CN115010800A

CN115010800A - Construction method and application of PVRIG gene humanized non-human animal

Info

Publication number: CN115010800A
Application number: CN202210655015.6A
Authority: CN
Inventors: 张译夫; 姚佳维; 刘重慧
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-06-10
Filing date: 2022-06-10
Publication date: 2022-09-06
Also published as: WO2022258049A1

Abstract

The invention provides a humanized PVRIG protein, a humanized PVRIG gene, a targeting vector of the PVRIG gene, a humanized non-human animal of the PVRIG gene, a construction method and application thereof in the field of biological medicine.

Description

Construction method and application of PVRIG 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 PVRIG gene humanized non-human animal and application thereof in the field of biomedicine.

Background

Pvrig (pvr related immunoglobulin domain binding), also known as CD112R, belongs to poliovirus receptor-like proteins, and is a new class of IGSF (Ig superfamily) with T cell co-signaling function. Expressed predominantly in T cells and NK cells. Its receptor, CD112(PVRL2), is widely expressed on tumor cells, such as acute myelogenous leukemia, multiple myeloma, and epithelial cancer tumor cells. Blockade of CD112R-CD112 resulted in a significant increase in the number of IFN- γ producing NK cells, suggesting that blockade of the PVRIG signaling pathway could enhance human NK cell function.

At present, the mechanism of action and the effect between CD112 and CD112R are studied, but not completely understood. COM701 was the first PVRIG antibody approved by the FDA in the united states for entry into clinical trials. The COM701 targeting PVRIG can effectively stimulate tumor immune response of certain cancers, has synergistic effect with PD-1 and TIGIT antibodies, and can enhance T cell function and improve anti-tumor response. The PVRIG can be used as a new immune target, and can be used for treating tumors independently or in combination with other targets;

with the continuous development and maturation of genetic engineering technology, the replacement or substitution of animal homologous genes with human genes has been realized, and the development of humanized experimental animal models in this way is the future development direction of animal models. The gene humanized animal model is one animal model with normal or mutant gene replaced with homologous gene in animal genome and similar physiological or disease characteristics. The gene humanized animal not only has important application value, for example, the humanized animal model of cell or tissue transplantation can be improved and promoted by gene humanization, but also more importantly, the human protein can be expressed or partially expressed in the animal body due to the insertion of the human gene segment, and the gene humanized animal can be used as a target of a drug which can only recognize the human protein sequence, thereby providing possibility for screening anti-human antibodies and other drugs at the animal level. However, due to differences in physiology and pathology between animals and humans, coupled with the complexity of genes, for example, the human and mouse PVRIG proteins are 39% identical, how to construct an "efficient" humanized animal model for new drug development remains the greatest challenge.

In view of the complicated mechanism of action of PVRIG and the great application value in the field of tumor therapy, there is an urgent need in the art to develop a non-human animal model of PVRIG-related signaling pathway in order to further explore its relevant biological properties, improve the effectiveness of preclinical pharmacodynamic tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

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

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

In a specific embodiment of the invention, the humanized PVRIG protein comprises all or part of the signal peptide and/or the extracellular region of the human PVRIG protein, wherein the humanized PVRIG protein comprises all or part of the extracellular region of the human PVRIG protein, preferably the extracellular region of the human PVRIG protein comprising at least 20 consecutive amino acids, such as the extracellular region of the human PVRIG protein comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, further preferably the extracellular region of the human PVRIG protein comprising 130 amino acids; preferably, the human PVRIG protein extracellular region comprises 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids removed from the C-terminus, and further preferably, the human PVRIG protein extracellular region comprises 3 amino acids removed from the C-terminus; more preferably comprises SEQ ID NO:2 from position 40 to 169 or from position 40 to 172; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 40-169 or 40-172, comprising substitution, deletion and/or insertion of one or more amino acid residues; the humanized PVRIG protein comprises all or part of a human PVRIG protein signal peptide, preferably a human PVRIG protein signal peptide comprising at least 5 consecutive amino acids, such as a human PVRIG protein signal peptide comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 amino acids, further preferably a human PVRIG protein signal peptide comprising 39 amino acids, more preferably the signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 1-39 of SEQ ID No. 2 or comprises an amino acid sequence as depicted in positions 1-39 of SEQ ID No. 2.

Preferably, the humanized PVRIG protein comprises SEQ ID NO:2, 1-169; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 1-169 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 1-169 do not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, 1-169, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized PVRIG protein further comprises all or part of a non-human animal PVRIG protein. Preferably comprising SEQ ID NO:1, amino acid sequence as shown at position 164-234; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at position 164 and 234 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, position 164-234, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at position 164-234, including substitutions, deletions and/or insertions of one or more amino acid residues.

Preferably, the humanized PVRIG protein comprises an amino acid sequence encoded by all or part of a human PVRIG gene.

Preferably, the humanized PVRIG protein comprises all or part of the amino acid sequence encoded by exons 1 to 6 of a human PVRIG gene. Further preferably, all or part of an amino acid sequence encoded by any one, two, three or more, two or three or more consecutive exons among exons 1 to 6 is contained. Even more preferably, it comprises all or part of the amino acid sequence encoded by exons 2 to 4. Still further preferably, the amino acid sequence encoded by a part of exon 2, all of exon 3 and a part of exon 4 is contained, wherein the part of exon 2 at least comprises a nucleotide sequence of 50bp, for example at least comprises a nucleotide sequence of 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp, further preferably comprises a nucleotide sequence of 118 bp; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp. Most preferably, the humanized PVRIG protein comprises a sequence identical to SEQ ID NO: 5 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to an amino acid sequence encoded by SEQ ID NO: 5, or a pharmaceutically acceptable salt thereof.

Preferably, the humanized PVRIG protein further comprises an amino acid sequence derived from a non-human animal.

Preferably, said humanized PVRIG protein further comprises all or part of an amino acid sequence encoded by a non-human animal PVRIG gene, preferably all or part of an amino acid sequence encoded by exons 4 to 5, and even more preferably also all or part of an amino acid sequence encoded by exon 3.

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

A) SEQ ID NO: 11;

B) and SEQ ID NO: 11 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or

D) And SEQ ID NO: 11, comprising the amino acid sequence of substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the invention, the humanized PVRIG protein comprises at least 25 amino acid sequences identical to the corresponding amino acid sequence of human PVRIG, wherein the non-human animal expresses the humanized PVRIG protein.

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

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 ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a second aspect of the invention, there is provided a nucleic acid encoding the humanized PVRIG protein described above.

In a third aspect of the invention, there is provided a humanized PVRIG gene comprising a portion of a human PVRIG gene.

Preferably, said humanized PVRIG gene encodes a humanized PVRIG protein as described above.

Preferably, the humanized PVRIG gene comprises a nucleotide sequence encoding all or part of a human PVRIG protein. Further preferably comprises all or part of the nucleotide sequence encoding the signal peptide, the extracellular region, the transmembrane region and/or the cytoplasmic region of the human PVRIG protein, more preferably the humanized PVRIG gene comprises all or part of the nucleotide sequence encoding the signal peptide and/or the extracellular region of the human PVRIG protein, wherein said extracellular region comprises all or part of the extracellular region of the human PVRIG protein, preferably the extracellular region of the human PVRIG protein comprising at least 20 consecutive amino acids, such as the extracellular region of the human PVRIG protein comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, further preferably the extracellular region of the human PVRIG protein comprising 130 consecutive amino acids; preferably, the human PVRIG protein extracellular region comprises a C-terminal with 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids removed, and more preferably, the human PVRIG protein extracellular region comprises a C-terminal with 3 amino acids removed; more preferably comprises SEQ ID NO:2 from position 40 to 169 or from position 40 to 172; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 40-169 or 40-172, comprising substitution, deletion and/or insertion of one or more amino acid residues; the signal peptide comprises all or part of a human PVRIG protein signal peptide, preferably a human PVRIG protein signal peptide comprising at least 5 consecutive amino acids, such as a human PVRIG protein signal peptide comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 consecutive amino acids, further preferably a human PVRIG protein signal peptide comprising 39 consecutive amino acids, more preferably the signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 1-39 of SEQ ID No. 2 or comprises an amino acid sequence as set forth in positions 1-39 of SEQ ID No. 2.

Preferably, the humanized PVRIG gene comprises all or part of exons 1 to 6 of a human PVRIG gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 6 is contained. Even more preferably, all or part of exons 2 to 4 are included. Even more preferably, the part comprising exon 2, all exon 3 and part of exon 4, preferably further comprises intron 2-3 and/or intron 3-4, wherein the part comprising exon 2 comprises at least 50bp nucleotide sequence, for example at least 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp nucleotide sequence, further preferably 118bp nucleotide sequence; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp.

In one embodiment of the invention, the humanized PVRIG gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, said humanized PVRIG gene further comprises all or part of a non-human animal PVRIG gene. Further preferably comprises all or part of exon 4 to exon 5 of the PVRIG gene of the non-human animal, and further preferably further comprises the nucleotide sequence of part of exon 1 and/or part of exon 3.

In one embodiment of the invention, the humanized PVRIG gene comprises a sequence encoding SEQ ID NO:2, 1-169 nucleotide sequence. Or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence identity between positions 1-169; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from 1 to 169 nucleotides differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO:2, 1-169, including nucleotide sequences with substitutions, deletions and/or insertions of one or more nucleotides.

Preferably, said humanized PVRIG gene further comprises a nucleotide sequence encoding all or part of a non-human animal PVRIG protein.

In a specific embodiment of the invention, the humanized PVRIG gene further comprises a sequence encoding SEQ ID NO:1 nucleotide sequence at position 164-234; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:1 at position 164-; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:1, nucleotide sequence at position 164-234 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO:1, nucleotide sequence at position 164-234, including nucleotide sequences in which one or more nucleotides are substituted, deleted and/or inserted.

In a specific embodiment of the present invention, the nucleotide sequence of the humanized PVRIG gene comprises any one of the following group:

A) the transcribed mRNA is SEQ ID NO: 10;

B) the transcribed mRNA is identical to SEQ ID NO: 10 is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) the transcribed mRNA is identical to SEQ ID NO: 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide;

D) the transcribed mRNA has the sequence of SEQ ID NO: 10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted; or the like, or, alternatively,

E) comprises the amino acid sequence of SEQ ID NO: 6 and/or 7.

Preferably, the humanized PVRIG gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible. 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. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, Rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

In a fourth aspect of the present invention, there is provided a targeting vector comprising a donor nucleotide sequence, preferably, the donor nucleotide sequence comprises one of the following groups:

A) a nucleotide sequence encoding a human or humanized PVRIG protein;

B) all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and/or a cytoplasmic region of a human PVRIG protein, preferably all or part of a nucleotide sequence encoding an extracellular region and/or a signal peptide of a human PVRIG protein, further preferably a nucleotide sequence encoding at least 20 consecutive amino acids of an extracellular region of a human PVRIG protein, such as a nucleotide sequence comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, more preferably a nucleotide sequence encoding at least 5 consecutive amino acids of a signal peptide of a human PVRIG protein, such as a nucleotide sequence comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 consecutive amino acids, even more preferably a nucleotide sequence encoding a sequence of SEQ ID NO:2, 1-169 nucleotide sequence;

C) a nucleotide sequence of a human or humanized PVRIG gene; or the like, or, alternatively,

D) all or part of exons 1 to 6 of the human PVRIG gene, preferably all or part of a combination of any one, two, three or more, two or more consecutive exons, or three or more consecutive exons, of exons 1 to 6. Even more preferably, all or part of exons 2 to 4 are included. Still further preferably, the part of exon 2, the whole exon 3 and the part of exon 4 are included, wherein the part of exon 2 at least includes nucleotide sequence of 50bp, such as nucleotide sequence of 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp, further preferably, nucleotide sequence of 118 bp; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp; further preferred comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the targeting vector further comprises a 5 'arm (5' homology arm) and/or a 3 'arm (3' homology arm).

The 5 'arm is a DNA fragment homologous to the 5' end of the transition region to be changed, and is selected from 100-10000 nucleotides in length of genome DNA of the PVRIG gene of the non-human animal. Preferably, the 5' arm has at least 90% homology with NCBI accession number NC _ 000071.7. Further preferably, the 5' arm sequence is as shown in SEQ ID NO: 3, respectively.

The 3 'arm is a second DNA fragment which is homologous with the 3' end of the switching region to be changed and is selected from 100-10000 nucleotides in length of the genome DNA of the PVRIG gene of the non-human animal; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000071.7; further preferably, the 3' arm sequence is as set forth in SEQ ID NO: 4, respectively.

Preferably, the targeting vector further comprises SEQ ID NO: 6. 7, 8 and/or 9.

Preferably, the transition region to be altered is located on exons 1 to 5 of the PVRIG gene of a non-human animal, and more preferably on exons 1 to 3 of the PVRIG gene of a non-human animal.

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

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

In one embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (a conventional LoxP recombination system can also be selected). The number of the specific recombination systems is 2, and the specific recombination systems are respectively arranged at two sides of the resistance genes.

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

In a sixth aspect of the invention, there is provided a use of the targeting vector and/or the cell in PVRIG gene editing, preferably, the use includes but is not limited to knock-out, insertion or substitution.

In a seventh aspect of the invention, there is provided a non-human animal humanized with a PVRIG gene, said non-human animal expressing a human or humanized PVRIG protein.

Preferably, said non-human animal has reduced or absent expression of endogenous PVRIG protein.

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

Preferably, the non-human animal comprises a portion of a human PVRIG gene, more preferably a humanized PVRIG gene as described above.

Preferably, the part of the human PVRIG gene or the nucleotide sequence of the humanized PVRIG gene is operably linked to endogenous regulatory elements at the endogenous PVRIG locus in at least one chromosome.

Preferably, the genome of said non-human animal comprises all or part of exons 1 to 6 of the human PVRIG gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 6 is contained. Even more preferably, all or part of exons 2 to 4 are included. Even more preferably, the part comprising exon 2, all exon 3 and part of exon 4, preferably further comprises intron 2-3 and/or intron 3-4, wherein the part comprising exon 2 comprises at least 50bp nucleotide sequence, for example at least 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp nucleotide sequence, further preferably 118bp nucleotide sequence; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp.

In a specific embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding all or part of a human PVRIG protein. Preferably comprises all or part of the nucleotide sequence encoding the signal peptide, the extracellular region, the transmembrane region and/or the cytoplasmic region of the human PVRIG protein, further preferably the genome of said non-human animal comprises all or part of the nucleotide sequence encoding the signal peptide and/or the extracellular region of the human PVRIG protein, wherein said extracellular region comprises all or part of the extracellular region of the human PVRIG protein, preferably the extracellular region of the human PVRIG protein comprising at least 20 consecutive amino acids, such as the extracellular region of the human PVRIG protein comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, further preferably the extracellular region of the human PVRIG protein comprising 130 consecutive amino acids; preferably, the human PVRIG protein extracellular region comprises a C-terminal with 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids removed, and more preferably, the human PVRIG protein extracellular region comprises a C-terminal with 3 amino acids removed; more preferably comprises SEQ ID NO:2 from position 40 to 169 or from position 40 to 172; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 40-169 or 40-172, comprising substitution, deletion and/or insertion of one or more amino acid residues; the signal peptide comprises all or part of a human PVRIG protein signal peptide, preferably a human PVRIG protein signal peptide comprising at least 5 consecutive amino acids, such as a human PVRIG protein signal peptide comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 consecutive amino acids, further preferably a human PVRIG protein signal peptide comprising 39 consecutive amino acids, more preferably the signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 1-39 of SEQ ID No. 2 or comprises an amino acid sequence as set forth in positions 1-39 of SEQ ID No. 2.

In one embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding SEQ ID NO:2, 1-169 nucleotide sequence. Or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% nucleotide sequence identity; or, comprises a nucleotide sequence identical to a sequence encoding SEQ ID NO:2 from 1 to 169 nucleotides differing by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, comprising a polypeptide having the sequence encoding SEQ ID NO:2, 1 st to 169 th nucleotide sequence, including nucleotide sequences with one or more nucleotide substitutions, deletions and/or insertions.

According to some embodiments of the invention, the non-human animal further comprises additional genetic modifications selected from at least one of PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4, and CD 226.

According to some embodiments of the invention, the PVRIG gene and/or the further gene is homozygous or heterozygous for the endogenous replaced locus.

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

In an eighth aspect of the invention, a method for constructing the above non-human animal is provided, wherein the human or humanized PVRIG protein is expressed in vivo in the non-human animal.

Preferably, the humanized PVRIG protein comprises the humanized PVRIG protein described above.

Preferably, the genome of the non-human animal further comprises a humanized PVRIG gene, and the humanized PVRIG gene comprises the humanized PVRIG gene.

Preferably, the genome of the non-human animal further comprises the nucleic acid.

Preferably, the construction method comprises introducing a nucleotide sequence encoding a human PVRIG protein or a humanized PVRIG protein as described above into the PVRIG locus of the non-human animal.

Preferably, the construction method comprises introducing into the PVRIG locus of the non-human animal a nucleotide sequence comprising all or part of a nucleotide sequence encoding a signal peptide, a transmembrane region, a cytoplasmic region and/or an extracellular region of the human PVRIG protein.

Preferably, the construction method comprises introducing the nucleotide sequence comprising the humanized PVRIG gene described above into the PVRIG locus of the non-human animal.

Preferably, the construction method comprises introducing a partial nucleotide sequence of the human PVRIG gene into the PVRIG locus of the non-human animal.

Preferably, the partial nucleotide sequence of the human PVRIG gene comprises all or part of exons 1 to 6 of the human PVRIG gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 6 is contained. Even more preferably, all or part of exons 2 to 4 are included. Even more preferably, the part comprising exon 2, all exon 3 and part of exon 4, preferably further comprises intron 2-3 and/or intron 3-4, wherein the part comprising exon 2 comprises at least 50bp nucleotide sequence, for example at least 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp nucleotide sequence, further preferably 118bp nucleotide sequence; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp. Preferably, the partial nucleotide sequence of the human PVRIG gene comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the construction method comprises introducing into the PVRIG locus of the non-human animal a sequence comprising all or part of a nucleotide sequence encoding a human PVRIG protein. Further preferably, the constructing method comprises introducing into the PVRIG locus of the non-human animal a nucleotide sequence comprising all or part of a signal peptide encoding the human PVRIG protein, a transmembrane region, a cytoplasmic region and/or an extracellular region; more preferably, the construction method comprises introducing into the non-human animal PVRIG locus a nucleotide sequence comprising all or part of the signal peptide encoding the human PVRIG protein and/or an extracellular region, wherein the extracellular region comprises all or part of the extracellular region of the human PVRIG protein, preferably, the extracellular region of the human PVRIG protein comprising at least 20 consecutive amino acids, for example, the extracellular region of the human PVRIG protein comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, further preferably, the extracellular region of the human PVRIG protein comprising 130 consecutive amino acids; preferably, the human PVRIG protein extracellular region comprises a C-terminal with 0-5 (e.g., 0, 1, 2, 3, 4, 5) amino acids removed, and more preferably, the human PVRIG protein extracellular region comprises a C-terminal with 3 amino acids removed; more preferably comprises SEQ ID NO:2 from position 40 to 169 or from position 40 to 172; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 40-169 or 40-172, comprising substitution, deletion and/or insertion of one or more amino acid residues; the signal peptide comprises all or part of a human PVRIG protein signal peptide, preferably a human PVRIG protein signal peptide comprising at least 5 consecutive amino acids, such as a human PVRIG protein signal peptide comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 consecutive amino acids, further preferably a human PVRIG protein signal peptide comprising 39 consecutive amino acids, more preferably the signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 1-39 of SEQ ID No. 2 or comprises an amino acid sequence as set forth in positions 1-39 of SEQ ID No. 2.

In one embodiment of the invention, the method of construction comprises introducing a donor nucleotide sequence into the PVRIG locus of a non-human animal.

Preferably, the donor nucleotide sequence comprises one of the following groups:

A) a nucleotide sequence encoding a human or humanized PVRIG protein.

B) A nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and/or a cytoplasmic region of a human PVRIG protein, preferably all or part of a nucleotide sequence encoding an extracellular region and/or a signal peptide of a human PVRIG protein, further preferably a nucleotide sequence encoding at least 20 consecutive amino acids of an extracellular region of a human PVRIG protein, such as a nucleotide sequence comprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 131, 132, 133 consecutive amino acids, more preferably a nucleotide sequence encoding at least 5 consecutive amino acids of a signal peptide of a human PVRIG protein, such as a nucleotide sequence comprising at least 5, 10, 11, 12, 13, 14, 15, 20, 30, 35, 36, 37, 38, 39 consecutive amino acids, even more preferably a nucleotide sequence encoding a sequence of SEQ ID NO:2, 1-169 nucleotide sequence.

D) all or part of exons 1 to 6 of the human PVRIG gene, preferably all or part of exons 1 to 6 of the human PVRIG gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 6 is contained. Even more preferably, all or part of exons 2 to 4 are included. Even more preferably, the part comprising exon 2, all exon 3 and part of exon 4, preferably further comprises intron 2-3 and/or intron 3-4, wherein the part comprising exon 2 comprises at least 50bp nucleotide sequence, for example at least 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp nucleotide sequence, further preferably 118bp nucleotide sequence; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp. Preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including substitution, deletion and/or insertion of one or more nucleotides.

In one embodiment of the invention, the PVRIG locus of the non-human animal is introduced with a cDNA sequence comprising a sequence encoding a human PVRIG protein.

In a specific embodiment of the invention, the PVRIG locus of the non-human animal is introduced with a nucleic acid sequence comprising a sequence encoding a humanized PVRIG protein.

In a specific embodiment of the invention, the nucleotide sequence comprising the humanized PVRIG gene is introduced into the PVRIG locus of a non-human animal.

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

Preferably, the donor nucleotide sequence is regulated in the non-human animal by regulatory elements. Further preferably, the regulatory element may be endogenous or exogenous.

Preferably, the regulatory element includes, but is not limited to, an endogenous promoter.

In a specific embodiment of the invention, the endogenous regulatory element is from a non-human animal PVRIG gene. The exogenous regulatory element is from human PVRIG gene.

The introduction is replacement or insertion, specifically, the introduction of the PVRIG locus of the non-human animal is replacement of a corresponding region of the non-human animal, and preferably replacement of all or part of a nucleotide sequence encoding an endogenous PVRIG protein in the genome of the non-human animal. Further preferably, the nucleotide sequence encoding positions 1-163 of SEQ ID NO. 1 in the genome of the non-human animal is replaced.

Preferably, exon 2 of the PVRIG gene of the non-human animal is replaced, and further preferably, a portion further comprising exon 1 and/or a portion of exon 3 of the PVRIG gene of the non-human animal is replaced.

Preferably, said PVRIG gene and/or said further gene is homozygous or heterozygous for the endogenous replaced locus.

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

In a specific embodiment of the invention, the construction method comprises modifying the coding frame of the PVRIG gene of the non-human animal, and inserting the nucleotide sequence encoding the human or humanized PVRIG protein or the nucleotide sequence of the humanized PVRIG gene after the endogenous regulatory element of the PVRIG gene of the non-human animal. Wherein, the coding frame of the modified non-human animal PVRIG gene can adopt a functional region for knocking non-human animal PVRIG gene or adopts a sequence inserted so that the non-human animal PVRIG protein is not expressed or the expression is reduced or the expressed protein is not functional. Further preferably, the coding frame of the modified non-human animal PVRIG gene may be all or part of the nucleotide sequence from exon 1 to exon 5 of the non-human animal PVRIG gene.

In a specific embodiment of the present invention, the construction method comprises inserting a nucleotide sequence encoding a human or humanized PVRIG protein or a nucleotide sequence of a humanized PVRIG gene and/or an auxiliary sequence into an endogenous regulatory element of a PVRIG gene of a non-human animal. Preferably, the auxiliary sequence may be a stop codon, such that the PVRIG gene expresses the human PVRIG protein in a humanized animal model and does not express the non-human animal PVRIG protein. Further preferably, the helper sequence is WPRE and/or polyA.

In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the encoded human PVRIG protein (preferably part of the signal peptide and/or extracellular region of the human PVRIG protein) into the corresponding region of the PVRIG gene of the non-human animal.

In a specific embodiment of the present invention, the constructing method comprises that a portion of the human PVRIG gene (preferably exon 1 to 6 of the human PVRIG gene, more preferably exon 2 to 4 of the human PVRIG gene, even more preferably all or part of exon 2 to 4 of the human PVRIG gene, even more preferably a portion comprising exon 2, all of exon 3 and part of exon 4, preferably further comprising intron 2-3 and/or intron 3-4, wherein the portion of exon 2 comprises at least a nucleotide sequence of 50bp, such as at least a nucleotide sequence of 50bp, 70 bp, 100 bp, 110 bp, 115 bp, 116 bp, 118, 119 bp, 120 bp, 130 bp, 150 bp, 170 bp, 200 bp, 220 bp, 250 bp, 270 bp, 300 bp, 310 bp, 316bp, even more preferably 118bp, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, for example, at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, and more preferably, the nucleotide sequence comprising 38bp is inserted or substituted into the corresponding region of the PVRIG gene of the non-human animal (preferably exon 1 to 5, more preferably exon 1 to 3, and still more preferably part of exon 1, all of exon 2, and part of exon 3).

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

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector into a cell of a non-human animal, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal humanized with the PVRIG gene.

According to some embodiments of the invention, the building method further comprises: the PVRIG gene humanized non-human animal is mated with other gene modified non-human animals, in vitro fertilized or directly subjected to gene editing, and then screened to obtain the multi-gene modified non-human animal.

Preferably, the other gene is a non-human animal genetically modified with at least one of PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4, and CD 226.

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.

Preferably, the non-human animal further comprises genetic modifications of one or more of PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4, and CD 226.

Preferably, the non-human animal further expresses a human or humanized PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4 or CD226 protein.

In a ninth aspect of the invention, there is provided a cell, tissue or organ which expresses the above-described humanized PVRIG protein. Alternatively, the cell, tissue or organ is derived from the above-mentioned non-human animal, or a non-human animal obtained by the above-mentioned construction method. Preferably, the cell or cell line or primary cell culture comprises a cell or cell line or primary cell culture that can develop into an animal subject or that cannot develop into an animal subject.

In the tenth aspect of the invention, a tumor tissue after tumor bearing is provided, wherein the tumor tissue expresses the humanized PVRIG protein. Alternatively, the tumor tissue after tumor bearing is derived from the above non-human animal, or the non-human animal obtained by the above construction method.

The eleventh aspect of the present invention provides a non-human animal (including a non-human animal humanized with a PVRIG gene or a non-human animal modified with multiple genes) obtained by the above construction method.

In a twelfth aspect of the present invention, an animal model is provided, wherein the animal model is derived from the above non-human animal or the non-human animal obtained by the above construction method. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a thirteenth aspect of the present invention, there is provided a method of constructing an animal model, which is performed by using the above-described non-human animal, non-human animal or progeny thereof. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a fourteenth aspect of the present invention, there is provided an application of the above non-human animal and the non-human animal obtained by the above construction method in constructing an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a fifteenth aspect of the present invention, an application of the above non-human animal, the non-human animal obtained by the above construction method, or the above animal model in the preparation of a medicament for treating tumor or immune-related diseases is provided.

In a sixteenth aspect of the invention, there is provided a cell humanized of a PVRIG gene, said cell expressing a human or humanized PVRIG protein.

Preferably, the cell expresses the humanized PVRIG protein described above.

Preferably, the genome of said cell comprises part of the human PVRIG gene. More preferably, the cell comprises the humanized PVRIG gene described above. Preferably, the cells include cells that can develop into an individual animal or that cannot develop into an individual animal.

In a seventeenth aspect of the present invention, there is provided a method of constructing a cell as described above, said method comprising introducing a donor nucleotide sequence into the PVRIG locus of the cell.

A) a nucleotide sequence encoding a human or humanized PVRIG protein.

D) all or part of exons 1 to 6 of the human PVRIG gene, preferably all or part of a combination of any one, two, three or more, two or more consecutive exons, or three or more consecutive exons, of exons 1 to 6. Even more preferably, all or part of exons 2 to 4 are included. Even more preferably, the part comprising exon 2, all exon 3 and part of exon 4, preferably further comprises intron 2-3 and/or intron 3-4, wherein the part comprising exon 2 comprises at least 50bp nucleotide sequence, for example at least 50, 70, 100, 110, 115, 116, 117, 118, 119, 120, 130, 150, 170, 200, 220, 250, 270, 300, 310, 316bp nucleotide sequence, further preferably 118bp nucleotide sequence; preferably, the part of exon 2 comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2, and the part of exon 4 comprises at least 10bp, such as at least 10, 20, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 127bp, more preferably 38 bp. Further preferred comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the introduced PVRIG locus is a nucleotide sequence that replaces the corresponding region of the PVRIG gene of the cell, preferably the extracellular region of the endogenous PVRIG protein encoded in the genome. Further preferably, the nucleotide sequence encoding positions 1-163 of SEQ ID NO. 1 in the PVRIG gene of the cell is replaced.

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

Preferably, the construction of the cell is carried out using the targeting vector described above.

In an eighteenth aspect of the invention, there is provided a construct comprising the humanized PVRIG gene described above or a construct expressing the humanized PVRIG protein described above. Preferably, the construct may be a plasmid.

In a nineteenth aspect of the present invention, there is provided a cell comprising the above construct.

In a twentieth aspect of the invention, there is provided a tissue comprising the above-described cell.

Preferably, any one of the above cells, tissues or organs or tumor tissue after tumor bearing includes cells, tissues or organs or tumor tissue after tumor bearing which can or cannot develop into animal individuals.

In a twenty-first aspect of the present invention, there is provided a use of the above-mentioned humanized PVRIG protein, the above-mentioned humanized PVRIG gene, the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method, the use comprising:

A) use in the development of products involving PVRIG-related immune processes of human cells;

B) use in model systems related to PVRIG as pharmacological, immunological, microbiological and medical research;

C) to the production and use of animal experimental disease models for the study of PVRIG-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) the application in screening, drug effect detection, curative effect evaluation, verification or evaluation of human PVRIG signal channel regulator is studied in vivo; alternatively, the first and second electrodes may be,

E) the function of the PVRIG gene is researched, the medicine and the drug effect aiming at the target site of the PVRIG of human are researched, and the application in the aspects of the medicine for immune-related diseases related to the PVRIG and the anti-tumor medicine is researched. Preferably, the use includes therapeutic and diagnostic purposes for both disease and non-disease.

In a twenty-second aspect of the present invention, there is provided a method for screening a human PVRIG-specific modulator, which comprises screening the human PVRIG-specific modulator with the non-human animal derived from the above-described non-human animal, the non-human animal obtained by the above-described construction method, or the above-described tumor-bearing or inflammatory animal model.

In a twenty-third aspect of the present invention, there is provided a method of screening for a human PVRIG-specific modulator, said method comprising administering the modulator to an individual implanted with tumor cells, and detecting tumor suppression; wherein, the individual is selected from the non-human animal or the non-human animal constructed by the method or the animal model with tumor or inflammation.

Preferably, the modulator is selected from CAR-T, a drug. Further preferably, the drug is an antibody, and specifically, the drug may be an anti-PVRIG antibody.

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

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

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

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

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

Preferably, the screening method is not a therapeutic method. The screening method detects and evaluates the effect of the modulator to determine whether the modulator has a therapeutic effect, i.e., the therapeutic effect is not necessary but only a possibility.

In a twenty-fourth aspect of the present invention, there is provided a method for screening or evaluating a human drug, said method comprising transplanting human tumor cells into an individual, administering a candidate drug to the animal into which the human tumor cells have been transplanted, and performing efficacy testing and/or comparison on the individual to whom the candidate drug has been administered. Wherein the individual is selected from the group consisting of a non-human animal obtained by the aforementioned construction method and humanized with a PVRIG gene, a multi-gene-modified non-human animal obtained by the aforementioned construction method and a progeny thereof, or the aforementioned animal model with a tumor or inflammation.

Preferably, the method of drug screening or evaluation includes both therapeutic and non-therapeutic methods.

In one embodiment, the method is used to screen or evaluate drugs, test and compare the potency of candidate drugs to determine which candidate drugs can act as drugs and which cannot act as drugs, or to compare the potency sensitivity of different drugs, i.e., the therapeutic effect is not necessarily but only a possibility.

Preferably, the drug candidate comprises a targeted drug. Further preferably, the targeted drug is an antigen binding protein. In one embodiment of the invention, the antigen binding protein is an antibody.

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

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, any of the above non-human animals 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, any of the above non-human animals may be selected from any non-human animal that can be genetically modified by gene editing, such as pig, rabbit, monkey.

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

The "tumor" according to the present invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In one embodiment of the present invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, liver cancer.

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

The "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 "PVRIG locus" refers to a DNA fragment of any one of exons 1 to 5 of the PVRIG gene. In one embodiment of the invention, the PVRIG locus to be replaced may be a DNA fragment of an optional stretch of exons 1 to 5 of the PVRIG gene. In one embodiment of the invention, the PVRIG locus to be replaced may be a DNA fragment of an optional stretch of exons 1 to 3 of the PVRIG gene.

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

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

The "humanized PVRIG protein" of the present invention comprises a portion derived from a human PVRIG protein and a portion of a non-human PVRIG protein.

The "humanized PVRIG protein" of the present invention comprises a portion derived from a human PVRIG protein. Wherein the human PVRIG protein is identical to the human PVRIG protein, namely the amino acid sequence of the human PVRIG protein is identical to the full-length amino acid sequence of the human PVRIG protein. The 'part of the human PVRIG protein' is a continuous or alternate sequence of 5-326 (preferably 10-169) amino acids which is consistent with the amino acid sequence of the human PVRIG protein or has more than 70% of homology with the amino acid sequence of the human PVRIG protein.

The "humanized PVRIG gene" of the present invention includes a portion derived from a human PVRIG gene and a portion derived from a non-human PVRIG gene. Wherein the human PVRIG gene is identical to the human PVRIG gene in all aspects, namely the nucleotide sequence of the human PVRIG gene is consistent with the full-length nucleotide sequence of the human PVRIG gene. The 'part of the human PVRIG gene' is a continuous or alternate nucleotide sequence of 20-2866bp (preferably 20-674bp or 20-507bp) which is consistent with the nucleotide sequence of the human PVRIG or has more than 70 percent of homology with the nucleotide sequence of the human PVRIG.

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.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the part of exon 2 of human PVRIG gene comprises consecutive or spaced nucleotide sequences of 5-316bp, preferably 10-118bp, identical to the nucleotide sequence of exon 3 of human PVRIG gene. In a specific embodiment of the invention, the "part of exon 2" contained in the "humanized PVRIG gene" at least comprises the nucleotide sequence from the start codon to the last nucleotide of exon 2.

The "cell" of the present invention may be a fertilized egg cell or other somatic cell, preferably including but not limited to platelets, monocytes, microglia and endothelial cells, neutrophils, activated macrophages, B cell precursors, dendritic cells, natural killer cells, late B cells or plasma cells, and the like. Thus, depending on the source of the cells, a portion of the cells described herein may develop into individual animals and a portion may not.

The "PVRIG protein" of the present invention, e.g., "human PVRIG protein", "non-human animal PVRIG protein" or "humanized PVRIG protein", comprises a signal peptide, an extracellular region, an intracellular region and/or a transmembrane region.

The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, and not substantially affect the presence of other stated elements or steps. However, when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.

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

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

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); 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. week and c.c. blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal. In one embodiment, the non-human animal to which the PVRIG gene is humanized is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the crimyspascimyscimysciaenopsis (for example of the crimysciaeidae (for example of the hamsters, the new world rats and the new world rats, the rats and the rats, the. In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily muridae), 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 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, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola C57BL, C58 NOBr, A/Ca, PrCBA/34/CBA, PrCBA J, CBA/CBD, SCID-SCID strain ^scid IL-2rg ^null Background mice.

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

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention.

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

Drawings

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

FIG. 1: schematic comparison of mouse PVRIG locus and human PVRIG locus (not to scale);

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

FIG. 3: PVRIG gene targeting strategies and targeting vector design schematic (not to scale);

FIG. 4 is a schematic diagram showing the result of a positive clone in Southern Blot assay, WT being a wild type;

FIG. 5: schematic representation of FRT recombination process for PVRIG gene humanized mice (not to scale);

FIG. 6: PVRIG gene humanized mouse F1 mouse tail PCR identification result, wherein WT is wild type, H ₂ O is water control, PC is positive control, and M is marker;

FIG. 7: results of detection of PVRIG mRNA in spleen of C57BL/6 wild-type mouse (+/+) and PVRIG Gene humanized homozygote mouse (H/H) ₂ O is water control;

FIG. 8: flow detection results of leukocyte subgroup proportion in the spleen;

FIG. 9: flow assay results for T cell subpopulation ratios in spleen;

FIG. 10: flow detection results of leukocyte subset ratio in peripheral blood;

FIG. 11: flow detection results of the proportion of T cell subsets in peripheral blood;

FIG. 12: flow detection results of the proportion of leukocyte subsets in lymph nodes;

FIG. 13: flow detection results of the proportion of the T cell subset in the lymph nodes;

FIG. 14: implanting mouse colon cancer cell MC38 into humanized PVRIG mouse body, and performing anti-tumor effect test to obtain the weight result of the mouse;

FIG. 15: implanting mouse colon cancer cell MC38 into humanized PVRIG mouse body, and performing anti-tumor drug effect test to obtain the weight change result of the mouse;

FIG. 16: the results of the tumor volume of the mice after the mouse colon cancer cell MC38 is implanted into the humanized PVRIG mice and the anti-tumor efficacy test is carried out are shown in a graph.

Detailed Description

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

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

SacI, EcoNI, NdeI enzymes were purchased from NEB under the respective accession numbers R3156V, R0521V, R0111V;

c57BL/6 mice and Flp tool mice were purchased from the rodent experimental animal seed center of China food and drug testing institute;

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Biolegend, cat # 103138;

PE anti-human PVRIG Antibody was purchased from R & D, cat # MAB 93651;

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

Example 1 humanized mouse of PVRIG Gene

A schematic comparison of the mouse PVRIG Gene (NCBI Gene ID: 102640920, Primary source: MGI:5596028, UniProt: A0A1B0GS01, from position 138340193 to 138346953 on chromosome 5 NC-000071.7, based on transcript NM-001378438.1 and its encoded protein NP-001365367.1 (SEQ ID NO: 1)) and the human PVRIG Gene (NCBI Gene ID: 79037, Primary source: HGNC:32190, UniProt ID: Q6DKI7, from position 100218625 to 100221490 on chromosome 7 NC-000007.14, based on transcript NM-024070.3 and its encoded protein NP-076975.2 (SEQ ID NO: 2)) is shown in FIG. 1.

To achieve the object of the present invention, a nucleotide sequence encoding a human PVRIG protein can be introduced at an endogenous PVRIG locus of a mouse, so that the mouse expresses the human or humanized PVRIG protein. Specifically, the humanized PVRIG locus diagram is shown in figure 2 and the humanized modification of the mouse PVRIG gene is realized by replacing the coding region of the mouse PVRIG genome with the coding region of the human PVRIG genome or replacing the partial sequence of the mouse exon 1 to the partial sequence of the exon 3 with the partial sequence from exon 2 to exon 4 of the human PVRIG gene about 0.65kb under the control of the regulatory element of the mouse PVRIG gene by using a gene editing technology.

The targeting strategy was designed as shown in figure 3, with the homologous arm sequences on the targeting vector containing the upstream and downstream of the mouse PVRIG gene, and the a-fragment containing the human PVRIG sequence shown in figure 3. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from position 138336113 to 138340230 of NCBI accession No. NC-000071.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence from position 138342219 to 138345377 of NCBI accession No. NC-000071.7. The nucleotide sequence of human PVRIG on fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence 100219911 to 100220584 of NCBI accession No. NC-000007.14; the ligation of the upstream human PVRIG sequence to the mouse was designed as:

(SEQ ID NO: 6) wherein the sequence

The last "C" in (A) is the last nucleotide, sequence, of the mouse "ATGA"the first" A "in" is the first human nucleotide. The connection of the downstream of the human PVRIG sequence and the mouse is designed as

(SEQ ID NO: 7) in which

The last "G" in (A) is the last nucleotide, sequence, of a human "GCAGThe first "G" of "is the first nucleotide of the mouse sequence.

The targeting vector also comprises a targeting vector for positive clone screeningThe resistance gene, i.e. neomycin phosphotransferase coding sequence Neo, is provided with two site-specific recombination system Frt recombination sites which are arranged in the same direction 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 gene is designed as

(SEQ ID NO: 8) in which

Wherein "G" is the last nucleotide, sequence "of the mouse"CCTA"the first" C "of is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo box and the mouse gene is designed as

(SEQ ID NO: 9), wherein sequence "TATG"middle" G "is the last nucleotide, sequence, of the Neo cassette"CTTCThe "first" C "in" is the first nucleotide in the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse PVRIG after being transformed is shown as SEQ ID NO: 10, and the expressed protein sequence is shown as SEQ ID NO: shown at 11.

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

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The method comprises the steps of transfecting a targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse by electroporation, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technology, screening correct positive clone cells, detecting clones which are identified as positive by PCR, further detecting Southern Blot (cell DNA is digested by SacI, EcoNI and NdeI enzymes and hybridized by using 3 probes, the lengths of the probes and target fragments are shown in table 1), detecting the clones with the detection results shown in figure 4, wherein the clones are positive by 1-B08, 3-E06, 3-F11 and 3-G06, and further performing next-step experiments on the clones which are verified as positive by sequencing and have no random insertion.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment	Recombinant sequence fragment
				SacI	3’Probe	10.2kb	7.7kb
EcoNI	5’Probe	9.8kb	7.8kb
				NdeI	Neo Probe	—	9.0kb

Wherein the PCR assay comprises the following primers:

WT-F：5’-GTGTAGGGCTGAATGGGATAAGGGC-3’(SEQ ID NO：12)

Mut-R：5’-GTGACACACAAGGTCAGCAGCAC-3’(SEQ ID NO：13)；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

5’Probe-F：5’-AAGGCCCCTCCATAGACTTCTCAGC-3’(SEQ ID NO：14)，

5’Probe-R：5’-GGGGCTGCTAACGTCAATCACCTAC-3’(SEQ ID NO：15)；

3 'Probe (3' Probe):

3’Probe-F：5’-TCCATGGAACGTTGAATACCAGAAGT-3’(SEQ ID NO：16)，

3’Probe-R：5’-TGGTGCAAGTCTATAATTACAGCAGGTC-3’(SEQ ID NO：17)；

neo Probe (Neo Probe):

Neo Probe-F：5’-GGATCGGCCATTGAACAAGAT-3’(SEQ ID NO：18)，

Neo Probe-R：5’-CAGAAGAACTCGTCAAGAAGGC-3’(SEQ ID NO：19)；

the selected correctly positive clone cells (black mouse) are introduced into the separated blastocyst according to the technology known in the art (white mouse), the obtained chimeric blastocyst is transferred into the culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous child mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene (the process is schematically shown in figure 5), and then mated with each other to obtain the PVRIG gene humanized homozygote mice. The somatic genotypes of the progeny mice were identified by PCR (primers shown in Table 2), and the results of identification of exemplary F1 generation mice (with the Neo marker gene removed) are shown in FIG. 6, in which three mice numbered F1-01, F1-02 and F1-03 were all positive heterozygous mice. This indicates that the method can be used for constructing the PVRIG gene humanized mouse which can be stably passaged and has no random insertion.

Table 2: name and specific sequence of primer

Example 2 detection of expression of PVRIG humanized mice

The expression of the humanized PVRIG mRNA in the positive mouse is confirmed by a conventional detection method such as RT-PCR or the like. Specifically, 1 mouse of 5-week-old female C57BL/6 wild-type mice and 1 mouse of the PVRIG gene humanized homozygote prepared in example 1 were each taken, spleen of the mice was harvested after cervical-free euthanasia, cellular RNA was extracted according to the instructions of Trizol kit, and RT-PCR detection was performed after reverse transcription into cDNA, and the detection results are shown in FIG. 7. As can be seen from FIG. 7, murine PVRIG mRNA was detected in the spleen of the C57BL/6 wild-type mouse, and humanized PVRIG mRNA was not detected; only humanized PVRIG mRNA was detected in the spleen of the humanized homozygote mouse for the PVRIG gene, and no mouse PVRIG mRNA was detected.

The RT-PCR primer sequence comprises:

PCR-F1：5’-TGACCTTGTGTGTCACTGCG-3’(SEQ ID NO：25)

PCR-R1：5’-GGCTGAGATTTACTCAAACACCA-3’(SEQ ID NO：26)

PCR-F2：5’-GTCTCTGAAGCAAGCCCTGAG-3’(SEQ ID NO：27)

PCR-R2：5’-GGTGCTAGTGAGAGATGGCTG-3’(SEQ ID NO：28)

GAPDH-F：5’-TCACCATCTTCCAGGAGCGAGA-3’(SEQ ID NO：29)

GAPDH-R：5’-GAAGGCCATGCCAGTGAGCTT-3’(SEQ ID NO：30)

positive mice can be confirmed by conventional detection methodsExpression of the internal humanized PVRIG protein, e.g., flow cytometry, etc. Specifically, 1 mouse of 5-week-old female C57BL/6 wild-type mice and 1 mouse of 5-week-old female PVRIG gene humanized homozygote were each taken, spleen tissues were taken after cervical dislocation, and anti-human PVRIG Antibody PE anti-human PVRIG Antibody (hPGRIG) and mouse leukocyte-recognizing Antibody Brilliant Violet 510 were used respectively ^TM Flow detection is carried out after recognition staining of anti-mouse CD45(mCD45) and anti-mouse CD16/32 antibody Purified anti-mouse CD16/32, and the like, and the result shows that immune cells (characterized by mCD45+) in the spleen of a C57BL/6 mouse contain 0.99% of hPGRIG positive cells (characterized by mCD45+ hPGRIG +). The immune cells in the spleen of PVRIG homozygote mice had 30.3% hPGRIG positive cells (mCD45+ hPGRIG +). The results show that the spleen cells of the C57BL/6 mouse do not express the humanized PVRIG protein, and the spleen cells of the PVRIG humanized mouse can successfully express the humanized PVRIG protein.

White blood cells and T cell immunophenotyping in spleen, peripheral blood and lymph nodes of wild type C57BL/6 mouse (C57BL/6) and PVRIG humanized homozygote mouse (B-hPGRIG) were further examined by flow cytometry. The results of detecting leukocyte subtypes and T-cell subtypes in the spleen are shown in FIGS. 8 and 9, respectively, and the results of detecting leukocyte subtypes and T-cell subtypes in peripheral blood are shown in FIGS. 10 and 11, respectively. As can be seen from the figure, leukocyte subtypes such as B cells (B cells), T cells (T cells), NK cells (NK cells), CD4+ T cells (CD4+ T cells), CD8+ T cells (CD8+ T cells), Granulocytes (Granulocytes), DC cells (Dendritic cells), Macrophages (Macrophages) and Monocytes (Monocytes) in spleen and peripheral blood samples of the PVRIG gene humanized homozygote mouse are substantially identical to those of the C57BL/6 wild-type mouse (fig. 8, fig. 10), and T cell subtype percentages such as CD4+ T cells, CD8+ T cells and Tregs cells are substantially identical to those of the C57BL/6 wild-type mouse (fig. 9, fig. 11).

The results of detecting the leukocyte subtypes and the T-cell subtypes in the lymph nodes are shown in FIG. 12 and FIG. 13, respectively, and it can be seen from the graphs that the leukocyte subtypes such as B-cells, T-cells, NK-cells, CD4+ T-cells, CD8+ T-cells, etc. in the lymph node sample of the humanized homozygote mouse of the PVRIG gene are substantially identical to those of the wild-type C57BL/6 mouse (FIG. 12), and the percentages of the T-cell subtypes such as CD4+ T-cells, CD8+ T-cells, Tregs-cells, etc. are substantially identical to those of the wild-type C57BL/6 mouse (FIG. 13).

The above results indicate that the humanization of the PVRIG gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice in spleen, peripheral blood and lymph nodes.

Example 3 preparation of double humanized or multiple double humanized mice

The method or the prepared PVRIG mouse can also be used for preparing a double-humanized or multi-humanized mouse model. For example, in the aforementioned 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, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4 and/or CD226, or can be obtained from humanized PVRIG mice by using isolated mouse ES embryonic stem cells and gene recombination targeting technology to obtain a mouse model of PVRIG and other gene modifications, bi-or multi-gene modifications. The homozygote or heterozygote of the PVRIG mouse obtained by the method can also be mated with homozygote or heterozygote modified by other genes, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of the humanized PVRIG and heterozygote modified by other genes can be obtained with a certain probability according to Mendel genetic rules, the homozygote can be mutually mated to obtain homozygote modified by double genes or multiple genes, and the in vivo efficacy verification of targeted human PVRIG and other gene regulators can be carried out by utilizing the homozygote or the heterozygote modified by the double genes or the multiple genes.

Example 4 efficacy test

The PVRIG gene humanized mouse prepared by the invention is used for constructing a tumor model and can be used for testing the drug effect of a drug targeting human PVRIG. Specifically, the PVRIG gene humanized homozygote mouse (6 weeks old) prepared in example 1 was selected and subcutaneously inoculated with mouse colon cancer cell MC38 (5X 10) ⁵ One), the tumor volume is about 100- ³ Then, the tumor volume was counted as control group or treatment group (n-5/group). The control group was injected with hIgG (purchased from: Changsheng Biotechnology, Ltd., Beijing ancient city; cat # AG-0011), and the treatment group used COM-701(G2, pharmaceutical information see patent document (publication No. US10213505B2)) and SRF-813 antibody (G3,drug information see patent text (publication No. US20200040081a 1)). Specific groups and dosing are shown in table 3. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of a single mouse reached 3000mm ³ Euthanasia is performed. The results of the measurement of the body weight and the tumor volume of the mice in the experimental period are shown in FIGS. 14 to 16, respectively.

Table 3: grouping and administration of drugs

The main data and analysis results of each experiment are listed in table 4, and specifically include Tumor volume at the time of grouping, 14 days after grouping, 17 days after grouping, survival of mice, Tumor-free mice, Tumor (volume) Inhibition rate (TGI) at Tumor Growth Inhibition value _TV ) And the statistical difference in tumor volume (P-value) between the treated and control mice.

Table 4: tumor volume, survival and tumor inhibition rate

As shown in FIGS. 14 and 15 and Table 4, the health status of the animals was good in the course of the experiment in each group, and there was no significant difference (P) in the weight gain tendency of the animals (FIGS. 14 and 15) at day 17 in all the treatment groups (G2, G3) compared with the control group (G1) (P in each group, and P is not significantly different in all the treatment groups (G2, G3)>0.05), which shows that the animal has good tolerance to human PVRIG antibodies COM-701 and SRF-813, does not generate obvious toxic effect on the animal and has better safety. As shown in FIG. 16 and Table 4, from the tumor volume results, the tumor volumes of the treated groups were smaller than those of the control group at each stage, and the tumor volumes of mice in groups G2 and G3 were 1662. + -. 312mm on day 17 ³ And 2058 + -433 mm ³ All are smaller than 2388 mm plus or minus 243mm of the control group ³ . The experimental result shows that the PVRIG humanized mouse prepared by the method provided by the embodiment of the invention can be used for in vivo efficacy detection of the drug targeting human PVRIG.

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

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

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tcttctacat gttggtaaat ggtgtcaacc aaagcagaga ggactgcccc ttcagtactt 1440

gaagttagcc tgtttgccac tgcagcaaag aatttgattg ctggcatcac agcctgcctg 1500

ctgtgacagc cattcaagta ggaagtgcca ccctaaggca gctatctgga ggtgatgaaa 1560

caggcaggat ctcagcaagt gggagatgga gtcctttata cgtaggtgat tgacgttagc 1620

agccccttgt gccccaggga ttgttgatag aaaaaaaaaa aagagacata acactcacgt 1680

caaagatgag aaatagttta ttctggagcc aaggtcatgg cctggggaca caggttgaag 1740

ttgctccaaa ttccatgctc ctacatggaa gcaggtttgt gaaatcttta tagtaacaga 1800

agaaagccaa atcaagatac tttaaaaata tgttggtgga accattaagt caacagtaga 1860

ggtaggcaaa gggggaaacc ttgtgttact aggccgcaga ggctatgtga cagtgtttta 1920

agcatttggt tggctggcaa gctagcacta tgtttgacca ttccaagtgg ttccaaatct 1980

aaagtcaagt acagaagtaa gttggtctta aggtggctaa cgatggcaca agataatgta 2040

gctctggact taaaacattc caaactctca ataatctctg acgttccaat cagcttatta 2100

atcttgcagt cattcatgaa aggcagagct ttgttatggg aacttttttt tccatttttt 2160

tattagatat tttctttatt tacatttcaa atgctatccc gaaagttccc tataacctgc 2220

gcccctcccc cgccccaccc tgctccacta cccacccact cccacttctt agccctggtg 2280

ttcccctgta ctggggcata taaagtttgc aagaccaagg ggcctctctt cccgatgatg 2340

gccaattagg ccatcttctg ctgcataact tttaaagatc ctttagccta cgtagcctga 2400

cctcaggtta gtcagctttg tcacattaac tggtgaaagc aggtctctgt caggtctctg 2460

ggcctcagag gagatcaaag aatgaagaga atgctggcca gaccaggaac tgtggggaca 2520

aaggcctgga gaccactctg gtaggcactg tgagggctag tttatatgtc tgcagcggca 2580

aggataaaga gacaaaatag tgttcagata gagagagccc gggctctggt gtagacctgg 2640

gattctggtt ctcagtacac agcgttctga tccctggaca gggtcggtca cctgtcacca 2700

catataccgc agcatcctgg tcaatttgtg gcattctcta aattactttg tgtaggacaa 2760

agagcagtgg caatgcttgt ggaaagaaaa aacaatttgg tggggacagt gactgcctcc 2820

atgacacaat tcatgggcaa gggccaacct gcccttggga cttcccaatt caataaagca 2880

atatgtcaca tagcaatatg acacatgggg gtggggagag ttaagaacag aagcaggtct 2940

gagagaaact tcaatgtttt ggttcctaga gaaataagga agagcaagca aaggaatgat 3000

ccgtgtgttt tgagggtcac ctgggctgct gcagtgaagc caaggttgat gtggggtgat 3060

cagcaagcca atttgttatt ttgctcttcc cagcagatga cattaatagg tagatggtgg 3120

taagggaggc agagagaaat cagttggtca gaatgggagt cagcaggatg gagatgaaca 3180

aacagcttca gggctaagca gtttgaagtt aatgtgctaa caagcagagc tctgaatggg 3240

acaagtgaag accactctta ttagtgttat gaaatataaa atataaattg tggtataaca 3300

tggtaggagt aatacttggc aggacagtgc cgagatgttc ctctgaggaa tcacaccatg 3360

cagcatgcct gtataaagat ttattgtgga gatgggagtg ggaacctgga aagaggtaga 3420

ggcagagaaa ggataggaca gagaaagaca ggaagagaga aagagagcaa gagaggcctg 3480

ccaggaacac atgggaactg agagagtggg aaagggagaa gtgggggtag ggctgtgagc 3540

agtcctcttt agatgatgct gcgctgccag gcacctgcct cacaccgggt ggggcagaga 3600

caagcttttg ctgggtagcc attgggcaga attgcctgtt tccaggtgga catgggacag 3660

gagctgggaa gggatggtga attgcagatt caaaccaggg actactagta tgtaatactg 3720

agaacacacc ttggggtgtg ctgggtcact caggttggct cctgactgcc tccacaatgg 3780

ctttcctgat tttaggatct tgcaatctcc agttgttcca ttccctgccc cttagctgtg 3840

tagggctgaa tgggataagg gctaagttcc tcaggggcag ccatggaccc tcctctctct 3900

gaggcaaaga gacgattgac ctcagcgacc tctgcagctt tcctcactac tactgcagat 3960

ctaggaagta gcaagcaaag cctggtactg taactttaag agggacaacc cattaccctg 4020

agttcactac cactgaggcc tgcaaaaagg aaataaggct aggttgtggg aagttaccac 4080

agtgcctcta actgctgaca atgtgaaaac ctccagcc 4118

<210> 4

<211> 3159

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cttccagaat aacagctctg agacttaaat atatttacaa atgcctaggc caaagctctg 60

gcttgttttt tgattatctc ataatttaat aacctatttg ccttattctt tgtaaaccac 120

atgggtggct acttctgctc aggttctata catctgtcct tcttagagtc cccagggtga 180

atcctcccac acttgactct atctcagaat tactctctct gtactgaaac ttccacctcc 240

ctatatccta cctaagctaa taggctaaca gcatttttat agacaagtga tgtttccatt 300

caagagattc tctctacaga ggcaggcaga gagagacctt gaagacacag ggttgactat 360

aggctcatac acctgaaatt gttctaccta ggagctgtgc tcctctgcag gagacaagca 420

ccctgggctc ctgtaccatc cacagagaag acaaaatgag ctgtaagcct tgtgagaacc 480

ttgtgagccg taaggaccct gggtttggat tcctgggcca ctctagcctg gcaaaccaca 540

tagcttgaat acatgggagg ttccccttgc ttgtggggta catccggagg ctaaggaagc 600

cttaggaaca tgaagattgc aaacagggag actaaactgt tgtttggtct gtttaggctt 660

taatgccctt gaaccaaacc tgaaagatgg agaagcttgg gatcctgtac agggtagaga 720

atcaggaagt cttttaagga aaaatctgag cataagcatt cctagtgggc atcctgggaa 780

accctgttag tggttttgtt cctaccagct gacaactaac cccactgctg aggggccaga 840

tggtccccac agggccatag cacaagaagc ccagaagcca ctggcaaggg gataacttca 900

cagcagctgt gagttagggc agggtgacct ggaattcata gcccagccct ctgctgggag 960

ggggagctgg accatcctca ggcactctcc ggaagctgcc actggctgtt gcttttccca 1020

actactcacc tctgggaaga ccagcaacta ggcccccaag aatcctccac agactcctct 1080

gcctccagtc accccgagtc tgacttcctt ggtatagtct ctgccaccct cagccttatt 1140

cccttgacaa ttctgcttgt ggtgtctcca tagtctttct caccttccct atgtctgctg 1200

catgtcccct tgcaacttgc tgggtgtctg taccccagca tagccctcct ggcagtctag 1260

attcataact accacatcct gagaagcctg taccacattc tgaattggcc caagatcttt 1320

tcttcctcag tccaacctca ctccaaccag tggcaagcct atggaagctg tgggactgag 1380

gggttgtgtg accttgaatc aatggctagc ctttagacct caaaccattg cctagtgcat 1440

tgttctcagg ttctcaactc cagcttcctg gggccaccat gcataggaag tatccaaaag 1500

actgccaaca atgacaacaa gctatggaat atgaaaagaa aatcaggtct gagatcttgg 1560

ccagcaggac cagggagaag gcccttgtca gggtggtgtg gtgtgggaaa ggtctgattg 1620

gaacagtgga gccttcctcc ttctttctat tgcatgagat tttccaaagc cccacagggg 1680

agtcacttta accatatccc ccaaagcaca ggacattagg tctgttagca aagttatggt 1740

gaagagggac aaggtttcca aaccgaggtt ccagccaacc tctgcagaac aggggtcagt 1800

ggcagaaagg attctgtaga tccactctta aaccgattca gcatctgtga aataaaataa 1860

cttgctcaca agacagtgat ggctggaaag agtccagggt ctgagagctg ccacaaacag 1920

ctcttccaga agagtgaagg acaagataag gcagaaggtc aaagtgagag agaggaggac 1980

cagccagcta tggcatgcct gttggtggcc agctatggtg ccttgccctc catatcctcc 2040

ttcaggatgt ctaagcacgg atgcatactg ggctggaaaa atgaagcaga gtccagaatg 2100

ggtggggtct gtaggattct ggtcttttct ggctgggagt gagtgccaag gggtagaagg 2160

ctttctctaa ggattgtagt gttacagttc ttttagacaa tactcaatga aacagctctc 2220

ttagacgctc ttcagtgaaa cagctggtgt gcatacactt tatttggggt ggacaatggt 2280

tgtatatgaa tcttggagag ttggaaggag ttttcaggtt gaatgtaaaa tcattggcca 2340

gagtttaatc ccaggttctt gctggatggg tccttatttg gaaagatgtt gaacttatag 2400

tttgccaagt tgaacttgta ggtttgcaac cttctccaga tagagggtgt gggggtcagg 2460

ctccccagac aaggtcagag aggggaaacc catgctggaa aaaaaggagt ccctcatttt 2520

gcgccaagct cagatgagtg agctatctgg acatggtaga cttggacctt aagtagcaag 2580

gtttccaaac atctcccccc cccccccccg ttttatttat aatggagaga tgtcatagta 2640

gccttcagcc cctgtgttgg gaacagtttg gtactgagct agaacccagt tggcaatggt 2700

ttttgtaatg ctactcatct gctgttttaa aaattggaca aggcagggtg ccaggaggag 2760

ggctctgatg gccagtacag ggccaaggag ggcagggaga agccaagcca caagggggtt 2820

tgagtaccat gaggttgggg tgttagacac atagcaggct tggagattcc cagtgcagat 2880

gagcatttgt atgtcttgtt ccaccagaca gatttgttaa catagaatca gcattccttt 2940

cccaatataa tgcaggtcct tcactgtttg gcagtcatca agtccagagc aactcccact 3000

aggaagttta tgtgttgttg aaggttgaag ggactcaaga ccatctgtag tagaagcctg 3060

gtcaagcttg tcctggaagt ctctggccaa atggcgtgct tgggctgttt cagctgctcc 3120

tgtggcaccc agtgagactg taagacccag tctgatgaa 3159

<210> 5

<211> 674

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagaacag aggcacaggt gccggccctg cagcccccag aacctggact ggagggggcc 60

atggggcacc ggaccctggt cctgccctgg gtgctgctga ccttgtgtgt cactgcgggt 120

gagtgccggc accagagagg ggcaggggct gcagggaggg tgatgtagga caacagccca 180

ccgaccttgc tgctgttcca cagggacccc ggaggtgtgg gttcaagttc ggatggaggc 240

caccgagctc tcgtccttca ccatccgttg tgggttcctg gggtctggct ccatctccct 300

ggtgactgtg agctgggggg gccccaacgg tgctgggggg accacgctgg ctgtgttgca 360

cccagaacgt ggcatccggc aatgggcccc tgctcgccag gcccgctggg aaacccagag 420

cagcatctct ctcatcctgg aaggctctgg ggccagcagc ccctgcgcca acaccacctt 480

ctgctgcaag tttgcgtcct tccctgaggg ctcctgggag gcctgtggga gcctcccgcc 540

cagctcagac ccaggtgggg ccggggcgag gggtccagga gggcagggag gggctcggga 600

actggccacc catctgattc ttgtctccgt gcccagggct ctctgccccg ccgactcctg 660

cccccattct gcgg 674

<210> 6

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctgacaatg tgaaaacctc cagccatgag aacagaggca caggtgccgg cc 52

<210> 7

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccgccgactc ctgcccccat tctgcgggca gacctggtga ggatcttggg aacctc 56

<210> 8

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggtgttttct ctcatggcct caagcctatc gaaggagtac tgaattccga agttcctatt 60

ctctagaaag 70

<210> 9

<211> 88

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaaagtatag gaacttcatc agtcaggtac ataatggtgg atctgatatc catatgcttc 60

cagaataaca gctctgagac ttaaatat 88

<210> 10

<211> 1283

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgacctctgc agctttcctc actactactg cagatctagg aagtagcaag caaagcctgg 60

tactgtaact ttaagaggga caacccatta ccctgagttc actaccactg aggcctgcaa 120

aaaggaaata aggctaggtt gtgggaagtt accacagtgc ctctaactgc tgacaatgtg 180

aaaacctcca gccatgagaa cagaggcaca ggtgccggcc ctgcagcccc cagaacctgg 240

actggagggg gccatggggc accggaccct ggtcctgccc tgggtgctgc tgaccttgtg 300

tgtcactgcg gggaccccgg aggtgtgggt tcaagttcgg atggaggcca ccgagctctc 360

gtccttcacc atccgttgtg ggttcctggg gtctggctcc atctccctgg tgactgtgag 420

ctgggggggc cccaacggtg ctggggggac cacgctggct gtgttgcacc cagaacgtgg 480

catccggcaa tgggcccctg ctcgccaggc ccgctgggaa acccagagca gcatctctct 540

catcctggaa ggctctgggg ccagcagccc ctgcgccaac accaccttct gctgcaagtt 600

tgcgtccttc cctgagggct cctgggaggc ctgtgggagc ctcccgccca gctcagaccc 660

agggctctct gccccgccga ctcctgcccc cattctgcgg gcagacctgg tgaggatctt 720

gggaacctca ggagttttcc tgtttggttt catctttata ctatgtctcc ggtggcagca 780

gaggcattgg tgtttgagta aatctcagcc atctctcact agcacccagg cacaggtgga 840

gactcagcct ccccacttgg cctctacaca cagcagcttc atctccatgg agaatggact 900

gtatgctctg gcatgagtga aacttcccca cactcttccc aacttccacc gactgtctgg 960

agcacagtac cctggaaaaa tgcttagaag ttcaatgaca ggtacccttg gtccactaca 1020

ggcctttgga acatctatct actgatgtct tcaacctggg caccaggctt cttcatgaat 1080

cttgctctat tcttaagctt ctgatcagtg tcatcaaacc ctgctagcca ggatgtggtt 1140

attgagtgtg cataaatgga cacaagtgtg agccgaagag tacgacaaag gttcagttac 1200

atcctgtttt tgcacacatt tccaaacacc cagatgattt ctttaccaat gtccataaag 1260

tggtccttct gtttgggact tta 1283

<210> 11

<211> 240

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Arg Thr Glu Ala Gln Val Pro Ala Leu Gln Pro Pro Glu Pro Gly

1 5 10 15

Leu Glu Gly Ala Met Gly His Arg Thr Leu Val Leu Pro Trp Val Leu

20 25 30

Leu Thr Leu Cys Val Thr Ala Gly Thr Pro Glu Val Trp Val Gln Val

35 40 45

Arg Met Glu Ala Thr Glu Leu Ser Ser Phe Thr Ile Arg Cys Gly Phe

50 55 60

Leu Gly Ser Gly Ser Ile Ser Leu Val Thr Val Ser Trp Gly Gly Pro

65 70 75 80

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

85 90 95

Ile Arg Gln Trp Ala Pro Ala Arg Gln Ala Arg Trp Glu Thr Gln Ser

100 105 110

Ser Ile Ser Leu Ile Leu Glu Gly Ser Gly Ala Ser Ser Pro Cys Ala

115 120 125

Asn Thr Thr Phe Cys Cys Lys Phe Ala Ser Phe Pro Glu Gly Ser Trp

130 135 140

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

145 150 155 160

Pro Pro Thr Pro Ala Pro Ile Leu Arg Ala Asp Leu Val Arg Ile Leu

165 170 175

Gly Thr Ser Gly Val Phe Leu Phe Gly Phe Ile Phe Ile Leu Cys Leu

180 185 190

Arg Trp Gln Gln Arg His Trp Cys Leu Ser Lys Ser Gln Pro Ser Leu

195 200 205

Thr Ser Thr Gln Ala Gln Val Glu Thr Gln Pro Pro His Leu Ala Ser

210 215 220

Thr His Ser Ser Phe Ile Ser Met Glu Asn Gly Leu Tyr Ala Leu Ala

225 230 235 240

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtgtagggct gaatgggata agggc 25

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtgacacaca aggtcagcag cac 23

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaggcccctc catagacttc tcagc 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggggctgcta acgtcaatca cctac 25

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tccatggaac gttgaatacc agaagt 26

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tggtgcaagt ctataattac agcaggtc 28

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggatcggcca ttgaacaaga t 21

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cagaagaact cgtcaagaag gc 22

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gagatgaggc tatatcctaa gaccc 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttcagaagc agagaggaga gggca 25

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aacctgagca gaagtagcca cccat 25

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gacaagcgtt agtaggcaca tatac 25

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gctccaattt cccacaacat tagt 24

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgaccttgtg tgtcactgcg 20

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggctgagatt tactcaaaca cca 23

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gtctctgaag caagccctga g 21

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggtgctagtg agagatggct g 21

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tcaccatctt ccaggagcga ga 22

<210> 30

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gaaggccatg ccagtgagct t 21

Claims

1. A humanized PVRIG protein, comprising a portion of a human PVRIG protein.

2. The humanized PVRIG protein of claim 1, wherein said humanized PVRIG protein comprises all or part of the signal peptide, extracellular, transmembrane and/or cytoplasmic region of a human PVRIG protein, preferably said part of a human PVRIG protein comprises all or part of the extracellular region of a human PVRIG protein, further preferably comprises at least 20 contiguous amino acids of the extracellular region of a human PVRIG protein, preferably comprises the amino acid sequence of SEQ ID NO:2 from position 40 to 169 or from position 40 to 172; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2 from position 40-169 or 40-172, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 40-169 or 40-172, including substitutions, deletions and/or insertions of one or more amino acid residues.

3. The humanized PVRIG protein of claim 1 or 2, further comprising all or part of a signal peptide of a human PVRIG protein, preferably comprising at least 5 consecutive amino acids of the signal peptide of a human PVRIG protein, wherein said signal peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 1-39 of SEQ ID No. 2 or comprises an amino acid sequence as depicted in positions 1-39 of SEQ ID No. 2.

4. The humanized PVRIG protein of any of claims 1-3, comprising the amino acid sequence of SEQ ID NO:2, 1-169; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 1-169, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 1-169 do not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, 1-169, an amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues,

preferably, said humanized PVRIG protein further comprises a portion of a non-human animal PVRIG protein.

5. The humanized PVRIG protein of any of claims 1-4, wherein the amino acid sequence of the humanized PVRIG protein comprises any of the following group:

A) SEQ ID NO: 11;

6. A nucleic acid encoding the humanized PVRIG protein of any of claims 1-5.

7. A humanized PVRIG gene comprising a portion of a human PVRIG gene.

8. The humanized PVRIG gene of claim 7, wherein the humanized PVRIG gene comprises all or part of exons 1 to 6, preferably all or part of exons 2 to 4, more preferably part of exons 2, 3 and 4, wherein part of exons 2 comprises at least a 50bp nucleotide sequence and part of exons 4 comprises at least a 10bp nucleotide sequence.

9. The humanized PVRIG gene of claim 7 or 8, comprising the sequence of SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

10. The humanized PVRIG gene of any of claims 7-9, further comprising all or a portion of a non-human animal PVRIG gene, further preferably comprising all or a portion of exons 4 to 5, further preferably further comprising the nucleotide sequence of part of exon 1 and/or part of exon 3 of a non-human animal PVRIG gene.

11. The humanized PVRIG gene of any of claims 7-10, wherein the nucleotide sequence of the humanized PVRIG gene comprises any of the following group:

A) the transcribed mRNA is SEQ ID NO: 10;

E) comprises the amino acid sequence of SEQ ID NO: 6 and/or 7.

12. A targeting vector, wherein said targeting vector comprises a donor nucleotide sequence, preferably wherein said donor nucleotide sequence comprises one of the group consisting of:

A) a nucleotide sequence encoding a human or humanized PVRIG protein;

B) all or part of a nucleotide sequence encoding a signal peptide, an extracellular region, a transmembrane region and/or a cytoplasmic region of a human PVRIG protein, preferably all or part of a nucleotide sequence encoding an extracellular region and/or a signal peptide of a human PVRIG protein, further preferably a nucleotide sequence encoding at least 20 consecutive amino acids of an extracellular region of a human PVRIG protein, more preferably still further comprising a nucleotide sequence encoding at least 5 consecutive amino acids of a signal peptide of a human PVRIG protein, still further preferably a nucleotide sequence encoding SEQ ID NO:2, 1-169 nucleotide sequence;

D) all or part of exons 1 to 6 of human PVRIG gene, preferably all or part of exons 2 to 4 of human PVRIG gene, further preferably part of exon 2, all of exon 3 and part of exon 4, wherein part of exon 2 comprises at least a 50bp nucleotide sequence, part of exon 4 comprises at least a 10bp nucleotide sequence, further preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or, comprises a polypeptide having the sequence of SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

13. The targeting vector according to claim 12, wherein said targeting vector further comprises a 5 'arm and/or a 3' arm, preferably wherein said 5 'arm has at least 90% homology to NCBI accession No. NC _000071.7, further preferably wherein said 5' arm sequence is as set forth in SEQ ID NO: 3 is shown in the specification; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000071.7; further preferably, the 3' arm sequence is as set forth in SEQ ID NO: 4, respectively.

14. A method of constructing a non-human animal, wherein a human or humanized PVRIG protein is expressed in said non-human animal.

15. The method of claim 14, wherein the humanized PVRIG protein is the humanized PVRIG protein of any one of claims 1 to 5.

16. The method of claim 14 or 15, wherein the genome of the non-human animal comprises the nucleic acid of claim 6 or the humanized PVRIG gene of any of claims 7-11.

17. The method of construction according to any one of claims 14 to 16 comprising introducing a donor nucleotide sequence into the PVRIG locus of the non-human animal, preferably wherein the donor nucleotide sequence comprises one of the following group:

A) a nucleotide sequence encoding a human or humanized PVRIG protein;

D) all or part of exons 1 to 6 of human PVRIG gene, preferably all or part of exons 2 to 4 of human PVRIG gene, further preferably part of exon 2, all of exon 3 and part of exon 4, wherein part of exon 2 comprises at least a 50bp nucleotide sequence, part of exon 4 comprises at least a 10bp nucleotide sequence, further preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

18. The method of construction of claim 17, wherein the donor nucleotide sequence is operably linked to an endogenous regulatory element.

19. Construction method according to any one of claims 17-18, wherein said introducing is a substitution or insertion, optionally said introducing into the PVRIG locus is a substitution of the corresponding region of a non-human animal, preferably exon 2 of a non-human animal PVRIG gene is substituted, further preferably a part comprising exon 1 and/or a part comprising exon 3 of a non-human animal PVRIG gene is substituted.

20. The method of construction according to any one of claims 14 to 19, wherein the targeting vector according to any one of claims 12 to 13 is used for construction of a non-human animal.

21. The method of any one of claims 14 to 20, further comprising mating the PVRIG gene-humanized non-human animal with another genetically modified non-human animal, in vitro fertilization or direct gene editing, and screening to obtain a polygenetically modified non-human animal, preferably wherein said other gene is selected from at least one of PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPA, CTLA4, and CD 226.

22. A cell, tissue or organ which expresses a humanized PVRIG protein of any of claims 1-5 or which comprises a nucleic acid of claim 6 or a humanized PVRIG gene of any of claims 7-11 or which is a non-human animal obtained by a method of construction of any of claims 14-21.

23. A tumorous tissue bearing a tumor expressing the humanized PVRIG protein of any of claims 1 to 5 or comprising the nucleic acid of claim 6 or the humanized PVRIG gene of any of claims 7 to 11 or a non-human animal obtained by the construction method of any of claims 14 to 21.

24. Use of a humanized PVRIG protein according to any of claims 1 to 5, a nucleic acid according to claim 6, a humanized PVRIG gene according to any of claims 7 to 11 or a non-human animal obtained by the construction method according to any of claims 14 to 21, comprising:

E) the function of PVRIG gene, the medicine and drug effect for PVRIG target site, and the application of medicine for immune related diseases and antitumor medicine.

25. The humanized PVRIG protein of any one of claims 4 to 5, the humanized PVRIG gene of any one of claims 10 to 11, the method of construction of any one of claims 14 to 21, the cell, tissue or organ of claim 22, the neoplastic tissue of claim 23, wherein the non-human animal is a non-human mammal, more preferably the non-human mammal is a rodent, even more preferably the rodent is a rat or a mouse.