CN114751973A

CN114751973A - Construction method and application of SIGLEC15 gene humanized non-human animal

Info

Publication number: CN114751973A
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: 2022-07-15
Anticipated expiration: 2042-03-11
Also published as: CN114751973B; WO2022188871A1

Abstract

The invention provides a construction method of a SIGLEC15 gene humanized non-human animal, humanized SIGLEC15 protein, humanized SIGLEC15 gene, a targeting vector of SIGLEC15 gene and application thereof in the field of biomedicine, wherein a nucleotide sequence coding human SIGLEC15 protein is introduced into the genome of the non-human animal by using a homologous recombination mode, the animal can normally express human or humanized SIGLEC15 protein, and the animal can be used as an animal model for researching the signal mechanism of the SIGLEC15 and screening tumor and autoimmune diseases medicaments, and has important application value for research and development of new medicaments of immune targets.

Description

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

Technical Field

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

Background

SIGLEC15(Sialic acid-binding immunoglobulin-like lectin 15) belongs to the SIGLEC family, is a type I transmembrane protein, and mRNA of the SIGLEC is rarely expressed in most normal human tissues and various immune cell subsets, but is relatively high in expression level on macrophages. In 2007, a Japanese scientist Takashi AngataMayo firstly discovers that the protein can recognize sialic acid, so that the protein is classified into a SIGLEC family, and plays an important regulation and control role in autoimmune diseases, inflammatory reactions and tumors of a body by regulating and controlling innate immunity and adaptive immune response.

SIGLEC15 is a related immunoreceptor of DAP12(DNAX activating protein 12kDa), DAP12 is a linker protein based on an ITAM (instant-based activation motif) signal pathway, and SIGLEC15 can cause the transmission of the ITAM signal pathway after being combined with DAP12 and participate in the differentiation of osteoclasts. The Anti-SIGLEC15 antibody can inhibit osteoclast differentiation, and can down-regulate the expression of RANKL (important factor for promoting osteoclast differentiation), so as to achieve the effect of treating osteoporosis. In addition, it was pointed out in 2019 that SIGLEC15 screened using the high-throughput functional screening system TCAA was able to continuously inhibit T cell activity and showed main characteristics satisfying normalization of cancer immunotherapy. In most patients with PD-L1 negative tumors, SIGLEC15 and PD-L1 are mutually exclusively expressed, and particularly, the immunosuppression of the protein is independent of PD-1, so that a new candidate scheme is provided for patients who do not respond to the current immunotherapy, particularly patients who do not respond to anti-PD-1/PD-L1 therapy. The analysis of the TCGA database Meta shows that the mRNA of SIGLEC15 is up-regulated in various human cancers, such as colon cancer, endometrioid cancer and thyroid cancer, and is remarkably up-regulated in bladder cancer, kidney cancer, lung cancer and liver cancer, and can be a candidate target of a tumor immunotherapy normalization strategy.

The experimental animal disease model is an indispensable research tool for researching the etiology and pathogenesis of human diseases and developing prevention and treatment technologies and medicines. In view of the huge application value of SIGLEC15 in the field of autoimmune diseases and tumor immunotherapy, in order to further research relevant biological characteristics, improve the effectiveness of preclinical drug efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure, there is an urgent need in the art to develop SIGLEC 15-related non-human animal models.

Disclosure of Invention

In a first aspect of the invention, a humanized SIGLEC15 protein is provided, wherein the humanized SIGLEC15 protein comprises 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 the human SIGLEC15 protein.

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

In one 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 part of the extracellular region of human SIGLEC15 protein, preferably at least 50 consecutive amino acids of the extracellular region of human SIGLEC15 protein, such as an extracellular region of human SIGLEC15 protein 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, more preferably an extracellular region of human SIGLEC15 protein comprising 197, 216, 223, or 233 consecutive amino acids; preferably, the human SIGLEC15 protein extracellular region comprising an N-terminal deletion of 0 to 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 deletion of 0 to 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) amino acids, further preferably, the human SIGLEC15 protein extracellular region comprising an N-terminal deletion of 0, 7, 9, or 11 amino acids and a C-terminal deletion of 4, 12, 47, or 17 amino acids, more preferably, the human SIGLEC15 protein extracellular region comprising an amino acid sequence substantially identical to SEQ ID NO:2 or an amino acid sequence having at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO:2 from position 31 to 246.

In some embodiments, the humanized SIGLEC15 protein comprises amino acids 31-246 of SEQ ID NO. 2 flanked by 6-15 amino acids, such as positions 27-259, 29-251, and 20-216 of SEQ ID NO. 2.

Preferably, the humanized SIGLEC15 protein comprises all or part of the amino acid sequence encoded by exons 1 to 6 of the human SIGLEC15 gene, further preferably comprises all or part of the amino acid sequence encoded by exons 2 to 4 of the human SIGLEC15 gene, further preferably comprises all or part of the amino acid sequence encoded by any one, two, three, two or three consecutive exons of exons 2 to 4, further preferably comprises all or part of the amino acid sequence encoded by a combination of exons 2 to 4, and further preferably comprises all or part of the amino acid sequence encoded by part of exon 2, all of exon 3 and part of exon 4, wherein part of exon 2 comprises at least 5bp of nucleotide sequence, such as at least 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 25, 40. 45, 50, 55, 60bp nucleotide sequence, more preferably 22bp nucleotide sequence; preferably, the part of exon 2 comprises at least the nucleotide sequence starting from the nucleotide sequence encoding 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the N-terminus of the extracellular domain of human SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably, the part of exon 2 comprises the nucleotide sequence starting from the nucleotide sequence encoding 12 amino acids from the N-terminus of the extracellular domain of human SIGLEC15 protein to the last nucleotide sequence of exon 2; the part of exon 4 comprises at least 100bp of nucleotide sequence, for example at least 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 of nucleotide sequence, and more preferably 242bp of nucleotide sequence; preferably, the part of exon 4 comprises at least the 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 from the C-terminus of the extracellular domain of human SIGLEC15 protein, preferably the nucleotide sequence from the first nucleotide of exon 4 to 18 amino acids from the C-terminus of the extracellular domain of human SIGLEC15 protein, and further 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 the amino acid sequence encoded by SEQ ID No. 5.

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

A) comprises all or part of the amino acid sequence from position 31 to 246, from position 27 to 259, from position 20 to 263, from position 20 to 216 or from position 29 to 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 the amino acid sequence of SEQ ID NO 2 at positions 31-246, 27-259, 20-263, 20-216 or 29-251;

C) an amino acid sequence comprising NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 amino acid different from the amino acid sequence of SEQ ID NO 2 at positions 31-246, 27-259, 20-263, 20-216 or 29-251; or

D) Comprises an amino acid sequence shown in the positions 31-246, 27-259, 20-263, 20-216 or 29-251 of SEQ ID NO. 2, and comprises substitution, deletion and/or insertion 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, an extracellular region, a transmembrane region and/or a cytoplasmic region of a non-human animal SIGLEC15 protein.

Preferably, at most 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 the extracellular domain of the SIGLEC15 protein of the non-human animal, wherein the extracellular domain comprises 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids and/or 0 to 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 sequences derived from the extracellular domain of the SIGLEC15 protein of the non-human animal, and further preferably, the humanized SIGLEC15 protein comprises 3 amino acid sequences derived from the extracellular domain of SIGLEC15 protein of the non-human animal, 5 or 7 amino acids and C-

terminal

5, 10 or 17 amino acid sequences.

Preferably, the humanized SIGLEC15 protein further comprises a part of an amino acid sequence encoded by the endogenous SIGLEC15 gene of the non-human animal, and further preferably comprises all or part of an amino acid sequence encoded by all of exon 1, exon 2, exon 4, exon 5 and all of exon 6 of the endogenous SIGLEC15 gene of the non-human animal, wherein the part of exon 2 of the endogenous SIGLEC15 gene of the non-human animal at least comprises a nucleotide sequence of 20bp, for example at least comprises a nucleotide sequence of 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55 and 60bp, and further preferably comprises a nucleotide sequence of 38 bp; preferably, the part of the exon 2 comprises at least the nucleotide sequence of the exon 2 starting from the first nucleotide and encoding 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular domain of SIGLEC15 protein, preferably, the part of the exon 2 comprises the nucleotide sequence of the exon 2 starting from the first nucleotide and encoding 7 amino acids from the N-terminus of the extracellular domain of SIGLEC15 protein; the part of the exon 4 of the non-human animal SIGLEC15 gene at least comprises a nucleotide sequence of 100bp, such as at least comprises 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 and 375bp, and further preferably comprises a nucleotide sequence of 136 bp; preferably, the part of exon 4 comprises at least the nucleotide sequence starting from the nucleotide sequence encoding 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 from the C-terminal of the extracellular domain of SIGLEC15 protein and ending at the last nucleotide sequence of exon 4, and preferably comprises the nucleotide sequence starting from the nucleotide sequence encoding 17 amino acids from the C-terminal of the extracellular domain of SIGLEC15 protein and ending at the last nucleotide sequence of exon 4.

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

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

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

c) an amino acid sequence comprising NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 amino acid difference from the amino acid sequence set forth in SEQ ID No. 10; or

d) Comprises an amino acid sequence shown as SEQ ID NO. 10 and comprises substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the non-human animal can be selected from any non-human animal such as rodent, pig, rabbit, monkey, etc. which can be genetically modified by gene editing.

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

The non-human animal is an immunodeficient non-human mammal, 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, even more preferably the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mouse, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

In a second aspect of the invention, there is provided a humanized SIGLEC15 gene, wherein the humanized SIGLEC15 gene comprises a portion of the human SIGLEC15 gene.

Preferably, said humanized SIGLEC15 gene comprises all or part of the nucleotide sequence encoding the signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of the human SIGLEC15 protein; preferably, the humanized SIGLEC15 gene comprises all or part of the nucleotide sequence of an extracellular region encoding human SIGLEC15 protein, wherein the extracellular region comprises all or part of an extracellular region of human SIGLEC15 protein, preferably at least 50 consecutive amino acids of the extracellular region of human SIGLEC15 protein, for example, an extracellular region of human SIGLEC15 protein 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 identical to the extracellular region of human SIGLEC15 protein, further preferably 197, 223, 216, or 233 consecutive amino acids; preferably, the human SIGLEC15 protein extracellular region comprising an N-terminal deletion of 0 to 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 deletion of 0 to 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 region comprising an N-terminal deletion of 0, 7, 9 or 11 amino acids and a C-terminal deletion of 4, 12, 47, or 17 amino acids, more preferably, the extracellular region comprises an amino acid sequence substantially identical to SEQ ID NO:2 or 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 from position 31 to 246 or from position 27 to 259.

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

Preferably, the humanized SIGLEC15 gene comprises all or part of exons 1 to 6 of human SIGLEC15 gene, further preferably comprises all or part of exons 2 to 4 of human SIGLEC15 gene, further preferably comprises all or part of any one, two, three, two consecutive or a combination of three exons 2 to 4, further preferably comprises part of exon 2, all of exon 3 and part of exon 4, further preferably further comprises intron 2 to 3 and/or intron 3 to 4, further preferably comprises any intron between exons 2 to 4, wherein part of exon 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 nucleotide sequence, more preferably 22bp nucleotide sequence; preferably, the part of exon 2 comprises at least a nucleotide sequence from 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the N-terminus of the extracellular domain of the human SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably a nucleotide sequence from 12 amino acids from the N-terminus of the extracellular domain of the human SIGLEC15 protein, and said part of exon 4 comprises at least a nucleotide sequence of 100bp, e.g. a nucleotide sequence of at least 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 378, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, bp, further preferably a nucleotide sequence of 242 bp; preferably, the part of exon 4 comprises at least the nucleotide sequence starting from the first nucleotide of exon 4 and extending from 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 from the C-terminus of the extracellular domain of human SIGLEC15 protein, preferably the part comprises the nucleotide sequence starting from the first nucleotide of exon 4 and extending from 18 amino acids from the C-terminus of the extracellular domain of human SIGLEC15 protein, more preferably the humanized SIGLEC15 gene comprises a nucleotide sequence having at least 85%, 90%, 95% or at least 99% identity with SEQ ID No. 5 or comprises a nucleotide sequence shown in SEQ ID No. 5.

In one embodiment of the present invention, the human SIGLEC15 gene included in 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. 5;

(B) comprises a nucleotide sequence having 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) a nucleotide sequence comprising NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or NO more than 1 nucleotide difference from the nucleotide sequence set forth in SEQ ID No. 5;

(D) 5, including 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, and/or a nucleotide sequence encoding at most 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 the extracellular domain of SIGLEC15 of a non-human animal, wherein the extracellular domain comprises 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids of the N-terminal domain and/or 0 to 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 of SIGLEC-human animal SIGLEC15 protein, further preferably, the recombinant human SIGLEC15 protein comprises an extracellular region derived from a non-human animal, 3, 5 or 7 amino acids from the N-terminal and 5, 10 or 17 amino acids from the C-terminal.

Preferably, the humanized SIGLEC15 gene further comprises all exon 1, part exon 2, part exon 4, all exon 5 and/or all exon 6 of endogenous SIGLEC15 gene of the non-human animal, wherein the exon 2 part of endogenous SIGLEC15 gene of the non-human animal comprises at least 20bp nucleotide sequence, for example at least 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 38bp nucleotide sequence; preferably, the part of the exon 2 at least comprises the nucleotide sequence of the exon 2 starting from the first nucleotide to 1-10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminal of the extracellular region of the SIGLEC15 protein, preferably, the nucleotide sequence of the exon 2 starting from the first nucleotide to 7 amino acids from the N-terminal of the extracellular region of the SIGLEC15 protein, and the part of the exon 4 of the endogenous SIGLEC15 gene of the non-human animal at least comprises the nucleotide sequence of 100bp, for example, the nucleotide sequence of 136bp, 110 bp, 120 bp, 230 bp, 132 bp, 133, 134 bp, 135 bp, 137, 138, 139, 140, 145, 150, 200, 250, 300 bp, 310 bp, 320, 330, 340 bp, 350, 360, 370 bp, and further preferably, the nucleotide sequence of 136 bp; preferably, the part of exon 4 comprises at least the nucleotide sequence starting from the nucleotide sequence encoding 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 from the C-terminal of extracellular domain of SIGLEC15 protein to the last nucleotide sequence of exon 4, and preferably comprises the nucleotide sequence starting from the nucleotide sequence encoding 17 amino acids from the C-terminal of extracellular domain of SIGLEC15 protein to the last nucleotide sequence of exon 4.

In a specific embodiment of the invention, the humanized SIGLEC15 gene at least comprises the nucleotide sequence shown in

SEQ ID NOs

6, 7 and 8, or comprises a nucleotide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with

SEQ ID NOs

6, 7 and 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 difference from the nucleotide sequence set forth in SEQ ID No. 9; or

(d) Comprises a nucleotide sequence shown as SEQ ID NO. 9 and comprises substitution, deletion and/or insertion of one or more nucleotides.

Preferably, the humanized SIGLEC15 gene further comprises a specific inducer or repressor, and further preferably, the specific inducer or repressor can be a substance which can be induced or repressed conventionally.

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

Preferably, the non-human animal is an immunodeficient non-human mammal, more 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, even more preferably, the immunodeficient mouse is a NOD-Prkdc^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mice, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

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

Preferably, the part of the human SIGLEC15 gene comprises all or part of exons 2 to 4 of the human SIGLEC15 gene, more preferably comprises all or part of any one, two, three, two consecutive or three combinations of exons 2 to 4, even more preferably comprises part of exon 2, all of exon 3 and part of exon 4, more preferably further comprises intron 2 to 3 and/or intron 3 to 4, and even more preferably comprises any intron 2 to 4, wherein the part of exon 2 comprises at least 5bp of nucleotide sequence, such as at least 5, 7, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 35, 40, 55. A nucleotide sequence of 60bp, further preferably, a nucleotide sequence comprising 22 bp; preferably, the part of exon 2 comprises at least the nucleotide sequence encoding 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the N-terminus of the extracellular domain of human SIGLEC15 protein to the last nucleotide sequence of exon 2, preferably the nucleotide sequence encoding 12 amino acids from the N-terminus of the extracellular domain of human SIGLEC15 protein, and the part of exon 4 comprises at least 100bp nucleotide sequence, e.g. at least 100, 150, 200, 210, 220, 230, 240, 241, 242, 243, 244, 245, 378, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, bp nucleotide sequence, further preferably 242bp nucleotide sequence; preferably, the part of exon 4 comprises at least the nucleotide sequence starting from the first nucleotide of exon 4 and extending from 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 from the C-terminus of the extracellular domain of human SIGLEC15 protein, and 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 the nucleotide sequence shown 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.the 5' arm, selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal SIGLEC15 gene; preferably, said 5' arm is at least 90% homologous to the NCBI accession number NC _ 000084.6; further preferably, the 5' arm sequence has at least 90% homology with SEQ ID NO 3 or 11, or 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 transition region to be altered, i.e., the 3' arm, selected from the group consisting of nucleotides of 100-10000 in length of the genomic DNA of the non-human animal SIGLEC15 gene; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 3' arm sequence has at least 90% homology with SEQ ID NO. 4 or 12, or is as shown in SEQ ID NO. 4 or 12.

Preferably, the targeting vector further comprises a nucleotide sequence as shown in

SEQ ID NOs

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 NOs

6, 7, 8.

Preferably, the transition region to be altered is located at the SIGLEC15 locus of the non-human animal, and further preferably, the transition region to be altered is located on exons 2 to 4 of SIGLEC15 gene of the non-human animal.

Preferably, the targeting vector is used for constructing a non-human animal humanized by the SIGLEC15 gene.

Preferably, the non-human animal is an immunodeficient non-human mammal, more 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, even more preferably, the immunodeficient mouse is a NOD-Prkdc ^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mice, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

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

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

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

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

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

Preferably, the target site sequence of the 5' end 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 NO 21-28.

In a fifth aspect of the invention, a DNA molecule encoding the sgRNA described above is provided. Preferably, the double strands of the DNA molecules are upstream and downstream sequences of the sgRNA, or forward and reverse oligonucleotide sequences after the addition of the enzyme cleavage site.

In one embodiment of the invention, the DNA molecule duplexes are SEQ ID NO 29 and 31, and SEQ ID NO 30 and SEQ ID NO 32, respectively.

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

In a sixth aspect of the present invention, a sgRNA vector is provided, which includes the sgRNA described above.

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

An eighth aspect of the present invention provides use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector, or the cell for SIGLEC15 gene modification. Preferably comprising the use of knocking out, inserting or replacing the SIGLEC15 gene.

In a ninth aspect of the invention, there is provided a SIGLEC15 gene humanized non-human animal, wherein the non-human animal comprises:

(1) expressing a human or humanized SIGLEC15 protein; and/or the presence of a gas in the atmosphere,

(2) comprising a human or humanized SIGLEC15 gene.

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

Preferably, the non-human animal expresses the humanized SIGLEC15 protein in vivo.

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

Preferably, the human SIGLEC15 gene portion or the humanized SIGLEC15 gene is operably linked to endogenous regulatory elements.

According to some embodiments of the invention, the non-human animal further comprises additional genetic modifications, the additional genes 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 additional gene is homozygous or heterozygous for the endogenous modified locus.

Preferably, the non-human animal can be selected from any non-human animal such as rodent, pig, rabbit, monkey, etc. which can be genetically edited to make a gene humanized.

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

Preferably, the non-human animal is an immunodeficient non-human mammal, more 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, even more preferably, the immunodeficient mouse is a NOD-Prkdc^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mouse, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

In the tenth aspect of the invention, the invention provides a construction method of a SIGLEC15 gene humanized non-human animal, wherein the non-human animal expresses human or humanized SIGLEC15 protein.

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

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

Preferably, the humanized SIGLEC15 protein is the humanized SIGLEC15 protein.

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

The human or humanized SIGLEC15 gene is regulated in non-human animals by regulatory elements including, but not limited to, promoters. The regulatory elements may be endogenous or exogenous. For example, the exogenous regulatory element can be 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 exons 1 to 6 of the human SIGLEC15 gene into the locus of the SIGLEC15 non-human animal; it is further preferred that the nucleotide sequence comprising all or part of exons 2 to 4 of the human SIGLEC15 gene is introduced into the locus of SIGLEC15, more preferably that the nucleotide sequence comprising all or part of a combination of any one, two, three, two or three consecutive exons of exons 2 to 4 is introduced into the locus of SIGLEC15, still more preferably that the nucleotide sequence comprising part of exon 2, all of exon 3 and part of exon 4 is introduced into the locus of SIGLEC15, still more preferably further comprising introns 2-3 and/or 3-4, yet more preferably any intron between exons 2-4, wherein part of exon 2 comprises at least 5bp of nucleotide sequence, e.g.comprising at least 5, 7, 9, 10, 11, 12. 13, 14, 15, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60bp nucleotide sequence, more preferably 22bp nucleotide sequence; preferably, the part of exon 2 comprises at least the nucleotide sequence from 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids from the N-terminus of the extracellular domain of human SIGLEC15 to the last nucleotide sequence of exon 2, preferably from the nucleotide sequence of 12 amino acids from the N-terminus of the extracellular domain of human SIGLEC15, and the part of exon 4 comprises at least 100bp nucleotide sequence, e.g. at least 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, bp nucleotide sequence, further preferably 242bp nucleotide sequence; preferably, the part of exon 4 comprises at least the nucleotide sequence starting from the first nucleotide of exon 4 and extending from 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 the C-terminus of the extracellular domain of human SIGLEC15 protein, preferably the nucleotide sequence starting from the 1 st nucleotide of exon 4 and extending from 18 amino acids from the C-terminus of the extracellular domain of human SIGLEC15 protein, more preferably the nucleotide sequence comprising at least 85%, 90%, 95% or at least 99% identity to SEQ ID No. 5 or the nucleotide sequence comprising SEQ ID No. 5 is introduced into the 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 the non-human animal SIGLEC15 gene.

Preferably, said introduced position is located after the endogenous regulatory elements of the SIGLEC15 gene.

Preferably, said introducing is a substitution or insertion, and in a particular embodiment of the invention, said introducing the locus of the SIGLEC15 gene is a substitution of the corresponding region of the non-human animal, further preferably, all or part of exon 2 to exon 4 of the SIGLEC15 gene of the non-human animal is substituted, and further preferably, all of exon 2, exon 3 and part of exon 4 of the SIGLEC15 gene of the non-human animal is substituted.

Preferably, the nucleic acid sequence encoding SEQ ID NO: 1 at positions 31-245 by a nucleotide sequence.

In some embodiments, the nucleic acid encoding SEQ ID NO: 1 at position 29-252 or at position 27-257.

Preferably, the construction method comprises introducing into the non-human animal SIGLEC15 locus a nucleotide sequence comprising all or part of a signal peptide encoding human SIGLEC15 protein, a transmembrane region, a cytoplasmic region and/or an extracellular region; further preferably, the nucleotide sequence comprising all or part of the extracellular region encoding the human SIGLEC15 protein is introduced into the non-human animal SIGLEC15 locus, preferably, at least 50 consecutive amino acids of the extracellular region encoding the human SIGLEC15 protein is introduced into the non-human animal SIGLEC15 locus, for example, 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 of the extracellular region encoding the human SIGLEC15 protein is introduced into the non-human animal SIGLEC15 locus, further preferably, the extracellular region of the human SIGLEC15 protein comprising 197, 223, 216 or 233 consecutive amino acids; the extracellular region comprises all or part of the extracellular region of the human SIGLEC15 protein, preferably, the extracellular region of the human SIGLEC15 protein comprising 0-15 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids deleted from the N-terminus and/or 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 deleted from the C-terminus, further preferably, the extracellular region of the human SIGLEC15 protein comprising 0, 7, 9, or 11 amino acids deleted from the N-terminus and 4, 12, 47, or 17 amino acids deleted from the C-terminus, more preferably, the extracellular region of the human SIGLEC15 protein comprising an amino acid sequence at least 85%, 90%, 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO:2, or comprising amino acid sequence corresponding to positions 31-246 of SEQ ID NO:2 The SIGLEC15 locus was introduced into a non-human animal.

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

Preferably, the construction method comprises introducing a nucleotide sequence encoding the humanized SIGLEC15 into the SIGLEC15 locus of the non-human animal.

Preferably, the insertion or substitution site is after an endogenous regulatory element of the SIGLEC15 gene.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous SIGLEC15 gene of the non-human animal and then performing the insertion operation, or the insertion step can be performed by both causing a frame shift mutation to the endogenous SIGLEC15 gene and performing the step of inserting the human sequence.

Preferably, the genome of the non-human animal comprises a humanized SIGLEC15 gene on at least one chromosome.

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

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

Preferably, the targeting vector described above is used for the construction of non-human animals.

Preferably, to improve recombination efficiency, a non-human animal can be constructed using sgRNA targeting SIGLEC15 gene together with the above-described targeting vector. Wherein the sgRNA targets the SIGLEC15 gene of the non-human animal, and the sequence of the sgRNA is on the target sequence of the SIGLEC15 gene to be changed.

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

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

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

Preferably, the 3' target site sequence of the sgRNA is shown in any one of SEQ ID NO 21-28.

In a specific embodiment of the invention, the construction method comprises the steps of introducing the targeting vector, sgRNA targeting the SIGLEC15 gene and Cas9 into cells of the non-human animals, culturing the cells (preferably fertilized eggs), transplanting the cultured cells into oviducts of female non-human animals, allowing the cells to develop, and identifying and screening the non-human animals humanized by the SIGLEC15 gene.

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

Preferably, the other genes are at least one genetically modified non-human animal selected from the group consisting 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 polygenic modified non-human animal is homozygous or heterozygous for the endogenous replaced locus.

In an eleventh aspect of the invention, there is provided a SIGLEC15 gene knockout non-human animal, wherein the non-human animal lacks all or part of the nucleotide sequence of the SIGLEC15 gene.

Preferably, the non-human animal lacks all or part of exon 2 to exon 4 of SIGLEC15 gene, further preferably lacks all or part of the nucleotide sequence of any one, two, three, two or three consecutive exons of exon 2 to exon 4, preferably lacks part of exon 2, all exon 3 and part of exon 4, preferably also lacks intron 2-3 and/or intron 3-4, further preferably lacks any intron 2-4, wherein the deleted part of exon 2 comprises at least 10bp of nucleotide sequence, such as at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60bp nucleotide sequence, further preferably, the deleted part of exon 2 comprises a 22bp nucleotide sequence; preferably, the deleted part in exon 2 at least comprises the nucleotide sequence from 0-15 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus of the extracellular domain of the SIGLEC15 protein of non-human animal to the last nucleotide sequence of exon 2, preferably comprises the nucleotide sequence from 7 amino acids from the N-terminus of the extracellular domain of SIGLEC15 protein of non-human animal to the last nucleotide sequence of exon 2; preferably, the deleted exon 4 portion comprises at least 100bp of nucleotide sequence, for example at least 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 of nucleotide sequence, further preferably 239bp of nucleotide sequence; preferably, the deleted part of exon 4 at least comprises the nucleotide sequence from the first nucleotide of exon 4 to the C-terminal 1-20 (for example, 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 of SIGLEC15 protein, and preferably comprises the nucleotide sequence from the 1 st nucleotide of exon 4 to the C-terminal 18 amino acids of the extracellular domain of 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 a mouse.

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. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mice, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

In a twelfth aspect of the present invention, a method for constructing a SIGLEC15 gene-knocked-out non-human animal is provided, in which sgRNA is used to construct the non-human animal. Wherein the sgRNA targets the SIGLEC15 gene of the non-human animal, while 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 exon 1 to exon 6 sequences of the SIGLEC15 gene. Further preferably, the target site at the 5 'end of the sgRNA is located on exon 2 sequence of SIGLEC10 gene, and more preferably, the target site at the 3' end of the sgRNA is located on exon 4 sequence of SIGLEC10 gene.

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

I) providing a non-human animal with a humanized SIGLEC15 gene or a non-human animal with a SIGLEC15 gene knockout, or a non-human animal with a humanized SIGLEC15 gene obtained by the construction method;

II) mating the non-human animal provided in step I) with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a polygenic modified non-human animal.

Preferably, the other genetically modified non-human animals include, but are not limited to, genetically modified non-human animals with 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 two-gene humanized non-human animal, a three-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 polygenic 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 progeny thereof obtained by the above construction method, wherein the non-human animal or progeny thereof is selected from a non-human animal humanized with SIGLEC15 gene, a non-human animal with SIGLEC15 gene knockout, or a multi-gene modified non-human animal.

In a fifteenth aspect of the present invention, an animal model of a disease is provided, wherein the animal model is derived from the above non-human animal, the non-human animal obtained by the above construction method, or the above non-human animal or its progeny, preferably, the disease comprises an autoimmune disease, a tumor, or inflammation.

In a sixteenth aspect of the present invention, there is provided a method for producing an animal model with a disease, said method comprising the steps of humanizing the SIGLEC15 gene, knocking out the SIGLEC15 gene, or modifying a polygene in a non-human animal; preferably, the disease comprises an autoimmune disease, a tumor or inflammation, and further preferably, the step of implanting tumor cells is also included.

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

In an eighteenth aspect of the present invention, there is provided a use of the above non-human animal, the above non-human animal or its progeny, the non-human animal obtained by the above construction method, or the above animal model in the preparation of a medicament for treating autoimmune diseases, tumors and/or inflammation.

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

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

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

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

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

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

Preferably, the expression of 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 incapable of developing into an individual animal.

In a twenty-third aspect of the invention, there is provided a SIGLEC15 gene knock-out cell in which all or part of the nucleotide sequence of 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 or three consecutive exons from exon 2 to 4, preferably lacks all of exon 2, all of exon 3 and part of exon 4, preferably also lacks introns 2 to 3 and/or introns 3 to 4, further preferably lacks any intron between exons 2 to 4, wherein the deleted exon 2 part comprises at least 10bp of nucleotide sequence, such as at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60bp nucleotide sequence, further preferably, the deleted part of exon 2 comprises a 22bp nucleotide sequence; preferably, the deleted part of exon 2 at least comprises the nucleotide sequence from 0-15 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminal of extracellular domain of SIGLEC15 protein of non-human animal to the last nucleotide sequence of exon 2, preferably, the deleted part of exon 4 at least comprises 100bp nucleotide sequence from the nucleotide sequence from 7 amino acids from the N-terminal of extracellular domain of SIGLEC15 protein of non-human animal to the last nucleotide sequence of exon 2, for example, a nucleotide sequence comprising at least 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, and more preferably a nucleotide sequence comprising 239 bp; preferably, the deleted part of exon 4 at least comprises the nucleotide sequence from the first nucleotide of exon 4 to the C-terminal 1-20 (for example, 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 of SIGLEC15 protein, and preferably comprises the nucleotide sequence from the 1 st nucleotide of exon 4 to the C-terminal 18 amino acids of the extracellular domain of SIGLEC15 protein.

Preferably, SIGLEC15 gene-deleted cells are prepared using the sgRNA targeting the SIGLEC15 gene as described above. Preferably, the cells are incapable of developing into an individual animal.

In a twenty-fourth aspect of the invention, a construct for expressing the humanized SIGLEC15 protein is provided, and preferably, the construct comprises the 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 incapable of developing 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 incapable of developing into an individual animal.

In a twenty-seventh aspect of the present invention, there is provided a use of the humanized SIGLEC15 protein described above, the humanized SIGLEC15 gene described above, the non-human animal obtained by the above construction method, the above non-human animal or progeny thereof, the above animal model, the above cell or cell line or primary cell culture, the above tissue or organ or culture thereof, the above tumor-bearing tissue, the above cell, the above construct, the above cell or the above tissue, the use comprising:

In product development requiring immunological processes involving human cells, the manufacture of antibodies, or as model systems for pharmacological, immunological, microbiological, medical research;

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

or,

the application in the aspects of screening, verifying, evaluating or researching the function of SIGLEC15, the signal mechanism of human SIGLEC15, a human-targeting antibody, a human-targeting drug, a drug effect, an immune-related disease drug and an anti-tumor or anti-inflammatory drug, screening and evaluating the 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, the present invention provides a use of the SIGLEC15 gene-humanized non-human animal, the SIGLEC15 gene-knocked-out non-human animal, the SIGLEC15 gene-humanized non-human animal obtained by the above-described construction method, the SIGLEC15 gene-knocked-out non-human animal, the multi-gene-modified non-human animal, or a progeny thereof for producing a human SIGLEC 15-specific modulator or for screening a human SIGLEC 15-specific modulator. Preferably, the use is not a method of treatment and/or diagnosis of a disease.

In a twenty-ninth aspect of the invention, there is provided a method of screening for a modulator specific for human SIGLEC15, the method comprising administering the modulator to an individual and detecting the effect of the modulation; wherein the individual is selected from the group consisting of the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or a progeny thereof, and the above animal model.

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

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

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

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

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

Preferably, the detecting comprises assessing the weight, fat mass, activation pathways, neuroprotective activity or metabolic changes in the individual, including changes in food consumption or water consumption.

Preferably, the tumor cell is derived from a human or non-human animal.

Preferably, the method of screening for a modulator specific for human SIGLEC15 is not a method of treatment. The method is used for screening or evaluating drugs, and detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary but is only a possibility.

In a thirtieth aspect of the invention, an intervention scheme evaluation method is provided, the evaluation method comprises the steps of applying the intervention scheme to an individual, and detecting and evaluating the regulation effect of the individual after applying the intervention scheme; wherein the individual is selected from the non-human animal, the non-human animal obtained by the construction method, the non-human animal or the offspring thereof, or the animal model.

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

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

Preferably, the tumor cell is 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 is therapeutically effective, i.e. the effect of the treatment is not necessarily but only a possibility.

In a thirty-first aspect of the present invention, there is provided a use of the non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny or disease model in the preparation of a medicament for treating tumor, inflammation or autoimmune disease.

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

"transgene" as used herein refers to the introduction 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 genetic changes in the cell following incorporation of the exogenous genetic material, wherein vectors used for stable integration in this process include: plasmids, retroviral vectors and other animal viruses, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and the like.

"tumors" as referred to herein include, but are not limited to, lymphoma, B cell tumors, T cell tumors, myeloid/monocytic tumors, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndromes, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin lymphoma and non-Hodgkin 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 one embodiment of the invention, the tumor is selected from the group consisting of a B cell tumor, a T cell tumor, a bone marrow/monocyte tumor. Preferably B-or T-cell Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), non-Hodgkin's lymphoma (NHL) and Multiple Myeloma (MM), nasopharyngeal carcinoma, lung carcinoma.

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

The term "inflammation" as used herein includes acute inflammation and also chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

Preferably, the subject of the present invention, cell line or primary cell culture, tissue or organ or culture thereof, cannot be developed into animal, wherein the cell is not stem cell or fertilized egg cell, the cell can be somatic cell, lymphocyte (preferably T cell or B cell), tumor cell, etc., and the tissue can be spleen, lymph node, bone marrow, tumor or culture thereof, etc.

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 the target site of human SIGLEC15, and can accelerate the development process of new drugs and save time and cost. Provides effective guarantee for researching the function of SIGLEC15 protein and screening related disease drugs.

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

The humanized SIGLEC15 protein comprises a part derived from human SIGLEC15 protein. Wherein, the 'human SIGLEC15 protein' is the same as the 'whole human SIGLEC15 protein', namely the amino acid sequence of the protein is consistent with the full-length amino acid sequence of the human SIGLEC15 protein. The part of the human SIGLEC15 protein is a continuous or alternate amino acid sequence of 5-328 (preferably 10-216) which is consistent with the amino acid sequence of the human SIGLEC15 protein or has more than 70 percent of homology with the amino acid sequence of the human SIGLEC15 protein.

The humanized SIGLEC15 gene comprises a part derived from a human SIGLEC15 gene and a part derived from a non-human SIGLEC15 gene. Wherein, the 'human SIGLEC15 gene' is the same as the whole 'human SIGLEC15 gene', namely the nucleotide sequence of the 'human SIGLEC15 gene' is consistent with the full-length nucleotide sequence of the human SIGLEC15 gene. The 'part of the human SIGLEC15 gene' is a continuous or spaced nucleotide sequence of 20-18420bp (preferably 20-2948bp or 20-1893bp) which is consistent with the human SIGLEC15 gene or has more than 70 percent of homology with the human SIGLEC15 gene.

The "xx to xxx exons" or all of the "xx to xxx exons" in the present invention include nucleotide sequences of exons and introns therebetween, for example, the "2 to 3 exons" include all nucleotide sequences of exon 2, intron 2 to 3, and exon 3.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 2-3" means an intron between exon 2 and exon 3.

The "part of an exon" as referred to in the present invention means that several, several tens or several hundreds of nucleotide sequences are identical to all exon nucleotide sequences continuously or intermittently. For example, the portion of exon 2 of the human SIGLEC15 gene comprises consecutive or spaced nucleotide sequences of 5-60bp, preferably 10-22bp, identical to the nucleotide sequence of exon 2 of the human SIGLEC15 gene. In a specific embodiment of the invention, the "part of exon 2" contained in the "humanized SIGLEC15 gene" comprises at least the start codon to the stop codon of the amino acid sequence encoded by exon 2.

The "locus" of the present invention represents a position occupied by a gene on a chromosome in a broad sense, and represents a DNA fragment of a certain gene in a narrow sense, that is, a gene or a part of a gene. For example, the "SIGLEC 15 locus" refers to a DNA fragment of any one of exons 1 to 6 of 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 introns therebetween, or all or part of one or two or more thereof, 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, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the sign, symptom, or the like of a disease or pathological state after the disease has begun to develop.

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

In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as a muridae. 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 the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the cricotes (for example of the crimyspaidae (the hamsters, the new world rats and the rats, the mice, the new world of the rats, the rats. In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In a particular embodiment, the non-human animal is a rodent, a mouse selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10 Sn, C57BL/10Cr and C57BL/Ola C57BL, C5475, A/Br, CBA/Ca, CBA/CBA, CBA/4/Ill, CBNOSc/J, CBA/background strain, PrNORGD, and PrNORG 2 strain.

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

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

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

All patents and publications mentioned in this specification are herein incorporated by reference in their entirety. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention.

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

Drawings

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

FIG. 1: schematic comparison of 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 diagram one (not to scale);

FIG. 4: SIGLEC15 gene targeting strategy diagram two (not to scale);

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

FIG. 6: genotype identification of F0 mouse, in which WT was a wild-type control, H₂O is water control, F0-01, F0-02, F0-03, F0-04 and F0-05 are mouse numbers;

FIG. 7 is a schematic view of: genotype identification of F1 mouse, in which WT was a wild-type control, H₂O is water control, PC is positive control, F1-01, F1-02, F1-03 and F1-04 are mouse numbers;

FIG. 8: f1 Southern Blot detection results, 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: the result of detecting humanized SIGLEC15 mRNA, wherein +/+ is wild type C57BL/6 mouse, H/H is SIGLEC15 gene humanized homozygote mouse, H₂O is water control;

FIG. 10: the identification result of the genotype of the SIGLEC15 gene knock-out mouse, wherein WT is a wild type control, H₂O is water control, KO-01 is mouse number;

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

FIG. 12: mice with humanized homozygote of SIGLEC15 gene are inoculated with mouse colon cancer cells MC38 which over-express human SIGLEC15 protein subcutaneously, and are given with anti-human SIGLEC15 antibody AB1 or anti-mouse PD-1 antibody AB2, and the weight measurement results of each group of mice in the experimental period are obtained;

FIG. 13 is a schematic view of: the SIGLEC15 gene humanized homozygote mouse is subcutaneously inoculated with mouse colon cancer cells MC38 over-expressing human SIGLEC15 protein, and is given with anti-human SIGLEC15 antibody AB1 or anti-mouse PD-1 antibody AB2, and the tumor volume measurement results of each group of mice in the experimental period are obtained;

FIG. 14 is a schematic view of: the SIGLEC15 gene humanized homozygote mouse is subcutaneously inoculated with mouse colon cancer cells MC38 over-expressing human SIGLEC15 protein, and is given with anti-human SIGLEC15 antibodies Ab1 or Ab2, and the weight measurement results of each group of mice in the experimental period are obtained;

FIG. 15 is a schematic view of: the SIGLEC15 gene humanized homozygote mouse is subcutaneously inoculated with mouse colon cancer cells MC38 over-expressing human SIGLEC15 protein, and is given with anti-human SIGLEC15 antibody Ab1 or Ab2, and the weight measurement results of each group of mice in the experimental period are the tumor volume measurement results of each group of mice in the experimental period;

FIG. 16: flow detection results of leukocyte subpopulation proportion in spleen of C57BL/6 wild-type mice and SIGLEC15 gene humanized homozygote mice;

FIG. 17: flow detection results of T cell sub-population ratios in spleens of a C57BL/6 wild-type mouse and a SIGLEC15 gene humanized homozygote mouse;

FIG. 18 is a schematic view of: flow detection results of leukocyte subgroup ratios in lymph nodes of a C57BL/6 wild type mouse and a SIGLEC15 gene humanized homozygote mouse;

FIG. 19 is a schematic view of: flow detection results of the proportion of T cell subsets in lymph nodes of a C57BL/6 wild type mouse and a SIGLEC15 gene humanized homozygote mouse;

FIG. 20: flow detection results of leukocyte subpopulation ratios in blood of a C57BL/6 wild-type mouse and a SIGLEC15 gene humanized homozygote mouse;

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

FIG. 22: a flow analysis strategy diagram, wherein diagram a is a flow analysis strategy diagram of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells, Tregs cells, and diagram B is a flow analysis strategy diagram of DC cells, granulocytes (granulocytes), monocytes (monocytes), and macrophages (macrophages);

FIG. 23: alignment of the amino acid sequence of mouse SIGLEC15 (NP-001094508.1; SEQ ID NO:1) and human SIGLEC15 (NP-998767.1; SEQ ID NO: 2).

Detailed Description

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

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

BbsI, EcoRI, BamHI, BspHI, EcoNI enzymes were purchased from NEB under the respective accession numbers R0539S, R0101M, R0136M, R0517L, R0521L;

c57BL/6 mice and Flp tool mice were purchased from the rodent laboratory seed center, national institute for food and drug assay (TCM);

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

cas9mRNA source SIGMA, cat number CAS9MRNA-1 EA;

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

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

fixable visual Dye eFluor 506 was purchased from eBioscience, cat # 65-0866-14;

APC/Cy7 anti-mouse F4/80 was purchased from Biolegend, cat # 123118;

v450 Rat Anti-mouse CD11b from BD horizons, cat # 560455;

anti-mouse MHC II (I-A/I-E) Super Bright 600 from eBioscience, cat # 63-5321-80;

Anti-Mo CD206(MMR) eBioscience PE/cyanineTM7 from eBioscience, cat # 25-2061-80;

Alexa

647-conjugated affinity peptide F (ab') 2Fragment Goat Anti-Human IgG, Fc γ Fragment Specific from Jackson Immuno Research, cat # 109-;

Recombinant Murine M-CSF was purchased from PeproTech, cat # 315-02;

atture NxT Flow Cytometer is available from Thermo Fisher, model Atture NxT

Brilliant Violet 510TManti-mouse CD45 was purchased from Biolegend, cat # 103138;

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody was purchased from Biolegend under accession number 108426; brilliant Violet 421TManti-mouse CD4 was purchased from Biolegend, cat # 100438;

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

PE anti-mouse CD8a Antibody from Biolegend, cat # 100708;

PE/CyTM7Mouse anti-Mouse NK1.1 from BD Pharmingen, cat # 552878;

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

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

PerCP/Cy5.5 anti-mouse TCR β chain was purchased from Biolegend, cat # 109228.

Example 1 preparation of humanized mouse with SIGLEC15 Gene

The comparative scheme 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)) and 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; a schematic diagram of the alignment of human murine protein sequences is shown in FIG. 23.

To achieve the object of the present invention, a nucleotide sequence encoding human SIGLEC15 protein may be introduced at the endogenous SIGLEC15 locus of a mouse, so that the mouse expresses human or humanized SIGLEC15 protein. Specifically, a specific mouse SIGLEC15 gene sequence can be replaced by a DNA sequence of a human SIGLEC15 gene at an endogenous SIGLEC15 locus of a mouse through a gene editing technology, for example, a sequence of about 1.9kb including at least a mouse SIGLEC15 gene is replaced by a corresponding human DNA sequence to obtain a humanized SIGLEC15 locus (a schematic diagram is shown in fig. 2), so that humanized modification of a mouse SIGLEC15 gene is realized.

The targeting strategy as shown in FIG. 3 was further designed, in which the targeting vector contained a 5 'homology arm, a 3' homology arm, and an A fragment containing the sequence of the human SIGLEC15 gene on one side. Wherein the 5' homology arm (SEQ ID NO: 3) and the N CBI accession number are identical to the nucleotide sequence 78049252-78053412 of NC-000084.6; the 3' homology arm (SEQ ID NO: 4) is identical to the nucleotide sequence at positions 78042522-78046612 of NCBI accession number NC-000084.6; the sequence of the human SIGLEC15 gene (SEQ ID NO: 5) was identical to the nucleotide sequence at positions 45837067-45838959 of NCBI accession NC-000018.10. The connection between the downstream of the human SIGLEC15 sequence in the A fragment and the mouse is designed to be 5' -ccgctacacgtgtacggccgccaacagcctgggcc gctccgaggccagcgtctacctgttccgcttccacggcgcccccg-3' (SEQ ID NO: 6), wherein 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 first targeting vector also comprises a resistance gene for positive clone screening, namely a neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the connection between the 5' end of the Neo-box and the mouse is designed as

Wherein the last "t" of the sequence "gtact" is the last nucleotide of the mouse, sequence

The first "a" of (a) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo box and the mouse is designed as

Therein, the sequence "GCCGC"last" C "of" is the last nucleotide of the Neo cassette, sequence

"t" of (a) is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin A subunit coding gene (DTA)) is constructed at the downstream of the 3' homologous arm of the recombinant vector. The mRNA sequence of the humanized mouse SIGLEC15 after being transformed is shown as SEQ ID NO: 9, the expressed protein sequence is shown as SEQ ID NO: shown at 10.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. The constructed targeting vector is sent to a sequencing company for sequencing verification after being preliminarily verified by enzyme cleavage. And (3) performing electroporation transfection on the targeting vector which is verified to be correct by sequencing into embryonic stem cells of a wild mouse, screening the obtained cells by using a positive clone screening marker gene, and detecting the integration condition of an exogenous gene by using PCR (polymerase chain reaction) to screen out correct positive clone cells. The selected correctly positive clone cells (black mice) are introduced into the separated blastocyst according to the technology known in the art (white mice), the obtained chimeric blastocyst is transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mice), and F0 generation chimeric mice (black and white alternating) can be produced. F1 generation mice are obtained by backcrossing F0 generation chimeric mice with wild type mice, and F1 generation hybrid mice are mutually mated to obtain F2 generation homozygous child mice. Alternatively, the positive mice and Flp tool mice can be mated to remove the positive clone screening marker gene, and then mated with each other to obtain SIGLEC15 gene humanized homozygote mice.

In addition, a CRISPR/Cas system can be introduced for gene editing, so that humanized transformation of the mouse SIGLEC15 gene is realized. Designing the targeting strategy as shown in FIG. 4, which shows that the targeting vector II contains the homologous arm sequences of the upstream and downstream of the mouse SIGLEC15 gene and the human SIGLEC15 genomic DNA sequence, wherein the upstream homologous arm sequence (5 'homologous arm, SEQ ID NO: 11) is identical to the nucleotide sequence at position 78049252 and 78050600 of NCBI accession No. NC-000084.6, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 12) is identical to the nucleotide sequence at position 78600042 and 78047346 of NCBI accession No. NC-000084.6; the sequence of the human SIGLEC15 gene is identical to that of SIGLEC15 gene of fragment A in FIG. 3. The mRNA sequence and the protein sequence of the reconstructed humanized mouse SIGLEC15 are respectively compared with those of SEQ ID NO: 9 and SEQ ID NO: 10 are identical.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. The constructed targeting vector is sent to a sequencing company for sequencing verification after being preliminarily verified by enzyme cleavage. The correctly sequenced targeting vector was used for subsequent experiments.

The target sequence determines the targeting specificity of the sgRNA and the efficiency of Cas9 to induce cleavage of the gene of interest. Therefore, efficient and specific target sequence selection and design are a prerequisite for constructing sgRNA expression vectors. sgRNA sequences that recognize the target site were designed and synthesized. The target sites are located on

exons

2 and 4 of the SIGLEC15 gene, and the target site sequences of the sgRNAs on SIGLEC15 are 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 assay results

The activity of multiple sgrnas is detected by using a UCA kit, and the sgrnas have different activities as shown in the results, and the detection results are shown in table 1 and fig. 5. sgRNA2 and sgRNA12 were selected for subsequent experiments in combination with the activity, targeting location, and sequence specificity of each sgRNA. The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide and a reverse oligonucleotide (see table 2), and after annealing, the annealed products are respectively connected to pT7-sgRNA plasmids (plasmids are firstly linearized by BbsI), so as to obtain expression vectors pT7-SIGLEC15-2 and pT7-SIGLEC 15-12.

Table 2 sgRNA2 and sgRNA12 forward and reverse oligonucleotide sequences

pT7-sgRNA vector A fragment DNA (SEQ ID NO: 37) containing the T7 promoter and sgRNA scafffold was synthesized by a plasmid synthesis company, and was linked to a backbone vector (Takara, Cat. No. 3299) by digestion with EcoRI and BamHI in sequence, and sequencing by a professional sequencing company revealed that the objective plasmid was obtained.

Taking the fertilized egg of the C57BL/6 wild-type mouse at the prokaryotic stage, and injecting the in vitro transcription products of pT7-SIGLEC15-2 and pT7-SIGLEC15-12 plasmids (using an Ambion in vitro transcription kit and performing transcription according to the instruction method) and the targeting vector and Cas9 mRNA into the cytoplasm or nucleus of the fertilized egg of the mouse after being premixed by using a microinjector. Microinjection of fertilized eggs was performed according to the method in the manual of experimental manipulation of mouse embryos (third edition), published by chemical industry, 2006, and the injected fertilized eggs were transferred to a culture medium for short-term culture and then transplanted into the oviduct of a recipient mother mouse for development, and the obtained mice (generation F0) were crossed and selfed to expand the population number and establish a stable SIGLEC15 gene mutant mouse strain.

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

TABLE 3 PCR detection primer sequences and target fragment lengths

SIGLEC15 gene humanized mice identified as positive for F0 were mated with C57BL/6 wild-type mice to give F1 generation mice. The PCR primer pairs WT-F/WT-R and WT-F/Mut-R (Table 3) were used to genotype mice for the F1 generation, and exemplary results are shown in FIG. 7, which shows 4 mice numbered F1-01 through F1-04 as positive mice. The 4 mice identified as positive by F1 PCR were tested for Southern blot to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting the genome with BspHI enzyme or EcoNI enzyme, transferring membrane and hybridizing. Probes 5 ' Probe and 3 ' Probe are located on the left side of the 5 ' homology arm and on the human sequence, and the specific lengths of the probes and the target fragment are shown in Table 4.

TABLE 4 length of specific probes and target fragments

Restriction enzyme	Probe needle	Wild type 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 assay results are shown in FIG. 8. The results of 5 'Probe and 3' Probe were combined and further verified by sequencing that none of 4 mice numbered F1-01, F1-02, F1-03, F1-04 had a random insertion, confirming that these 4 mice were positive heterozygous mice and that there was no random insertion. The obtained positive heterozygous mice of the F1 generation are mutually mated to obtain humanized homozygous mice of the SIGLEC15 gene of the F2 generation. This shows that the method can construct the SIGLEC15 gene humanized gene engineering mouse which can be stably passaged and has no random insertion.

The expression of humanized SIGLEC15 mRNA in the positive mice obtained can be confirmed by conventional detection methods, e.g. using RT-PCR. Selecting 1 female wild type C57BL/6 mouse and 1 SIGLEC15 gene humanized homozygote mouse prepared in the embodiment, taking ovary tissues after depollution to detect the mRNA expression condition of humanized SIGLEC15, wherein the detection result (shown in figure 9) shows that only mouse SIGLEC15 mRNA is detected in the wild type C57BL/6 mouse (shown in figure 9A); only humanized SIGLEC15 mRNA was detected in the humanized homozygote mouse of SIGLEC15 gene (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’

in addition, since the cleavage of Cas9 causes double strand break of genomic DNA, insertion/deletion mutations are randomly generated by a repair mode of chromosomal homologous recombination, and it is possible to obtain a knockout mouse with loss of SIGLEC15 protein function. For this purpose, a pair of primers was designed for detecting knockout mice, wild type mice should have no PCR band, knockout mice should have 1 PCR band, and the product length should be about 569bp, and the results are shown in FIG. 10, wherein the mice with number KO-01 were further verified as SIGLEC15 knockout mice by sequencing. The primers are respectively positioned at the left side of a 5 'end target site and the right side of a 3' end target site, and have the following sequences:

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

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

The expression of the humanized SIGLEC15 protein in SIGLEC15 gene humanized mice and SIGLEC15 gene knockout mice can be detected by using a conventional method, such as flow cytometry and the like. Specifically, wild type C57BL/6 mice, 1 mouse of humanized homozygous for SIGLEC15 gene and 1 mouse of SIGLEC15 gene prepared in this example were selected, killed by cervical dislocation, bone marrow cells were harvested, erythrocytes were lysed, induced to differentiate into macrophages using M-CSF-containing medium, blocked antibodies Purified-mouse CD16/32, dead cells identified as the Dye, Purified visual Dye eFluor TM506, macrophage-labeled antibodies V450 Rat Anti-mouse CD11b, APC/Cy7 Anti-mouse F4/80, macrophage-labeled antibodies Anti-Mo CD206(MMR) E-bioscience PE/Cyaninetm7, macrophage-labeled antibodies M1, MHC 4624E-bioscience Anti-mouse (I-A/I-E) II, Super Bri 600, and MHC 4624-IgG 1 were obtained by conventional Anti-SIGLEC 1 method, The secondary antibody Alexa Fluor 647-conjugated Affinipure Goat Anti-Human IgG was subjected to recognition staining and then flow detection, Human IgG1 was used as an isotype control, and the detection results are shown in FIG. 11. As can be seen from the figures, the expression of SIGLEC15 protein was not detected on the surface of macrophages induced to differentiate by SIGLEC15 gene knock-out mice (fig. 11D, 11E, 11F), the expression of SIGLEC15 protein was detected on the surface of macrophages induced to differentiate by SIGLEC15 gene humanized homozygote mice (fig. 11A, 11B, 11C) and wild type C57BL/6 mice (fig. 11G, 11H, 11I), and the M2 type was higher than the M1 type. Combining the RT-PCR results of FIG. 9, the SIGLEC15 gene humanized homozygote mouse detected only humanized SIGLEC15 protein, while the wild-type C57BL/6 mouse detected only murine SIGLEC15 protein.

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

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared SIGLEC15 gene humanized mouse. For example, in the above example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40 and IL 10, or can be used to obtain a two-gene or multi-gene modified mouse model of SIGLEC15 and other gene modifications by using isolated mouse ES embryonic stem cells and gene recombination targeting technology on the basis of SIGLEC15 humanized mice. The SIGLEC15 mouse homozygote or heterozygote obtained by the method can also be mated with other gene modified homozygote or heterozygote mice, the offspring of the mouse is screened, the humanized SIGLEC15 and other gene modified double-gene or multi-gene modified heterozygote mice can be obtained with a certain probability according to Mendel genetic rule, then the heterozygote is mated with each other to obtain double-gene or multi-gene modified homozygote, and the double-gene or multi-gene modified mice can be used for in vivo efficacy verification of targeted human SIGLEC15 and other gene regulators and the like.

Example 3 SIGLEC15 Gene human mouse model drug effect experiment I

8 weeks from example 1 were selectedAged female SIGLEC15 gene humanized homozygous mice were subcutaneously inoculated with mouse colon cancer cells MC38 (5X 10) overexpressing human SIGLEC15 protein⁵One), the tumor volume is about 100 mm³Then, the tumor volume was counted as control group or treatment group (n-5/group). The control group was injected with phosphate buffered saline (PB S), and the treatment group used various doses of anti-human SIGLEC15 antibody AB1 or anti-mouse PD-1 antibody AB2 (both obtained by immunizing mice using conventional methods, see Janeway' S immunology (9th Edition)). The administration method comprises the following steps: intraperitoneal (i.p.) injection, the administration is started on the same day, 2 times per week and 6 times in total. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of a single mouse reached 3000mm³Euthanasia is performed. Specific groups and dosing are shown in table 5. The results of measurement of the body weight and tumor volume of the mice in the experimental period are shown in fig. 12 and fig. 13, respectively.

TABLE 5 grouping and dosing

The main data and analysis results of each experiment are listed in table 6, and specifically 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 (TGI) at Tumor Growth Inhibition value, TGI _TV) And the statistical difference in tumor volume (P-value) between the treated and control mice.

TABLE 6 tumor volume, survival and tumor inhibition

On the whole, in the experimental process, the health state of each group of animals is good, the weight average of the animals in all treatment groups (G2, G3 and G4) and the control group (G1) shows a growing trend (figure 12), which shows that the animals have good tolerance to the anti-human SIGLEC15 antibody AB1 and the anti-mouse PD-1 antibody AB2, and both AB1 and AB2 do not have obvious toxic effects on the animals, so that the safety is good. From tumor volume measurementsIn each period of the experiment, the tumor volume in the treated group was smaller than that in the control group (fig. 13); on day 20 post-dose, the tumor volumes of mice in treatment groups G2, G3, G4 were 1643. + -.278 mm, respectively³、1398±168mm³And 1277 + -274 mm³1735 + -173 mm from the control group³Compared with the reduction in tumor volume of the anti-mouse PD-1 antibody AB2 treated group (G4) which is smaller than that of the anti-human SIGLEC15 antibody treated group, the different antibodies show different tumor inhibition effects in SIGLEC15 gene humanized mice. For the anti-human SIGLEC15 antibody AB1 treatment group, the 30mg/kg dose group (G3) had less than 10mg/kg tumor volume (G2), indicating that different doses of AB1 had particularly different tumor suppression effects in SIGLEC15 gene humanized mice. The experimental results prove that the SIGLEC15 gene humanized mouse prepared by the method can be used for screening in-vivo efficacy detection of an anti-human SIGLEC15 antibody, can be used as a living body substitution model for in-vivo research, and is used for screening, evaluating and treating a human SIGLEC15 signal channel regulator.

Example 4 pharmaceutical experiment of SIGLEC15 Gene humanized mouse model

The 8-week-old female SIGLEC15 gene humanized homozygote mouse prepared in example 1 was selected and subcutaneously inoculated with mouse colon cancer cell MC38 (5X 10) overexpressing human SIGLEC15 protein⁵One), the tumor volume is about 100 mm³Then, the tumor volume was counted as control group or treatment group (n-5/group). The control group was injected with phosphate buffered saline (PB S) and the treatment group with a different anti-human SIGLEC15 antibody Ab1 or Ab2 (both obtained by immunizing mice using conventional methods, see Janeway' S immunology (9th Edition)). The administration mode comprises the following steps: p. administration was started on the day of the group, 2 times per week for a total of 6 times. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of 3000mm was achieved in a single mouse³Euthanasia is performed. Specific groups and dosing profiles are shown in table 7. The results of the measurement of the mouse body weight and the tumor volume in the experimental period are shown in fig. 14 and fig. 15, respectively.

TABLE 7 grouping and dosing

The main data and analysis results of each experiment are listed in table 8, and specifically 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 rate (TGI Growth Inhibition value, TGI) _TV) And the statistical difference in tumor volume (P-value) between the treated and control mice.

TABLE 8 tumor volume, survival and tumor inhibition

On the whole, in the experimental process, the health state of each group of animals is good, the weight average of all the animals in the treatment groups (G2 and G3) and the control group (G1) shows an increasing trend (figure 14), which indicates that the tolerance of the animals to the anti-human SIGLEC15 antibodies Ab1 and Ab2 is good, and both Ab1 and Ab2 do not have obvious toxic effects on the animals, so that the safety is good. From the tumor volume measurements, the tumor volumes in the treated groups were smaller than those in the control group at each stage of the experiment (fig. 15); on day 25 post-dose, the tumor volumes of mice in treatment groups G2 and G3 were 1801 + -181 mm, respectively³And 1220 + -234 mm³2528. + -.377 mm compared with the control group³Compared with the above-mentioned antibody, the tumor volume of the Ab 2-treated group (G3) was smaller than that of the Ab 1-treated group (G2), indicating that Ab2 has better tumor inhibition effect than Ab.

The results prove that the SIGLEC15 gene humanized mouse 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 the human SIGLEC15 signal channel regulator.

Example 5 immunotyping of SIGLEC15 Gene in human mice

And detecting the leukocyte immunoassay condition of the SIGLEC15 gene humanized mouse in vivo by adopting flow cytometry. Specifically, 3 mice each of wild-type C57BL/6 and SIGLEC15 gene humanized homozygote mice prepared in example 1 were selected, and spleen, lymph node and blood samples were taken after neck-off euthanasia and single cell suspensions were prepared for flow cytometry. The flow analysis strategy diagram is shown in FIG. 22. The results of detecting the leukocyte subtypes and the T-cell subtypes in the spleen are shown in FIGS. 16 and 17, respectively, the results of detecting the leukocyte subtypes and the T-cell subtypes in the lymph node are shown in FIGS. 18 and 19, respectively, and the results of detecting the leukocyte subtypes and the T-cell subtypes in the blood are shown in FIGS. 20 and 21, respectively. As can be seen from the figures, the leukocyte subtypes such as T cells, B cells, NK cells, DC cells (fig. 14), granulocytes (granuclyte), monocytes (Monocyte), macrophages (Macrophage) in spleen, lymph node and blood samples of the SIGLEC15 gene humanized homozygote mice were consistent with those of C57BL/6 wild-type mice (fig. 16, fig. 18, fig. 20), and the percentage of T cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells were consistent with those of C57BL/6 wild-type mice (fig. 17, fig. 19, fig. 21), indicating that the humanized modification of the SIGLEC15 gene did not affect the differentiation, development and distribution of leukocytes in the mice in the lymphoid tissues.

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

It should be noted that the various features described in the foregoing detailed description may be combined in any suitable manner without contradiction, and in order to avoid unnecessary repetition, various possible combinations will not be further described in the present invention.

In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

Sequence listing

<110> Baiosai Picture (Beijing) pharmaceutical science and technology, Inc

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

<130> 1

<150> 2021102674259

<151> 2021-03-12

<150> 2021104761497

<151> 2021-04-29

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

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

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

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

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

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

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

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

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

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

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

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

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Ser Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu

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Leu Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn

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

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

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

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

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

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

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Val Ile His His Glu Lys

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

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

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

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

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

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

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

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

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

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

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gtctcagtca gggagtcatg cctgacactt tttagcaggc actcacgggc tgccagaggt 60

gatggtaaat actaccagaa gccaggagct aggagctctg ccaaagcaat gtctttgttc 120

tctggtcaat acctgctaag gacttctcgc cttctgcctt gagtcctcag ggtggcccct 180

gaggattctg cctaagacag agcatctctc tcttttgaac ccactcagca catgagcagg 240

aagcactgta tctgctaatg gaagctaaaa cagagcctct gaatgaaggg tctcagtgct 300

cccttaggct cctcgagcca tctctacctg cgccatccat ttagagcaca ctttagaata 360

tttcacagca ggatgaagat ctgtgaggaa ttcacagagg catgctgcct gatgttagag 420

cgactaagtt aagcctctat taccattttc tcctggcaaa gccaactgaa gcgatatgtc 480

accagcatgc ctccgctggc agcagagccc tcacctacct gggatcttgg cctcctctga 540

ctgcttttgg cattgctggg gtatttcaga gttcctcata gagtgggtaa ctaggggaat 600

ttcccccaaa cttcaggaca aaaaggtatt gttttttctt gcaagccctc tccccctttg 660

ggggcttttc cattgaaaac cttaaatgtt actctcacaa gctgcaggcc tgccgggcaa 720

ggagcaggtg aggaggcagt ggagaagcaa acctgtgtga gcttctgaat gtagcatgta 780

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

gaggtagaga atgactcaca atgccccccc ccccgccttc ttcagcagtc tggcctccac 1320

agcccctcat gcattctctg ccccctctga gagccaggct tcatgcttgc ctttgccagt 1380

tttttcgaga cagggtccca agtatcttta actccctatg tagacccagg ctttggtggc 1440

gcatgcgcgc ctttaatccc agcactcggg aggcagaggc aggtggattt ctgagttcaa 1500

ggccagtctg gtctacagag tgagttccag gacagccagg gctacacaga gaaaccctgt 1560

ctcaaaaaaa caaaaaaaca aaaaaaaaac aaaaaaacca aaaaccaaaa cccaaaaccc 1620

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

atggaaggga aagggggatg ggggcaggtg agccatcctc tcccatcagg aaaaggtaat 2880

gagtccaaga gccaccataa tgagcttctg caagcggcag ctgggacctg tacatgttcc 2940

cgaaagcagt ggcagttccg gtgtggttct gtcgatccac aggtgtatgg gctctacagg 3000

agatggacta gcagcggtgg cttgagggca ttccaaccca agaagtcatt gaggctcact 3060

gaccacaaag agcactgggc ttagttagca tcaggaagaa ctggcttttc tctttccttg 3120

cctggatgtt ggccttcatg tacacatcta taacactctc tgccctgtct ttctcctctt 3180

gggtgctttc aggacagatg gcatggtggt gtggtataaa taaagggact gtgtataata 3240

caagcccaat tactccctgt aaaggtccct tttgtgcact gtgccccagg gcactgactg 3300

ggcaggctca accgcctgcc tcctcttggg gaggtaccac ccctggcata accttctgag 3360

actgaacacc tccataaaca cagcaacaca gagcccgatg gtttgccagc actgggtgaa 3420

ctggaagcag ggctcagctt ccacaaccct tggctccgca ttctgaagaa tgtggttcac 3480

caggtggtgg gcaaagtgag gctggagcag aggtgtcaac ctgtgggccg cacgcccttt 3540

tgggggggag ggtgttgact gaccctttcc taggagtcac ctaagactac ctgcatatca 3600

gatatttata ttatgattca taacagtaac aaaattacac caatgaagta gcaacaaaaa 3660

taattttatg gttggggtca ccacaacatg aggaactgta ttaaagggtc gcagcattag 3720

gaaggctgag aaccactggt cttgaggtac aatgcagtgt gtacccacca tgttaaagta 3780

cccaccacga gggtctttct ctgcctttta agcagccagc caggacgcag ttcacctggg 3840

ctggctctga ttctaaagga acttgtaact ctactggtgt gcttcaaagc accgcccccc 3900

cccccccccc cagggtctgg gtttctcaac tacaaataaa gactgttagc ccttcaggtg 3960

aatcacagct tcatgccttt agggcgttct aatacaggct ctgggtccag gcaggtctga 4020

gtgggacgca gactttgcct caacatcgca gttactccaa ttatttaact tttctgcgcc 4080

tcagtgtatt aacatgtaac aggctatcta catttgtctc taggatccct tgtgaaaact 4140

agaagagacg cttcggggga t 4161

<210> 4

<211> 4091

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tccaagctgc tgtccctgac cttgatgggt catctgctga gcacccagaa aagggaggga 60

ccttctcaga gcctctcagt cttggccaac caataaggtt agcctccaac tctccaggga 120

cacaacgcag aatttgtctc tgggggcccc agctgccatc cttgctgtga ccctgccatt 180

cttgctggtt cctcaaactt atcaaacatg ttgtgtttca gggtccccag aaaggcagtt 240

gtgttctctc ttaatatccc caccccgcct ccccgaccgg ggtcaagtta accctatcat 300

gtatcaggtt aagttgctcc agctagcaag ccatctgctg cacatttcct ggggactgcc 360

tctcactgtg gggcggggca atgtgtgaag ctgacaggtt ggctggagac cactgtgcaa 420

attcacacct cctctttctc catgtccctc atacagatca cctggtcccc caggacaccc 480

ctccacggta cgtactcact gcccttcccc catctctgcc taccacattg gcccaggcat 540

gccactttct cagaactggg agttcacaga gaaaaagcag gcatagggca aggctagggt 600

agtaggctgg actagggcct ggatcatacc caaaatgccc tgcccctcca tccagcacac 660

cccagagtgg gccaggctga cctgttactg catccagtac acaaggagct tgctgtctat 720

gaccccaaaa gcgaatacca ctaagctgtg tcctcctgtt gcctcccagc tttctagaaa 780

ttagccccag aaggatgcca ctgtaggaag actcatcccc caggcttaat gctgactgct 840

tccaaacaaa atcaccaaga gcctggctta gatgaaagat ctagaaagac attaagccag 900

agtccactct ttcctcttca gtgtctaccc cagtaaggac ccttgttgag ggagtaagaa 960

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 humanized SIGLEC15 protein, wherein the humanized SIGLEC15 protein comprises all or a portion of human SIGLEC15 protein.

2. The humanized SIGLEC15 protein according to claim 1, wherein the humanized SIGLEC15 protein comprises all or part of the signal peptide, transmembrane region, cytoplasmic region and/or extracellular region of the human SIGLEC15 protein, preferably the humanized SIGLEC15 protein comprises all or part of the extracellular region of the human SIGLEC15 protein, further preferably the humanized SIGLEC15 protein comprises at least 50 contiguous amino acids of the extracellular region of the human SIGLEC15 protein.

3. The humanized SIGLEC15 protein of claim 1 or 2, wherein the amino acid sequence of the human SIGLEC15 protein contained in the humanized SIGLEC15 protein comprises any one of the following groups:

A) comprises all or part of the amino acid sequence from position 31 to 246 or from position 20 to 263 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 the amino acid sequence of SEQ ID NO. 2 from position 31 to 246 or 20 to 263;

C) comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID No. 2 from position 31 to 246 or 20 to 263 by NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by NO more than 1 amino acid; or

D) An amino acid sequence comprising one or more amino acid residues shown in positions 31-246 or 20-263 of SEQ ID NO 2, including substitutions, deletions and/or insertions;

Preferably, the amino acid sequence of the humanized SIGLEC15 protein comprises any one of the following groups:

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

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

c) an amino acid sequence comprising NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or NO more than 1 amino acid difference from the amino acid sequence set forth in SEQ ID No. 10; or

4. A humanized SIGLEC15 gene, wherein the humanized SIGLEC15 gene comprises a portion of the human SIGLEC15 gene.

5. The humanized SIGLEC15 gene of claim 4, wherein the humanized SIGLEC15 gene encodes the humanized SIGLEC15 protein of any one of claims 1-3.

6. The humanized SIGLEC15 gene according to claim 4 or 5, wherein the humanized SIGLEC15 gene comprises all or part of exon 1 to 6 of human SIGLEC15 gene, preferably comprises part of exon 2, all of exon 3 and part of exon 4, wherein part of exon 2 comprises at least 5bp of nucleotide sequence and part of exon 4 comprises at least 100bp of nucleotide sequence.

7. The humanized SIGLEC15 gene according to any one of claims 4-6, wherein the human SIGLEC15 gene contained in the humanized SIGLEC15 gene comprises any one of the following group:

(A) comprises all or part of the nucleotide sequence shown in SEQ ID NO. 5;

8. The humanized SIGLEC15 gene of any one of claims 4-7, wherein 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;

(d) Comprises a nucleotide sequence shown in SEQ ID NO. 9 and comprises one or more nucleotides in substitution, deletion and/or insertion.

9. A targeting vector, wherein said targeting vector comprises a portion of the human SIGLEC15 gene, preferably, the part of the human SIGLEC15 gene comprises all or part of exons 2 to 4 of the human SIGLEC15 gene, more preferably comprises part of the exon 2, all of the exon 3 and part of the exon 4, wherein the part of exon 2 comprises at least a 5bp nucleotide sequence and said part of exon 4 comprises at least a 100bp nucleotide sequence, more preferably, the targeting vector comprises a targeting sequence identical to the sequence shown in SEQ ID NO:5 or a nucleotide sequence having at least 85%, 90%, 95%, or at least 99% identity to a nucleic acid comprising SEQ ID NO:5, even more preferably, the targeting vector comprises the humanized SIGLEC15 gene of any one of claims 4-8;

preferably, the targeting vector further comprises a 5' arm; preferably, said 5' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 5' arm sequence has at least 90% homology with SEQ ID NO 3 or 11, or is as shown in SEQ ID NO 3 or 11; and/or, the targeting vector further comprises a 3' arm; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 3' arm sequence has at least 90% homology with SEQ ID NO. 4 or 12, or is as shown in SEQ ID NO. 4 or 12.

10. A construction method of a SIGLEC15 gene humanized non-human animal is characterized in that the non-human animal expresses human or humanized SIGLEC15 protein, or the genome of the non-human animal comprises all or part of human or humanized SIGLEC15 gene,

preferably, the humanized SIGLEC15 protein is the humanized SIGLEC15 protein of any one of claims 1 to 3, or the humanized SIGLEC15 gene is the humanized SIGLEC15 gene of any one of claims 4 to 8.

11. The method of claim 10, comprising introducing into the non-human animal SIGLEC15 locus all or part of a vector comprising exons 1 to 6 of the human SIGLEC15 gene; preferably, the non-human animal SIGLEC15 locus is introduced with a gene comprising part of exon 2, all of exon 3 and part of exon 4, wherein part of exon 2 comprises at least a 5bp nucleotide sequence and said part of exon 4 comprises at least a 100bp nucleotide sequence; more preferably, the non-human animal SIGLEC15 locus is introduced with a nucleotide sequence comprising at least 85%, 90%, 95% or at least 99% identity to SEQ ID NO. 5 or a nucleotide sequence comprising SEQ ID NO. 5.

12. The method of construction according to any one of claims 10-11, wherein said introducing is a substitution or insertion, preferably said introducing is a substitution of the SIGLEC15 locus in a non-human animal to a corresponding region in a non-human animal, further preferably a substitution of all or part of exons 2 to 4 of the SIGLEC15 gene in a non-human animal, more preferably a substitution of part of exon 2, all of exon 3 and part of exon 4 in a non-human animal.

13. The method of constructing a recombinant human SIGLEC15 vector according to any one of claims 10-12, wherein the human or humanized SIGLEC15 gene is regulated by endogenous regulatory elements in a non-human animal.

14. The method of constructing a non-human animal according to any one of claims 10 to 13, wherein the targeting vector of claim 9 is used for the construction of a non-human animal.

15. The method of constructing a recombinant human antibody according to any one of claims 10 to 14, which further comprises mating a SIGLEC15 gene-humanized non-human animal with another genetically modified non-human animal, in vitro fertilization or direct gene editing, and screening the mating or in vitro fertilization or direct gene editing to obtain a polygenetically modified non-human animal,

preferably, the other genes are at least one selected from PD-1, PD-L1, IL6, TNF, 41BB, CD40, IL17, TNFR2, IL4, IL33, TIGIT, OX40 and IL 10.

16. A cell, tissue or organ expressing a humanized SIGLEC15 protein according to any one of claims 1-3, wherein the cell, tissue or organ comprises the humanized SIGLEC15 gene according to any one of claims 4-8, or is derived from a non-human animal obtained by the construction method according to any one of claims 10-15.

17. Use of a cell, tissue or organ derived from a humanized SIGLEC15 protein according to any one of claims 1-3, a humanized SIGLEC15 gene according to any one of claims 4-8, a non-human animal obtained by the construction method according to any one of claims 10-15, or a cell, tissue or organ according to claim 16, the use comprising:

or,

the application in the aspects of screening, verifying, evaluating or researching the function of SIGLEC15, the signal mechanism of human SIGLEC15, a human-targeting antibody, a human-targeting drug, a drug effect, an immune-related disease drug and an anti-tumor or anti-inflammatory drug, screening and evaluating human drugs and drug effect research.