttccatct tctgagactc gaagttctta 4080

tcatacagat c 4091

<210> 5

<211> 1893

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttgctcaaca cagaggtgca cagtaagtgc ttttattatt atcaccatct cggggatctt 60

gggagtctgt tttaaccacg agatcccagg gtttttccac agggcaggtt ttgatgggga 120

aaaactaagg gtaagaatat ggggtcaagg ggcctgcagg tgaattagga aacgaagagg 180

ggaatagtcc cagggaagag ccgcgggaac gcaggcactc ccaagtcgag ggagtgcagg 240

ccctgggggt gcaggcacca ggaattgccc ctcaagtcac aaggtggggt tccggctccc 300

ctggaagtgg gggacgatcc ctgagtcctg gggtttccag gctaggggtt gggggagcgt 360

ttcctgggtc gtggggtttc caggccccgg gtgcgggcgc ctcgacccca gggccccgag 420

cctgacgcag cccgccccgc cctcaggctc gccagcgcag cgctggtcca tgcaggtgcc 480

acccgaggtg agcgcggagg caggcgacgc ggcagtgctg ccctgcacct tcacgcaccc 540

gcaccgccac tacgacgggc cgctgacggc catctggcgc gcgggcgagc cctatgcggg 600

cccgcaggtg ttccgctgcg ctgcggcgcg gggcagcgag ctctgccaga cggcgctgag 660

cctgcacggc cgcttccggc tgctgggcaa cccgcgccgc aacgacctct cgctgcgcgt 720

cgagcgcctc gccctggctg acgaccgccg ctacttctgc cgcgtcgagt tcgccggcga 780

cgtccatgac cgctacgaga gccgccacgg cgtccggctg cacgtgacag gcgaggcggc 840

gtgggagcgg gtccccggcc tcccttcccg ccctcccgcc tgccccgccc caagggctac 900

gtgggtgcca ggcgctgtgc tgagccagga agggcaacga gacccagccc tctcctctac 960

cccagggatc tcacacctgg gggtagttta ggaccacctg ggagcttgac acaaatgcag 1020

aatccaggtc ccaggaaggg ctgaggtggg cccgggaata ggcattgccg tgactctcgt 1080

agagtgactg tccccagtgg ctctcagacg aagaggcgag aaagacaagt gaatggcaat 1140

cctaaatatg ccaagaggtg caatgtggtg tgtgctacca gcccggaaag acactcgcag 1200

cccctctacc caggggtgca cagacagccc accaagtagt gcctagcact ttgccagacc 1260

ctgatataca aagatgcctg aaccagggtc ccgtccctag agcagtggct ctccactcta 1320

gcccccaccc tgctctgcga caataatggc cacttagcat ttgctaggga gccgggacct 1380

agtccaagca cccacaagca tgaatttgcc aaatcttttc agcaacctct taaggcaact 1440

gctatcatga tcctcacttt acacatggag aagcagaagc agagatgata gaatctttcg 1500

cccaaggcca catctgtatt gggacggggg cagcctggca cccaagtgcc cattcctccc 1560

ttctgaccag cccccacccc tccggctctg gcgtccaaag ggctaagggg aggggtgccc 1620

ttgtgacagt cacccgcctt ctcccctgca gccgcgccgc ggatcgtcaa catctcggtg 1680

ctgcccagtc cggctcacgc cttccgcgcg ctctgcactg ccgaagggga gccgccgccc 1740

gccctcgcct ggtccggccc ggccctgggc aacagcttgg cagccgtgcg gagcccgcgt 1800

gagggtcacg gccacctagt gaccgccgaa ctgcccgcac tgacccatga cggccgctac 1860

acgtgtacgg ccgccaacag cctgggccgc tcc 1893

<210> 6

<211> 80

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ccgctacacg tgtacggccg ccaacagcct gggccgctcc gaggccagcg tctacctgtt 60

ccgcttccac ggcgcccccg 80

<210> 7

<211> 80

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctggcctggg aacccaggtt acttttagag tctcagtact aagcttgata tcgaattccg 60

aagttcctat tctctagaaa 80

<210> 8

<211> 80

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aggtacataa tggtggatcc actagttcta gagcggccgc tccaagctgc tgtccctgac 60

cttgatgggt catctgctga 80

<210> 9

<211> 2326

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

acaccgctgg ctacttggct gcggctgcct agctcctcca gtttgcttag agcccagcgg 60

ccctgcagac ttggcacaga gcacacccac ctgcctttgt cacagcacac taagaaggtt 120

ctctgtggtg accaggctgg gtagagggct gctgggtctg caggcgtcag agcatggagg 180

ggtccctcca actcctggcc tgcttggcct gtgtgctcca gatgggatcc cttgtgaaaa 240

ctagaagaga cgcttcgggg gatttgctca acacagaggt gcacagctcg ccagcgcagc 300

gctggtccat gcaggtgcca cccgaggtga gcgcggaggc aggcgacgcg gcagtgctgc 360

cctgcacctt cacgcacccg caccgccact acgacgggcc gctgacggcc atctggcgcg 420

cgggcgagcc ctatgcgggc ccgcaggtgt tccgctgcgc tgcggcgcgg ggcagcgagc 480

tctgccagac ggcgctgagc ctgcacggcc gcttccggct gctgggcaac ccgcgccgca 540

acgacctctc gctgcgcgtc gagcgcctcg ccctggctga cgaccgccgc tacttctgcc 600

gcgtcgagtt cgccggcgac gtccatgacc gctacgagag ccgccacggc gtccggctgc 660

acgtgacagc cgcgccgcgg atcgtcaaca tctcggtgct gcccagtccg gctcacgcct 720

tccgcgcgct ctgcactgcc gaaggggagc cgccgcccgc cctcgcctgg tccggcccgg 780

ccctgggcaa cagcttggca gccgtgcgga gcccgcgtga gggtcacggc cacctagtga 840

ccgccgaact gcccgcactg acccatgacg gccgctacac gtgtacggcc gccaacagcc 900

tgggccgctc cgaggccagc gtctacctgt tccgcttcca cggcgccccc ggaacctcga 960

ccctagcgct cctgctgggc gcgctgggcc tcaaggcctt gctgctgctt ggcattctgg 1020

gagcgcgtgc cacccgacgc cgactagatc acctggtccc ccaggacacc cctccacggt 1080

ctcaggctca ggagtccaat tatgaaaatt tgagccagat gagtcctcca ggccaccagc 1140

tgccacgtgt ttgctgtgag gaactcctca gccatcacca tctagtcatt caccatgaga 1200

aataaaggac agtaccaggc tagacctgga cagctcataa ttcccaggtt ccttggccag 1260

gcaacatcca aggggaccag gaccctgctc acagaggctc cgagcctcac ggtattataa 1320

caacagtgga gccagcaatc tgggtgtata tgtatgccga catgagcctc agcatgaaac 1380

ttctggaaaa actccccttg ttttattctt tcttagaact acgtagtaaa gcagacagga 1440

aactagccat aatgtctgga ggtccaggct ccagtctgct ctgtcatgga cttccagtgc 1500

ggaccaggac acttcaccta tctggggctc agctgaagaa atagaagatc tgaaagacct 1560

gcataaactc caacgctagc tcaaaggtgg gcctctggat tgctgaattc ttaggtaggt 1620

gtggcactgc tcagccctgt ggagaacaaa gcctgagact ggagctgtgc aaactgatca 1680

gaggtacagc aaaccctggg aatgttatag caagaagtgt ggagcccctg cctacctgtg 1740

atcatatcag ctgacccaca cttccttaat ctgggaccca ccaagagaag gagactgaag 1800

acccatagtc tcaaagcatc tgaaagctgt atgtcaaaac ctgggagacc tgaaggggct 1860

ctgggtccca gtagttaagg cagctgtggc tctgacccct cctggggagt gcactccctc 1920

ctaggatgtg aaagtccagg agatctggca gtgtcttccc acttgcccaa acccagccag 1980

gctaacttca agagtggtgc tttccttatt tacacttcgg gtgcatttgt tggtcctatt 2040

aaaggagaga ggtgtacttg ggaccaagtg agccacaaga caggaggcta aggagaccat 2100

gtttttctgg ggacggcatc tctatgtaca gggcaatcgg tttcaggctt cccactgcgc 2160

cagagcttca ggcgaggcgg agctcagcca agggcaatgg cagatcatag ttattcccag 2220

atccatcgag agctgttaag aaagcacatg ggagatcttt ccatcttctg agactcgaag 2280

ttcttatcat acagatcttt cacttgctta gttagagtca caccaa 2326

<210> 10

<211> 343

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

Gly Ala Pro Gly Thr Ser Thr Leu Ala Leu Leu Leu Gly Ala Leu Gly

260 265 270

Leu Lys Ala Leu Leu Leu Leu Gly Ile Leu Gly Ala Arg Ala Thr Arg

275 280 285

Arg Arg Leu Asp His Leu Val Pro Gln Asp Thr Pro Pro Arg Ser Gln

290 295 300

Ala Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Ser Pro Pro Gly

305 310 315 320

His Gln Leu Pro Arg Val Cys Cys Glu Glu Leu Leu Ser His His His

325 330 335

Leu Val Ile His His Glu Lys

340

<210> 11

<211> 1349

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ggacttgaat ggaagggaaa gggggatggg ggcaggtgag ccatcctctc ccatcaggaa 60

aaggtaatga gtccaagagc caccataatg agcttctgca agcggcagct gggacctgta 120

catgttcccg aaagcagtgg cagttccggt gtggttctgt cgatccacag gtgtatgggc 180

tctacaggag atggactagc agcggtggct tgagggcatt ccaacccaag aagtcattga 240

ggctcactga ccacaaagag cactgggctt agttagcatc aggaagaact ggcttttctc 300

tttccttgcc tggatgttgg ccttcatgta cacatctata acactctctg ccctgtcttt 360

ctcctcttgg gtgctttcag gacagatggc atggtggtgt ggtataaata aagggactgt 420

gtataataca agcccaatta ctccctgtaa aggtcccttt tgtgcactgt gccccagggc 480

actgactggg caggctcaac cgcctgcctc ctcttgggga ggtaccaccc ctggcataac 540

cttctgagac tgaacacctc cataaacaca gcaacacaga gcccgatggt ttgccagcac 600

tgggtgaact ggaagcaggg ctcagcttcc acaacccttg gctccgcatt ctgaagaatg 660

tggttcacca ggtggtgggc aaagtgaggc tggagcagag gtgtcaacct gtgggccgca 720

cgcccttttg ggggggaggg tgttgactga ccctttccta ggagtcacct aagactacct 780

gcatatcaga tatttatatt atgattcata acagtaacaa aattacacca atgaagtagc 840

aacaaaaata attttatggt tggggtcacc acaacatgag gaactgtatt aaagggtcgc 900

agcattagga aggctgagaa ccactggtct tgaggtacaa tgcagtgtgt acccaccatg 960

ttaaagtacc caccacgagg gtctttctct gccttttaag cagccagcca ggacgcagtt 1020

cacctgggct ggctctgatt ctaaaggaac ttgtaactct actggtgtgc ttcaaagcac 1080

cgcccccccc ccccccccca gggtctgggt ttctcaacta caaataaaga ctgttagccc 1140

ttcaggtgaa tcacagcttc atgcctttag ggcgttctaa tacaggctct gggtccaggc 1200

aggtctgagt gggacgcaga ctttgcctca acatcgcagt tactccaatt atttaacttt 1260

tctgcgcctc agtgtattaa catgtaacag gctatctaca tttgtctcta ggatcccttg 1320

tgaaaactag aagagacgct tcgggggat 1349

<210> 12

<211> 1345

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gaggccagcg tctacctgtt ccgcttccac ggcgcccccg gaacctcgac cctagcgctc 60

ctgctgggcg cgctgggcct caaggccttg ctgctgcttg gcattctggg agcgcgtgcc 120

acccgacgcc gactaggtgg gtgcatccca gatctgggtg ggtgagaggg ggagagaagg 180

ctccctgccc tttctgccgg tccacctgat gtggcacatc agaaatcctt tctgcacctg 240

catccttctc tctgcctgga gcccatgaga tggagctctt cgcagttagg aaatggtggc 300

tctgcaaagg attttgagtc acacagagac acaggcctgt aggctctcca acacttcttt 360

tactaactac cacgccacaa atcagtgtcc tgtaaaggtc gggtctccgg cttctccctt 420

acttttaact agcgtcttga ggtcctgagg ttatggacca ggatgctggg aacgcaaaga 480

tgctcagatg tgattccctg ctagccggta agaggctggg tctgcagaac tccttttcat 540

ggaggtactg cagaaggcag aaattgcaga agacaggggg aggggggata tcttgcaatt 600

ctgaggtctt agtaagttag gagggctcag ccaaaaaaaa aaaaaataaa taaaataaag 660

ccttcaggac acaggagctt ggaaatcata aatggctcac ctggcctggg aacccaggtt 720

acttttagag tctctccaag ctgctgtccc tgaccttgat gggtcatctg ctgagcaccc 780

agaaaaggga gggaccttct cagagcctct cagtcttggc caaccaataa ggttagcctc 840

caactctcca gggacacaac gcagaatttg tctctggggg ccccagctgc catccttgct 900

gtgaccctgc cattcttgct ggttcctcaa acttatcaaa catgttgtgt ttcagggtcc 960

ccagaaaggc agttgtgttc tctcttaata tccccacccc gcctccccga ccggggtcaa 1020

gttaacccta tcatgtatca ggttaagttg ctccagctag caagccatct gctgcacatt 1080

tcctggggac tgcctctcac tgtggggcgg ggcaatgtgt gaagctgaca ggttggctgg 1140

agaccactgt gcaaattcac acctcctctt tctccatgtc cctcatacag atcacctggt 1200

cccccaggac acccctccac ggtacgtact cactgccctt cccccatctc tgcctaccac 1260

attggcccag gcatgccact ttctcagaac tgggagttca cagagaaaaa gcaggcatag 1320

ggcaaggcta gggtagtagg ctgga 1345

<210> 13

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cccctcgact tccggaggca ggg 23

<210> 14

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ggtagtgtgg gtcacacgcc agg 23

<210> 15

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tccctgcctc cggaagtcga ggg 23

<210> 16

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tgtgctcccc cataactaaa agg 23

<210> 17

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ccactcccct cgacttccgg agg 23

<210> 18

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ttggacagtt tgtttcatcc tgg 23

<210> 19

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

aaagagttgc tgagatgcta tgg 23

<210> 20

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cccgtattac aagatcccta agg 23

<210> 21

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ttgcccgcgc tgacccgcga cgg 23

<210> 22

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gtgcacggcg gccaatagcc tgg 23

<210> 23

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

tggccgccgt gcacgtgtag cgg 23

<210> 24

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

tgcacggcgg ccaatagcct ggg 23

<210> 25

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

tcacctggta gccgtgaccc tgg 23

<210> 26

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

gcctggtcgg gtcccgcccc agg 23

<210> 27

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gcctggggcg ggacccgacc agg 23

<210> 28

<211> 23

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

agggcagcgg agctgttgcc tgg 23

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

ggtagtgtgg gtcacacgcc 20

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

taggggtagt gtgggtcaca cgcc 24

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

ggcgtgtgac ccacactacc 20

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

aaacggcgtg tgacccacac tacc 24

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

tgcacggcgg ccaatagcct 20

<210> 34

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

taggtgcacg gcggccaata gcct 24

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

aggctattgg ccgccgtgca 20

<210> 36

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

aaacaggcta ttggccgccg tgca 24

<210> 37

<211> 132

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

aagcctaaga acaggccgtg tgaaa 25

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

tagcacacac cacattgcac ctctt 25

<210> 40

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

cctctacccc agggatctca cacc 24

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

acatcaggca gtgttgtgac tctga 25

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

cagcttcatg cctttagggc gttct 25

<210> 43

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

gggccagaga cacatcagac ttagc 25

<210> 44

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

tgggactatt cccctcttcg tttcc 25

<210> 45

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

tggcctgaac gcctaataac tctcc 25

<210> 46

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 46

catggtcgcc agcctacttt cactt 25

<210> 47

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 47

ccaccctgct ctgcgacaat aatgg 25

<210> 48

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 48

gcaccgagat gttgacgatc cgc 23

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 49

gagagtcgcc atggggtccg 20

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 50

gctcggagcc tctgtgagca g 21

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 51

cgtccatgac cgctacgaga 20

<210> 52

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 52

tgactagatg gtgatggctg agg 23

<210> 53

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 53

tcaccatctt ccaggagcga ga 22

<210> 54

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 54

gaaggccatg ccagtgagct t 21

<210> 55

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 55

tttgcctcaa catcgcagtt actcca 26

<210> 56

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

ctcaaaatcc tttgcagagc cacca 25

Claims

1. A construction method of a non-human animal humanized by a SIGLEC15 gene is characterized in that the non-human animal expresses a humanized SIGLEC15 protein in vivo, or the genome of the non-human animal contains a humanized SIGLEC15 gene, the human SIGLEC15 protein in the humanized SIGLEC15 protein is all or part of an extracellular region of the human SIGLEC15 protein, wherein all or part of the extracellular region of the human SIGLEC15 protein is from 0 th to 15 th amino acids from the N end to 0 th to 50 th amino acids from the C end of the extracellular region, the construction method comprises introducing all or part of a nucleotide sequence encoding the extracellular region of the human SIGLEC15 protein into a non-human animal SIGLEC15 locus, the amino acid sequence of the humanized SIGLEC15 protein is the amino acid sequence shown in SEQ ID NO 10, and the non-human animal is a mouse.

2. The construction method according to claim 1, wherein the amino acid sequence of the human SIGLEC15 protein contained in the humanized SIGLEC15 protein is amino acid sequence 31-246 of SEQ ID No. 2.

3. The method of claim 1, wherein the humanized SIGLEC15 gene comprises a portion of a human SIGLEC15 gene, the humanized SIGLEC15 gene comprises a portion of exon 2, a portion of exon 3, and a portion of exon 4 of the human SIGLEC15 gene, wherein the portion of exon 2 comprises at least a 5bp nucleotide sequence and the portion of exon 4 comprises at least a 100bp nucleotide sequence.

4. The construction method according to claim 3, wherein the human SIGLEC15 gene has a nucleotide sequence shown in SEQ ID NO. 5.

5. The construction method according to claim 4, wherein the mRNA transcribed from the humanized SIGLEC15 gene is the nucleotide sequence shown in SEQ ID NO. 9.

6. The method of construction according to claim 1, wherein the method of construction comprises introducing a portion of exon 2, all of exon 3 and a portion of exon 4 of the human SIGLEC15 gene into the non-human animal SIGLEC15 locus, wherein the portion of exon 2 comprises at least a 5bp nucleotide sequence and the portion of exon 4 comprises at least a 100bp nucleotide sequence.

7. The construction method according to claim 6, wherein the nucleotide sequence shown in SEQ ID NO. 5 is used for introducing the non-human animal SIGLEC15 locus.

8. The method of claim 1, wherein the introducing is a substitution or insertion.

9. The method of claim 8, wherein the non-human animal SIGLEC15 locus is introduced as a replacement for a corresponding region of a non-human animal.

10. The method of construction according to claim 9, wherein all or part of exons 2 to 4 of the non-human animal SIGLEC15 gene are replaced.

11. The method of construction according to claim 10, wherein the non-human animal has a part of exon 2, all of exon 3 and a part of exon 4 replaced.

12. The method of construction according to any one of claims 1 to 11, wherein the human or humanized SIGLEC15 gene is regulated in a non-human animal by endogenous regulatory elements.

13. The method of claim 12, wherein the non-human animal is constructed using a targeting vector comprising a portion of the human SIGLEC15 gene.

14. The method of claim 13, wherein the targeting vector further comprises a 5' arm; the 5' arm sequence is shown as SEQ ID NO. 3 or 11; the targeting vector further comprises a 3' arm; the 3' arm sequence is shown as SEQ ID NO. 4 or 12.

15. The method of any one of claims 1-11, 13-14, further comprising mating, in vitro fertilization, or direct gene editing of a SIGLEC15 gene humanized non-human animal with other genetically modified non-human animals and screening to obtain a polygenic modified non-human animal.

16. The method of claim 15, wherein the additional gene is selected from at least one of PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40, and IL 10.

17. The humanized SIGLEC15 protein is characterized in that the amino acid sequence of the humanized SIGLEC15 protein is the amino acid sequence shown in SEQ ID NO. 10.

18. The humanized SIGLEC15 protein of claim 17, wherein the amino acid sequence of the human SIGLEC15 protein comprised in the humanized SIGLEC15 protein is amino acid sequence of SEQ ID No. 2 at positions 31-246.

19. A humanized SIGLEC15 gene, wherein said humanized SIGLEC15 gene comprises a portion of a human SIGLEC15 gene, said humanized SIGLEC15 gene encoding the humanized SIGLEC15 protein of any one of claims 17-18.

20. The humanized SIGLEC15 gene of claim 19, wherein the humanized SIGLEC15 gene comprises a portion of exon 2, a portion of exon 3, and a portion of exon 4 of the human SIGLEC15 gene, wherein the portion of exon 2 comprises at least a 5bp nucleotide sequence and the portion of exon 4 comprises at least a 100bp nucleotide sequence.

21. The humanized SIGLEC15 gene of claim 20, wherein the human SIGLEC15 gene comprised in the humanized SIGLEC15 gene is the nucleotide sequence set forth in SEQ ID No. 5.

22. The humanized SIGLEC15 gene of any one of claims 19-21, wherein the mRNA transcribed from the humanized SIGLEC15 gene is the nucleotide sequence set forth in SEQ ID No. 9.

23. The targeting vector is characterized by comprising a nucleotide sequence shown as SEQ ID NO. 5, and further comprising a 5' -arm; the 5' arm sequence is shown as SEQ ID NO. 3 or 11; the targeting vector further comprises a 3' arm; the 3' arm sequence is shown as SEQ ID NO. 4 or 12.

24. A cell, tissue or organ, wherein the cell, tissue or organ expresses the humanized SIGLEC15 protein of any one of claims 17-18, wherein the cell, tissue or organ comprises the humanized SIGLEC15 gene of any one of claims 19-22, or wherein the cell, tissue or organ is derived from a non-human animal obtained by the method of construction of any one of claims 1-16.

25. Use of a cell, tissue or organ according to claim 24 derived from a humanized SIGLEC15 protein according to any one of claims 17-18, a humanized SIGLEC15 gene according to any one of claims 19-22, a non-human animal obtained by a construction method according to any one of claims 1-16 or a cell, tissue or organ according to claim 24, said use comprising:

or,

the application in screening, verifying, evaluating or researching SIGLEC15 function, human SIGLEC15 signaling mechanism, human targeting antibody, human targeting drug, drug effect, immune related disease drug and anti-tumor or anti-inflammatory drug, screening and evaluating human drug and drug effect research.