NPR1 gene humanized non-human animal as well as construction method and application thereof

Abstract

The invention provides an NPR1 gene humanized non-human animal, a construction method thereof and an application of the NPR1 gene humanized non-human animal in the field of biological medicine. The invention also discloses a targeting vector of the NPR1 gene, a humanized NPR1 protein, and a humanized NPR1 gene. The non-human animal can express the human or humanized NPR1 protein, can be used as an animal model for human NPR1 signal mechanism research and drug screening of neurological diseases, tumors and immune related diseases, and has important application value for research and development of new drugs of immune targets.

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic engineering and gene genetic modification, and specifically relates to a non-human animal with humanized NPR1 gene and a construction method thereof and application in the field of biomedicine. Background Art

[0002] NPR1 stands for natriuretic peptide receptor 1, also known as NPRA. It belongs to the guanylate cyclase family and is a type I transmembrane receptor protein that generally exists in the form of a homodimer. Human NPR1 is expressed in a variety of tissues, among which it is highly expressed in the kidneys, lungs, adrenal glands, vascular system, brain, liver, endothelium and adipose tissue, and is expressed at a lower level in the heart.

[0003] NPR1 contains an extracellular ligand binding domain, a transmembrane region, a kinase homology domain, and a carboxyl-terminal catalytic domain. When it binds to a ligand (atrial natriuretic peptide ANP or brain natriuretic peptide BNP), NPR1 dimerizes and twists, thereby inducing the activation of the guanylate cyclase domain and the production of cGMP, and triggering subsequent intracellular signal transduction and physiological changes. NPR1 is involved in regulating a variety of physiological functions, most of which are related to reducing cardiac load, such as stimulating vascular relaxation, natriuresis, diuresis, endothelial permeability, and inhibiting the renin-angiotensin pathway. NPR1 is the target of two drugs approved for the treatment of congestive heart failure (carpiroxine and nesiritide) and a new chimeric natriuretic peptide CD-NP, which has a good therapeutic effect in clinical studies. In addition, there are also literature reports that it plays a role in tumors or is a potential therapeutic target.

[0004] In view of the great application value of NPR1 in the field of cardiovascular disease treatment and its potential value in the field of anti-tumor treatment, in order to further explore its related biological characteristics, improve the effectiveness of preclinical efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize research and development failures, the field urgently needs to develop non-human animal models of NPR1-related signaling pathways. In addition, the non-human animals obtained by this method can also be mated with other genetically humanized non-human animals to obtain multi-gene humanized animal models, which are used to screen and evaluate the efficacy of human drugs and combination drugs targeting this signaling pathway. The present invention has broad application prospects in academic and clinical research. Summary of the invention

[0005] This application uses gene editing technology to replace homologous genes in animal genomes with normal or mutant human genes, and establishes normal or mutant gene animal models that are closer to human physiological or disease characteristics. Gene humanization can improve and enhance humanized animal models for cell or tissue transplantation. More importantly, due to the insertion of human gene fragments, human proteins can be expressed or partially expressed in animals, which can be used as targets for drugs that can only recognize human protein sequences, making it possible to screen anti-human antibodies and other drugs at the animal level. Specifically:

[0006] In a first aspect, the present invention provides a humanized NPR1 protein, wherein the humanized NPR1 protein comprises all or part of a human NPR1 protein.

[0007] Preferably, the humanized NPR1 protein comprises at least 50 consecutive amino acids of human NPR1 protein, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 441, 450, 469, 470, 471, 472, 473, 474, 475, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or 1061 consecutive amino acids.

[0008] Preferably, the humanized NPR1 protein comprises all or part of the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human NPR1 protein.

[0009] Preferably, the humanized NPR1 protein comprises all or part of the extracellular region of the human NPR1 protein, further preferably comprises at least 50 consecutive amino acids, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441 consecutive amino acids, of the extracellular region of the human NPR1 protein, more preferably comprises the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2. An amino acid sequence that differs from the amino acid sequence shown at positions 33-473 of NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, an amino acid sequence that comprises a substitution, deletion and / or insertion of one or more amino acids from the amino acid sequence shown at positions 33-473 of SEQ ID NO:2.

[0010] Preferably, the humanized NPR1 protein comprises all or part of the signal peptide of the human NPR1 protein, further preferably comprises at least 10 consecutive amino acids of the signal peptide of the human NPR1 protein, such as at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32 consecutive amino acids, more preferably comprises the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2; or, comprises an amino acid sequence that differs from the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence that is identical to the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; The amino acid sequence shown in the amino acid sequence shown at positions 1 to 32 of NO: 2 includes the amino acid sequence of substitution, deletion and / or insertion of one or more amino acids.

[0011] Preferably, the humanized NPR1 protein comprises all or part of the transmembrane region of the human NPR1 protein, further preferably comprises at least one consecutive amino acid, such as at least 1, 2, 3, 5, 10, 15, 18, 19, 20, 21 consecutive amino acids, of the transmembrane region of the human NPR1 protein, more preferably comprises the amino acid sequence as shown in positions 474-494 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence as shown in positions 474-494 of SEQ ID NO: 2; or, comprises an amino acid sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence as shown in positions 474-494 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence as shown in positions 474-494 of SEQ ID NO: 2 The amino acid sequence shown in the amino acid sequence at positions 474-494 of NO:2 includes substitution, deletion and / or insertion of one or more amino acids.

[0012] Preferably, the humanized NPR1 protein comprises all or part of the cytoplasmic region of the human NPR1 protein, further preferably comprises at least 100 consecutive amino acids, such as at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 567 consecutive amino acids, of the cytoplasmic region of the human NPR1 protein, more preferably comprises the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; An amino acid sequence that differs from the amino acid sequence shown at positions 495-1061 of NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, an amino acid sequence that comprises a substitution, deletion and / or insertion of one or more amino acids from the amino acid sequence shown at positions 495-1061 of SEQ ID NO:2.

[0013] Preferably, the humanized NPR1 protein further comprises a portion of a non-human animal NPR1 protein.

[0014] Further preferably, the humanized NPR1 protein comprises all or part of the transmembrane region of the non-human animal NPR1 protein, and further preferably comprises at least 10 consecutive amino acids, such as at least 10, 15, 18, 19, 20, 21 consecutive amino acids, of the transmembrane region of the non-human animal NPR1 protein, and more preferably comprises the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; or, comprises an amino acid sequence that differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid from the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1 The amino acid sequence shown in the amino acid sequence at positions 470-490 of NO: 1 includes the amino acid sequence of substitution, deletion and / or insertion of one or more amino acids.

[0015] Further preferably, the humanized NPR1 protein comprises all or part of the cytoplasmic region of the non-human animal NPR1 protein, and further preferably comprises at least 50 consecutive amino acids, such as at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 567 consecutive amino acids, of the cytoplasmic region of the non-human animal NPR1 protein, and more preferably comprises the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; or, comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; An amino acid sequence that differs from the amino acid sequence shown at positions 491-1057 of NO:1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, an amino acid sequence that comprises a substitution, deletion and / or insertion of one or more amino acids from the amino acid sequence shown at positions 491-1057 of SEQ ID NO:1.

[0016] In a specific embodiment of the present invention, the humanized NPR1 protein comprises any one of the following groups:

[0017] a) the extracellular region derived from human NPR1 protein, and the signal peptide, transmembrane region and cytoplasmic region derived from non-human animal NPR1 protein;

[0018] b) signal peptide and extracellular region derived from human NPR1 protein, and transmembrane region and cytoplasmic region derived from non-human animal NPR1 protein;

[0019] c) transmembrane region and extracellular region derived from human NPR1 protein, and signal peptide and cytoplasmic region derived from non-human animal NPR1 protein;

[0020] d) the cytoplasmic region and extracellular region derived from human NPR1 protein, and the signal peptide and transmembrane region derived from non-human animal NPR1 protein;

[0021] e) a signal peptide, an extracellular region and a transmembrane region derived from a human NPR1 protein, and a cytoplasmic region derived from a non-human animal NPR1 protein;

[0022] f) a signal peptide, an extracellular region and a cytoplasmic region derived from a human NPR1 protein, and a transmembrane region derived from a non-human animal NPR1 protein;

[0023] g) the transmembrane region, the extracellular region and the cytoplasmic region derived from human NPR1 protein, and the signal peptide derived from non-human animal NPR1 protein;

[0024] h) a signal peptide, a human or humanized extracellular region, a human or humanized transmembrane region, and a human or humanized cytoplasmic region of an NPR1 protein derived from human or non-human animals;

[0025] i) a human or humanized signal peptide derived from the extracellular region, human or humanized transmembrane region and human or humanized cytoplasmic region of human NPR1 protein;

[0026] g) a human or humanized signal peptide, a human or humanized extracellular region, a transmembrane region of an NPR1 protein derived from human or non-human animals, and a human or humanized cytoplasmic region;

[0027] k) human or humanized signal peptide, human or humanized extracellular region, human or humanized transmembrane region and cytoplasmic region of NPR1 protein derived from human or non-human animals.

[0028] Among them, the humanized signal peptide includes the signal peptide portion of the human NPR1 protein and the portion of the signal peptide of the non-human animal NPR1 protein, as the signal peptide region of the complete protein, and maintains the function of the complete signal peptide. The humanized extracellular region includes the extracellular region portion of the human NPR1 protein and the portion of the extracellular region of the non-human animal NPR1 protein, as the region of the extracellular region of the complete protein, and maintains the function of the complete extracellular region. The humanized transmembrane region includes the transmembrane region portion of the human NPR1 protein and the portion of the transmembrane region of the non-human animal NPR1 protein, as the region of the transmembrane region of the complete protein, and maintains the function of the complete transmembrane region. The humanized cytoplasmic region includes the cytoplasmic region portion of the human NPR1 protein and the portion of the cytoplasmic region of the non-human animal NPR1 protein, as the region of the cytoplasmic region of the complete protein, and maintains the function of the complete cytoplasmic region.

[0029] In a specific embodiment of the present invention, the amino acid sequence of the human NPR1 protein contained in the humanized NPR1 protein comprises any of the following:

[0030] A) the amino acid sequence shown at positions 1 to 473 of SEQ ID NO: 2;

[0031] B) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence as set forth in positions 1-473 of SEQ ID NO:2;

[0032] C) an amino acid sequence that differs from the amino acid sequence shown in positions 1-473 of SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0033] D) an amino acid sequence represented by the amino acid sequence represented by positions 1 to 473 of SEQ ID NO: 2, including substitution, deletion and / or insertion of one or more amino acids.

[0034] In a specific embodiment of the present invention, the amino acid sequence of the non-human animal NPR1 protein contained in the humanized NPR1 protein comprises any of the following:

[0035] A) the amino acid sequence shown at positions 470-1057 of SEQ ID NO: 1;

[0036] B) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence set forth in positions 470-1057 of SEQ ID NO: 1;

[0037] C) an amino acid sequence that differs from the amino acid sequence shown in positions 470 to 1057 of SEQ ID NO: 1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

[0038] D) an amino acid sequence comprising substitution, deletion and / or insertion of one or more amino acids as shown in the amino acid sequence shown at positions 470-1057 of SEQ ID NO: 1.

[0039] Preferably, the humanized NPR1 protein comprises an amino acid sequence encoded by all or part of exons 1 to 22 of the human NPR1 gene; further preferably, it comprises an amino acid sequence encoded by any one, two or more than three, or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene; and even more preferably, it comprises an amino acid sequence encoded by all or part of exon 1 to all or part of exon 7 of the human NPR1 gene.

[0040] wherein the portion of exon 1 comprises at least 100 bp of continuous nucleotide sequence, for example, at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800,

[0041] 850, 900, 950, 1000, 1100, 1142 bp continuous nucleotide sequence. Alternatively, the portion of exon 1 at least includes the nucleotide sequence of the coding region. Alternatively, the portion of exon 1 at least includes the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the sequence from the start codon in exon 1 to the last nucleotide at the 3' end.

[0042] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0043] More preferably, the humanized NPR1 protein comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7; or, comprises an amino acid sequence that differs from the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprises an amino acid sequence as shown in the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7, including substitution, deletion and / or insertion of one or more amino acids.

[0044] Preferably, the humanized NPR1 protein comprises the amino acid sequence encoded by all or part of exons 1 to 22 of the NPR1 gene of a non-human animal; preferably comprises the amino acid sequence encoded by all or part of exon 7 to all or part of exon 22.

[0045] In a specific embodiment of the present invention, the humanized NPR1 protein comprises an amino acid sequence encoded by a portion of a human NPR1 gene and a portion of a non-human animal NPR1 gene, preferably a portion of exon 1 to exon 7 of a human NPR1 gene, and all or part of a portion of exon 7 to exon 22 of a non-human animal NPR1 gene.

[0046] In a specific embodiment of the present invention, the humanized NPR1 protein comprises any one of the following groups:

[0047] A) the amino acid sequence shown in SEQ ID NO: 9;

[0048] B) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:9;

[0049] C) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:9 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or,

[0050] D) an amino acid sequence comprising substitution, deletion and / or insertion of one or more amino acids as shown in the amino acid sequence shown in SEQ ID NO: 9.

[0051] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0052] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0053] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice or nude mice.

[0054] The second aspect of the present invention provides a complex, wherein the complex comprises the above-mentioned humanized NPR1 protein and its ligand. Preferably, the ligand may be atrial natriuretic peptide ANP or brain natriuretic peptide BNP.

[0055] The third aspect of the present invention provides a humanized NPR1 gene, wherein the humanized NPR1 gene encodes the above-mentioned humanized NPR1 protein.

[0056] The fourth aspect of the present invention provides a humanized NPR1 gene.

[0057] The humanized NPR1 gene comprises part of the human NPR1 gene. Preferably, it comprises all or part of exons 1 to 22 of the human NPR1 gene, and more preferably comprises any one, two or more than three, or two or more than three consecutive exons of exons 1 to 22 of the human NPR1 gene; and further preferably comprises part of exon 1 to part of exon 7 of the human NPR1 gene.

[0058] wherein the portion of exon 1 comprises at least 100 bp of continuous nucleotide sequence, for example, at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800,

[0059] 850, 900, 950, 1000, 1100, 1142 bp continuous nucleotide sequence. Alternatively, the portion of exon 1 at least includes the nucleotide sequence of the coding region. Alternatively, the portion of exon 1 at least includes the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the sequence from the start codon in exon 1 to the last nucleotide at the 3' end.

[0060] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0061] In a specific embodiment of the present invention, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, it comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, it comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotides; or, it comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides.

[0062] Preferably, the humanized NPR1 gene comprises part of the NPR1 gene of a non-human animal, more preferably comprises all or part of exons 1 to 22 of the NPR1 gene of a non-human animal; and further preferably comprises all or part of the nucleotide sequence from part of exon 7 to all or part of exon 22. The part of exon 7 at least comprises the nucleotide sequence encoding the transmembrane region and / or the cytoplasmic region.

[0063] In a specific embodiment of the present invention, it comprises part of exon 1 to part of exon 7 of human NPR1 gene, and all or part of part of exon 7 to exon 22 of non-human animal NPR1 gene.

[0064] Preferably, the humanized NPR1 gene comprises SEQ ID NO: 10 and / or 11.

[0065] Preferably, the humanized NPR1 gene comprises SEQ ID NO: 12 and / or 13.

[0066] The humanized NPR1 gene comprises all or part of a nucleotide sequence encoding a human NPR1 protein, preferably a nucleotide sequence encoding at least 50 consecutive amino acids of a human NPR1 protein, such as a nucleotide sequence of at least 50, 100, 150, 200, 250, 300, 350, 400, 441, 450, 469, 470, 471, 472, 473, 474, 475, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or 1061 consecutive amino acids.

[0067] Preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the signal peptide, extracellular region, transmembrane region and / or cytoplasmic region of the human NPR1 protein.

[0068] Preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the extracellular region of the human NPR1 protein, further preferably comprises a nucleotide sequence encoding at least 50 consecutive amino acids of the extracellular region of the human NPR1 protein, such as a nucleotide sequence of at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441 consecutive amino acids, more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2. A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 33-473 of NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding the amino acid sequence shown at positions 33-473 of SEQ ID NO:2, including substitution, deletion and / or insertion of one or more amino acids.

[0069] Preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the signal peptide of the human NPR1 protein, further preferably comprises a nucleotide sequence encoding at least 10 consecutive amino acids of the signal peptide of the human NPR1 protein, such as a nucleotide sequence of at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32 consecutive amino acids, more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 1-32 of SEQ ID NO: 2. A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 1-32 of NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding an amino acid sequence shown at positions 1-32 of SEQ ID NO:2, including a substitution, deletion and / or insertion of one or more amino acids.

[0070] Preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the transmembrane region of the human NPR1 protein, further preferably comprises a nucleotide sequence encoding at least one consecutive amino acid of the transmembrane region of the human NPR1 protein, such as a nucleotide sequence of at least 1, 2, 3, 5, 10, 15, 18, 19, 20, or 21 consecutive amino acids, more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 474 to 494 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 474 to 494 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 474 to 494 of SEQ ID NO: 2. A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 474 to 494 of NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding an amino acid sequence shown at positions 474 to 494 of SEQ ID NO: 2, including a substitution, deletion and / or insertion of one or more amino acids.

[0071] Preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the cytoplasmic region of the human NPR1 protein, further preferably comprises a nucleotide sequence encoding at least 100 consecutive amino acids of the cytoplasmic region of the human NPR1 protein, such as a nucleotide sequence of at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 567 consecutive amino acids, more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 495-1061 of SEQ ID NO: 2; A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 495-1061 of NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding an amino acid sequence shown at positions 495-1061 of SEQ ID NO:2, including a substitution, deletion and / or insertion of one or more amino acids.

[0072] Preferably, the humanized NPR1 gene further comprises a partial nucleotide sequence encoding a non-human animal NPR1 protein.

[0073] Further preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the transmembrane region of the non-human animal NPR1 protein, and further preferably comprises a nucleotide sequence encoding at least 10 consecutive amino acids of the transmembrane region of the non-human animal NPR1 protein, such as a nucleotide sequence of at least 10, 15, 18, 19, 20, or 21 consecutive amino acids, and more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 470-490 of SEQ ID NO: 1; A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 470-490 of NO:1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding the amino acid sequence shown at positions 470-490 of SEQ ID NO:1, including substitution, deletion and / or insertion of one or more amino acids.

[0074] Further preferably, the humanized NPR1 gene comprises all or part of the nucleotide sequence encoding the cytoplasmic region of the non-human animal NPR1 protein, and further preferably comprises a nucleotide sequence encoding at least 50 consecutive amino acids of the cytoplasmic region of the non-human animal NPR1 protein, such as a nucleotide sequence of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 567 consecutive amino acids, and more preferably comprises a nucleotide sequence encoding the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; or, comprises a nucleotide sequence encoding an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 491-1057 of SEQ ID NO: 1; A nucleotide sequence comprising an amino acid sequence that differs from the amino acid sequence shown at positions 491-1057 of NO:1 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, a nucleotide sequence encoding an amino acid sequence shown at positions 491-1057 of SEQ ID NO:1, including a substitution, deletion and / or insertion of one or more amino acids.

[0075] In a specific embodiment of the present invention, the humanized NPR1 gene comprises a nucleotide sequence encoding the above-mentioned humanized NPR1 protein.

[0076] In a specific embodiment of the present invention, the mRNA transcribed from the humanized NPR1 gene comprises any one of the following groups:

[0077] A) the nucleotide sequence shown in SEQ ID NO: 8;

[0078] B) a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:8;

[0079] C) a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or,

[0080] D) A nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides compared to the nucleotide sequence shown in SEQ ID NO: 8.

[0081] Preferably, the humanized NPR1 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor can be a conventional substance that can be induced or repressed. In a specific embodiment of the present invention, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0082] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0083] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0084] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγnull Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice or nude mice.

[0085] In a fifth aspect of the present invention, a targeting vector of NPR1 gene is provided, wherein the targeting vector comprises a donor DNA sequence, and the donor DNA sequence comprises any one of the following groups:

[0086] A) a portion of the human NPR1 gene, preferably including all or part of exons 1 to 22 of the human NPR1 gene, more preferably including the nucleotide sequence of one, two or more, or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene, and even more preferably including the nucleotide sequence of part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the portion of exon 1 includes at least 100 bp of continuous nucleotide sequence, such as at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800, 850, 900, 950, 1000, 1100, 1142 bp of continuous nucleotide sequence. Alternatively, the portion of exon 1 at least includes the nucleotide sequence of the coding region. Alternatively, the portion of exon 1 at least includes the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the sequence from the start codon in exon 1 to the last nucleotide at the 3' end.

[0087] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0088] More preferably, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides;

[0089] B) all or part of the nucleotide sequence encoding the human NPR1 protein, preferably comprising all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of the human NPR1 protein;

[0090] Preferably, the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, such as a nucleotide sequence comprising at least 50 (e.g., at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441) consecutive amino acids of the extracellular region of human NPR1 protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of human NPR1 protein, such as a nucleotide sequence comprising at least 10 (e.g., at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31 or 32) consecutive amino acids of the signal peptide of human NPR1 protein; further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2;

[0091] C) the above humanized NPR1 gene;

[0092] D) Nucleotide sequence encoding the above humanized NPR1 protein.

[0093] Preferably, the targeting vector further comprises a 5' arm and / or a 3' arm, wherein:

[0094] The 5' arm is a DNA fragment homologous to the 5' end of the conversion region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the NPR1 gene of non-human animals, preferably nucleotides with at least 90% homology with NCBI accession number NC_000069.7. Further preferably, the 5' arm sequence comprises SEQ ID NO: 3 or 5.

[0095] The 3' arm is a DNA fragment homologous to the 3' end of the conversion region to be changed, which is selected from 100-10000 nucleotides in length of the genomic DNA of the NPR1 gene of non-human animals, preferably nucleotides with at least 90% homology with NCBI accession number NC_000069.7. Further preferably, the 3' arm sequence comprises SEQ ID NO: 4 or 6.

[0096] Preferably, the targeting vector comprises SEQ ID NO: 10 and / or 11.

[0097] Preferably, the targeting vector comprises SEQ ID NO: 12 and / or 13.

[0098] Preferably, the switch region to be changed is located on exons 1 to 7 of the NPR1 gene of a non-human animal.

[0099] Preferably, the targeting vector comprises, from the 5' end to the 3' end, a 5' homology arm, a donor DNA sequence, and a 3' homology arm.

[0100] Preferably, the targeting vector further comprises a marker gene. Further preferably, the marker gene is a gene encoding a negative selection marker. Even further preferably, the gene encoding the negative selection marker is a gene encoding a diphtheria toxin A subunit (DTA).

[0101] In a specific embodiment of the present invention, the targeting vector further comprises a resistance gene for positive clone screening. Further preferably, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo.

[0102] In a specific embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is an Frt recombination site (a conventional LoxP recombination system may also be selected). There are two specific recombination systems, which are respectively installed on both sides of the resistance gene in the same direction.

[0103] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0104] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0105] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice or nude mice.

[0106] In a sixth aspect, the present invention provides an sgRNA targeting the NPR1 gene.

[0107] The sgRNA targets exons 1 to 7 of the NPR1 gene of non-human animals, preferably exon 1 and / or exon 7.

[0108] Preferably, the target site of the sgRNA on the NPR1 gene comprises the nucleotide sequence shown in SEQ ID NO: 14 and / or 15.

[0109] In a seventh aspect, the present invention provides a DNA molecule encoding the above-mentioned sgRNA targeting the NPR1 gene.

[0110] Preferably, the double-stranded DNA molecule is the upstream and downstream sequences of the sgRNA, or the forward oligonucleotide sequence or the reverse oligonucleotide sequence after adding the restriction site.

[0111] In a specific embodiment of the present invention, the sequence of the DNA molecule is shown in Table 1.

[0112] In an eighth aspect, the present invention provides a vector comprising the above-mentioned sgRNA targeting the NPR1 gene.

[0113] In a ninth aspect of the present invention, a cell comprising the targeting vector of the NPR1 gene, the sgRNA targeting the NPR1 gene, the DNA molecule and / or the vector is provided.

[0114] In a tenth aspect, the present invention provides a use of a targeting vector comprising the NPR1 gene, the sgRNA targeting the NPR1 gene, the DNA molecule, the vector and / or the cell in NPR1 gene editing.

[0115] Preferably, the application includes but is not limited to knockout, insertion or replacement.

[0116] In the eleventh aspect of the present invention, a non-human animal with humanized NPR1 gene is provided, wherein the non-human animal expresses human or humanized NPR1 protein in vivo, and / or the genome of the non-human animal contains part of the human NPR1 gene or the humanized NPR1 gene.

[0117] Preferably, the humanized NPR1 protein is the above-mentioned humanized NPR1 protein, and the humanized NPR1 gene is the above-mentioned humanized NPR1 gene.

[0118] Preferably, the expression of endogenous NPR1 protein in the non-human animal is reduced or absent.

[0119] Preferably, the portion of the human NPR1 gene contained in the genome of the non-human animal includes:

[0120] A) comprising all or part of exons 1 to 22 of the human NPR1 gene, preferably comprising a nucleotide sequence of one, two or more than three or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene, and further preferably comprising a nucleotide sequence of part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the part of exon 1 comprises at least 100 bp of continuous nucleotide sequence, for example, at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800, 850, 900, 950, 1000, 1100, 1142 bp of continuous nucleotide sequence. Alternatively, the part of exon 1 at least comprises the nucleotide sequence of the coding region. Alternatively, the part of exon 1 at least comprises the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the region from the start codon in exon 1 to the last nucleotide at the 3' end.

[0121] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0122] More preferably, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides; or,

[0123] B) comprises all or part of the nucleotide sequence encoding human NPR1 protein, preferably comprises all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of human NPR1 protein;

[0124] Preferably, the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, such as a nucleotide sequence comprising at least 50 (e.g., at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441) consecutive amino acids of the extracellular region of human NPR1 protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of human NPR1 protein, such as a nucleotide sequence comprising at least 10 (e.g., at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31 or 32) consecutive amino acids of the signal peptide of human NPR1 protein; further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; A nucleotide sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence of the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of NO:2; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides as shown in the nucleotide sequence encoding the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2.

[0125] Preferably, the nucleotide sequence encoding the human or humanized NPR1 protein, a portion of the human NPR1 gene or the humanized NPR1 gene is operably linked to an endogenous regulatory element of an endogenous NPR1 gene in at least one chromosome.

[0126] Preferably, the genome of at least one cell of the non-human animal comprises a nucleotide sequence encoding a portion of the human NPR1 protein, a portion of the human NPR1 gene, or the above-mentioned humanized NPR1 gene.

[0127] Preferably, the non-human animal is constructed by introducing any of the following nucleotide sequences into the NPR1 locus of the non-human animal:

[0128] A) a portion of the human NPR1 gene, preferably including all or part of exons 1 to 22 of the human NPR1 gene, more preferably including the nucleotide sequence of one, two or more, or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene, and even more preferably including the nucleotide sequence of part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the portion of exon 1 includes at least 100 bp of continuous nucleotide sequence, such as at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800, 850, 900, 950, 1000, 1100, 1142 bp of continuous nucleotide sequence. Alternatively, the portion of exon 1 at least includes the nucleotide sequence of the coding region. Alternatively, the portion of exon 1 at least includes the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the sequence from the start codon in exon 1 to the last nucleotide at the 3' end.

[0129] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0130] More preferably, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides;

[0131] B) all or part of the nucleotide sequence encoding the human NPR1 protein, preferably comprising all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of the human NPR1 protein;

[0132] Preferably, the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, such as a nucleotide sequence comprising at least 50 (e.g., at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441) consecutive amino acids of the extracellular region of human NPR1 protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of human NPR1 protein, such as a nucleotide sequence comprising at least 10 (e.g., at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31 or 32) consecutive amino acids of the signal peptide of human NPR1 protein; further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2;

[0133] C) the above humanized NPR1 gene;

[0134] D) Nucleotide sequence encoding the above humanized NPR1 protein.

[0135] Preferably, the non-human animal is constructed using the above-mentioned targeting vector.

[0136] Preferably, the non-human animal further comprises other gene modifications, and further preferably, the other genes are selected from at least one of PD-1, PD-L1, GLP1R, OX40, LAG3, CTLA4, 4-1BB, CD73, ANGPTL3 or TIM3.

[0137] Preferably, the human or humanized NPR1 gene and / or other genes are homozygous for the endogenous modified locus.

[0138] Preferably, the human or humanized NPR1 gene and / or other genes are heterozygous for the endogenous modified locus.

[0139] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0140] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0141] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice or nude mice.

[0142] The twelfth aspect of the present invention provides a method for constructing a non-human animal with a humanized NPR1 gene, wherein the non-human animal expresses human or humanized NPR1 protein in vivo, and / or the genome of the non-human animal contains a portion of the human NPR1 gene or a humanized NPR1 gene.

[0143] Preferably, the humanized NPR1 protein is the above-mentioned humanized NPR1 protein, and the humanized NPR1 gene is the above-mentioned humanized NPR1 gene.

[0144] Preferably, the expression of endogenous NPR1 protein in the non-human animal is reduced or absent.

[0145] Preferably, the portion of the human NPR1 gene contained in the genome of the non-human animal includes:

[0146] A) comprising all or part of exons 1 to 22 of the human NPR1 gene, preferably comprising a nucleotide sequence of one, two or more than three or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene, and further preferably comprising a nucleotide sequence of part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the part of exon 1 comprises at least 100 bp of continuous nucleotide sequence, for example, at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800, 850, 900, 950, 1000, 1100, 1142 bp of continuous nucleotide sequence. Alternatively, the part of exon 1 at least comprises the nucleotide sequence of the coding region. Alternatively, the part of exon 1 at least comprises the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the region from the start codon in exon 1 to the last nucleotide at the 3' end.

[0147] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0148] More preferably, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides; or,

[0149] B) comprises all or part of the nucleotide sequence encoding human NPR1 protein, preferably comprises all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of human NPR1 protein;

[0150] Preferably, the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, such as a nucleotide sequence comprising at least 50 (e.g., at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441) consecutive amino acids of the extracellular region of human NPR1 protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of human NPR1 protein, such as a nucleotide sequence comprising at least 10 (e.g., at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31 or 32) consecutive amino acids of the signal peptide of human NPR1 protein; further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; A nucleotide sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to the nucleotide sequence of the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of NO:2; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides as shown in the nucleotide sequence encoding the amino acid sequence as shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2.

[0151] Preferably, the non-human animal is the non-human animal mentioned above.

[0152] Preferably, the genome of at least one cell of the non-human animal comprises a nucleotide sequence encoding a portion of a human or humanized NPR1 protein, a portion of a human NPR1 gene, or the above-mentioned humanized NPR1 gene.

[0153] Wherein, the nucleotide sequence encoding part of human or humanized NPR1 protein, part of human NPR1 gene or nucleotide sequence of humanized NPR1 gene is operably linked to the endogenous regulatory element of endogenous NPR1 gene in at least one chromosome.

[0154] Preferably, the construction method comprises introducing any of the following nucleotide sequences into the NPR1 locus of a non-human animal:

[0155] A) a portion of the human NPR1 gene, preferably including all or part of exons 1 to 22 of the human NPR1 gene, more preferably including the nucleotide sequence of one, two or more, or two or more consecutive exons of exons 1 to 22 of the human NPR1 gene, and even more preferably including the nucleotide sequence of part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the portion of exon 1 includes at least 100 bp of continuous nucleotide sequence, such as at least 100, 200, 300, 400, 500, 600, 700, 710, 720, 721, 722, 723, 724, 725, 730, 750, 800, 850, 900, 950, 1000, 1100, 1142 bp of continuous nucleotide sequence. Alternatively, the portion of exon 1 at least includes the nucleotide sequence of the coding region. Alternatively, the portion of exon 1 at least includes the nucleotide sequence encoding the signal peptide and / or the extracellular region in exon 1. Alternatively, the portion of exon 1 at least includes the sequence from the start codon in exon 1 to the last nucleotide at the 3' end.

[0156] The portion of exon 7 comprises at least 5 bp of continuous nucleotide sequence, for example, at least 5, 10, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 85 bp of continuous nucleotide sequence. Alternatively, the portion of exon 7 comprises at least the nucleotide sequence encoding the extracellular region. Alternatively, the portion of exon 7 comprises at least the first nucleotide at the 5' end to the last nucleotide sequence encoding the extracellular region.

[0157] More preferably, it comprises the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:7; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO:7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, comprises a nucleotide sequence shown in the nucleotide sequence shown in SEQ ID NO:7, including substitution, deletion and / or insertion of one or more nucleotides;

[0158] B) all or part of the nucleotide sequence encoding the human NPR1 protein, preferably comprising all or part of the nucleotide sequence encoding the extracellular region, signal peptide, cytoplasmic region and / or transmembrane region of the human NPR1 protein;

[0159] Preferably, the nucleotide sequence comprises all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, such as a nucleotide sequence comprising at least 50 (e.g., at least 50, 100, 150, 200, 250, 300, 350, 400, 437, 438, 439, 440, 441) consecutive amino acids of the extracellular region of human NPR1 protein, and further preferably comprises all or part of the nucleotide sequence encoding the signal peptide of human NPR1 protein, such as a nucleotide sequence comprising at least 10 (e.g., at least 10, 15, 20, 25, 26, 27, 28, 29, 30, 31 or 32) consecutive amino acids of the signal peptide of human NPR1 protein; further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; or, comprises a nucleotide sequence encoding the amino acid sequence shown in positions 33-473, 1-32 or 1-473 of SEQ ID NO: 2; a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2; or, a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; or, a nucleotide sequence that comprises a substitution, deletion and / or insertion of one or more nucleotides as set forth in the nucleotide sequence encoding the amino acid sequence as set forth at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2;

[0160] C) the above humanized NPR1 gene;

[0161] D) Nucleotide sequence encoding the above humanized NPR1 protein.

[0162] Preferably, any one of the nucleotide sequences A)-D) is expressed on a plasmid or on a chromosome.

[0163] The introduction is insertion or replacement.

[0164] Preferably, the introduction position is after the endogenous regulatory element.

[0165] The insertion is to place the target fragment directly between two adjacent bases without deleting nucleotides, or to place the target fragment at the deletion position after deleting part of the nucleotide sequence. According to the needs of the specific implementation scheme, the insertion may also include destroying the coding frame of the endogenous NPR1 gene of the non-human animal or destroying the coding frame of the endogenous NPR1 gene after the insertion sequence, followed by the insertion operation, or the insertion step can both cause a frameshift mutation to the endogenous NPR1 gene and achieve the step of inserting the sequence, the insertion sequence includes an auxiliary sequence, preferably, the auxiliary sequence can be a stop codon, a flip sequence or a knockout sequence, and further preferably, the auxiliary sequence is selected from a WPRE sequence, a 3'UTR, a polyA sequence and / or a STOP sequence.

[0166] The replacement is a replacement of a corresponding position or a replacement of a non-corresponding position. The replacement of a corresponding position does not only mechanically represent a direct corresponding replacement of the base sites of the NPR1 gene of human and non-human animals, but also includes the replacement of the corresponding functional region.

[0167] In a specific embodiment of the present invention, the introduction of the non-human animal NPR1 locus is to replace the corresponding region of the non-human animal NPR1 gene.

[0168] It is further preferred that all or part of exons 1 to 7 of the non-human animal NPR1 gene are replaced. That is, the introduction into the non-human animal NPR1 locus is to replace part of the nucleotide sequence from exon 1 to exon 7 of the non-human animal NPR1 gene.

[0169] It is further preferred that all or part of the nucleotide sequence encoding the NPR1 protein of the non-human animal is replaced. It is further preferred that all or part of the nucleotide sequence encoding the extracellular region and / or the signal peptide is replaced.

[0170] It is further preferred that the nucleotide sequence encoding positions 1 to 469 of SEQ ID NO: 1 is replaced.

[0171] Preferably, the nucleotide sequence encoding the human or humanized NPR1 protein, part of the human NPR1 gene or the humanized NPR1 gene is regulated in a non-human animal by a regulatory element. The regulatory element may be endogenous or exogenous. Preferably, the regulatory element includes but is not limited to a promoter.

[0172] In a specific embodiment of the present invention, the regulatory element is an endogenous regulatory element.

[0173] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the non-human animal NPR1 protein with all or part of the nucleotide sequence encoding the human NPR1 protein.

[0174] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the extracellular region of non-human animal NPR1 protein with all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein.

[0175] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the signal peptide and extracellular region of non-human animal NPR1 protein with all or part of the nucleotide sequence encoding the signal peptide and extracellular region of human NPR1 protein.

[0176] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the signal peptide, extracellular region and transmembrane region of the non-human animal NPR1 protein with all or part of the nucleotide sequence encoding the signal peptide, extracellular region and transmembrane region of the human NPR1 protein.

[0177] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of the nucleotide sequence encoding the signal peptide, extracellular region, cytoplasmic region and transmembrane region of the non-human animal NPR1 protein with all or part of the nucleotide sequence encoding the signal peptide, extracellular region, cytoplasmic region and transmembrane region of the human NPR1 protein.

[0178] In a specific embodiment of the present invention, the construction method comprises inserting or replacing the nucleotide sequence encoding positions 1-469 of SEQ ID NO: 1 in a non-human animal with a nucleotide sequence encoding positions 1-473 of SEQ ID NO: 2.

[0179] In a specific embodiment of the present invention, the construction method comprises inserting or replacing a portion of a non-human animal NPR1 gene with a portion comprising a human NPR1 gene.

[0180] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of exons 1 to 22 of the non-human animal NPR1 gene with all or part of exons 1 to 22 of the human NPR1 gene.

[0181] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of exons 1 to 7 of the non-human animal NPR1 gene with all or part of exons 1 to 7 of the human NPR1 gene.

[0182] In a specific embodiment of the present invention, the construction method comprises inserting or replacing all or part of exons 1 to 7 of the non-human animal NPR1 gene with a part of exon 1 to exon 7 of the human NPR1 gene.

[0183] In a specific embodiment of the present invention, the construction method comprises inserting or replacing the nucleotide sequence encoding positions 1-469 of SEQ ID NO: 1 in a non-human animal with the nucleotide sequence comprising SEQ ID NO: 7.

[0184] In one embodiment of the present invention, the construction method comprises introducing a genomic fragment of a non-human animal NPR1 gene with a portion of a human NPR1 gene at an endogenous NPR1 locus of a non-human animal to form a modified NPR1 gene.

[0185] The modified NPR1 gene encodes a humanized NPR1 protein.

[0186] The expression of the modified NPR1 gene is regulated by endogenous regulatory elements of non-human animals.

[0187] Preferably, gene editing technology is used to construct non-human animals with humanized NPR1 gene, and the gene editing technology includes gene targeting technology using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

[0188] Preferably, the non-human animal is constructed using the above-mentioned targeting vector and / or the above-mentioned sgRNA.

[0189] In a specific embodiment of the present invention, the construction method includes introducing the above-mentioned targeting vector into non-human animal cells (preferably embryonic stem cells), screening out the correct positive clone cells and introducing them into the isolated blastocysts, culturing the blastocysts, and then transplanting the cultured blastocysts into the fallopian tubes of female non-human animals, allowing them to develop, and identifying and screening to obtain non-human animals with humanized NPR1 genes.

[0190] Preferably, in order to improve the recombination efficiency, the above-mentioned NPR1 gene targeting vector and the above-mentioned sgRNA targeting the NPR1 gene can also be used together to construct non-human animals.

[0191] In a specific embodiment of the present invention, the construction method comprises introducing the aforementioned NPR1 gene targeting vector and the aforementioned sgRNA and Cas9 targeting the NPR1 gene into non-human animal cells, culturing the cells (preferably fertilized eggs), and then transplanting the cultured cells into the fallopian tubes of female non-human animals, allowing them to develop, and identifying and screening to obtain non-human animals with humanized NPR1 genes.

[0192] Preferably, the construction method further comprises mating, in vitro fertilization or direct gene editing of the NPR1 gene humanized non-human animal with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0193] Preferably, the other genes are selected from at least one of PD-1, PD-L1, GLP1R, OX40, LAG3, CTLA4, 4-1BB, CD73, ANGPTL3 or TIM3.

[0194] Preferably, the human or humanized NPR1 gene and / or other genes are homozygous for the endogenous modified locus.

[0195] Preferably, the human or humanized NPR1 gene and / or other genes are heterozygous for the endogenous modified locus.

[0196] Preferably, each of the multiple genes in the genome of the multi-gene modified non-human animal is homozygous for the endogenous modified locus.

[0197] Preferably, each of the multiple genes in the genome of the multi-gene modified non-human animal is heterozygous for the endogenous modified locus.

[0198] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0199] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0200] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / - mice, NOD / SCID mice or nude mice.

[0201] A thirteenth aspect of the present invention provides a non-human animal lacking the NPR1 gene, wherein the non-human animal lacks all or part of the NPR1 gene.

[0202] It is preferred to delete all or part of exons 1 to 7 of the NPR1 gene. It is further preferred to delete part of exon 1 to part of exon 7 of the NPR1 gene.

[0203] Preferably, the nucleotide sequence encoding the extracellular region and / or signal peptide of the NPR1 gene is deleted.

[0204] In a fourteenth aspect of the present invention, a method for constructing a non-human animal with a NPR1 gene deletion is provided, wherein the construction method comprises using the above-mentioned targeting vector and / or sgRNA to construct the non-human animal.

[0205] According to a fifteenth aspect of the present invention, a cell lacking the NPR1 gene is provided, wherein the cell lacks all or part of the NPR1 gene.

[0206] It is preferred to delete all or part of exons 1 to 7 of the NPR1 gene. It is further preferred to delete part of exon 1 to part of exon 7 of the NPR1 gene.

[0207] Preferably, the nucleotide sequence encoding the extracellular region and / or signal peptide of the NPR1 gene is deleted.

[0208] In a sixteenth aspect of the present invention, a method for constructing a cell lacking the NPR1 gene is provided, wherein the construction method comprises constructing the cell using the above-mentioned targeting vector and / or sgRNA.

[0209] The seventeenth aspect of the present invention provides a cell with humanized NPR1 gene.

[0210] The cell expresses human or humanized NPR1 protein, and / or the cell genome contains part of the human NPR1 gene or the humanized NPR1 gene.

[0211] Preferably, the cells express the above-mentioned humanized NPR1 protein.

[0212] Preferably, the cell genome comprises the above-mentioned humanized NPR1 gene.

[0213] In an eighteenth aspect of the present invention, a method for constructing NPR1 gene humanized cells is provided, the construction method comprising using the above-mentioned NPR1 gene targeting vector and / or the above-mentioned sgRNA targeting NPR1 gene to construct NPR1 gene humanized cells.

[0214] The nineteenth aspect of the present invention provides a genome of a non-human animal with humanized NPR1 gene.

[0215] Preferably, the genome comprises all or part of the human or humanized NPR1 gene, and / or comprises all or part of the nucleotide sequence encoding the human or humanized NPR1 protein.

[0216] Preferably, the humanized NPR1 gene is the above-mentioned humanized NPR1 gene.

[0217] Preferably, the humanized NPR1 protein is the above-mentioned humanized NPR1 protein.

[0218] Preferably, the genome comprises a genomic fragment of a human NPR1 gene (preferably a partial nucleotide sequence of a human NPR1 gene) introduced into the non-human animal NPR1 gene locus at the endogenous NPR1 locus of the non-human animal to form a modified NPR1 gene.

[0219] Preferably, the genome comprises a genomic fragment of a non-human animal NPR1 gene, in which all or part of exons 1 to 7 of the human NPR1 gene are introduced into the endogenous NPR1 locus of the non-human animal to form a modified NPR1 gene.

[0220] Preferably, the genome comprises a genomic fragment of a non-human animal NPR1 gene, in which a portion of exon 1 to a portion of exon 7 of a human NPR1 gene is introduced into the endogenous NPR1 locus of the non-human animal to form a modified NPR1 gene.

[0221] The modified NPR1 gene encodes a humanized NPR1 protein.

[0222] Preferably, the introduction is insertion or replacement.

[0223] Preferably, the genome comprises replacing a genomic fragment of a non-human animal NPR1 gene with a genomic fragment of a human NPR1 gene at the endogenous NPR1 locus of the non-human animal to form a modified NPR1 gene.

[0224] The genomic fragment of the human NPR1 gene comprises all or part of exons 1 to 7 of the human NPR1 gene, preferably comprising part of exon 1 to part of exon 7 of the human NPR1 gene.

[0225] The replaced genome fragment of the non-human animal NPR1 gene comprises all or part of exons 1 to 7 of the non-human animal NPR1 gene.

[0226] The modified NPR1 gene encodes a humanized NPR1 protein.

[0227] Preferably, the expression of the modified NPR1 gene is controlled by endogenous regulatory elements of the non-human animal.

[0228] Preferably, the genome comprises a humanized endogenous NPR1 locus, in which a segment of the endogenous NPR1 locus has been deleted and replaced with a corresponding human NPR1 sequence.

[0229] Preferably, the humanized endogenous NPR1 locus comprises an endogenous NPR1 promoter, wherein the human NPR1 sequence is operably linked to the endogenous NPR1 promoter.

[0230] Preferably, at least one intron and / or exon of the endogenous NPR1 locus has been deleted and replaced with the corresponding human NPR1 sequence.

[0231] In one embodiment of the present invention, a portion of exon 1 to a portion of exon 7 of the endogenous NPR1 locus has been deleted and replaced with the corresponding human NPR1 sequence.

[0232] In one embodiment of the present invention, the nucleotide sequence encoding the signal peptide and / or the extracellular region of the endogenous NPR1 locus has been deleted and replaced with the corresponding human NPR1 sequence.

[0233] Preferably, the expression of the modified NPR1 gene is controlled by endogenous regulatory elements of the non-human animal.

[0234] Preferably, the non-human animal can be selected from any non-human animal that can be gene-edited to prepare humanized genes, such as rodents, zebrafish, pigs, chickens, rabbits, monkeys, etc.

[0235] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent. Even more preferably, the rodent is a rat or a mouse.

[0236] 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. Still further preferably, the immunodeficient mouse is a NOD-Prkdc scid IL-2rγ null Mouse, NOD-Rag 1 - / - -IL2rg - / - Mouse, Rag 2 - / - -IL2rg - / -mice, NOD / SCID mice or nude mice.

[0237] The twentieth aspect of the present invention provides cells, tissues or organs comprising the genome of the non-human animal with humanized NPR1 gene as described above.

[0238] The twenty-first aspect of the present invention provides a method for constructing a multi-gene modified non-human animal, the method comprising the following steps:

[0239] 1) Providing any of the above-mentioned non-human animals, or a non-human animal obtained by any of the above-mentioned construction methods;

[0240] 2) The non-human animal provided in step 1) is mated with other genetically modified non-human animals, fertilized in vitro or directly gene-edited, and screened to obtain multi-gene modified non-human animals.

[0241] Preferably, the other genetically modified non-human animals include at least one of the non-human animals modified with genes PD-1, PD-L1, GLP1R, OX40, LAG3, CTLA4, 4-1BB, CD73, ANGPTL3 or TIM3.

[0242] Preferably, the multi-gene modified non-human animal is a two-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal, a five-gene humanized non-human animal, a six-gene humanized non-human animal, a seven-gene humanized non-human animal, an eight-gene humanized non-human animal or a nine-gene humanized non-human animal.

[0243] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal may be homozygous for the endogenous modified locus;

[0244] Preferably, each of the multiple genes modified in the genome of the multi-gene modified non-human animal can be heterozygous for the endogenous modified locus.

[0245] The twenty-second aspect of the present invention provides a non-human animal and its offspring obtained by any of the above-mentioned construction methods.

[0246] The twenty-third aspect of the present invention provides a cell, tissue or organ, the genome of which contains the above-mentioned humanized NPR1 gene, or the cell, tissue or organ expresses the above-mentioned humanized NPR1 protein, or the cell, tissue or organ is derived from the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method.

[0247] The twenty-fourth aspect of the present invention provides a tumor tissue, wherein the tumor tissue is derived from the non-human animal described above, or the non-human animal obtained by the above-mentioned construction method.

[0248] The twenty-fifth aspect of the present invention provides an animal model, which is derived from the non-human animal mentioned above, or the non-human animal obtained by the above-mentioned construction method. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0249] The twenty-sixth aspect of the present invention provides a method for constructing an animal model, wherein the construction method is carried out using the non-human animal mentioned above, or the non-human animal obtained by the above construction method. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

[0250] The twenty-seventh aspect of the present invention provides a use of any of the above-mentioned non-human animals, the non-human animals obtained by any of the above-mentioned construction methods, or the above-mentioned animal models in the preparation of drugs for the treatment and / or prevention of cardiovascular diseases, tumors, inflammation or immune-related diseases.

[0251] The twenty-eighth aspect of the present invention provides an application of the above-mentioned humanized NPR1 protein, or the above-mentioned humanized NPR1 gene, or the above-mentioned non-human animal, or the non-human animal obtained by the above-mentioned construction method, or the above-mentioned cells, tissues or organs, or the above-mentioned tumor tissues, or the above-mentioned animal models.

[0252] Preferably, the application may be for non-disease diagnosis and / or treatment purposes, or for disease diagnosis and / or treatment purposes.

[0253] The applications described include:

[0254] A) Application in product development involving NPR1-related immune processes in human cells;

[0255] B) Application as a model system related to NPR1 for pharmacology, immunology, microbiology and medical research;

[0256] C) Applications involving the production and use of animal experimental disease models for NPR1-related etiology studies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0257] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human NPR1 signaling pathway modulators; or,

[0258] E) Study the function of NPR1 gene, study the drugs and efficacy targeting human NPR1 target sites, and study the application of drugs related to NPR1 in inflammation, cardiovascular disease, immune-related diseases and anti-tumor drugs.

[0259] The twenty-ninth aspect of the present invention provides a non-human animal derived from any of the above-mentioned non-human animals, a non-human animal obtained by any of the above-mentioned construction methods, or the above-mentioned animal model for use in screening NPR1-specific regulators.

[0260] The thirtieth aspect of the present invention provides a method for screening NPR1-specific regulators, the method comprising applying the regulator to an individual implanted with tumor cells and detecting tumor inhibition; wherein the individual is the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method or the above-mentioned animal model.

[0261] Preferably, the regulator is selected from CAR-T and drugs. Further preferably, the drug is an antibody, specifically, the drug can be an anti-NPR1 antibody.

[0262] Preferably, the regulator is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0263] Preferably, said detecting comprises measuring the size and / or proliferation rate of tumor cells.

[0264] Preferably, the detection method includes vernier caliper measurement, flow cytometry detection and / or in vivo animal imaging detection.

[0265] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0266] Preferably, the tumor cells are derived from humans or non-human animals.

[0267] Preferably, the screening method can be for therapeutic or non-therapeutic purposes. The screening method detects and evaluates the effect of the modulator to determine whether the modulator has a therapeutic effect, that is, the therapeutic effect is not inevitable, but only a possibility.

[0268] The thirty-first aspect of the present invention provides a method for screening or evaluating human drugs, the method comprising constructing an animal model, administering a candidate drug to the animal model, and performing efficacy testing and / or comparison on the animal model administered with the candidate drug.

[0269] Preferably, the method for screening or evaluating human drugs can be for therapeutic purposes or non-therapeutic purposes. The method is used to screen or evaluate drugs, detect and compare the efficacy of candidate drugs to determine which candidate drugs can be used as drugs and which cannot be used as drugs, or compare the sensitivity of the efficacy of different drugs, that is, the therapeutic effect is not inevitable, but only a possibility.

[0270] Preferably, the candidate drug includes a targeted drug. Further preferably, the targeted drug is an antigen binding protein. In a specific embodiment of the present invention, the antigen binding protein is an antibody.

[0271] Preferably, the candidate drug is a monoclonal antibody or a bispecific antibody or a combination of two or more drugs.

[0272] Preferably, the detection includes determining the size and / or proliferation rate of tumor cells; preferably, the detection method includes vernier caliper measurement, flow cytometry and / or in vivo animal imaging detection.

[0273] Preferably, the testing comprises assessing an individual's body weight, fat mass, activated pathways, neuroprotective activity or metabolic changes, including changes in food consumption or water consumption.

[0274] The "cardiovascular disease" mentioned in the present invention refers to ischemic or hemorrhagic diseases of the heart, brain and systemic tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension, etc. For example, atherosclerosis, hypertension, hyperlipidemia, diabetes or heart failure.

[0275] The "immune-related diseases" described in the present invention include, but are not limited to, GVHD (graft versus host disease), psoriasis, allergies, asthma, atopic dermatitis, 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 disorders, etc.

[0276] The "tumor" described in 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, described leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia and chronic myeloid leukemia; described lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, T cell lymphoma and Waldenstrom macroglobulinemia; described sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma. In a specific embodiment of the present invention, described tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

[0277] The "inflammation" described in the present invention includes both acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

[0278] As used herein, "treat" means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0279] The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene or a part of a gene. For example, the "NPR1 locus" refers to a DNA fragment selected from exons 1 to 22 of the NPR1 gene. In a specific embodiment of the present invention, the replaced NPR1 locus can be a DNA fragment selected from exons 1 to 7 of the NPR1 gene.

[0280] The "nucleotide sequence" of the present invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences, preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA.

[0281] The “all or part” mentioned in the present invention, “all” refers to the whole, and “part” refers to a part of the whole, or an individual that makes up the whole.

[0282] The "humanized NPR1 protein" of the present invention comprises a portion derived from a human NPR1 protein. The "human NPR1 protein" is the same as the "entire human NPR1 protein", that is, its amino acid sequence is consistent with the full-length amino acid sequence of the human NPR1 protein. The "portion of the human NPR1 protein" is 5-1061 (preferably 10-473 or 10-442) consecutive or intermittent, such as 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 442, 450, 473, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1061, and the amino acid sequence is consistent with the amino acid sequence of the human NPR1 protein or has more than 80% homology with the amino acid sequence of the human NPR1 protein.

[0283] The "humanized NPR1 gene" of the present invention includes a portion derived from the human NPR1 gene. The "human NPR1 gene" is the same as the "entire human NPR1 gene", that is, its nucleotide sequence is consistent with the full-length nucleotide sequence of the human NPR1 gene. The "portion of the human NPR1 gene" is a continuous or intermittent 20bp-15305bp (preferably 20bp-1419bp, 20-4185bp, 20bp-1840bp), for example, a nucleotide sequence of 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1419, 1500, 1840, 2000, 2500, 3000, 3500, 4000, 4185, 4500, 5000, 10000, 15000, 15305bp, which is consistent with the nucleotide sequence of the human NPR1 gene or has more than 80% homology with the nucleotide sequence of the human NPR1 gene.

[0284] The "exons xx to xxx" or "all of exons xx to xxx" described in the present invention include the nucleotide sequences of exons and introns therebetween. For example, the "exons 1 to 7" described herein include the entire nucleotide sequences of exon 1, introns 1-2, exon 2, introns 2-3, exon 3, introns 3-4, exon 4, introns 4-5, exon 5, introns 5-6, exon 6, introns 6-7 and exon 7.

[0285] The "part of exon xx to part of exon xxx" described in the present invention represents part of exon xx, intron xx-(xx+1), all of exon xx+1 to exon xxx-1, intron (xxx-1)-xxx, and part of exon xxx. For example, the "part of exon 1 to part of exon 7" includes part of exon 1, introns 1-2, exon 2, introns 2-3, exon 3, introns 3-4, exon 4, introns 4-5, exon 5, introns 5-6, exon 6, introns 6-7, and part of exon 7.

[0286] The "intron x-xx" of the present invention refers to the intron between exon x and exon xx. For example, "intron 1-2" refers to the intron between exon 1 and exon 2.

[0287] The "cell" described in the present invention may be a fertilized egg cell or other somatic cell, etc. Therefore, depending on the source of the cell, some of the cells described in the present application can develop into individual animals, while some cannot.

[0288] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0289] The term "comprising" or "including" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. However, when used to describe a protein or nucleic acid sequence, 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 in the present invention.

[0290] The "homology" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (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.

[0291] One skilled in the art is able to determine and compare sequence elements or degrees of identity to distinguish between additional mouse and human sequences.

[0292] In one aspect, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as the family of Jerboas. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family of Muridae. In one embodiment, the genetically modified animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Malvinas (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormouseidae (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the family of Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from the group consisting of a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0293] In a specific embodiment, the non-human animal is a rodent selected from the group consisting of BALB / c, A, A / He, A / J, A / WySN, AKR, 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 of C57BL, C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, CBA / H strains and NOD, NOD / SCID, NOD-Prkdc scid IL-2rg null Background of mice.

[0294] Unless otherwise indicated, the practice of the present invention will employ 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 literature. For example: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (DNGlovered., 1985); Oligonucleotide Synthesis (MJGaited., 1984); Mullisetal. US Pat. No. 4, 683, 195; Nucleic Acid Hybridization (BDHames&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RIFreshney, 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.Abelsonand M.Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols.154and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes V (DMWeir and CCBlackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0295] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0296] All patents and publications mentioned in this specification are incorporated herein by reference as a whole. Those skilled in the art will recognize that certain changes may be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention in detail and should not be considered to limit the scope of the present invention or the specific methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0297] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0298] Figure 1 : Schematic diagram comparing the mouse NPR1 locus and the human NPR1 locus (not to scale);

[0299] Figure 2 : Schematic diagram of the humanization transformation of the mouse NPR1 locus (not to scale);

[0300] Figure 3 : Schematic diagram of NPR1 gene targeting strategy and targeting vector V1 design (not to scale);

[0301] Figure 4 : Schematic diagram of the FRT recombination process of NPR1 gene humanized mice (not to scale);

[0302] Figure 5 : Schematic diagram of NPR1 gene targeting strategy and targeting vector V2 design (not to scale);

[0303] Figure 6 :Southern blot detection results, where WT is the wild-type control;

[0304] Figure 7:Western Blot test results, where + / + is wild-type C57BL / 6 mice, H / H is NPR1 gene humanized homozygous mice, GAPDH is glyceraldehyde-3-phosphate dehydrogenase internal reference;

[0305] Figure 8 : RT-PCR test results, where + / + represents wild-type C57BL / 6 mice, H / H represents NPR1 gene humanized homozygous mice, H2O represents water control, and GAPDH represents glyceraldehyde-3-phosphate dehydrogenase internal reference. DETAILED DESCRIPTION

[0306] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0307] In each of the following examples, equipment and materials were obtained from the following companies:

[0308] C57BL / 6 mice were purchased from the National Rodent Laboratory Animal Seed Center of the China Food and Drug Administration;

[0309] BbsI, EcoRI, BamHI, ScaI, and AvrII enzymes were purchased from NEB with catalog numbers: R3539S, R3101S, R3136S, R3122S, and R0174S, respectively;

[0310] NPR1 Polyclonal Antibody was purchased from thermofisher, catalog number PA5-99835;

[0311] Anti-NPR-A antibody was purchased from abcam, catalog number ab14356;

[0312] GAPDH(D16H11) Rabbit mAb was purchased from cell signaling, catalog number 5174.

[0313] Example 1 NPR1 gene humanized mice

[0314] A schematic diagram of the comparison between the mouse NPR1 gene (NCBI Gene ID: 18160, Primary source: MGI: 97371, UniProt ID: P18293, located at positions 90357898 to 90373235 of chromosome 3 NC_000069.7, based on transcript NM_008727.5 and its encoded protein NP_032753.5 (SEQ ID NO: 1)) and the human NPR1 gene (NCBI Gene ID: 4881, Primary source: HGNC: 7943, UniProt ID: P16066, located at positions 153678688 to 153693992 of chromosome 1 NC_000001.11, based on transcript NM_000906.4 and its encoded protein NP_000897.3 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0315] In order to achieve the purpose of the present invention, a nucleotide sequence encoding a human NPR1 protein can be introduced into the mouse endogenous NPR1 locus, so that the mouse expresses a human or humanized NPR1 protein. Specifically, using gene editing technology, under the control of the mouse NPR1 gene regulatory element, a partial sequence of exon 1 to a partial sequence of exon 7 of about 4.7 kb of the human NPR1 gene is used to replace a partial sequence of exon 1 to a partial sequence of exon 7 of about 4.3 kb of the mouse, and a schematic diagram of the humanized NPR1 locus is obtained as shown in FIG. Figure 2 As shown, the humanization transformation of the mouse NPR1 gene was achieved.

[0316] according to Figure 2 Further design such as Figure 3 The targeting strategy shown in the figure shows that the V1 targeting vector contains homology arm sequences upstream and downstream of the mouse NPR1 gene, as well as a human NPR1 fragment. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 90372811 to 90376786 of the NCBI accession number NC_000069.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 90363920 to 90367931 of the NCBI accession number NC_000069.7. The nucleotide sequence of the human NPR1 fragment (SEQ ID NO: 7) is identical to the nucleotide sequence of positions 153679109 to 153683759 of the NCBI accession number NC_000001.11; the connection design of the upstream of the human NPR1 fragment sequence and the mouse is: 5'-GACCATGGTGACAGCGCTGCTCCGTCGCTGCGCTCGCTGA GGCC CGGGGCCCCGGCGCCCCGCTGGCTCCCGCCTGCGCCTGCTC-3' (SEQ ID NO: 10), wherein the sequence " GGCC The last "C" in the sequence is the last nucleotide in the mouse. The "A" in the sequence is the first nucleotide of the human sequence. The downstream connection of the human NPR1 fragment sequence and the mouse sequence is designed as: 5'-TCTTGCTGCAATATGGACTCTCTCCTGCAGATCACCTTTCCACCCT GGAG TGGCTTTGGTGGGCAGCCTCTCTCTGGTTAGCTTT-3' (SEQ ID NO: 11), wherein the sequence " GGAG The last "G" in the sequence is the last nucleotide in humans. The "G" in is the first nucleotide in the mouse sequence.

[0317] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the mouse gene is designed as: 5'-TTCAACTAAAGGAGTTGGTTCTGGATGACAATTCAG CTGA TCGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAG-3' (SEQ ID NO: 12), wherein the sequence " CTGA "A" is the last nucleotide in the mouse sequence. The "G" in the Neo box is the first nucleotide; the connection between the 3' end of the Neo box and the mouse gene is designed as: 5'-GAACTTCATCAGTCAGGTACATAATGGTGGATCCAT GCAT TAGGTGCTGACGCTAAAAGAAGGTGAGCGA-3' (SEQ ID NO: 13), wherein the sequence " GCAT "T" in the sequence is the last nucleotide of the Neo box. The first "G" in is the first nucleotide of the mouse. The mRNA sequence of the modified humanized mouse NPR1 is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.

[0318] Conventional methods can be used to construct the targeting vector, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified to be correct by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen out the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to techniques known in the art. The obtained chimeric blastocysts are transferred to the culture medium and cultured briefly before being transplanted into the oviducts of the recipient mother mice (white mice), which can produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and the F1 generation heterozygous mice are then mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene (see the schematic diagram of this process). Figure 4 ) and then mating with each other to obtain NPR1 gene humanized homozygous mice.

[0319] In addition, CRISPR / Cas9 system can be used for gene editing to further design Figure 5 The targeting strategy shown in the figure shows that the targeting vector V2 contains the homology arm sequences upstream and downstream of the mouse NPR1 gene, as well as the human NPR1 fragment. The schematic diagram of the humanized NPR1 locus after construction is shown in Figure 2 As shown. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 90372811 to 90374218 of NCBI accession number NC_000069.7, and the downstream 3' homology arm sequence (SEQ ID NO: 6) is identical to the nucleotide sequence of positions 90367007 to 90368461 of NCBI accession number NC_000069.7. The nucleotide sequence of the human NPR1 fragment (SEQ ID NO: 7) is identical to the nucleotide sequence of positions 153679109 to 153683759 of NCBI accession number NC_000001.11. The mRNA sequence of the modified humanized mouse NPR1 is shown in SEQ ID NO: 8, and the expressed protein sequence is shown in SEQ ID NO: 9.

[0320] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0321] The target sequence determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of an exemplary sgRNA on the NPR1 gene is as follows:

[0322] sgRNA1 target site (SEQ ID NO: 14): 5'-GAGCGACCTGACCGTGGCCGTGG-3';

[0323] sgRNA2 target site (SEQ ID NO: 15): 5'-ACTGGGGTTAGACATTAGGAAGG-3';

[0324] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 1. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-NPR1-1 and pT7-NPR1-2.

[0325] Table 1: Sequence list of sgRNA1 and sgRNA2

[0326]

[0327] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 24) of the T7 promoter and sgRNA scaffold and connected to the backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. The sequencing was verified by a professional sequencing company, and the results showed that the target plasmid was obtained. Take the pronuclear fertilized eggs of mice, such as C57BL / 6 mice, and use a microinjector to pre-mix the in vitro transcription products of pT7-NPR1-1 and pT7-NPR1-2 plasmids (using the Ambion in vitro transcription kit, according to the instructions for transcription), the targeting vector and Cas9 mRNA and inject them into the cytoplasm or nucleus of the mouse fertilized eggs. Microinjection of fertilized eggs was performed according to the method in the Mouse Embryo Manipulation Experiment Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable NPR1 gene humanized mouse strain.

[0328] Table 2: Primer sequences and recombinant fragment sizes for PCR detection of F0 genotypes

[0329]

[0330] PCR technology can be used to screen F0 generation positive mice, and the primers are shown in Table 2. The NPR1 gene humanized mice identified as positive in F0 are mated with wild-type mice to obtain F1 generation mice. The F1 generation mice are mated with each other to obtain NPR1 gene humanized homozygous mice. Positive clones are screened by PCR (primers are shown in Table 2). The clones identified as positive by PCR are then subjected to Southern blot detection (cell DNA is digested with ScaI or AvrII and hybridized with 2 probes, and the probes and target fragment lengths are shown in Table 3) to confirm whether there is random insertion. Exemplary results are shown in Figure 6 As shown, combined with the PCR and sequencing results, the six mice numbered F1-01, F1-02, F1-03, F1-04, F1-05 and F1-07 had no random insertions. This indicates that the present method can be used to construct NPR1 gene humanized mice that can be stably propagated and have no random insertions.

[0331] Table 3: Specific probe and target fragment lengths

[0332] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size ScaI 5'Probe-A 5.1kb 5.8kb AvrII AProbe(3') - 6.2kb

[0333] 5'Probe-F (SEQ ID NO: 27): 5'-TCATTGTCCCCAAACTCCAGCGTTA-3',

[0334] 5'Probe-R (SEQ ID NO: 28): 5'-CCCATGTCCCTTACAGCACCATCAA-3';

[0335] 3'Probe-F (SEQ ID NO: 29): 5'-ACCATGCCTGGACACTTACAAGAGC-3',

[0336] 3'Probe-R (SEQ ID NO: 30): 5'-ACCCGATCTCTACACTCAGGGAGTT-3';

[0337] In addition, the expression of humanized NPR1 (hNPR1) protein in NPR1 humanized mice can be detected by conventional methods such as Western Blot. Specifically, 8-week-old female C57BL / 6 mice (+ / +) and humanized homozygous NPR1 genes (H / H) prepared in this embodiment were taken, and the heart, liver, spleen, lung, brain and ovarian tissues were taken after euthanasia by cervical dislocation. Human-mouse cross antibody NPR1 Polyclonal Antibody and human-rat cross antibody Anti-NPR-A antibody, as well as GAPDH (D16H11) were used. Rabbit mAb was used for Western Blot detection. Figure 7 As shown in the results, it can be seen that the expression of hNPR1 protein can be detected in the livers of wild-type C57BL / 6 mice and NPR1 gene humanized homozygous mice using human-mouse cross antibodies. This result shows that the transformed humanized mice can normally express hNPR1 protein.

[0338] The expression of mRNA in NPR1 gene humanized mice was further detected by RT-PCR. Specifically, 8-week-old female C57BL / 6 mice (+ / +) and homozygous NPR1 gene humanized mice (H / H) prepared in this embodiment were selected, and lung tissues were obtained after euthanasia by cervical dislocation. RT-PCR detection was performed using the primer sequences shown in Table 4. The test results are shown in FIG. Figure 8 As shown in the figure, it can be seen that only mouse NPR1 mRNA was detected in wild-type C57BL / 6 mice, but no humanized NPR1 mRNA was detected; in NPR1 gene homozygous mice, only humanized NPR1 mRNA was detected, but no mouse NPR1 mRNA was detected.

[0339] Table 4 RT-PCR primer sequences and target fragment sizes

[0340]

[0341] Example 2 Pharmacodynamic Model

[0342] The NPR1 humanized mice prepared by this method can be used to evaluate the efficacy of modulators targeting human NPR1. For example, NPR1 humanized homozygous mice are taken, angiotensin II is used to induce the mice to increase their blood pressure, and the mice are divided into a control group or a treatment group according to the blood pressure. The treatment group randomly selects drugs targeting human NPR1, and the control group is injected with an equal volume of normal saline. The blood pressure and body weight of the mice are measured regularly, and the in vivo safety and in vivo efficacy of the compound can be effectively evaluated by comparing the changes in the weight and blood pressure of the mice.

[0343] Example 3 Preparation of double-gene or multi-gene humanized mice

[0344] The humanized NPR1 gene mice prepared by this method can also be used to prepare multi-humanized mouse models. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice modified with genes such as PD-1, PD-L1, GLP1R, OX40, LAG3, CTLA4, 4-1BB, CD73, ANGPTL3 or TIM3, or, on the basis of humanized NPR1 mice, separate mouse ES embryonic stem cells and gene recombination targeting technology can be used to obtain double humanized or multi-humanized mouse models. The homozygous or heterozygous NPR1 mice obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendel's genetic laws, there is a certain probability of obtaining multi-gene mice modified with humanized NPR1 genes and other genes, and then mating heterozygotes with each other can obtain homozygous double-gene or multi-gene modified.

[0345] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0346] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0347] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a non-human animal with humanized NPR1 gene, It is characterized in that The non-human animal expresses human or humanized NPR1 protein in vivo, and / or the genome of the non-human animal contains part of the human NPR1 gene or the humanized NPR1 gene.

2. The construction method according to claim 1, It is characterized in that The construction method comprises introducing any of the following nucleotide sequences into the NPR1 locus of a non-human animal: A) a portion of the human NPR1 gene, preferably comprising all or part of exons 1 to 22 of the human NPR1 gene, further preferably comprising a portion of the nucleotide sequence of exon 1 to exon 7 of the human NPR1 gene, more preferably comprising the nucleotide sequence shown in SEQ ID NO: 7; or comprising a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 7; B) a partial nucleotide sequence encoding human NPR1 protein, preferably comprising all or part of the nucleotide sequence encoding the extracellular region of human NPR1 protein, for example, comprising a nucleotide sequence encoding at least 50 consecutive amino acids of the extracellular region of human NPR1 protein, further preferably comprising a nucleotide sequence encoding the amino acid sequence shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2; or, comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with the nucleotide sequence encoding the amino acid sequence shown at positions 33-473, 1-32 or 1-473 of SEQ ID NO:2; C) Humanized NPR1 gene; D) Nucleotide sequence encoding humanized NPR1 protein.

3. The construction method according to claim 2, It is characterized in that The introduction is insertion or replacement; preferably, the introduction into the non-human animal NPR1 locus is replacement of a portion of the nucleotide sequence from exon 1 to exon 7 of the non-human animal NPR1 gene.

4. The method according to any one of claims 1 to 3, It is characterized in that The method comprises replacing a nucleotide sequence encoding an extracellular corresponding region of endogenous mouse NPR1 with a nucleotide sequence encoding an extracellular corresponding region of human NPR1 at an endogenous mouse NPR1 locus.

5. The method according to any one of claims 1 to 4, It is characterized in that The humanized NPR1 protein comprises a human or humanized extracellular region, an endogenous transmembrane region and an endogenous cytoplasmic region. Preferably, the humanized NPR1 protein is as shown in SEQ ID NO:

9.

6. The construction method according to any one of claims 1 to 5, It is characterized in that The nucleotide sequence encoding the human or humanized NPR1 protein, the portion of the human NPR1 gene, or the humanized NPR1 gene is regulated in a non-human animal by an endogenous regulatory element.

7. The construction method according to any one of claims 1 to 6, It is characterized in that The construction method also includes mating, in vitro fertilization or direct gene editing of non-human animals with humanized NPR1 genes with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals; Preferably, the other genes are selected from at least one of PD-1, PD-L1, GLP1R, OX40, LAG3, CTLA4, 4-1BB, CD73, ANGPTL3 or TIM3.

8. The construction method according to any one of claims 1 to 7, It is characterized in that The non-human animal is a non-human mammal, preferably a rodent, and more preferably a rat or a mouse.

9. A humanized NPR1 protein, It is characterized in that The humanized NPR1 protein comprises all or part of the human NPR1 protein, and part of the endogenous NPR1 protein of a non-human animal.

10. The humanized NPR1 protein according to claim 9, It is characterized in that The humanized NPR1 protein comprises all or part of the extracellular region, transmembrane region and / or cytoplasmic region of the human NPR1 protein, preferably comprises the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; or, comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO: 2; more preferably, comprises the amino acid sequence shown at positions 470-490 and / or 491-1057 of SEQ ID NO: 1; or, comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown at positions 33-473 of SEQ ID NO:

2. NO:1 at positions 470-490 and / or 491-1057, which are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:1; more preferably, it comprises the amino acid sequence shown in SEQ ID NO:9; or, it comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:

9.

11. A humanized NPR1 gene, It is characterized in that The humanized NPR1 gene encodes the humanized NPR1 protein according to any one of claims 9 to 10, or the humanized NPR1 gene comprises a portion of a human NPR1 gene.

12. The gene according to claim 11, It is characterized in that The humanized NPR1 gene comprises all or part of exons 1 to 22 of the human NPR1 gene, preferably comprises part of exon 1 to part of exon 7 of the human NPR1 gene, wherein the part of exon 1 comprises at least 100 bp of continuous nucleotide sequence, and the part of exon 7 comprises at least 5 bp of continuous nucleotide sequence. Further preferably, the humanized NPR1 gene comprises part of a non-human animal NPR1 gene, preferably comprises all or part of the nucleotide sequence from part of exon 7 to exon 22. More preferably, the humanized NPR1 gene comprises the nucleotide sequence shown in SEQ ID NO: 7 or 8; or, comprises a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 7 or 8.

13. A cell, tissue or organ, It is characterized in that The genome of the cell, tissue or organ contains the humanized NPR1 gene described in any one of claims 11-12, or the cell, tissue or organ expresses the humanized NPR1 protein described in any one of claims 9-10, or the cell, tissue or organ is derived from a non-human animal obtained by the construction method described in any one of claims 1-8.

14. A use of the humanized NPR1 protein according to any one of claims 9 to 10, or the humanized NPR1 gene according to any one of claims 11 to 12, or the non-human animal obtained by the construction method according to any one of claims 1 to 8, or the cell, tissue or organ according to claim 13, It is characterized in that The applications described include: A) Application in product development involving NPR1-related immune processes in human cells; B) Application as a model system related to NPR1 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for NPR1-related etiology studies and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human NPR1 signaling pathway modulators; or, E) Study the function of NPR1 gene, study the drugs and efficacy targeting human NPR1 target sites, and study the application of drugs related to NPR1 in inflammation, cardiovascular disease, immune-related diseases and anti-tumor drugs.

Images