CN113234139A

CN113234139A - TNFSF9 gene humanized non-human animal and construction method and application thereof

Info

Publication number: CN113234139A
Application number: CN202110414462.8A
Authority: CN
Inventors: 沈月雷; 姚佳维; 郭雅南; 白阳; 赵磊
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd; Biocytogen Jiangsu Gene Biotechnology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd; Biocytogen Jiangsu Gene Biotechnology Co ltd
Priority date: 2020-04-17
Filing date: 2021-04-16
Publication date: 2021-08-10
Also published as: WO2021209050A1

Abstract

The invention provides a construction method of a TNFSF9 gene humanized non-human animal and application in the field of biomedicine. The non-human animal body constructed by the method can normally express human or humanized TNFSF9 protein, can be used for research on human TNFSF9 signal path mechanism and multiple aspects of human immune system, and has important application value for research and development of new immune-related medicines. The invention also provides a humanized TNFSF9 protein, a humanized TNFSF9 gene, a targeting vector and the like.

Description

TNFSF9 gene humanized non-human animal and construction method and application thereof

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a genetically modified non-human animal which expresses human or humanized TNFSF9 protein, in particular to a TNFSF9 gene humanized non-human animal, a construction method thereof and application thereof in the field of biomedicine.

Background

4-1BB/TNFSF9 is an important member of the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) superfamily, and is also a pair of co-stimulatory molecules which are currently concerned, and the mediated bidirectional signal plays an important role in T lymphocyte, monocyte and DC mediated immune regulation.

4-1BB, also known as CD137, is a type I transmembrane glycoprotein with a molecular weight of 30kD, which is mainly expressed on the surfaces of activated CD4+ T cells, CD8+ T cells and NK cells, and also expressed on the surfaces of monocytes, activated macrophages and DCs. In addition, 4-1BB expression was also detected on part of the non-immune cell surface, such as malignant vascular endothelial cells. In the presence of TCR signaling, the 4-1BB costimulatory signal can cooperate with the costimulatory signal of CD28 to maintain the activated state of T cells and inhibit activation-induced Apoptosis (AICD). The CD28 costimulatory signal acts primarily during the pre-phase of T cell activation, promoting T cell proliferation and maintaining short-term survival, while the 4-1BB costimulatory signal acts primarily during the post-phase of T cell activation.

TNFSF9, also known as 4-1BBL, is a ligand for 4-1BB, is a type II transmembrane glycoprotein expressed on the surface of various activated Antigen Presenting Cells (APC), such as IFN-. gamma.activated macrophages, CD40 ligand-activated B cells, monocytes, DCs, T cells, and the like. TNFSF9 on the surface of cell membrane can transmit reverse signal to inhibit proliferation of activated T cell and induce its apoptosis; the reverse signal can also induce the activation of monocytes, promote the secretion of IL-6, IL-8 and TNF-Ade and prolong the survival of the monocytes; and also stimulates maturation of CD34+ hematopoietic stem cell-derived DCs.

The transmission of 4-1BB/TNFSF9 signal is bidirectional, i.e., after 4-1BB binds to its ligand TNFSF9, co-stimulatory signals (positive signals) are transmitted through the receptor 4-1BB, resulting in a series of biological effects on the cell; meanwhile, the reverse signal can be transmitted through the ligand TNFSF9, and the negative regulation effect is realized in the activation process of T cells. The characteristic enables the 4-1BB/TNFSF9 signal channel to promote the proliferation of antigen-sensitized T cells, particularly CD8+ T cells, control the development of diseases such as tumors and virus infection, and simultaneously inhibit the damage of autoreactive CD4+ T cells to organisms and prevent the occurrence of autoimmune diseases. In view of the wide regulation effect of the 4-1BB/TNFSF9 bidirectional signal channel, the wide expression on the surfaces of various immune cells and the co-expression of some cells, the 4-1BB/TNFSF9 molecules on the surfaces of various cells can form a complex regulation system of an organism, and the method has important significance on the precise regulation of immune regulation and response, the development of the immune system and other processes.

The experimental animal disease model is an indispensable research tool for researching human disease pathogenesis, developing prevention and treatment technologies and medicines. Due to the significant difference between the amino acid sequences of human 4-1BB and TNFSF9 and the corresponding proteins in rodents, for example, the protein sequences of human 4-1BB and mouse 4-1BB are only 60% identical, and the protein sequences of human TNFSF9 and mouse TNFSF9 are only 36% identical, antibodies recognizing human 4-1BB or TNFSF9 proteins generally cannot recognize mouse 4-1BB or TNFSF9 proteins, i.e., ordinary mice cannot be used to screen and evaluate the efficacy of drugs targeting the 4-1BB/TNFSF9 signal pathway. In view of the extremely important utility value of the 4-1BB/TNFSF9 signaling pathway, there is an urgent need in the art to develop 4-1BB and/or TNFSF9 humanized non-human animal models in order to make preclinical testing more effective and minimize the rate of development failures.

Disclosure of Invention

In a first aspect of the present invention, there is provided a humanized TNFSF9 protein, said humanized TNFSF9 protein comprising all or part of a human TNFSF9 protein.

Preferably, the humanized TNFSF9 protein comprises all or part of the transmembrane, extracellular and/or cytoplasmic domain of human TNFSF9 protein.

Further preferably, the humanized TNFSF9 protein comprises a transmembrane region and/or an extracellular region of human TNFSF9 protein.

Still further preferably, said humanized TNFSF9 protein further comprises a portion of the cytoplasmic region of human TNFSF9 protein; preferably, the portion of the cytoplasmic domain of human TNFSF9 protein is identical to SEQ ID NO: 4 or at least 90% identical to the amino acid sequence at positions 26-28 of SEQ ID NO: 4, 26 th to 28 th amino acid sequence.

In one embodiment of the invention, the humanized TNFSF9 protein comprises the transmembrane region of human TNFSF9 protein; or an extracellular region comprising human TNFSF9 protein; or an extracellular region and a transmembrane region of the human TNFSF9 protein; or an extracellular region and a cytoplasmic region comprising the human TNFSF9 protein; or comprises the transmembrane and cytoplasmic domains of the human TNFSF9 protein.

Preferably, said humanized TNFSF9 protein further comprises a portion of a non-human animal TNFSF9 protein. Further preferred is a cytoplasmic domain of TNFSF9 protein in a non-human animal.

In one embodiment of the invention, the humanized TNFSF9 protein comprises an extracellular domain, a transmembrane domain, and a non-human animal cytoplasmic domain derived from a human TNFSF9 protein.

Preferably, the humanized TNFSF9 protein comprises all or part of the amino acid sequence encoded by exons 1 to 3 of a human TNFSF9 gene. Further preferably, said humanized TNFSF9 protein comprises all or part of an amino acid sequence encoded by one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene. Still more preferably, the humanized TNFSF9 protein comprises a portion of exon 1, an amino acid sequence encoded by exons 2 to 3 of the human TNFSF9 gene, wherein the portion of exon 1 preferably comprises a nucleotide sequence encoding a transmembrane region and/or an extracellular region, and further preferably further comprises a nucleotide sequence encoding SEQ ID NO: 4, 26-28.

Preferably, said humanized TNFSF9 protein further comprises a portion of the amino acid sequence encoded by exon 1 of a non-human animal TNFSF9 gene, preferably said portion of the amino acid sequence encoded by exon 1 of a non-human animal TNFSF9 gene that hybridizes to SEQ ID NO: 2 or at least 90% identical to the amino acid sequence at positions 1-82 of SEQ ID NO: 2, 1-82 amino acid sequence.

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

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

Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is an immunodeficient rodent, an immunodeficient pig, an immunodeficient rabbit or an immunodeficient monkey. Still further preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is a NOD-Prkdcscid IL-2r γ nul mouse, a NOD-Rag 1-/- -IL2RG-/- - (NRG) mouse, a Rag 2-/- -IL2RG-/- - (RG) mouse, a NOD/SCID mouse, or a nude mouse.

Preferably, the amino acid sequence of the humanized TNFSF9 protein derived from the human TNFSF9 protein has the amino acid sequence shown in SEQ ID NO: 4 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

Preferably, the amino acid sequence of the humanized TNFSF9 protein derived from the non-human animal TNFSF9 protein is identical to the amino acid sequence of SEQ ID NO: 2 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

In one embodiment of the present invention, the humanized TNFSF9 protein comprises a portion of the amino acid sequence of human TNFSF9 protein comprising one of the following groups:

A) is SEQ ID NO: 4, all or part of the amino acid sequence from position 26 to 254;

B) and SEQ ID NO: 4, at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% amino acid sequence identity between positions 26-254;

C) and SEQ ID NO: 4 from position 26 to 254, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or

D) And SEQ ID NO: 4, 26-254, including substitution, deletion and/or insertion of one or more amino acid residues.

In one embodiment of the present invention, the amino acid sequence of the humanized TNFSF9 protein is selected from one of the following groups:

a) is SEQ ID NO: 12 amino acid sequence, in whole or in part;

b) and SEQ ID NO: 12 are at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical in amino acid sequence;

c) and SEQ ID NO: 12 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 amino acid; or

d) And SEQ ID NO: 12, comprising substitution, deletion and/or insertion of one or more amino acid residues.

Optionally, the humanized TNFSF9 protein recognizes and \ or binds a humanized 4-1BB protein, said humanized 4-1BB protein comprising all or a portion of a human 4-1BB protein; further preferably, the polypeptide comprises all or part of the extracellular region of human 4-1BB protein, and further preferably further comprises a signal peptide; more preferably, the polypeptide comprises a sequence identical to SEQ ID NO: 54, 1-184 or an amino acid sequence having 90% homology to SEQ ID NO: 54 from position 1 to 184.

In a second aspect of the present invention, there is provided a humanized TNFSF9 gene, said humanized TNFSF9 gene comprising a portion of the human TNFSF9 gene.

Preferably, the humanized TNFSF9 gene comprises all or part of a nucleotide sequence encoding a transmembrane, cytoplasmic, and/or extracellular region of human TNFSF9 protein. Further preferably, said humanized TNFSF9 gene comprises all or part of a nucleotide sequence encoding a transmembrane region and/or an extracellular region of human TNFSF9 protein. Still more preferably, said humanized TNFSF9 gene further comprises a partial nucleotide sequence encoding a cytoplasmic region of human TNFSF9 protein, wherein said portion of the cytoplasmic region of human TNFSF9 protein is identical to SEQ ID NO: 4 or at least 90% identical to the amino acid sequence at positions 26-28 of SEQ ID NO: 4 amino acid sequence at positions 26-28

Preferably, said humanized TNFSF9 gene further comprises a portion of a non-human animal TNFSF9 gene. Further preferably, the nucleotide sequence of all or a part of the cytoplasmic region of TNFSF9 protein is encoded in a non-human animal. Further preferred is a polypeptide encoding SEQ ID NO: 2 or at least 90% identical to the amino acid sequence at positions 1-82 of SEQ ID NO: 2, 1-82 amino acid sequence.

In one embodiment of the invention, the humanized TNFSF9 gene encodes the humanized TNFSF9 protein described above.

Preferably, the humanized TNFSF9 gene comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene. Further preferably, the humanized TNFSF9 gene comprises all or part of the nucleotide sequence of one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene. Still more preferably, the humanized TNFSF9 gene comprises the partial nucleotide sequence of exon 1, all exon 2 and partial nucleotide sequence of exon 3 of human TNFSF9 gene, and preferably, the partial nucleotide sequence of exon 1, all exon 2 and partial nucleotide sequence of exon 3 of human TNFSF9 gene has the same nucleotide sequence as SEQ ID NO: 3, all or part of the corresponding exon 1 to 3, have at least 60%, 70%, 80%, 90% or at least 95% identity. More preferably, the humanized TNFSF9 gene further includes introns 1-2 and/or intron 2-3. Wherein the portion of exon 1 of said human TNFSF9 gene comprises a nucleotide sequence encoding the extracellular and/or transmembrane region of human TNFSF9, preferably further comprising a nucleotide sequence encoding SEQ ID NO: 4, 26-28, and the part of exon 3 is preferably the nucleotide sequence encoding the extracellular region.

Preferably, the nucleotide sequence derived from the human TNFSF9 gene in the humanized TNFSF9 gene is identical to the nucleotide sequence of SEQ ID NO: 3 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

Preferably, the nucleotide sequence of the humanized TNFSF9 gene derived from the non-human animal TNFSF9 gene is identical to SEQ ID NO: 1 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

In one embodiment of the present invention, the humanized TNFSF9 gene comprises a partial nucleotide sequence of human TNFSF9 gene comprising one of the following group:

(A) is SEQ ID NO: 7, or a portion or all of the nucleotide sequence set forth in seq id no;

(B) and SEQ ID NO: 7 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

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

(D) Has the sequence shown in SEQ ID NO: 7, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Optionally, the humanized TNFSF9 gene encodes a protein capable of recognizing and/or binding at least a portion of a human 4-1BB protein, further wherein the human 4-1BB protein is encoded by a nucleotide sequence comprising one, two, three, two or a combination of three or more of exons 1 to 9 of the human 4-1BB gene; further preferably, said human 4-1BB protein is encoded by a nucleotide sequence comprising all or part of exon 3 to exon 8 of human 4-1BB gene; still more preferably, the human 4-1BB protein consists of a nucleotide sequence comprising a signal peptide encoded by exon 3 and an extracellular domain of the human 4-1BB gene, all of exons 4 to 7, and exon 8 encoded by SEQ ID NO: 54 at position 182-184.

Preferably, the humanized TNFSF9 gene further includes a portion of exon 1 and a portion of exon 3 of the non-human animal TNFSF9 gene. Further preferably, the part of exon 1 is a nucleotide sequence encoding cytoplasmic domain of TNFSF9 gene of non-human animal, and the part of exon 3 is a non-coding region of exon 3 of TNFSF9 gene of non-human animal. Still more preferably, the part of exon 1 and the part of exon 3 of said non-human animal TNFSF9 gene are identical to SEQ ID NO:

exons

1 and 3, respectively, 1 are at least 60%, 70%, 80%, 90%, or at least 95% identical.

Preferably, the nucleotide sequence of the humanized TNFSF9 gene comprises a nucleotide sequence identical to SEQ ID NO: 8, or a nucleotide sequence comprising at least 60%, 70%, 80%, 90%, or at least 95% identity to SEQ ID NO: 8.

In one embodiment of the present invention, the mRNA transcribed from the nucleotide sequence of the humanized TNFSF9 gene is selected from one of the following groups:

(a) is SEQ ID NO: 11, or a portion or all of a nucleotide sequence set forth in seq id no;

(b) and SEQ ID NO: 11 is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

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

(d) And SEQ ID NO: 11, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

Preferably, the humanized TNFSF9 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible.

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

In a third aspect of the present invention, there is provided a non-human animal humanized with TNFSF9 gene, said non-human animal expressing human or humanized TNFSF9 protein.

Preferably, the expression of endogenous TNFSF9 protein in said non-human animal is reduced or absent.

Preferably, the humanized TNFSF9 protein is selected from the humanized TNFSF9 proteins described above.

Preferably, the genome of the non-human animal comprises a portion of the human TNFSF9 gene. More preferably, the genome of the non-human animal includes the humanized TNFSF9 gene.

Preferably, the nucleotide sequence of said human or humanized TNFSF9 gene is operably linked to a non-human animal endogenous regulatory element.

Preferably, the non-human animal also expresses a human or humanized 4-1BB protein.

Further preferably, the humanized 4-1BB protein comprises all or part of a human 4-1BB protein. Still further preferably, the humanized 4-1BB protein comprises all or part of the extracellular domain of human 4-1BB protein. Still further preferably, the kit further comprises a signal peptide.

Further preferably, the humanized 4-1BB protein comprises the transmembrane region and/or cytoplasmic region of a non-human animal 4-1BB protein.

In one embodiment of the invention, the humanized 4-1BB protein comprises a sequence identical to SEQ ID NO: 54, 1-184 or an amino acid sequence having 90% homology to SEQ ID NO: 54 from position 1 to 184.

In another embodiment of the invention, the humanized 4-1BB protein comprises a sequence identical to SEQ ID NO: 52, position 184 and 256, or an amino acid sequence having 90% homology to SEQ ID NO: 54 at amino acid positions 184 and 256.

Still further preferably, the humanized 4-1BB protein comprises all or part of an amino acid sequence encoded by exons 3 to 8 of human 4-1BB protein.

In a specific embodiment of the invention, the humanized 4-1BB protein comprises the amino acid sequence encoded by exons 3 to 7 and the amino acid sequence encoded by all or part of exon 8 of human 4-1BB protein. Wherein the portion of exon 8 comprises a nucleotide sequence encoding SEQ ID NO: 54 at position 182-184.

In one embodiment of the invention, the amino acid sequence of the humanized 4-1BB protein is identical to the amino acid sequence of SEQ ID NO: 56 is 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical or identical to SEQ ID NO: 56 are identical.

Preferably, the genome of the non-human animal comprises a portion of the human 4-1BB gene. Further preferably, the nucleotide sequence of an exon that comprises one, two, three, two or more consecutive exons among exons 1 to 9 of the human 4-1BB gene in combination. Still further preferably, the nucleotide sequence comprises all or part of exon 3 to 8 of the human 4-1BB gene.

In one embodiment of the invention, the nucleotide sequence comprising exon 3 encoding the signal peptide and the extracellular region of the human 4-1BB gene, all of exons 4-7 and exon 8 encoding the amino acid sequence of SEQ ID NO: 54 at position 182-184.

In one embodiment of the invention, the genome of the non-human animal comprises mRNA transcribed from the human 4-1BB gene that comprises a nucleotide sequence identical to SEQ ID NO: 57 is 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical or identical to SEQ ID NO: 57 are identical.

In a specific embodiment of the present invention, the genome of the non-human animal comprises a nucleotide sequence encoding the humanized 4-1BB protein described above.

In another embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence identical to SEQ ID NO: 55 is 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical or identical to SEQ ID NO: 55 are identical.

In a fourth aspect of the invention, there is provided a method of constructing a humanized non-human animal which expresses human or humanized TNFSF9 protein.

Preferably, the non-human animal is a humanized non-human animal of the TNFSF9 gene described above.

Preferably, the humanized TNFSF9 gene is regulated in a non-human animal by endogenous or exogenous regulatory elements. Further preferably, the regulatory element is a promoter.

Preferably, the constructing method comprises introducing a partial nucleotide sequence comprising the human TNFSF9 gene into the non-human animal TNFSF9 locus. Further preferably, the nucleotide sequence comprising all or part of exons 1 to 3 of human TNFSF9 gene is introduced into the TNFSF9 locus of a non-human animal. Still more preferably, all or part of a nucleotide sequence comprising a combination of one, two, three or two consecutive exons from exon 1 to exon 3 of the human TNFSF9 gene is introduced into the TNFSF9 locus of a non-human animal. Still more preferably, the humanized TNFSF9 gene is formed by introducing a partial nucleotide sequence comprising part of exon 1, all of exon 2 and part of exon 3 of the human TNFSF9 gene into the non-human animal TNFSF9 locus. Optionally, the introduction is insertion or substitution.

In one embodiment of the present invention, a partial nucleotide sequence comprising part of exon 1, intron 1-2, all of exon 2, intron 2-3 and exon 3 of the human TNFSF9 gene is inserted or substituted into or at the TNFSF9 locus of a non-human animal; to form a humanized TNFSF9 gene.

Preferably, the method of construction comprises insertion or substitution at the non-human animal TNFSF9 locus with a nucleotide sequence comprising all or part of the transmembrane, cytoplasmic, and/or extracellular region encoding the human TNFSF9 protein. Further preferably, the non-human animal TNFSF9 locus is inserted or substituted with a nucleotide sequence comprising a transmembrane region and/or extracellular region encoding a human TNFSF9 protein. In one embodiment of the invention, the polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 4, 26-254, following insertion or substitution of an endogenous regulatory element at the TNFSF9 locus in a non-human animal. Optionally, at least a partial region of exon 1 through exon 3 at the TNFSF9 locus of said non-human animal, preferably, a transmembrane region and an extracellular region of exon 1 through exon 3 at the TNFSF9 locus of said non-human animal. Preferably, the method of construction comprises insertion or substitution of a nucleotide sequence comprising all or part of the sequence encoding human TNFSF9 protein into the non-human animal TNFSF9 locus.

Preferably, the method of construction comprises insertion or substitution of a cDNA sequence comprising a protein encoding human TNFSF9 into the non-human animal TNFSF9 locus.

Preferably, said method of construction comprises insertion or substitution of a nucleotide sequence comprising said humanized TNFSF9 gene at the non-human animal TNFSF9 locus.

Preferably, said method of construction comprises insertion or substitution into the non-human animal TNFSF9 locus of a nucleotide sequence comprising a nucleotide sequence encoding said humanized TNFSF9 protein.

Preferably, the insertion or substitution site is after an endogenous regulatory element of the TNFSF9 gene.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous TNFSF9 gene in the non-human animal, followed by insertion. Or the insertion step can cause frame shift mutation to the endogenous TNFSF9 gene and can realize the step of inserting the human sequence.

Optionally, the construction method further comprises humanization of the 4-1BB, PD-1, PD-L1, or OX40 genes.

Preferably, the non-human animal is homozygous or heterozygous.

Preferably, the genome of the non-human animal comprises a humanized TNFSF9 gene on at least one chromosome.

Preferably, at least one cell in the non-human animal expresses a human or humanized TNFSF9 protein.

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

Preferably, the targeting vector is used for the construction of non-human animals. Wherein the targeting vector comprises a donor DNA sequence comprising a portion of the human TNFSF9 gene. Preferably, the donor DNA sequence comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene. Further preferably, the donor DNA sequence comprises all or part of a nucleotide sequence of one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene. Still more preferably, the donor DNA sequence comprises a partial nucleotide sequence of exon 1, all of exon 2 and a partial nucleotide sequence of exon 3 of human TNFSF9 gene. Most preferably, the donor DNA sequence further comprises the nucleotide sequences of intron 1-2 and intron 2-3 of the human TNFSF9 gene.

Preferably, the donor DNA sequence comprises all or part of a nucleotide sequence encoding a cytoplasmic, extracellular and/or transmembrane region. Further preferably, the donor DNA sequence comprises all or part of the nucleotide sequence encoding the extracellular domain and/or the transmembrane domain. Still further preferably, said donor DNA sequence comprises a nucleotide sequence encoding an extracellular region, a transmembrane region and a partial cytoplasmic region. The nucleotide sequence of said partial cytoplasmic region preferably encodes SEQ ID NO: 4, 26-28.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 5 'end of the switch region to be altered, i.e., the 5' arm, selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the TNFSF9 gene of a non-human animal. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000083.6. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 5 or SEQ ID NO: 50 or as shown in SEQ ID NO: 5 or SEQ ID NO: shown at 50.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 3 'end of the transition region to be altered, i.e., the 3' arm, selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the TNFSF9 gene of a non-human animal. Further preferred are nucleotides having at least 90% homology in the 3' arm with NCBI accession No. NC _ 000083.6. Still more preferably, the 3' arm sequence is identical to SEQ ID NO: 6 or SEQ ID NO: 13 or as shown in SEQ ID NO: 6 or SEQ ID NO: shown at 13.

Preferably, the targeting vector further comprises a non-human animal 3' UTR.

In another embodiment of the invention, the sgRNA is used for the construction of a non-human animal. Wherein the sgRNA targets the TNFSF9 gene of a non-human animal, and the sequence of the sgRNA is unique on the target sequence on the TNFSF9 gene to be changed.

Preferably, the target site of the sgRNA is located on exon 1 and/or exon 3 sequences of the TNFSF9 gene.

Further preferably, the sequence of the target site at the 5' end targeted by the sgRNA is shown in SEQ ID NO: 14-21, the 3' end target site sequence is shown in SEQ ID NO: 21-28.

Preferably, the non-human animal 4-1BB locus is inserted or substituted with a nucleotide sequence comprising all or part of the nucleotide sequence encoding the human 4-1BB protein using gene editing techniques. Further preferably, the non-human animal 4-1BB locus is inserted or substituted with a nucleotide sequence comprising all or part of the extracellular region encoding human 4-1BB protein, preferably further comprising a signal peptide.

In one embodiment of the invention, the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 54 at positions 1-184 is inserted or substituted into the non-human animal 4-1BB locus.

Preferably, the non-human animal 4-1BB locus is inserted or substituted with a partial nucleotide sequence comprising the human 4-1BB gene using gene editing techniques. Further preferably, the nucleotide sequence comprising one, two, three, two or more combined exons among exons 1 to 9 of the human 4-1BB gene is inserted or substituted into or at the non-human animal 4-1BB locus. Still further preferably, the non-human animal 4-1BB locus is inserted or substituted with a nucleotide sequence comprising all or part of exons 3 to 8 of the human 4-1BB gene.

In one embodiment of the invention, the nucleotide sequence comprising exon 3 of the human 4-1BB gene encoding the signal peptide and the extracellular domain, all of exons 4-7 and exon 8 encoding the amino acid sequence of SEQ ID NO: 54 at position 182-184 is inserted or replaced into the non-human animal 4-1BB locus.

Preferably, the insertion site is after the endogenous regulatory elements of the 4-1BB gene.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous 4-1BB gene of the non-human animal, followed by insertion. Or the insertion step can not only cause frame shift mutation to the endogenous 4-1BB gene, but also realize the step of inserting the human sequence. Preferably, the non-human animal is homozygous or heterozygous.

Preferably, the genome of the non-human animal comprises a humanized 4-1BB gene on at least one chromosome.

Preferably, at least one cell in the non-human animal expresses a human or humanized 4-1BB protein.

Preferably, the gene editing technology comprises 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.

Preferably, a non-human animal humanized with the TNFSF9 gene described above is mated or inseminated in vitro with a non-human animal humanized with a 4-1BB gene expressing a human or humanized 4-1BB protein.

In a fifth aspect of the present invention, there is provided a method for constructing a non-human animal humanized with TNFSF9 gene and 4-1BB gene, comprising:

one) providing the above-mentioned TNFSF9 gene humanized non-human animal, or the TNFSF9 gene humanized non-human animal obtained by the above-mentioned construction method;

second) mating the non-human animal humanized with the TNFSF9 gene provided in the first step with a non-human animal humanized with a 4-1BB gene, in vitro fertilization, or directly editing the gene of the non-human animal humanized with the TNFSF9 gene, and screening.

Preferably, the 4-1BB gene humanized non-human animal body expresses human or humanized 4-1BB protein.

Preferably, the humanized 4-1BB protein comprises all or part of a human 4-1BB protein. Further preferably, the polypeptide comprises all or part of the extracellular region of human 4-1BB protein, and further preferably further comprises a signal peptide. Most preferably, the polypeptide comprises a sequence identical to SEQ ID NO: 54, 1-184 or an amino acid sequence having 90% homology to SEQ ID NO: 54 from position 1 to 184.

Preferably, said gene editing of a non-human animal humanized with TNFSF9 gene comprises: the partial nucleotide sequence of the human 4-1BB gene was substituted at the non-human animal 4-1BB locus.

Preferably, the nucleotide sequence comprising one, two, three, two consecutive or more combined exons from exon 1 to exon 9 of the human 4-1BB gene is substituted at the non-human animal 4-1BB locus. Further preferably, the non-human animal 4-1BB locus is replaced with a nucleotide sequence comprising all or part of exons 3 to 8 of the human 4-1BB gene.

In one embodiment of the invention, the nucleotide sequence comprising exon 3 of the human 4-1BB gene encoding the signal peptide and the extracellular domain, all of exons 4-7 and exon 8 encoding the amino acid sequence of SEQ ID NO: 54 at position 182-184, to the non-human animal 4-1BB locus.

In a sixth aspect of the present invention, there is provided a targeting vector comprising a donor DNA sequence comprising a portion of the human TNFSF9 gene.

Preferably, the donor DNA sequence comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene. Further preferably, the donor DNA sequence comprises all or part of a nucleotide sequence of one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene. Still more preferably, the donor DNA sequence comprises a partial nucleotide sequence of exon 1, all of exon 2 and a partial nucleotide sequence of exon 3 of human TNFSF9 gene. Most preferably, the donor DNA sequence comprises the nucleotide sequences of intron 1-2 and intron 2-3 of the human TNFSF9 gene.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 5 ' end of the switch region to be altered, i.e., the 5 ' arm (or 5 ' homologous arm), which is selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the TNFSF9 gene of a non-human animal. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000083.6. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 5 or SEQ ID NO: 50 or as shown in SEQ ID NO: 5 or SEQ ID NO: shown at 50.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 3 ' end of the transition region to be altered, i.e., the 3 ' arm (or 3 ' homologous arm), which is selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the TNFSF9 gene of a non-human animal. Further preferred are nucleotides having at least 90% homology in the 3' arm with NCBI accession No. NC _ 000083.6. Still more preferably, the 3' arm sequence is identical to SEQ ID NO: 6 or SEQ ID NO: 13 or as shown in SEQ ID NO: 6 or SEQ ID NO: shown at 13.

Preferably, the targeting vector further comprises a non-human animal 3' UTR.

Preferably, the switch region to be altered is at the non-human animal TNFSF9 locus. Further preferably, the transition region to be altered is located on exons 1 to 3 of the TNFSF9 gene of a non-human animal.

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

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

In one embodiment of the present invention, the targeting vector further comprises a specific recombination system. Further preferably, the specific recombination system is a Frt recombination site (a conventional LoxP recombination system can also be selected). The specific recombination system is provided with two Frt recombination sites which are respectively connected to two sides of the resistance gene.

In a seventh aspect of the present invention, there is provided a sgRNA targeting a non-human animal TNFSF9 gene, while the sequence of the sgRNA is unique on a target sequence on the TNFSF9 gene to be altered.

In a specific embodiment of the invention, the sgRNA target 5' end site sequence is as shown in SEQ ID NO: 21, and the 3' end target site sequence is shown as SEQ ID NO: shown at 30.

In an eighth aspect of the invention, a DNA molecule encoding the sgRNA is provided.

Preferably, the double strand of the DNA molecule is an upstream and downstream sequence of the sgRNA, or a forward oligonucleotide sequence or a reverse oligonucleotide sequence after the addition of the enzyme cleavage site. Further preferably, TAGG is added to the 5' end of the sgRNA sequence, and AAAC is added to the complementary strand thereof.

In one embodiment of the present invention, the DNA molecule may be SEQ ID NO: 35 and SEQ ID NO: 37, SEQ ID NO: 36 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 41, or, SEQ ID NO: 40 and SEQ ID NO: 42.

in a ninth aspect of the present invention, there is provided a sgRNA vector including the sgRNA or the DNA molecule.

In a tenth aspect of the present invention, a method for preparing a sgRNA vector is provided, which includes:

(i) providing the sgRNA, and preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence, wherein the sgRNA targets a TNFSF9 gene, the sgRNA is unique on a target sequence on a TNFSF9 gene to be changed, and a target site of the sgRNA is positioned on a No. 1 exon and/or a No. 3 exon of the TNFSF9 gene;

(ii) synthesizing fragment DNA containing a T7 promoter and sgRNA scaffold, carrying out enzyme digestion on the fragment DNA through EcoRI and BamHI to be connected to a skeleton vector, and carrying out sequencing verification to obtain a pT7-sgRNA vector;

(iii) (iii) denaturing and annealing the forward and reverse oligonucleotides obtained in step (i) to form a double strand into which the pT7-sgRNA vector of step (ii) can be ligated;

(iv) and (5) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step (iii) with pT7-sgRNA vectors, and screening to obtain the sgRNA vectors.

Preferably, the T7 promoter and sgRNA scaffold fragment DNA in step (ii) are as shown in SEQ ID NO: shown at 43.

In an eleventh aspect of the invention, there is provided a cell comprising the targeting vector, the sgRNA, the DNA molecule, or the sgRNA vector, preferably, wherein the cell is incapable of developing into an individual animal.

In a specific embodiment of the invention, the cell comprises the targeting vector and sgRNA described above.

In a twelfth aspect of the present invention, there is provided an application of the targeting vector, the sgRNA, the DNA molecule or the sgRNA vector, the sgRNA vector obtained by the preparation method, or a cell containing the targeting vector or the sgRNA to TNFSF9 gene modification. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In a thirteenth aspect of the present invention, there is provided a TNFSF9 gene knock-out non-human animal, which lacks all or part of the nucleotide sequence of TNFSF9 gene.

Preferably, the non-human animal lacks all or part of exons 1 to 3 of the TNFSF9 gene. Further preferably, all or part of the nucleotide sequence of one, two, three or a combination of two consecutive exons from exons 1 to 3 is deleted. Still more preferably, part of the nucleotide sequence of exon 1, all of exon 2 and part of exon 3 are deleted. Most preferably, the nucleotide sequence of part of exon 1, intron 1-2, all of exon 2, intron 2-3 and part of exon 3 are deleted.

In one embodiment of the invention, the deletion is at least from the first nucleotide sequence of exon 1 encoding the transmembrane region to the last nucleotide sequence of exon 3 encoding the extracellular region.

Preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the cytoplasmic, transmembrane and/or extracellular region. Further preferably, the non-human animal lacks all or part of the nucleotide sequence encoding the extracellular domain and/or the transmembrane domain. In one embodiment of the invention, the non-human animal lacks the nucleotide sequences encoding the extracellular and transmembrane regions.

In a fourteenth aspect of the present invention, there is provided a method for constructing a TNFSF9 gene-knocked-out non-human animal, the method comprising constructing the non-human animal using sgRNA. Wherein the sgRNA targets the TNFSF9 gene of a non-human animal, and the sequence of the sgRNA is unique on the target sequence on the TNFSF9 gene to be changed.

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

I) providing the non-human animal or the non-human animal obtained by the construction method;

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

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized with the genes PD-1, PD-L1 or OX 40.

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

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

In a sixteenth aspect of the present invention, there is provided a non-human animal humanized with TNFSF9 gene, a non-human animal knockout with TNFSF9 gene, or a multi-gene modified non-human animal or progeny thereof obtained by the above construction method.

In a seventeenth aspect of the present invention, there is provided a tumor-bearing or inflammation model of an animal, wherein the tumor-bearing or inflammation model is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, or the above-mentioned non-human animal or its progeny.

In an eighteenth aspect of the present invention, there is provided a method for producing a tumor-bearing or inflammatory model in an animal, said method comprising the step of constructing the above-mentioned TNFSF9 gene-humanized non-human animal, TNFSF9 gene-knockout non-human animal or polygene-modified non-human animal or progeny thereof. Preferably, the method further comprises the step of implanting the tumor cells.

In a nineteenth aspect of the present invention, there is provided a use of the above-mentioned TNFSF9 gene-humanized non-human animal, TNFSF9 gene-knocked out non-human animal, multi-gene-modified non-human animal or progeny thereof, or TNFSF9 gene-humanized non-human animal obtained by the above-mentioned construction method, TNFSF9 gene-knocked out non-human animal, multi-gene-modified non-human animal or progeny thereof for preparing a tumor-bearing or inflammatory model of an animal.

In a twentieth aspect of the present invention, there is provided a cell or cell line or primary cell culture derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, or the above-mentioned tumor-bearing or inflammation model. Preferably, the cell or cell line or primary cell culture is not capable of developing into an individual animal.

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

In a twenty-second aspect of the present invention, there is provided a tumor tissue after tumor bearing, wherein the tumor tissue is derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, or the above-mentioned tumor bearing or inflammation model. Preferably, said tumor-bearing tumor tissue is incapable of developing into an individual animal.

In a twenty-third aspect of the present invention, there is provided a cell in which the TNFSF9 gene is humanized, said cell expressing a human or humanized TNFSF9 protein. Preferably, the cell humanized with TNFSF9 gene cannot develop into animal individual.

Preferably, the humanized TNFSF9 protein is a humanized TNFSF9 protein according to the first aspect of the invention.

Preferably, the expression of endogenous TNFSF9 protein is reduced or absent in said cell.

Preferably, the genome of said cell comprises all or part of the human TNFSF9 gene. Further preferably, the cell comprises the humanized TNFSF9 gene described above.

In a twenty-fourth aspect of the present invention, there is provided a TNFSF9 gene knock-out cell, wherein all or part of the nucleotide sequence of the TNFSF9 gene is deleted. Preferably, the TNFSF9 knockout cell cannot be developed into an individual animal.

Preferably, the cell lacks all or part of exons 1 to 3 of the TNFSF9 gene. Further preferably, all or part of the nucleotide sequence of one, two, three or a combination of two consecutive exons from exons 1 to 3 is deleted. Still more preferably, part of the nucleotide sequence of exon 1, all of exon 2 and part of exon 3 are deleted. Most preferably, the nucleotide sequence of part of exon 1, intron 1-2, all of exon 2, intron 2-3 and part of exon 3 are deleted.

Preferably, the cell lacks all or part of the nucleotide sequence encoding the cytoplasmic, transmembrane and/or extracellular region. Further preferably, the cell lacks all or part of the nucleotide sequence encoding the extracellular domain and/or transmembrane region. In one embodiment of the invention, the cell lacks the nucleotide sequence encoding the extracellular and transmembrane regions.

In a twenty-fifth aspect of the present invention, there is provided a construct expressing the humanized TNFSF9 protein described above. Preferably, the construct comprises a humanized TNFSF9 gene.

In a twenty-sixth aspect of the invention, there is provided a cell comprising the above construct, preferably, the cell is incapable of developing into an individual animal.

In a twenty-seventh aspect of the invention, there is provided a tissue comprising cells as described above, preferably said tissue is not capable of developing into an individual animal.

A twenty-eighth aspect of the present invention provides use of a protein derived from the above-mentioned humanized TNFSF9, the above-mentioned humanized TNFSF9 gene, the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, the above-mentioned tumor-bearing or inflammatory model, the above-mentioned cell or cell line or primary cell culture, the above-mentioned tissue or organ or its culture, the above-mentioned tumor-bearing tissue, the above-mentioned cell, the above-mentioned construct, the above-mentioned cell or the above-mentioned tissue in a product development requiring an immune process involving human cells, for producing an antibody, or as a model system for pharmacological, immunological, microbiological and medical research; or in the production and use of animal experimental disease models for the development of new diagnostic and/or therapeutic strategies; or screening, verifying, evaluating or researching TNFSF9 function, TNFSF9 signal mechanism, human-targeting antibody, human-targeting drug, drug effect, immune-related disease drug and anti-tumor or anti-inflammatory drug, screening and evaluating human drug and drug effect research.

Preferably, the use is not a method of treatment and/or diagnosis of a disease.

In a twenty-ninth aspect of the present invention, there is provided a screening method for a human TNFSF 9-specific modulator, said screening method comprising applying the modulator to an individual implanted with tumor cells, and detecting tumor suppressivity; wherein the individual is selected from the group consisting of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned tumor-bearing or inflammation model.

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

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

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

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

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

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

Preferably, the screening method for a human TNFSF 9-specific modulator is not a therapeutic method. The method is used for screening or evaluating drugs, and detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary and is only a possibility.

In a thirtieth aspect of the present invention, there is provided an evaluation method of an intervention program, the evaluation method comprising implanting tumor cells into an individual, applying the intervention program to the individual in which the tumor cells are implanted, and detecting and evaluating a tumor suppression effect of the individual after the application of the intervention program; wherein the individual is selected from the group consisting of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, or the above-mentioned tumor-bearing or inflammation model.

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

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

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

In a thirty-first aspect, the present invention provides a use of the non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, the above tumor-bearing or inflammation model for preparing a human TNFSF 9-specific modulator.

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

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

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

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

The TNFSF9 gene humanized non-human animal of the present invention can express human or humanized TNFSF9 protein normally in vivo, and may be used in medicine screening, medicine effect evaluation, immunological diseases and tumor treatment of human TNFSF9 target site, and this can speed up the development of new medicine and save time and cost. Provides effective guarantee for researching the function of the TNFSF9 protein and screening related disease drugs.

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

The humanized TNFSF9 protein of the present invention comprises a portion derived from human TNFSF9 protein and a portion of non-human TNFSF9 protein. Wherein, the ' human TNFSF9 protein ' is identical to the whole human TNFSF9 protein, namely the amino acid sequence of the ' human TNFSF9 protein is identical to the full-length amino acid sequence of the human TNFSF9 protein. The 'part of the human TNFSF9 protein' is a continuous or alternate 5-254 amino acid sequence consistent with the amino acid sequence of the human TNFSF9 protein. Preferably 10 to 229 amino acid sequences are consecutive or separated, more preferably 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 229, 230, 240, 250, 254 amino acid sequences are identical to the amino acid sequence of the human TNFSF9 protein.

The "whole transmembrane region of human TNFSF9 protein", "whole cytoplasmic region of human TNFSF9 protein" or "whole extracellular region of human TNFSF9 protein" according to the present invention means that the amino acid sequence thereof is identical to the full-length amino acid sequence of the transmembrane region, cytoplasmic region or extracellular region of human TNFSF9 protein, respectively.

The "part of the transmembrane region of the human TNFSF9 protein" of the invention is that the continuous or spaced 5-21 amino acid sequences are consistent with the transmembrane region amino acid sequence of the human TNFSF9 protein, preferably the continuous 5, 10, 15, 20 and 21 amino acid sequences are consistent with the transmembrane region amino acid sequence of the human TNFSF9 protein.

The "part of cytoplasmic region of human TNFSF9 protein" of the present invention is that the continuous or separated 5-28 amino acid sequences are identical to the amino acid sequence of cytoplasmic region of human TNFSF9 protein, preferably the continuous 5, 10, 15, 20, 25, 28 amino acid sequences are identical to the amino acid sequence of cytoplasmic region of human TNFSF9 protein.

The "part of the extracellular region of the human TNFSF9 protein" of the present invention is a sequence of consecutive or 5-205 amino acids apart, which is identical to the amino acid sequence of the extracellular region of the human TNFSF9 protein, preferably, the sequence of consecutive 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205 amino acids is identical to the amino acid sequence of the extracellular region of the human TNFSF9 protein.

The "part of the non-human animal TNFSF9 protein" of the present invention is a sequence of consecutive or 5-309 amino acids identical to the amino acid sequence of the non-human animal TNFSF9 protein, preferably consecutive or 10-82 amino acids, more preferably consecutive 5, 10, 20, 30, 40, 50, 60, 70, 80, 82, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 309 amino acids identical to the amino acid sequence of the non-human animal TNFSF9 protein. In one embodiment of the invention, the portion of the non-human animal TNFSF9 protein that constitutes the humanized TNFSF9 protein comprises at least the amino acid sequence of the cytoplasmic domain of the non-human animal.

The "humanized TNFSF9 gene" according to the present invention comprises a portion derived from the human TNFSF9 gene and a portion of the non-human TNFSF9 gene. Wherein, the 'human TNFSF9 gene' is identical to the whole human TNFSF9 gene, i.e. the nucleotide sequence is identical to the full-length nucleotide sequence of the human TNFSF9 gene. The 'part of the human TNFSF9 gene' is that the continuous or spaced 20-4899bp nucleotide sequence is consistent with the nucleotide sequence of the human TNFSF9 gene, preferably 20-3955 or 20-690, more preferably 20, 50, 100, 200, 300, 400, 500, 600, 690, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3955, 4000 or 4899bp nucleotide sequence is consistent with the nucleotide sequence of the human TNFSF9 gene.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the portion of exon 1 of human TNFSF9 gene comprises consecutive or spaced nucleotide sequences of 5-278bp, preferably 10-192 bp, identical to the nucleotide sequence of exon 1 of human TNFSF9 gene. For example, the portion of exon 3 of human TNFSF9 gene comprises consecutive or spaced nucleotide sequences of 5-1325bp, preferably 10-467bp, identical to the nucleotide sequence of exon 3 of human TNFSF9 gene. In one embodiment of the present invention, the "part of exon 1" contained in the "human TNFSF9 gene" includes at least a first nucleotide sequence encoding the transmembrane region and a last nucleotide sequence of exon 1. In one embodiment of the present invention, the "part of exon 3" contained in the "humanized TNFSF9 gene" includes at least a part from the first nucleotide sequence of exon 3 to a stop codon.

The "xx to xxx exon" or "all of the xx to xxx exons" of the present invention comprise nucleotide sequences of exons and introns therebetween, for example, the "1 to 3 exons" comprise all nucleotide sequences of exon 1, intron 1-2, exon 2, intron 2-3 and exon 3.

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

The 'part of the non-human animal TNFSF9 gene' is a nucleotide sequence which is continuous or spaced by 20-2472bp and is consistent with the nucleotide sequence of the non-human animal TNFSF9 gene, preferably is continuous or spaced by 20-546 bp, and more preferably is continuous by 20, 50, 100, 200, 300, 400, 500, 546, 600, 690, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 and 2472 nucleotide sequence and is consistent with the nucleotide sequence of the non-human animal TNFSF9 gene. In one embodiment of the invention, the portion of the non-human animal TNFSF9 gene that comprises the humanized TNFSF9 gene comprises a portion of exon 1 and/or a portion of exon 3 of the non-human animal TNFSF9 gene. Preferably, the portion of exon 1 of TNFSF9 gene in non-human animal comprises at least a non-coding region and/or a nucleotide sequence encoding all or part of a cytoplasmic region. Preferably, the portion of exon 3 of TNFSF9 gene of non-human animal comprises at least a nucleotide sequence of a non-coding region.

The "humanized 4-1BB protein" according to the present invention comprises a portion derived from a human 4-1BB protein and a portion derived from a non-human 4-1BB protein. The part of the human 4-1BB protein is a continuous or alternate 5-255 amino acid sequence which is consistent with the amino acid sequence of the human 4-1BB protein. Preferably, the amino acid sequence of the human 4-1BB protein is identical to the amino acid sequence of the human 4-1BB protein in a sequence or interval of 10-184, more preferably in a sequence of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 184, 190, 200, 210, 220, 230, 240, 250, or 255. In one embodiment of the invention, the humanized 4-1BB protein comprises all or part of the amino acid sequence of the signal peptide and/or the extracellular domain of the human 4-1BB protein. In another embodiment of the invention, the humanized 4-1BB protein comprises all or part of an amino acid sequence encoded by exons 3 to 8 of human 4-1BB protein.

The 4-1BB gene humanized non-human animal is a non-human animal expressing a human or humanized 4-1BB protein, and the genome of the non-human animal comprises a part of a human 4-1BB gene. Preferably, the genome of the non-human animal comprises nucleotide sequences of one, two, three, two or more combined exons in the exons 1 to 9 of the human 4-1BB gene; further preferably, the nucleotide sequence comprises all or part of exon 3 to exon 8 of human 4-1BB gene.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "TNFSF 9 locus" refers to a DNA fragment of an optional stretch of exons 1 to 3 of the TNFSF9 gene. In one embodiment of the invention, the replaced TNFSF9 locus may be a DNA fragment of an optional stretch of exon 1 to exon 3 of TNFSF9 gene.

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

The term "more than three" includes, but is not limited to, three, four, five, six, seven or eight, etc.

The expression "three or more in succession" in the present invention includes, but is not limited to, three in succession, four in succession, five in succession, six in succession, seven in succession, eight in succession, nine in succession, and the like. Wherein "three or more consecutive exons from exon 1 to exon 9" includes three, four, five, six, seven, eight or nine consecutive exons, and also includes intron nucleotide sequences in between.

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

"homology" in the context of the present invention refers to the fact that, in the context of using amino acid sequences or nucleotide sequences, a person skilled in the art can adjust the sequences to 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.

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

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

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

In a particular embodiment, the non-human animal is a rodent, a mouse strain selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10 Sn, C57BL/10Cr and C57BL/Ola, C57 cscs, C58, A/Br, CBA/Ca, CBA/J, CBA/CBA, PrCBD/NOrgD, and SCID NORG.

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

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

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

FIG. 1: schematic comparison of mouse TNFSF9 gene and human TNFSF9 locus (not to scale);

FIG. 2: mouse TNFSF9 locus humanization schematic (not to scale);

FIG. 3: schematic representation of TNFSF9 gene targeting strategy (not to scale);

FIG. 4: the Southern detection result of the cell after TNFSF9 recombination, wherein CL-01, CL-02, CL-03 and CL-04 are clone numbers, and WT is a wild type control;

FIG. 5: schematic representation (not to scale) of mouse FRT recombination process for humanization of TNFSF9 gene;

FIG. 6: f1 TNFSF9 humanized mouse genotype test results, wherein F1-01 to F1-10 are mouse numbers, PC is positive control, WT is wild type control, H₂O is water control;

FIG. 7: CRISPR method TNFSF9 gene targeting strategy schematic (not to scale);

FIG. 8: the sgRNA1-sgRNA16 activity detection results, wherein Con is a negative control, PC is a positive control, and fig. (a) shows activity detection results of sgrnas 1 and 3-7 at 5 '-end target sites, and (B) shows activity detection results of sgrnas 9-16 at 3' -end target sites;

FIG. 9: exemplary PCR assay results for humanized mice with the F0-generation TNFSF9 gene, wherein F0-01 to F0-04 are mouse numbers, and H₂O is water control and WT is wild type control;

FIG. 10: exemplary PCR assay results for humanized mice with the F1-generation TNFSF9 gene, wherein F1-01 to F1-07 are mouse numbers, and H₂O is water control and WT is wild type control;

FIG. 11: an exemplary Southern detection result of the humanized mouse with the F1-generation TNFSF9 gene, wherein F1-01, F1-02 and F1-03 are mouse numbers, and WT is a wild-type control;

FIG. 12: performing PCR identification on mouse tail of knockout mouse, wherein KO-1, KO-2 and KO-3 are mouse numbers, PC is a positive control, WT is a wild type control, and H is₂O is water control;

FIG. 13: schematic comparison of mouse 4-1BB gene and human 4-1BB locus (not to scale);

FIG. 14: schematic representation of humanization of mouse 4-1BB locus (not to scale);

FIG. 15: implanting mouse colon cancer cell MC38 into 4-1BB gene humanized homozygous mouse, and performing antitumor effect experiment with human 4-1BB antibody Ab2(3mg/kg), wherein the figure is the measurement result of mouse weight in the experiment period;

FIG. 16: implanting mouse colon cancer cell MC38 into 4-1BB gene humanized homozygous mouse, and performing antitumor effect experiment with human 4-1BB antibody Ab2(3mg/kg), wherein the figure is the change result of mouse body weight in the experimental period;

FIG. 17: implanting mouse colon cancer cell MC38 into 4-1BB gene humanized homozygous mouse, and performing antitumor effect experiment with human 4-1BB antibody Ab2(3mg/kg), wherein the figure is the measurement result of mouse tumor volume in the experiment period;

FIG. 18: implanting a mouse colon cancer cell MC38 into a TNFSF9/4-1BB double-gene humanized homozygous mouse, and performing an anti-tumor drug effect experiment by using a human 4-1BB antibody Ab2(3mg/kg), wherein the figure is a measurement result of the weight of the mouse in an experimental period;

FIG. 19: implanting mouse colon cancer cell MC38 into TNFSF9/4-1BB double-gene humanized homozygous mouse, and performing anti-tumor drug effect experiment by using Urelumab, wherein the figure is the change result of mouse weight in the experiment period;

FIG. 20: implanting mouse colon cancer cell MC38 into TNFSF9/4-1BB double-gene humanized homozygous mouse, and performing anti-tumor effect experiment by using Urelumab, wherein the figure is the measurement result of the tumor volume of the mouse in the experiment period;

FIG. 21: based on a 4-1BB gene humanized homozygous mouse and a TNFSF9/4-1BB double gene humanized homozygous mouse, a toxicology experiment is carried out by using Urelumab, and the figure is a serum detection result on the 21 st day after the first administration;

FIG. 22: based on a 4-1BB gene humanized homozygous mouse and a TNFSF9/4-1BB double gene humanized homozygous mouse, a toxicology experiment is carried out by using Urelumab, and the figure is a schematic diagram of liver HE staining microscopic results on the 21 st day after the first administration.

Detailed Description

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

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

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

the UCA kit is sourced from Beijing Baiosai chart gene biotechnology limited company with the cargo number of BCG-DX-001;

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

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

StuI, EcoRI, BamHI, NcoI were purchased from NEB under the respective accession numbers R0187M, R0101M, R0136M, R0193M.

Example 1 mouse humanized of TNFSF9 Gene

This example modifies a non-human animal (e.g., a mouse) to include a nucleic acid sequence encoding human TNFSF9 protein in the non-human animal, resulting in a genetically modified non-human animal that expresses human or humanized TNFSF9 protein in vivo. A schematic comparison of the mouse TNFSF9 Gene (NCBI Gene ID: 21950, Primary source: MGI: 1101058, UniProt ID: P41274, NM-009404.3 → NP-033430.1 based transcript with mRNA sequence shown in SEQ ID NO: 1 and the corresponding protein sequence shown in SEQ ID NO: 2) and the human TNFSF9 Gene (NCBI Gene ID: 8744, Primary source: HGNC:11939, UniProt ID: P41273, NM-003811.4 → NP-003802.1 based transcript with mRNA sequence shown in SEQ ID NO: 3 and the corresponding protein sequence shown in SEQ ID NO: 4) is shown in FIG. 1.

To achieve the object of the present invention, the gene sequence of human TNFSF9 may be introduced at the endogenous TNFSF9 locus of a mouse, so that the mouse expresses a human or humanized TNFSF9 protein. Specifically, the mouse TNFSF9 gene sequence can be replaced by the human TNFSF9 gene sequence at the endogenous TNFSF9 locus of the mouse through a gene editing technology, for example, the 1827bp sequence from the No. 1 exon part sequence to the No. 3 exon part sequence of the mouse TNFSF9 gene is replaced by the corresponding human DNA sequence to obtain a humanized TNFSF9 gene sequence (the schematic diagram is shown in fig. 2), so that the humanized transformation of the mouse TNFSF9 gene is realized.

Further designing the targeting strategy as shown in FIG. 3, the targeting vector contains the upstream and upstream of mouse TNFSF9 geneA downstream homology arm sequence, and an a fragment comprising a nucleotide sequence encoding human TNFSF9 protein. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 5) is the same as the nucleotide sequence from position 57095327 to 57105677 of NCBI accession No. NC-000083.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is the same as the nucleotide sequence from position 57108062 to 57112881 of NCBI accession No. NC-000083.6; a 3955bp genomic DNA sequence (SEQ ID NO: 7) comprising the sequence of the No. 1 exon portion through the sequence of the No. 3 exon portion of the human TNFSF9 gene on the A fragment, which is identical to the nucleotide sequence of 6531112 to 6535066 of NCBI accession No. NC-000019.10; the ligation of the downstream human TNFSF9 sequence to mouse TNFSF9 in fragment A was designed to

Wherein the sequence "aataa"the last" a "of" is the last nucleotide, sequence, of a human

"g" of (a) is the first nucleotide of a mouse.

The targeting vector also comprises a resistance gene used for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the connection between the 5' end of the Neo box and the mouse sequence is designed as

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

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

Wherein the sequence "GATCC"the last" C "of" is the last nucleotide, sequence, of the Neo cassette

The first "g" of (a) is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker, namely a coding gene (DTA) of diphtheria toxin A subunit is constructed at the downstream of the 3' homologous arm of the recombinant vector. The mRNA sequence of the humanized mouse TNFSF9 after being transformed is shown as SEQ ID NO: 11, the expressed protein sequence is shown as SEQ ID NO: shown at 12.

The construction of the targeting vector can be carried out by a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The targeting vector with correct sequencing verification is transfected into embryonic stem cells of a C57BL/6 mouse by electroporation, the obtained cells are screened by using a positive clone screening marker gene, the integration condition of an exogenous gene is detected and confirmed by using PCR and Southern Blot technology, and correct positive clone cells are screened. The screened positive clones were identified by PCR using the primers CL-F and CL-R, and the clones identified as positive were tested by Southern Blot (cell DNA digested with StuI and hybridized using probes having the length shown in Table 1), with the results shown in FIG. 4, wherein 4 clones numbered CL-01, CL-02, CL-03, and CL-04 were positive clones without random insertion, and all 4 clones were further verified by sequencing.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment
				StuI	P1	——	13.8kb

The PCR assay included the following primers:

CL-F(SEQ ID NO：39)：5’-AGCTCAGTAGGTTCCATGCCCAGTA-3’

CL-R(SEQ ID NO：40)：5’-GAAGGTGCTGGGAGGAGTGTCTTG-3’

the Southern Blot detection comprises the following probe primers:

P1-F(SEQ ID NO：43)：5’-CCGACCCTCGGTAGCTGGTCTC-3’

P1-R(SEQ ID NO：44)：5’-CTCCCGTGCAAGACGGAGAAGGAG-3’

the selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 5 for a schematic diagram), and then mated with each other to obtain a homozygote mouse humanized with the TNFSF9 gene. The somatic genotypes of the progeny mice can be identified by PCR (see Table 2 for primer sequences and target fragment lengths), and the identification results of exemplary F1 generation mice (with the Neo marker gene removed) are shown in FIG. 6, wherein 10 mice numbered F1-01 to F1-10 are all positive heterozygous mice.

Table 2: f1 generation genotype identifying primer sequence and target fragment length

In addition, a CRISPR/Cas system can be introduced for gene editing, and a targeting strategy as shown in fig. 7 is designed by taking a mouse humanized with the TNFSF9 gene as shown in fig. 2 as an example. The figure shows the homology arm sequences of the targeting vector containing the upstream and downstream of mouse TNFSF9, and a fragment of the human TNFSF9 sequence. Wherein the sequence of the upstream homology arm (5' homology arm, SEQ ID NO: 50) is identical to the nucleotide sequence from position 57104184 to 57105677 of NCBI accession No. NC-000083.6; the sequence of the above downstream homology arm (3' homology arm, SEQ ID NO: 13) is identical to the nucleotide sequence 57107505 to 57108979 of NCBI accession No. NC-000083.6; the human TNFSF9 sequence is identical to SEQ ID NO: 7 are identical. The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, direct synthesis and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The correct targeting vector was verified by sequencing for subsequent experiments.

sgRNA sequences that recognize the 5 'target site (sgRNA1-sgRNA8), the 3' target site (sgRNA9-sgRNA16) were designed and synthesized. The 5 'end target site and the 3' end target site are respectively positioned on the No. 1 exon and the No. 3 exon sequences of the TNFSF9 gene, and the target site sequence of each sgRNA is as follows:

sgRNA1 target site sequence (SEQ ID NO: 14): 5'-GCTCTATGGCCTAGTCGCTTTGG-3'

sgRNA2 target site sequence (SEQ ID NO: 15): 5'-GGCTCGGTGCGGGTGAAGATAGG-3'

sgRNA3 target site sequence (SEQ ID NO: 16): 5'-TCGGGTACCCAGGTTGGGCGAGG-3'

sgRNA4 target site sequence (SEQ ID NO: 17): 5'-GCGCTGGCCGAGGCTCGGTGCGG-3'

sgRNA5 target site sequence (SEQ ID NO: 18): 5'-TCTTCACCCGCACCGAGCCTCGG-3'

sgRNA6 target site sequence (SEQ ID NO: 19): 5'-TGTGAGCGCTGGCCGAGGCTCGG-3'

sgRNA7 target site sequence (SEQ ID NO: 20): 5'-TCCCGCCACCCAAAGCTCTATGG-3'

sgRNA8 target site sequence (SEQ ID NO: 21): 5'-GGTACCCAGGTTGGGCGAGGTGG-3'

sgRNA9 target site sequence (SEQ ID NO: 22): 5'-ACAAGTTAGTGGACCGTTCCTGG-3'

sgRNA10 target site sequence (SEQ ID NO: 23): 5'-TGTGAAACCCGACAACCCATGGG-3'

sgRNA11 target site sequence (SEQ ID NO: 24): 5'-TTGTGAAACCCGACAACCCATGG-3'

sgRNA12 target site sequence (SEQ ID NO: 25): 5'-GCTGGCCACCGCCTCAGTGTGGG-3'

sgRNA13 target site sequence (SEQ ID NO: 26): 5'-GGCTGGCCACCGCCTCAGTGTGG-3'

sgRNA14 target site sequence (SEQ ID NO: 27): 5'-CTCCATGGAGAACAAGTTAGTGG-3'

sgRNA15 target site sequence (SEQ ID NO: 28): 5'-GGTCTGAGGGCTTATCTGCATGG-3'

sgRNA16 target site sequence (SEQ ID NO: 29): 5'-CCCAGGATGCATACAGAGACTGG-3'

Table 3: UCA assay results

The results of detecting the activity of each sgRNA by using the UCA kit are shown in table 3 and fig. 8, and it can be seen that the sgrnas have different activities, wherein although the sgrnas 9, 14, and 16 have relatively low activities, which may be caused by the specificity of the target site sequence, according to our experiments, the values of sgrnas 9, 14, and 16 are still significantly higher than those of the control group, and it can be determined that sgrnas 9, 14, and 16 are active, and the activities meet the requirements of gene targeting experiments, and

sgrnas

1 and 10 are randomly selected from the sgrnas for subsequent experiments. The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide and a reverse oligonucleotide (the sequences are shown in a table 4), and after annealing, the annealed products are respectively connected to pT7-sgRNA plasmids (the plasmids are firstly linearized by BbsI), so that expression vectors pT7-sgRNA1 and pT7-sgRNA10 are obtained.

Table 4: list of sgRNA1 and sgRNA10 sequences

pT7-sgRNA vector was synthesized by plasmid synthesis company as a fragment DNA (SEQ ID NO: 38) containing the T7 promoter and sgRNA scaffold, and ligated to a backbone vector (Takara, cat. No. 3299) by enzyme digestion (EcoRI and BamHI) in sequence, and sequencing by the professional sequencing company was verified, and the result indicated that the objective plasmid was obtained.

Taking mouse prokaryotic fertilized eggs, such as C57BL/6 mice, and injecting in-vitro transcription products of pT7-sgRNA1 and pT7-sgRNA10 plasmids (transcription is carried out by using an Ambion in-vitro transcription kit according to a method of an instruction) and a targeting vector and Cas9mRNA into cytoplasm or nucleus of the mouse fertilized eggs after being premixed by using a microinjector. Microinjection of fertilized eggs was performed according to the method in the manual of experimental manipulation of mouse embryos (third edition), published by chemical industry, 2006, and the injected fertilized eggs were transferred to a culture medium for short-term culture and then transplanted into the oviduct of a recipient mother mouse for development, and the obtained mice (generation F0) were crossed and selfed to expand the population number and establish a stable TNFSF9 gene-mutated mouse strain.

The somatic cell genotype of F0 mouse can be identified by conventional detection methods (e.g., PCR analysis), and the results of partial F0 mouse identification are shown in FIG. 9. Combining the detection results of the 5 'end primer and the 3' end primer (see Table 5), 4 mice numbered from F0-01 to F0-04 in FIG. 9 were positive mice, and the 4 mice were further determined to be positive mice by sequencing. The PCR analysis included the following primers:

table 5: f0 generation PCR detection primer sequence and target fragment length

Humanized mice identified as positive for F0 were mated with wild type mice to give F1 generation mice. The same PCR method can be used for genotyping F1 mice, and the PCR primers are the same as those used for genotyping F0. The results are shown in FIG. 10, which shows that 7 mice numbered F1-01 to F1-07 were all positive mice.

Further, F1 mice positive for PCR were subjected to Southern blot analysis (see Table 6 for specific probes and length of the target fragment) to confirm the presence of random insertions. Extracting genome DNA from the mouse tail, digesting the genome with NcoI or StuI enzyme, transferring the membrane, and hybridizing. Probes P1, P2 were located outside the human sequence and 3' homology arm, respectively. The probe synthesis primers were as follows:

P1-F(SEQ ID NO：43)：5’-CCGACCCTCGGTAGCTGGTCTC-3’，

P1-R(SEQ ID NO：44)：5’-CTCCCGTGCAAGACGGAGAAGGAG-3’；

P2-F(SEQ ID NO：45)：5’-TGAGCTGTTGGGAGACCTTGACTTA-3’，

P2-R(SEQ ID NO：46)：5’-GGAGTTGACTCAGTGGTCAGCACTTA-3’；

table 6: length of specific probes and target fragments

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment
				StuI	P1	——	13.8kb
NcoI	P2	9.3kb	11.3kb

Exemplary Southern blot assays are shown in FIG. 11. The results of the P1 and P2 probes showed that 3 mice numbered F1-01, F1-02 and F1-03 had no random insertions. This shows that the method can construct the TNFSF9 humanized gene engineering mouse which can be stably passaged and has no random insertion.

The expression of the humanized TNFSF9 protein in the humanized mouse of the TNFSF9 gene can be confirmed by a conventional detection method, for example, by flow cytometry.

In addition, since the cleavage of Cas9 causes double-strand break of genomic DNA, insertion/deletion mutations are randomly generated by the repair mode of chromosome homologous recombination, and it is possible to obtain a knockout mouse with TNFSF9 protein loss of function. For this purpose, a pair of primers (see Table 7) was designed for detecting knockout mice, which wild-type knockout mice should contain 1 PCR band of about 420bp in length, and the results are shown in FIG. 12. Among them, 3 mice numbered KO-1, KO-2 and KO-3 were TNFSF9 knock-out mice. The primers are respectively positioned on the left side of a5 'end target site and the right side of a 3' end target site, and have the following sequences:

table 7: KO mouse PCR detection primer sequence and target fragment length

Example 24-1 BB Gene-humanized mouse

A comparison scheme between mouse 4-1BB Gene (NCBI Gene ID: 21942, Primary source: MGI:1101059, UniProtKB: P20334, from position 150920155 to 150946104 on chromosome 4 NC-000070.6, based on transcript NM-011612.2 (SEQ ID NO: 51) and its encoded protein NP-035742.1 (SEQ ID NO: 52)) and human 4-1BB Gene (NCBI Gene ID: 3604, Primary source: HGNC: 11924, UniProtKB: Q07011, from position 7915871 to 7941607 on chromosome 1 NC-000001.11, based on transcript NM-001561.5 (SEQ ID NO: 53) and its encoded protein NP-001552.2 (SEQ ID NO: 54)) is shown in FIG. 13.

On the endogenous 4-1BB locus of a mouse, a part of sequences of exons 2 to 7 of the mouse 4-1BB gene are replaced by 6706bp sequences of exons 3 to 8 of the human 4-1BB gene to obtain an F1 generation 4-1BB gene humanized heterozygous mouse, the heterozygous mouse is mutually mated, and the 4-1BB gene humanized homozygous mouse is obtained by multi-generation screening. The schematic diagram of the transformed humanized mouse 4-1BB gene is shown in FIG. 14, and the mRNA sequence and the protein sequence coded by the mRNA sequence are respectively shown in SEQ ID NO: 55 and SEQ ID NO: as shown at 56.

4-1BB gene humanized homozygous mice (4-8 weeks old) were inoculated subcutaneously with mouse colon cancer cells MC38 (5X 10)⁵/100. mu.L PBS), until the tumor volume grows to about 100mm³Then divided into control or treatment groups (n-6/group) according to tumor volume. Treatment groups randomly selected 1 anti-human 4-1BB antibody Ab2 (obtained by immunizing mice using conventional methods, see Janeway's immunology (9)^thEdition)) was injected at a dose of 3mg/kg, and the control group was injected with an equal volume of physiological saline. The frequency of administration was 1 dose every 3 days for a total of 6 doses. Tumor volume was measured 2 times per week and mice were weighed, and after inoculation the tumor volume of a single mouse reached 3000mm³An euthanasia end test should be performed.

The animals in each group were in good health during the experiment, at the end of the experiment (day 25 after grouping), all the treatment and control groups showed weight gain, and there was no obvious difference in weight and weight change in the whole experimental period (fig. 15, 16); however, from the results of tumor volume measurement (FIG. 17), the tumors of the mice in the control group continued to grow during the experimental period, while the tumor volume increased significantly in the treated group compared to the control group. The main data and analysis results are listed in Table 8, including tumor volume at and 18 days after grouping, end of experiment (25 days after grouping)Tumor volume of (1), survival of mice, Tumor free mice (Tumor free), Tumor Growth Inhibition value (TGI)_TV) And the statistical differences (P-values) between the body weight and tumor volume of mice in the treated group and the control group.

TABLE 8 tumor volume, survival and tumor inhibition

As can be seen from table 8, in conjunction with fig. 15, at the end point of the experiment (day 25 after grouping), from the tumor volume measurement results, all mice in the control group continued to grow the tumor during the experiment, while 3 mice (50%) in the treated group of 6 mice disappeared the tumor at the end point of the experiment. At the end of the experiment, the mean tumor volume of the control group (G1) was 3501. + -. 458mm³The mean tumor volume of the treatment group G2 was 84. + -. 76mm³The tumor volume of the mice in the treatment group is obviously smaller than that of the control group, and the tumor volume is obviously different; TGI_TV102.7%, indicating that the anti-human 4-1BB antibody Ab1 has obvious inhibition effect on Tumors (TGI)_TV＞60％)。

The research results show that the humanized 4-1BB mouse can be used as a living model for in vivo efficacy research, and is used for screening, evaluating and treating human 4-1BB signaling pathway modulators, evaluating the effectiveness of targeted 4-1BB drugs in vivo and evaluating the treatment effect of the targeted 4-1 BB.

Example 3 preparation of double-or multiple-humanized mice

A double-gene modified or multiple-gene modified mouse model can be prepared by using the method or the prepared TNFSF9 mouse. For example, in example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing other gene modifications such as 4-1BB, PD-1, PD-L1, OX40, etc., or can be obtained from two-gene or multi-gene modified mouse models of TNFSF9 and/or 4-1BB and other gene modifications by using isolated mouse ES embryonic stem cells and gene recombination targeting technology on the basis of humanized TNFSF9 mice. The homozygote or heterozygote of the TNFSF9 mouse obtained by the method can be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, humanized TNFSF9 and heterozygote mouse of double gene or multiple gene modification of other gene modification can be obtained with a certain probability according to Mendel genetic rule, then the heterozygote is mated with each other to obtain homozygote of double gene or multiple gene modification, and the in vivo efficacy verification of targeted TNFSF9 and other gene regulators can be carried out by utilizing the mouse of double gene or multiple gene modification. Specifically, TNFSF9/4-1BB double-gene humanized mice can be obtained by mating TNFSF9 and 4-1BB homozygous mice and carrying out multi-generation screening.

The mouse can be used for inducing and preparing multiple human disease models for testing the in vivo efficacy of the human specific antibody. For example, mice humanized with TNFSF9 and/or 4-1BB genes can be used to assess the pharmacodynamics, pharmacokinetics, and in vivo therapeutic efficacy of human specific TNFSF9 and/or 4-1BB signaling pathway drugs in various disease models known in the art.

Example 4 evaluation of drug efficacy Using TNFSF9/4-1BB double-Gene humanized mouse to construct tumor model

The TNFSF9 and/or 4-1BB gene humanized mouse prepared by the invention is used for constructing a tumor model, and can be used for testing the drug effect of a drug targeting human TNFSF9 and/or 4-1 BB. Specifically, 8-week-old female TNFSF9/4-1BB double-gene humanized homozygote mice prepared in example 3 were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5X 10)⁵One), the volume of the tumor to be treated is about 100mm³Then, the tumor volume was counted as control group or treatment group (n-5/group). Control groups were injected with isotype control antibody (hIgG4) and treatment groups used varying doses of homemade Urelumab (for drug information see patent text (publication No. WO2005035584a 1)). The administration mode comprises the following steps: intraperitoneal (i.p.) injection, the administration is started on the same day, 2 times per week and 6 times in total. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of a single mouse reached 3000mm³And performing euthanasia. Specific groups and dosing are shown in table 9. The results of measurement of the body weight and tumor volume of the mice in the experimental period are shown in fig. 18 and fig. 19, respectively.

Table 9: grouping and administration of drugs

The main data and analysis results of each experiment are listed in table 10, and specifically include Tumor volume at the time of grouping, 14 days after grouping, 21 days after grouping, survival of mice, Tumor-free mice condition, Tumor (volume) Inhibition rate (TGI) at Tumor Growth Inhibition value, TGI_TV) And the statistical difference in tumor volume (P-value) between the treated and control mice.

Table 10: tumor volume, survival and tumor inhibition rate

As shown in FIGS. 18 and 19 and Table 10, the health status of the animals was good in the experimental process of each group, and the weight of the animals showed a tendency to increase (FIG. 18) at day 21 in all the treatment groups (groups G2, G3 and G4) compared to the control group (group G1), and there was no significant difference (P1)>0.05), which shows that the animal has good tolerance to the anti-human 4-1BB antibody Urelumab, does not generate obvious toxic effect on the animal and has better safety. As shown in FIG. 20 and Table 10, from the tumor volume results, the tumor volumes of the treatment groups were smaller than those of the control group at each stage, and the tumor volumes of the mice of the groups G2, G3 and G4 were 1906. + -. 219mm on day 21, respectively³、1437±184mm³796. + -. 181mm³1983. + -. 329mm from control group³The comparison is reduced, especially the G4 group has significant difference compared with the control group (P)<0.05), indicating that different doses of Urelumab have particularly different tumor suppression effects in TNFSF9/4-1BB humanized animals and show dose dependence.

Example 5 construction of tumor drug toxicity evaluation model Using 4-1BB humanized mouse and TNFSF9/4-1BB double-Gene humanized mouse

The TNFSF9 and/or 4-1BB gene humanized mouse prepared by the invention is used for constructing a tumor model, and can be used for testing the toxicity of a medicament targeting human TNFSF9 and/or 4-1 BB. Specifically, 8-week-old female 4-1BB humanized homozygote mice prepared in example 2 and 8-week-old female TNFSF9/4-1BB double-gene humanized homozygote mice prepared in example 3 were randomly selected as a control group or a treatment group (n-5/group). Control groups were injected with isotype control antibody (hIgG4) and treatment groups were administered with different doses of Urelumab. The administration mode comprises the following steps: intraperitoneal (i.p.) administration was started on the same day, 3 times per week for 4 times. Specific groups and dosing are shown in table 11. Blood was collected on day 21 after the first administration, serum was centrifuged to detect alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) indices, and liver tissue was fixed and HE stained for pathological analysis.

Table 11: grouping and administration of drugs

Group of

Number of animals

Species of animal

Treatment method

Dosage to be administered

Mode of administration

Frequency and number of administrations

G1

5

4-1BB mice

hIgG4

20mg/kg

i.p.

TIW×4

G2

5

4-1BB mice

Urelumab

1mg/kg

i.p.

TIW×4

G3

5

4-1BB mice

Urelumab

20mg/kg

i.p.

TIW×4

G4

5

TNFSF9/4-1BB mice

hIgG4

20mg/kg

i.p.

TIW×4

G5

5

TNFSF9/4-1BB mice

Urelumab

1mg/kg

i.p.

TIW×4

G6

5

TNFSF9/4-1BB mice

Urelumab

20mg/kg

i.p.

TIW×4

Table 12: pathological evaluation table

Note: evaluation criteria: perihepatic vascular cell infiltration or chronic inflammation.

Slight (+): the lesion site accounts for about 5% of the total area

Mild (++): the lesion part accounts for about 5 to 20 percent of the total area

Moderate (+++): the lesion part accounts for about 20 to 40 percent of the total area

Severe (+++): the lesion site accounts for about 40% of the total area

In the experimental process of each group, the animal health state is good, the animal weight is in the growth trend, and no obvious difference exists (P is more than 0.05). ALT and AST detection results are shown in FIG. 21, in 4-1BB mice, compared with a control group (G1), 20mg/kg of Urelumab can significantly improve ALT, the average value of the G3 group is 54.91, the average value of the G1 group is 35.85, but AST has no obvious change; in TNFSF9/4-1BB mice, compared with a control group, 20mg/kg of Urelumab can obviously improve AST, the average value of a G6 group is 113.3, the average value of a G1 group is only 58.95, and the ALT is not obviously influenced; ALT and AST did not change significantly in the 1mg/kg dose groups (groups G2 and G5) in 4-1BB mice and TNFSF9/4-1BB mice. The above results indicate that TNFSF9/4-1BB mouse double-gene humanized mouse is more sensitive to hepatotoxicity than 4-1BB humanized mouse at the dose of 20mg/kg and the frequency described above for Urelumab.

The HE staining micrograph and pathology score are shown in fig. 22 and table 12, respectively, and in 4-1BB mice, no significant abnormal change was seen in the liver of group G2, and abnormal changes were seen in all of the mice of group G3, manifested by infiltration of perihepatic cells or chronic inflammation, with less lesions. In TNFSF9/4-1BB mice, 3/5 mice in group G5 showed pathological changes (mild 2/5, mild 1/5), all mice in group G6 showed moderate changes in liver (5/5), and the degree and incidence of liver lesions were significantly higher in group G6 than in group G5. The above results suggest that, at the same frequency of administration as described above, the 20mg/kg group is more susceptible to perihepatic vascular cell infiltration or chronic inflammation than the 1mg/kg group, and TNFSF9/4-1BB mice appear to be more sensitive to toxic effects than the 4-1BB mice.

The experiments prove that the TNFSF9/4-1BB humanized mouse prepared by the method can be used for screening anti-human TNFSF9 and/or 4-1BB antibodies and detecting in-vivo drug effects, can be used as a living body substitution model for in-vivo research, and is used for screening, evaluating and treating a human TNFSF9/4-1BB signal channel regulator.

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

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

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

Sequence listing

<110> Baiosai map Jiangsu Gene Biotechnology Co., Ltd, Baiosai map (Beijing) pharmaceutical science and technology Co., Ltd

<120> TNFSF9 gene humanized non-human animal and construction method and application thereof

<130> 1

<160> 57

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1230

<212> DNA/RNA

<213> Mouse (Mouse)

<400> 1

ataaagcacg ggcactggcg ggagacgtgc actgaccgac cgtggtaatg gaccagcaca 60

cacttgatgt ggaggatacc gcggatgcca gacatccagc aggtacttcg tgcccctcgg 120

atgcggcgct cctcagagat accgggctcc tcgcggacgc tgcgctcctc tcagatactg 180

tgcgccccac aaatgccgcg ctccccacgg atgctgccta ccctgcggtt aatgttcggg 240

atcgcgaggc cgcgtggccg cctgcactga acttctgttc ccgccaccca aagctctatg 300

gcctagtcgc tttggttttg ctgcttctga tcgccgcctg tgttcctatc ttcacccgca 360

ccgagcctcg gccagcgctc acaatcacca cctcgcccaa cctgggtacc cgagagaata 420

atgcagacca ggtcacccct gtttcccaca ttggctgccc caacactaca caacagggct 480

ctcctgtgtt cgccaagcta ctggctaaaa accaagcatc gttgtgcaat acaactctga 540

actggcacag ccaagatgga gctgggagct catacctatc tcaaggtctg aggtacgaag 600

aagacaaaaa ggagttggtg gtagacagtc ccgggctcta ctacgtattt ttggaactga 660

agctcagtcc aacattcaca aacacaggcc acaaggtgca gggctgggtc tctcttgttt 720

tgcaagcaaa gcctcaggta gatgactttg acaacttggc cctgacagtg gaactgttcc 780

cttgctccat ggagaacaag ttagtggacc gttcctggag tcaactgttg ctcctgaagg 840

ctggccaccg cctcagtgtg ggtctgaggg cttatctgca tggagcccag gatgcataca 900

gagactggga gctgtcttat cccaacacca ccagctttgg actctttctt gtgaaacccg 960

acaacccatg ggaatgagaa ctatccttct tgtgactcct agttgctaag tcctcaagct 1020

gctatgtttt atggggtctg agcaggggtc ccttccatga ctttctcttg tctttaactg 1080

gacttggtat ttattctgag catagctcag acaagacttt atataattca ctagatagca 1140

ttagtaaact gctgggcagc tgctagataa aaaaaaattt ctaaatcaaa gtttatattt 1200

atattaatat ataaaaataa atgtgtttgt 1230

<210> 2

<211> 309

<212> PRT

<213> Mouse (Mouse)

<400> 2

Met Asp Gln His Thr Leu Asp Val Glu Asp Thr Ala Asp Ala Arg His

1 5 10 15

Pro Ala Gly Thr Ser Cys Pro Ser Asp Ala Ala Leu Leu Arg Asp Thr

20 25 30

Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr

35 40 45

Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg

50 55 60

Asp Arg Glu Ala Ala Trp Pro Pro Ala Leu Asn Phe Cys Ser Arg His

65 70 75 80

Pro Lys Leu Tyr Gly Leu Val Ala Leu Val Leu Leu Leu Leu Ile Ala

85 90 95

Ala Cys Val Pro Ile Phe Thr Arg Thr Glu Pro Arg Pro Ala Leu Thr

100 105 110

Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala Asp Gln

115 120 125

Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln Gln Gly

130 135 140

Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser Leu Cys

145 150 155 160

Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser Ser Tyr

165 170 175

Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu Val Val

180 185 190

Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu Ser Pro

195 200 205

Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val Ser Leu Val

210 215 220

Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala Leu Thr

225 230 235 240

Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp Arg Ser

245 250 255

Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser Val Gly

260 265 270

Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg Asp Trp Glu

275 280 285

Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu Val Lys Pro

290 295 300

Asp Asn Pro Trp Glu

305

<210> 3

<211> 1634

<212> DNA/RNA

<213> human (human)

<400> 3

agtctctcgt catggaatac gcctctgacg cttcactgga ccccgaagcc ccgtggcctc 60

ccgcgccccg cgctcgcgcc tgccgcgtac tgccttgggc cctggtcgcg gggctgctgc 120

tgctgctgct gctcgctgcc gcctgcgccg tcttcctcgc ctgcccctgg gccgtgtccg 180

gggctcgcgc ctcgcccggc tccgcggcca gcccgagact ccgcgagggt cccgagcttt 240

cgcccgacga tcccgccggc ctcttggacc tgcggcaggg catgtttgcg cagctggtgg 300

cccaaaatgt tctgctgatc gatgggcccc tgagctggta cagtgaccca ggcctggcag 360

gcgtgtccct gacggggggc ctgagctaca aagaggacac gaaggagctg gtggtggcca 420

aggctggagt ctactatgtc ttctttcaac tagagctgcg gcgcgtggtg gccggcgagg 480

gctcaggctc cgtttcactt gcgctgcacc tgcagccact gcgctctgct gctggggccg 540

ccgccctggc tttgaccgtg gacctgccac ccgcctcctc cgaggctcgg aactcggcct 600

tcggtttcca gggccgcttg ctgcacctga gtgccggcca gcgcctgggc gtccatcttc 660

acactgaggc cagggcacgc catgcctggc agcttaccca gggcgccaca gtcttgggac 720

tcttccgggt gacccccgaa atcccagccg gactcccttc accgaggtcg gaataacgtc 780

cagcctgggt gcagcccacc tggacagagt ccgaatccta ctccatcctt catggagacc 840

cctggtgctg ggtccctgct gctttctcta cctcaagggg cttggcaggg gtccctgctg 900

ctgacctccc cttgaggacc ctcctcaccc actccttccc caagttggac cttgatattt 960

attctgagcc tgagctcaga taatatatta tatatattat atatatatat atatttctat 1020

ttaaagagga tcctgagttt gtgaatggac ttttttagag gagttgtttt gggggggggg 1080

gggtcttcga cattgccgag gctggtcttg aactcctgga cttagacgat cctcctgcct 1140

cagcctccca agcaactggg attcatcctt tctattaatt cattgtactt atttgcttat 1200

ttgtgtgtat tgagcatctg taatgtgcca gcattgtgcc caggctaggg ggctatagaa 1260

acatctagaa atagactgaa agaaaatctg agttatggta atacgtgagg aatttaaaga 1320

ctcatcccca gcctccacct cctgtgtgat acttgggggc tagctttttt ctttctttct 1380

tttttttgag atggtcttgt tctgtcaacc aggctagaat gcagcggtgc aatcatgagt 1440

caatgcagcc tccagcctcg acctcccgag gctcaggtga tcctcccatc tcagcctctc 1500

gagtagctgg gaccacagtt gtgtgccacc acacttggct aactttttaa tttttttgcg 1560

gagacggtat tgctatgttg ccaaggttgt ttacatgcca gtacaattta taataaacac 1620

tcatttttcc tccc 1634

<210> 4

<211> 254

<212> PRT

<213> human (human)

<400> 4

Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

1 5 10 15

Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

20 25 30

Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

35 40 45

Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser

50 55 60

Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

65 70 75 80

Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

85 90 95

Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

100 105 110

Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

115 120 125

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

130 135 140

Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

145 150 155 160

Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

165 170 175

Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

180 185 190

Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

195 200 205

Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

210 215 220

Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

225 230 235 240

Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

245 250

<210> 5

<211> 10351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tttttggtcc aacagggtag atcagatatg cggtacacac tctcacacca aaagatacac 60

acctgtgtat gctaagtgtg atgggctgga agctatgcag agtccctttc tccagctgac 120

cttcgatgac acaaacacac gttggttgca ttccacatga atgggtaggc acacagtcca 180

agtgcacacc ctggggcagg cagcctcccc gtccccatca tcccacagag agtgggactc 240

aaggctgggc catatgcagg tatggtggtc ttctgaattg ggggcggggg ctaaggaaat 300

ggctcagtgg ctaagagcac ttgctgctct tgtagcggct tagagttcag tccccagcac 360

tcaatccctg ctcataatta ctctagttcc agggaatctg accccctcct ctggcctctc 420

tgaacacttg tagcatccgc tcatagacaa cgcattacac attaataaaa gcaaactaaa 480

ttttaaagag aataagggtg ggcgtgtcca gcagctggaa tgagaatttg taaggcacag 540

aaggaggtgg aggcatttta gactgccttg ccgttaacca ggtcccaggt ggacgaagga 600

gtaaagtagt ctctgaggaa ggtccgatgg gaaacaagct ttctttccta ggctgttaga 660

gtccggtgga atagggagaa cagcggggga tgtgggaagg accagggcgg gctctgggag 720

gctgatagag aagtctgtta agcctacccg gtagggcctg agcagggggg gggggggggg 780

cggagggggg tctttggaga ttggcaggga agcaggcgag gcaagagaac tgaaccgcct 840

gggaagctaa gggagcggga agtaggctgg aagaatgacg tcaaaagtgg gaatccacaa 900

cctcgaggaa gctgttgact ggggaaagtg ggcagggcgt agagagaatg acagagcagc 960

tgggaggggc taagaaagtg ggaggaacct atagagattg agaagtgaga gggacctggg 1020

aagtacacag ctgaaaagta aggatgtggg gctagagaaa gtgaaggagc cattgggagg 1080

gactgcaaat gaaggtttag agagtaacaa ctgggaagtg gacggagcct aaggagaagt 1140

gtatgggctt ggaaaccaga gtctactctc aagtggcacc atgggctctg gccactttcg 1200

tgttgctgat gctggccctg taggaaccat agtgacctgc ctcctcctac aacatcctca 1260

ctgatcgtat ctcgcggtca tacaggcttc tctgggttcc ggtcctcggg ctggtggtcc 1320

tcccggcttc caggttctga tgtcaagttc ttccaaggct cggcaagttt ggggggcaga 1380

cccttccacc tgcatctcat acagcctagg gcgaggatgc cgtccacaag ggggcgcgtg 1440

tgtcccgcgc tgggatccaa ggctgagtag gagggagttc tgttcacaga tctggtacct 1500

ggatgtttta ttggagtatt ggttttgttc ttgttgtttg tttgcttttt tttttaattt 1560

attttatttt gtctttatag gtgtttcgcc tgcctgtatg tctgtgtgaa agtgtcataa 1620

gccttggaac tggagttaca gacaggtctg agctgccata tagctgctgg gaattgaact 1680

tgagtcctct gcaagagcag ccagtgttct taaccatgga gccacctaat cctcactgtt 1740

atttattttt ttttttttta agatagaatc tctcactggt ctgaggagtc cagctatttg 1800

tctagaatag tttgccaatg agttccagga atcctccttt gtctaccttc ccagactaag 1860

attataagca cttgacagct tgtctttttt tttttttttt tttttttcca gacagggttt 1920

ctctgtatag ccctggctgt cctggagctc actttgtaga ccagactggc ctcgaactca 1980

gaaatcggcc tgcctctgcc tcccaagtgc tgggattaaa ggcgtgtggc gccaccacgc 2040

ccggctctta ttattattat taaacaaaac aacaagccag gggtgctggg aaatcaaatc 2100

aggtccccat gcttatgaaa caagtgtcac tggctgagct atgtatgcct ggcatattaa 2160

aaaaaagttt taaagtttta cttgtattta tgtgtatatg tgtgtctcag ggtaagtgca 2220

ggtgagtgta gtgcctattg ctgccagaag aggtgtcaca tccccttgga tctggaatta 2280

caggaggttg tgagccacca tgtgagggtt ggggacttaa ctctggttat ctgaaagagc 2340

agagtaggag tactcttacc tcaacccttt ccatacctct tttatttccc tttctcctct 2400

ctttctgtcc tcacgagaca gggtctatct ctgtagccaa ggctagcctg aaactataca 2460

taggctggcc tcagacttgt gtaatgtcct accttagcat cccaaatgct caggtgacag 2520

atatgaacca ccacaccttg gctgttcagt gtgtttgaca atgatctctc tctctctctc 2580

tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc cccctgccca 2640

gggggctatg gtgtatttgt ggttgctgtg tctgttctat gtcctggctg tgggtgtctt 2700

tgtgtttctg tctaattgtg gctgtctaat ggctacttcc cctggcagca ctggggaatc 2760

ccttacagat gatagtcagg ctgggattcc ctttctactc agcttggttg atttcccatc 2820

actgagttag ctcttatcaa cctctgaggg gtccccagcc tgctcagcca ctcccccatc 2880

tttctcaggg aagaacttat tgttgatgtt aaatttttcc ttcttccaga aagctatggt 2940

caccttttct ttctttctgg gtgcttagtt gactacaggt tgttcatggc taaaaggaag 3000

gaggaggaag atagaaggag aaaaggagga agaggaagag gaggaggagg cagaggaaga 3060

agaggaaccc aaacaaaact ttgtacacat ggaaatgcag gctagagcca taccacagac 3120

tccagtgtgg acaaacctcc ggaagcaatt gtaatgctga ctctttgctc aaactggaag 3180

atcctgtccc cgtggttcaa aggcatgagc tgtctttgct gttgtgtgac tggtgatgtc 3240

atcaatggta atctgaacga ttcccttttg aagactggag agatcatttc cgttctctgc 3300

actttttgct gttatgggct ttgctgttgg tcaaagggtc tcacatagac caggttggcc 3360

tccaactaga cttttaaccg aggctgatct tgaactcctt cctgatcctc ctaactctgc 3420

ctccagagtt ctggctctgt gtagatcctt tcaaacatcc tcacgggtca acccagaggc 3480

aggatttgtg tactggattt cagtgtttca aagtccacat gtagtcactg acacatgcta 3540

ggctgtgtag gctgccagtg gcaaggatgg ctgaacttac tgagtgactg ctgtatgtca 3600

caacatcctg gctccctcac tgtggacacc ttgaattttg aggtagtatt tttttttttt 3660

tttcgagaca aggtttctct gtatagccct ggctgtcctg gaactcactt tgtagatcag 3720

gctggcctcg aactcagaaa tccgcctgcc tctgcctccc aagtgctggg attaagtatt 3780

tttttttaaa gatttatttt atttatatga gtatactgta actgccttca gacacaccag 3840

aagagggcat cagatcccat taccccatta cagatggttg tgagccacca tgtggttgct 3900

ggaaattgaa ctcaggacat ctggaagagc agtcagtgct cttaaccgct gaaccatctc 3960

tccagcctaa gatagtattg ttttaatttc atttcacaga tggggaaatt gaggcaccaa 4020

gaggtaaagg aattccctca ggaggtgggg ctgggatttg aacctatgct gtgtagtttc 4080

catgtgccca ccaggggaga gtcagggaag ttgactttta agcctggctt ctggtatgtg 4140

tatgtaagct cagtacattg gagtctgagg ctgccctggg ctgatagcaa tatcctcact 4200

caagacagga aagcagttga cactcctggc tatgctggtg gccagtacct gtcatcctag 4260

acatttggaa cactgaggca ggaggtgggc atgaacctag gacttggagg tcagcctggg 4320

aaacttacat agactctaca tcaaaaaata aggcaaaaca aaaataaaaa gttaatacta 4380

aattgcttat ggtagtacat atataacatc ccagtttggg ggcttagcat agagcctctg 4440

cctagaatct cccagtgaag ggctgggggc gtggccatag ggtggagccc cgcctattga 4500

ccgccaatga ggggccaggg gcgtgattca gggaatggag cttttgccta ccgcgtatga 4560

gctcattatg gctgacggga gagtgacagt gaaatagaac ggaaccctgc catagaaacc 4620

ttgggataga aacctttact tttcgtctta gaaaatacca agtcatgcct gcctgttctg 4680

gggacctgag aggtgggaga ggagcatcta agccaacact gtagggcggg tccctctaac 4740

tacttgcctc ccttgagttt ttccatgctg tgaggacagc tagggttaac agtcatttca 4800

ggaagacaac tgagtacacc acaggggggg ctattggact ggggttggag gagggagctc 4860

agctctgcat ggtggataga gcttcctgcc cctgcaagtc cctcagcatt gtcagctagg 4920

ctgtagccca gaaagggtgt tccagagctc tgtgaaggca cacagctcag aacatctctg 4980

ggacaccttg gtcagcttgc ttcatagttt acagtgaatc tctggatcta catgcatgtg 5040

ggttgatgtc cccctaggta gaaaccagag agttattttc acttaagttc ctcgcagccc 5100

aagcatgcca ttccagcact tgggaggtgg aggcaggagg aacaagaagt agttggaggc 5160

cattctttgt taagaagtga gctgcctaga ctgggttacg cgaaacccag tctgaagaaa 5220

tatttaggaa gaggaagggg actgatgaga gcgaaagcgg ggcaagagag gatcatgggt 5280

gtgtaggtgg acaggcagcc tgtggtggag ggtgctgagg acctaggttc agttcctggc 5340

actcacatgg catctcacag ttgcctgtaa ctctacttct ggcctctcag aaggcatgta 5400

catggactac agatagacag acagacagac agacagacag acacacacac acacacacac 5460

acacacacac acacacagac ggacctgaga gatggttcag tggctgagag catttgttgc 5520

tatcacagag gacctgggtt ctgttccttg cacgcacatg gtggctcaca actatcctta 5580

attgcaattc caggagatct gacctgggta ctggacctgc acgttggtgc atgcacatac 5640

atgcaggcaa aacagtcata cgcttaaaat atataaatac aaaaacataa ggcacagaaa 5700

caaaccattc ggggtgaata tgatcaaaat gaattatata cataaatgaa aatgtccagg 5760

ctggagagat ggttcagcag ttaagagcac tgactgctct tctaaaggtc ctgagctcaa 5820

atcccagcaa ccacatggtg gctcacagcc attcggaatg agatctgatg ccctcttctg 5880

gtgtgtctga aaacagctat agtgtactta catataataa ataaataaat ctttaaaaga 5940

aaagaaaaga aaatgtccta aagggactca ttgttacata tagctaatat atagctagta 6000

tatattcata aaatatgaaa aacaactcaa acattataga tctcctttgc tacgaatgta 6060

ttctatgtgc aacactgcac acagtggtgc tgggatggaa tccaggtgag tgcttgccat 6120

agagcctgac cctcagaccc tgcgattgaa tgtagtacct gagtgtaatg tattcaagct 6180

taagcatcca gcattgcaag acccatgctg tttaccacag taggcatgga actatacctg 6240

ctttacccac agtcctgcag aatcagcacc aagtttgagc tcttctccat ataccttctt 6300

ctttccctct tatccactct tacacctgcc tgtccatcca ttcacccata caaccatcaa 6360

tccatctatc atccatccac ctgtccatcc atccatccat ccatccatcc atccatccat 6420

ccatccatct actcacttat ccatctgtct gtatgtccat ccatccatct gtctgctcac 6480

ctatctaccc actcactgac tcatatgtca atccatccat ccatccatcc atccatccat 6540

ccatccatcc atccatccat ctgtctgtcc atctgcctgc ctgtccatat agctacctac 6600

tcaccgatcc atctatccat ctgtccatcc atctatccat ctgtctatcc atctacccac 6660

ccacccatct attcatccac ccacctaccc atccatccat ctatccatac attcacccac 6720

acactcaccc atccctgttc cttccttttc ttttttctca cccttcttgc attccgtctt 6780

cccataaata tcatggagac actttttgct ccgtatactg tgggtacctg ggaatacagt 6840

ggatagagac acttttgagg atgccataca tcagtagggg tgggaggcaa tgataagcaa 6900

ggaaagcaat taataaacag tcagtcaagg tgggtgatag ggccagcata acattcttgt 6960

ggcaccagga gtgtctaagt acacttgcta tcctgcacac agttttggga ggctgaggaa 7020

ggaaggtcag ggcaatgtgg caagcctgta attgaaaaca aaaacaaaga gctcatgggg 7080

tggtgtacca cagacctttg agggtggggt tctgtcatct gcgttattgt agatctagat 7140

gctttcctcc cagttcttgt cactgccagc ttaggcaata cttggctgtg ggctgtggtt 7200

ttaattgtaa acagattggc tcaggatctg ggagctagct gctgctgctg ctgctgctgc 7260

tactactact actactacta ctactactac tactactact actactacca ctacggtact 7320

actactacta ctactactac tactactact actactacta ctactaccac tacggtcctg 7380

agatgctaaa tctttcttct tagcaataag gggcaggggg tggactttgt gtgtgagcca 7440

ggggggaggg ggagacttga gttctatccc cagcatctta tacaccagac atggtggcat 7500

ggacctgtca atttagcact ccagaaatgg aggcaggagg attagaagtt tattctttac 7560

tacataggga atgtcaggcc agccttgact acacgagatc ctgttcaaaa acaaattaat 7620

tttaatatta aaatccccct tgtctgtctg tttgtctgtc tgtctgtctc tgtgtgtgtt 7680

tatctgtctg tctcactatt tagccctggc tggtccattc tcattatgta gttcaaggtg 7740

gccttgaatt caccaagacc catctgcttc tacctcctga gtgcaataca taccccctgc 7800

atccagtttt gttttgtttt gtttttttag acagtgtctc ttcatgtagc ccaggctggt 7860

ttttgaactc caaccttcaa acttcagctt cctgaaggct gggctcacag gtgcatacca 7920

tgcatggctt gttctaaatg gtatgaactc tctctctccg tgtgtgtgtg tgttttctgt 7980

ccagaaactc caccaattcc cccccccccc aataataaag caccttcagg ctctgcagga 8040

gagaaagatg gctgagtgga gttactttca gctgagtcag gattccagtt ccctggtgac 8100

atggatgatg agtctgtgag gtggaagtct tcaaactgtg gctttgaaga agttatagct 8160

ggagagagag agagaaagtc ttcatctgcc tttttttttt tctcactgtg atgatggcag 8220

ggatacaaat gaggatgaga caatgtgttc actgtgcagc cgaggcaggg aggggtaaac 8280

tagtccaggc attgaggggt cccttttcgt gagagtcatg taggtgtggg ctgactctga 8340

gcagtgtgtg tccagcagag agaggttgga ccactgtctg cgtgagggcc agagttccca 8400

cagctgagtc accgctggtg ccctgcagct gctccttggg tggaaccttc aatttctaga 8460

tcacccaagt gtcttttgtg tcattttaat gtcccacctc ccccacctct cccctgaccc 8520

caagctgggg ttttcctgta attcccaggg gaagttgatg tggaataaaa cagaaggaag 8580

gggtggctgg tgtctccaga gggggctccg tgcacgtctc ccaggctggg agctcagtag 8640

gttccatgcc cagtatggaa aaaaagcagg tggggcctgg gctgggtctc tgttgtaaga 8700

atcttggccc agtaagctcg aggctttgcg ctccttcccc aacacagaag aagctggatt 8760

tgagggtgta tgcctgtctg tgtctgtatc ccaatatact ggggagggtc agaagttcag 8820

agtcatccta ggctgtctag tgagttggag ggctgcaagg cgaagaccag accctatgta 8880

gaaacaaaca actccccatt ttatagcttg ttgtttcctg ctgagataaa tggtctggtt 8940

atatcagtac tttttttttt tttaattatg tttactgatg gtatgcatgt cgtggtcaaa 9000

gggcactttc aggacaaatt tctgggtgtc agttctttct tcccaccatg taggtcctga 9060

ggactgaact caggccacca ggcttgtgat tatactatta ctggctgagc catctcagca 9120

gatctaacca gatcagaaca ttgaggagtc tgtctccaag gtttgcccag agccagtcat 9180

ctctcagttc ctcttttggg gggtcttctc tgcgcccccc ccttttgcct ctacctcagg 9240

caagaacagt gaaagaatgg gaaattggaa atgtgtccgc tgcctggggg aatttctcta 9300

tctggactct ccagccttct gtttctggag tcctaagaga ctactaacag gaagccccaa 9360

gaaagtgggt ggggacacac caggaaaggc ccagggtaga gatggggtcc tggggaggaa 9420

gaggctgagt gagtgggtgc ctagcaggtc tctttgattc cgctggggat gggtgggcag 9480

ttttggatgt ccctagagtc ctcgacaaaa caacaacaac aaaaaacaaa acaaaacaaa 9540

acaaaacaac aacaaaaccc cctgcattta gggcgtccca gggatgggga gctaggggtt 9600

ggaagaaaaa gggagcagga gagatcagga gagagacaga agtttgagag acagaaaaac 9660

aggaatgaaa atcagcagcg gagaaataac ctaccagtgg ggaacacaga aaaagaaggg 9720

agacagagct gtggattcag gagaggccag ggagggaatg gattccgata tcacatggtt 9780

cccatcatat aaaaagctct ttaatgtgtc agtgttaaac caaatcaaac acacacacac 9840

acacacacac acacacacac acacacacac acacaaaaag ggaagaagga cgggcgggca 9900

cagggcctgg acagggaagg ggaaaggggc gagaaggggg aggggaaggg gggaggagga 9960

gagaaagttc cgggagtcga ggggcggagc gcggagcccg ggaaccccgc ggctgccctc 10020

cctcccttcc ctccccggaa aaggcgcctc ggtagcctat aaagcacggg cactggcggg 10080

agacgtgcac tgaccgaccg tggtaatgga ccagcacaca cttgatgtgg aggataccgc 10140

ggatgccaga catccagcag gtacttcgtg cccctcggat gcggcgctcc tcagagatac 10200

cgggctcctc gcggacgctg cgctcctctc agatactgtg cgccccacaa atgccgcgct 10260

ccccacggat gctgcctacc ctgcggttaa tgttcgggat cgcgaggccg cgtggccgcc 10320

tgcactgaac ttctgttccc gccacccaaa g 10351

<210> 6

<211> 4820

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggctgaagtg gctctgtacc actatccctt ttttgagaca agaaagtatg tagcccaggc 60

tgacctagaa gtccatatat agcctagatt ggccttaaac tcaggccagc atcctgatac 120

ctgagtgctc agttgacagg gtggcagatg tgctgagata gagccagagt ctctcatgca 180

tgtgaggccc atatgcttta tcacagaggg ctctgttcct tggaatccag ctacgaataa 240

gcgaaaaacc aaaaccaaaa caacccccta cctcaaccat ccagtgtgaa agtctgtgat 300

ttctgatgtg acggtactgg actctgtgtg ctatttcgag agacagaacc tgttggagca 360

atacaattat agatacagat aaatgaagga ggagttgtta aggaaattcc cccaacggtg 420

gtcatgtgga ctgagaagtc cctcagctga agactgcagg tgaaggctgg aggaactgga 480

gttcaaggcg ggacagacca ggagcacaag accctgtctc aaaacaaaca gcaaaataaa 540

tcaaactaaa aagaaacaga gacgtcccac aaatcagcta gatctctgta aattcacagg 600

attggaaggt agaggctatc cttgggagtt caaaggctgc ctgggctact tacacaacaa 660

aacaaccacc agcaaacgga ggctgggcta ctcctctgtg agcctgagca tgggtctcac 720

aaggaaggcc tcccaaccat ccaagctgat gtgccagggg ccggcagtgt atttgtaaac 780

tcaactgata agttaagcat ttcctcactc cgtgtgagtc acccagactt gaaacccaca 840

gccactctgg gcctcaacag atttttgaaa aatttccatt tcagtatttc ctagctctga 900

cttcaggtga tttacctcgc tgcctatgag agggtagtgg aaatcccttt gtccctccca 960

cccaatctgg attccgtgtt tagtcacatc acagcctgag gtcaggccca aaggaaacag 1020

tagtgttgaa gttggtttta ctgtattctt caggggtgac agtggaacag cttgagaaac 1080

ctgtcattca gcaaagaagt ggctcactga ggtgagagag gctgggctgg gactgacaaa 1140

ggaggccctg acttccattc cagcacttca taaattactg cataaatgaa catatatgtc 1200

tagatttgca taagaaaaga attcaggact ttgcctcccc cctccagacc ccccctcttt 1260

ttttgtgtgt gtgtgtgtgt tccatgcatc aagtcttcct taatccccct ctattttatc 1320

atttggggca gtgttggtct tctgtccaga ctccaccccc acaattacct ggcaacagcc 1380

aggttaggtt ggcccactat aataggaact gcttggcccc tcttccctct cttactctct 1440

tgtctccctt actcttgctt cccgcttgcc ccctctctcc ccattccctt ctcccctctc 1500

tccacgtggc catagccagc ctctacttct ctactctcta cttctctctg cctttctctg 1560

cctcttaact ctcctcccca tgccctgaat aaactctatt ctatactata ccctcatgtg 1620

gctggttcct caggggaaaa ggatgccttg gcatgggctg ccggggcatc tccttccctc 1680

atacccctgc cagaacatat cttaatagct ctttctcttt ttatgatcac aacaggcagg 1740

gtctttctta ttcctattat tgaacataga ttcttttctc atacaatata tcctgattac 1800

aattttttct ccctctgctt ctcccagttc ttcccctccc tcccctccag atccagtccc 1860

tttctgtctc tcagtaggaa agaacaggct tctaagagat aacaactgga cacgacaaaa 1920

tcaaatataa taagacaaag caaaaccttt catactgcag ttggtcaggt ctgagttcaa 1980

accctgcttc agctagtacc agctaagctg tgtgtacttg ggcaagtgtc tgacttctct 2040

gaacctactg ttcacaataa tgagcataga agctgagttt accgcaaggc tcttggggat 2100

gttaactgag ataataggct ttatgacaca gtgtctgaca ctaaattaaa gcatgaaggt 2160

tgtggtgata agatagacag agagagagag agagagagag agagagagag agagagagag 2220

atatggtttc agaggttcta tcaccatgtc ctgaggcagg caggagatgg gctgaaagat 2280

caggtagcaa tcaagagggc agtgtttcag accactgtaa atctggattc tttggtttct 2340

ggccctggga caagggactg aagtgcatat tttgcatacc aaagcctacc tggcctccag 2400

attatcccag catccctaag tccctacagt ggtatgccct gcctccaatt ctgaactttc 2460

cagcccagag gctgggctct cctttcccag aggctcttcc ctataaaatc cagacactct 2520

ccttccaacc ccccaccaca cacacccttt tctctttctc ttcccctctc ccatctctcc 2580

ccactgtgtc ttcccaagtc tagtccttgg gccagtgaac tcaccagaga gcagtttacc 2640

agtatacctg cccataatat tttctaatct ggcttgaact ggctcatttt gctagcagag 2700

aaataactct ccaaagcctt tcatcaaaac tctagagagg cccggggaag tggctcggtt 2760

aggaaagtgt ttctcgaaag cttgagggcc tgactttgaa tccccagaac ccattttcaa 2820

atgctgaatg tgtagctgaa tgtgtgtcag ggtgcaggcc tttgttccta gcactctgga 2880

ggcaaaggca ggaggatctc tgagtgtgag gccagcttgt ttacagagca tgttccagga 2940

cagccagaac ctcccccatc tctcttcctt ccttccttcc ttccttcctt ccttccttcc 3000

ttccttcctt ccttccttcc tccctccctt cctccctcct tccttccccc tctctcccct 3060

ctctttcccc cctctctctc actcacacac acacacacac acacacacac acacacacac 3120

acacacacac cagcccaaaa aaaaaaaaaa aagctgagtg tggctctgga gagagggtta 3180

atggttccga gagcctgcag ctgttacaga gaaccccagg ttacttccta gcacccattc 3240

caagtggctc accaacactt gtaactccat ctgatgctct cttttgcctc tactggcatt 3300

gtacacatgt gtacacattc cccatttccc atacacacag tacaaagact tcttaaagaa 3360

cattgggcct gaagatgcac aggtgtaact ggcctgtctg tatgagtaat tacaaagaca 3420

caggggagct tcctgtttgg tatcagacta tcgaaaatag ggtatccccg aagttagaat 3480

ctccccagga atctccagct gctggaggtc ccagggctga agcttgtctc ctggagaaag 3540

attgacactt ggttgggcct tgcatggggg gagggagccg agagagtgat gggcgggacc 3600

gtatggtggc aggctatgtg cttatcattt cttactcttt ttgagattta ttttatttta 3660

tgtggatgag tgttatctgt gtgtatctat gcatgctaag tttgtgcctg gtgcccacag 3720

aggccagaag aggatgccag cgtgactgtg agctgctgtg tgggtgcggg tgcaagaaca 3780

gtgggtactc taaccgctgg tatcttcagc ccttgtttct cctgccctct acttaaacct 3840

tggtgccatg ttaggccctc tcaagatgag cttggagggg ggtggctgtc atggatgtct 3900

ttgtctcttt tgccagtgtg gccacataca tttttttctg cttttcacta ttaatttatc 3960

tatttattcg agatagggtc tcaccatgca gccccagatg cctggaccgg gctggcttgg 4020

aactcagaga tatctgcttg ccctttcctc cctagtgcta agattaaaag cgggtgccac 4080

cacatttagt taatttggct gttttagtag gcttattgaa ttaggcttat taaattgaat 4140

ggctgaacct gtgttactgg ggcacagatt gtgatctcta aggccagtag taaatttaca 4200

ctcaaagcac tgggaggtag acacatgaag gtccctgagg aggggtggga tttagaaatg 4260

cacctgaggt tgacctgtgg cctctgcatg cacctgcaca tacctgtgca cacccacaac 4320

acctgctcag agtgatgcgt tgccaagcag aacagacaca tctagttagc ttattggagg 4380

actgcttagt ttgcaagatg agcttgaaag gggttggggg ggagcaggat ggaggcagag 4440

atttatggaa gaggctgtgg aatgttctag aagaggggta gttgaatgaa cagtacagct 4500

gagagtcgat attatttatt tatttattta tttatttatt tatttattca tctatctatc 4560

tatctatcta tctatctatc tatggagaca tctatctatc tatctatcta tctatctatc 4620

tatctatcta tctatctatc tatctatcta tctatctatt tgacaaaggg tctctctata 4680

gctctggctg tcctgtagtt tgggctatag accaggctgg ccttggactc agagatctgc 4740

ctgcctctga ctcccaagtc ctgggaataa aggcgtgtgc cattactacc acctcagtgt 4800

ggtggtgaca tttgtcccat 4820

<210> 7

<211> 3955

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtactgcctt gggccctggt cgcggggctg ctgctgctgc tgctgctcgc tgccgcctgc 60

gccgtcttcc tcgcctgccc ctgggccgtg tccggggctc gcgcctcgcc cggctccgcg 120

gccagcccga gactccgcga gggtcccgag ctttcgcccg acgatcccgc cggcctcttg 180

gacctgcggc aggtgagacg tgccccgacc ctcggtagct ggtctcgcgg gagaccccta 240

ccgcccctct gggactccct tctccctccc gcacccccag ggacacctgt tctacactcc 300

cggccgggga gaggagaccc accggggctc ccattctcca tctagcaccg aggccggggg 360

ggcacacctt tcatcccgca cccctacgac acctcatctg taccccaggc cgggggcggg 420

gaggagaccg cccaacagtt ctttttggac cccccaaggg tctccttctt ctagtccggg 480

gggcacccct ttcatcctgc gcccgctacg agaccctatc tgtatcccgg gagggggaga 540

ggagaccccc acaagttctt tttggacctc caagggctct ccttctccgt cttgcacggg 600

agggcacccc tttcattccg catcccgaga ccccatcagc accccaggcc aggaggagga 660

gacccccaca gtcctcaacc cctcagggat acgtcttctg caccccgggt cggagaaagg 720

ctggcttccc tccatgtggc agtgggtggg ggcacccctc ttcattccgc attcccaagg 780

caccccatct gcaccctgga cgggggaccc tccgcggttc tccaaccctc gacggcttcc 840

tttctctatg taggacttag gacattgagg gcacctcttt ttaccccgca cccccacaag 900

ctctgcatct ctggggggaa ccttttttcc atccccgatc cgaggggggc acttcctctt 960

tatctgggac cgccaaggag acccccagtt cctttgctaa ctcccaaaga gccttatccc 1020

caacatctga ggtacccctc tccctttcaa gacccccagg ggaatacccc caagggggct 1080

ggaaagaaga gggatcgctt ttccctcctg aattcttgga gatctgtcct tggggcaggt 1140

tgagccacca gcttcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttcg tgtttgtgtg 1200

ggtgtttgtg ttcgtgtgtg tgtgttcgtg tgggggtgcg tatgtgtgtg tgttcgtggg 1260

tgtgtttgtg ttcgtgtggg tgtgtgttcg tgtgggtgtt tgtgtttgtt cgttcgtgtg 1320

ggtgtttgtg ttcatgtggg tgtgtttgga tatttgtgtt cgtgtgtgtc tgtgtgtgtt 1380

cgtgtgggtg tttgtgtgtg tttgtgtttg cgttcgtgtg tgtgttcgtg tttgttcgtg 1440

tgtctgtgtg ttaatgtggg tgttcgtgtg tgtgttcgtt tgtgtttgtg ttcgtgtgtt 1500

tgtgtgtttg tgtgtgtgtg tgtgcggtct ctgttcttta gttgggaggg aagggaagcg 1560

taggcttcag gtcggcacag actctgggga ccctgacagc tgaagtgagt ggggacagaa 1620

cctccatttt ctaggggaac ccccatccac tttcctcctt tctactttta acagggcatg 1680

tttgcgcagc tggtggccca aaatggtaag tatcctccgc cacttccggt ccctggcccc 1740

ccaccatccc caccccggga tacgaagaag ggggaacctg gagagtgagg ctctgccgca 1800

cactggcttt gacccttgac cgctgctgtc tctgaaagct gctacttccc ctctttgaac 1860

gccaccgatc cctctttctg acttgctctg actctctgga tgccatggcc tccccatcag 1920

acccccatct gggcccacct gggacccctg ccctctcaga gctggggctt gacaccccca 1980

acccccagcc tggttttgtg tctctggcct ccttcttttc gaaaccctct tccccgctgc 2040

ttttcttccc tctttcctgc cccttcccca ctctcccagc ctctcttccc tacctcttcc 2100

cctcgcccag agcctcctcc ttactcccat tctcttcccc atccccaggc cctcctcctg 2160

tccccttccc tccccctaga ccctcctccc tgccccttcc ccaaaccctc cccagcccca 2220

tgctctccca gtttccaaga cactcctccc agcaccttcc ctccccgtcc agagacttct 2280

tccccgctcg agacctcttc ccccttcccc tcccagagac ccccttcccc cttcccctct 2340

ccatctagag atcacttccc cctttcccct ccccctccag aaaccccttc ccccttccct 2400

ctcccctcca gagacccctt ccccctccaa agaccccttc ccccttcctt ccccctccag 2460

agaccccttt ccccttccct ccccttccag agaccccttc cccctccccc tccaaagacc 2520

ccttcctcct tcccctccct ctccagagac cccttccccc ttccctctcc ctccagagac 2580

cccttccccc ttccctgccc cctccagagt ccccttcccc tccagaggcc ccttccccct 2640

tccccctccc cctccagaga caccttctcc cttctccccc tccagagacc ccttccccct 2700

tccctccccc tgcctaagac tcccctccct gccatcttct ctccttggtg cttccttctc 2760

cctaacccct cccccacccc ttcctgctcc ctttgccttt cttcgaagtc ctctcttacc 2820

aggtcccctg tccacccctc ttaaagttcc cttgattccc tgatggatcc ttcctctaga 2880

acttcctcct ttcctaacag tcttccctct ccctcctagc tccctgtcca cctctcctct 2940

cccctttcct tccttctctc ccttctcctc cccccatctc catcctgtcc gagatctgct 3000

gctccctcca accccctcac cgccctctcc aggagtcccc ttacccctcc ccgaggcctc 3060

ttccagccag actcgcctgg ccttttgctt ttcaccccct cttcctcttc ctgcatgtct 3120

ttccccgcac ccgcatcact ccttccccac ccaacctctt ggagttttgc cctgtcccga 3180

ctcttgtcct tctccctgga ttcccttcct ctgttctatc cccgtctttc cctcccctag 3240

ctctacccct gctcagagtc ccgcccccaa caacctcagt tctcttcaat cagcaccccc 3300

ccaacgcccc ccccacccag gctccccgct ctgctcccct gtcccgctcc ctgccccgcc 3360

attccccgcc ccccggcccc tgacccgttc ttttctccca gggctgcgct gacatgttcg 3420

gtgctcagcc acgctcagct gtgctgggac atgctcagct aagctaagtg catgctttcc 3480

tcccacagtt ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg 3540

cgtgtccctg acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa 3600

ggctggagtc tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg 3660

ctcaggctcc gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc 3720

cgccctggct ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt 3780

cggtttccag ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca 3840

cactgaggcc agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact 3900

cttccgggtg acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 3955

<210> 8

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgaaatccca gccggactcc cttcaccgag gtcggaataa gaactatcct tcttgtgact 60

cctagttgct aagtcctcaa 80

<210> 9

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

attccccagg ggagtgccta taccagatct taaaataatt gatatcgaat tccgaagttc 60

ctattctcta gaaagtatag 80

<210> 10

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtataggaac ttcatcagtc aggtacataa tggtggatcc ggctgaagtg gctctgtacc 60

actatccctt ttttgagaca 80

<210> 11

<211> 1236

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ataaagcacg ggcactggcg ggagacgtgc actgaccgac cgtggtaatg gaccagcaca 60

cacttgatgt ggaggatacc gcggatgcca gacatccagc aggtacttcg tgcccctcgg 120

atgcggcgct cctcagagat accgggctcc tcgcggacgc tgcgctcctc tcagatactg 180

tgcgccccac aaatgccgcg ctccccacgg atgctgccta ccctgcggtt aatgttcggg 240

atcgcgaggc cgcgtggccg cctgcactga acttctgttc ccgccaccca aaggtactgc 300

cttgggccct ggtcgcgggg ctgctgctgc tgctgctgct cgctgccgcc tgcgccgtct 360

tcctcgcctg cccctgggcc gtgtccgggg ctcgcgcctc gcccggctcc gcggccagcc 420

cgagactccg cgagggtccc gagctttcgc ccgacgatcc cgccggcctc ttggacctgc 480

ggcagggcat gtttgcgcag ctggtggccc aaaatgttct gctgatcgat gggcccctga 540

gctggtacag tgacccaggc ctggcaggcg tgtccctgac ggggggcctg agctacaaag 600

aggacacgaa ggagctggtg gtggccaagg ctggagtcta ctatgtcttc tttcaactag 660

agctgcggcg cgtggtggcc ggcgagggct caggctccgt ttcacttgcg ctgcacctgc 720

agccactgcg ctctgctgct ggggccgccg ccctggcttt gaccgtggac ctgccacccg 780

cctcctccga ggctcggaac tcggccttcg gtttccaggg ccgcttgctg cacctgagtg 840

ccggccagcg cctgggcgtc catcttcaca ctgaggccag ggcacgccat gcctggcagc 900

ttacccaggg cgccacagtc ttgggactct tccgggtgac ccccgaaatc ccagccggac 960

tcccttcacc gaggtcggaa taagaactat ccttcttgtg actcctagtt gctaagtcct 1020

caagctgcta tgttttatgg ggtctgagca ggggtccctt ccatgacttt ctcttgtctt 1080

taactggact tggtatttat tctgagcata gctcagacaa gactttatat aattcactag 1140

atagcattag taaactgctg ggcagctgct agataaaaaa aaatttctaa atcaaagttt 1200

atatttatat taatatataa aaataaatgt gtttgt 1236

<210> 12

<211> 311

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Asp Gln His Thr Leu Asp Val Glu Asp Thr Ala Asp Ala Arg His

1 5 10 15

Pro Ala Gly Thr Ser Cys Pro Ser Asp Ala Ala Leu Leu Arg Asp Thr

20 25 30

Gly Leu Leu Ala Asp Ala Ala Leu Leu Ser Asp Thr Val Arg Pro Thr

35 40 45

Asn Ala Ala Leu Pro Thr Asp Ala Ala Tyr Pro Ala Val Asn Val Arg

50 55 60

Asp Arg Glu Ala Ala Trp Pro Pro Ala Leu Asn Phe Cys Ser Arg His

65 70 75 80

Pro Lys Val Leu Pro Trp Ala Leu Val Ala Gly Leu Leu Leu Leu Leu

85 90 95

Leu Leu Ala Ala Ala Cys Ala Val Phe Leu Ala Cys Pro Trp Ala Val

100 105 110

Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg

115 120 125

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

130 135 140

Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile

145 150 155 160

Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser

165 170 175

Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val

180 185 190

Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg

195 200 205

Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu

210 215 220

Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val

225 230 235 240

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

245 250 255

Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His

260 265 270

Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly

275 280 285

Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly

290 295 300

Leu Pro Ser Pro Arg Ser Glu

305 310

<210> 13

<211> 1475

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaactatcct tcttgtgact cctagttgct aagtcctcaa gctgctatgt tttatggggt 60

ctgagcaggg gtcccttcca tgactttctc ttgtctttaa ctggacttgg tatttattct 120

gagcatagct cagacaagac tttatataat tcactagata gcattagtaa actgctgggc 180

agctgctaga taaaaaaaaa tttctaaatc aaagtttata tttatattaa tatataaaaa 240

taaatgtgtt tgtaaataag ccttttatta ttttccttgt ctggttttaa attttcatgt 300

ctatgggtat ggtgttcatt tatgaatgtg tctttgtata cgtgtgtgtg tgtgtgtgtg 360

tgtgtgtgtg tgtgtgtgtg tgtgtatttg tagagaggat agagtttggc tcagtgtctt 420

cttcagtcat tctccatctt gttttttgag acaagttctc ttactgagct tggaattcac 480

ggtttctgca aggctgtctg gccattgggc cccaagaatt ccccagggga gtgcctatac 540

cagatcttaa aataattggc tgaagtggct ctgtaccact atcccttttt tgagacaaga 600

aagtatgtag cccaggctga cctagaagtc catatatagc ctagattggc cttaaactca 660

ggccagcatc ctgatacctg agtgctcagt tgacagggtg gcagatgtgc tgagatagag 720

ccagagtctc tcatgcatgt gaggcccata tgctttatca cagagggctc tgttccttgg 780

aatccagcta cgaataagcg aaaaaccaaa accaaaacaa ccccctacct caaccatcca 840

gtgtgaaagt ctgtgatttc tgatgtgacg gtactggact ctgtgtgcta tttcgagaga 900

cagaacctgt tggagcaata caattataga tacagataaa tgaaggagga gttgttaagg 960

aaattccccc aacggtggtc atgtggactg agaagtccct cagctgaaga ctgcaggtga 1020

aggctggagg aactggagtt caaggcggga cagaccagga gcacaagacc ctgtctcaaa 1080

acaaacagca aaataaatca aactaaaaag aaacagagac gtcccacaaa tcagctagat 1140

ctctgtaaat tcacaggatt ggaaggtaga ggctatcctt gggagttcaa aggctgcctg 1200

ggctacttac acaacaaaac aaccaccagc aaacggaggc tgggctactc ctctgtgagc 1260

ctgagcatgg gtctcacaag gaaggcctcc caaccatcca agctgatgtg ccaggggccg 1320

gcagtgtatt tgtaaactca actgataagt taagcatttc ctcactccgt gtgagtcacc 1380

cagacttgaa acccacagcc actctgggcc tcaacagatt tttgaaaaat ttccatttca 1440

gtatttccta gctctgactt caggtgattt acctc 1475

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctctatggc ctagtcgctt tgg 23

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggctcggtgc gggtgaagat agg 23

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tcgggtaccc aggttgggcg agg 23

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcgctggccg aggctcggtg cgg 23

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tcttcacccg caccgagcct cgg 23

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tgtgagcgct ggccgaggct cgg 23

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tcccgccacc caaagctcta tgg 23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ggtacccagg ttgggcgagg tgg 23

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acaagttagt ggaccgttcc tgg 23

<210> 23

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tgtgaaaccc gacaacccat ggg 23

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ttgtgaaacc cgacaaccca tgg 23

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gctggccacc gcctcagtgt ggg 23

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggctggccac cgcctcagtg tgg 23

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctccatggag aacaagttag tgg 23

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggtctgaggg cttatctgca tgg 23

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cccaggatgc atacagagac tgg 23

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gctctatggc ctagtcgctt 20

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

taggctctat ggcctagtcg ctt 23

<210> 32

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aagcgactag gccatagag 19

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aaacaagcga ctaggccata gag 23

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

tgtgaaaccc gacaacccat 20

<210> 35

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

taggtgtgaa acccgacaac ccat 24

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

atgggttgtc gggtttcaca 20

<210> 37

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

aaacatgggt tgtcgggttt caca 24

<210> 38

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 39

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

agctcagtag gttccatgcc cagta 25

<210> 40

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gaaggtgctg ggaggagtgt cttg 24

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ccctctcaga gctggggctt ga 22

<210> 42

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

tctctcacct cagtgagcca cttct 25

<210> 43

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ccgaccctcg gtagctggtc tc 22

<210> 44

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

ctcccgtgca agacggagaa ggag 24

<210> 45

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tgagctgttg ggagaccttg actta 25

<210> 46

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ggagttgact cagtggtcag cactta 26

<210> 47

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

acacacttga tgtggaggat accgc 25

<210> 48

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

agctgcccag cagtttacta atgcta 26

<210> 49

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

cgagaccagc taccgagggt cg 22

<210> 50

<211> 1494

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

aggcgaagac cagaccctat gtagaaacaa acaactcccc attttatagc ttgttgtttc 60

ctgctgagat aaatggtctg gttatatcag tacttttttt ttttttaatt atgtttactg 120

atggtatgca tgtcgtggtc aaagggcact ttcaggacaa atttctgggt gtcagttctt 180

tcttcccacc atgtaggtcc tgaggactga actcaggcca ccaggcttgt gattatacta 240

ttactggctg agccatctca gcagatctaa ccagatcaga acattgagga gtctgtctcc 300

aaggtttgcc cagagccagt catctctcag ttcctctttt ggggggtctt ctctgcgccc 360

cccccttttg cctctacctc aggcaagaac agtgaaagaa tgggaaattg gaaatgtgtc 420

cgctgcctgg gggaatttct ctatctggac tctccagcct tctgtttctg gagtcctaag 480

agactactaa caggaagccc caagaaagtg ggtggggaca caccaggaaa ggcccagggt 540

agagatgggg tcctggggag gaagaggctg agtgagtggg tgcctagcag gtctctttga 600

ttccgctggg gatgggtggg cagttttgga tgtccctaga gtcctcgaca aaacaacaac 660

aacaaaaaac aaaacaaaac aaaacaaaac aacaacaaaa ccccctgcat ttagggcgtc 720

ccagggatgg ggagctaggg gttggaagaa aaagggagca ggagagatca ggagagagac 780

agaagtttga gagacagaaa aacaggaatg aaaatcagca gcggagaaat aacctaccag 840

tggggaacac agaaaaagaa gggagacaga gctgtggatt caggagaggc cagggaggga 900

atggattccg atatcacatg gttcccatca tataaaaagc tctttaatgt gtcagtgtta 960

aaccaaatca aacacacaca cacacacaca cacacacaca cacacacaca cacacacaaa 1020

aagggaagaa ggacgggcgg gcacagggcc tggacaggga aggggaaagg ggcgagaagg 1080

gggaggggaa ggggggagga ggagagaaag ttccgggagt cgaggggcgg agcgcggagc 1140

ccgggaaccc cgcggctgcc ctccctccct tccctccccg gaaaaggcgc ctcggtagcc 1200

tataaagcac gggcactggc gggagacgtg cactgaccga ccgtggtaat ggaccagcac 1260

acacttgatg tggaggatac cgcggatgcc agacatccag caggtacttc gtgcccctcg 1320

gatgcggcgc tcctcagaga taccgggctc ctcgcggacg ctgcgctcct ctcagatact 1380

gtgcgcccca caaatgccgc gctccccacg gatgctgcct accctgcggt taatgttcgg 1440

gatcgcgagg ccgcgtggcc gcctgcactg aacttctgtt cccgccaccc aaag 1494

<210> 51

<211> 2134

<212> DNA/RNA

<213> Mouse (Mouse)

<400> 51

ttcctgaaat tcaggtgctg caggcagccc tcagcacaga gagctgacag ggaccctggg 60

tcaggggttc tgagttccag ctgccactat tcttcttcac ctttggtgtc ctgtgcatgt 120

gacatttcgc catgggaaac aactgttaca acgtggtggt cattgtgctg ctgctagtgg 180

gctgtgagaa ggtgggagcc gtgcagaact cctgtgataa ctgtcagcct ggtactttct 240

gcagaaaata caatccagtc tgcaagagct gccctccaag taccttctcc agcataggtg 300

gacagccgaa ctgtaacatc tgcagagtgt gtgcaggcta tttcaggttc aagaagtttt 360

gctcctctac ccacaacgcg gagtgtgagt gcattgaagg attccattgc ttggggccac 420

agtgcaccag atgtgaaaag gactgcaggc ctggccagga gctaacgaag cagggttgca 480

aaacctgtag cttgggaaca tttaatgacc agaacggtac tggcgtctgt cgaccctgga 540

cgaactgctc tctagacgga aggtctgtgc ttaagaccgg gaccacggag aaggacgtgg 600

tgtgtggacc ccctgtggtg agcttctctc ccagtaccac catttctgtg actccagagg 660

gaggaccagg agggcactcc ttgcaggtcc ttaccttgtt cctggcgctg acatcggctt 720

tgctgctggc cctgatcttc attactctcc tgttctctgt gctcaaatgg atcaggaaaa 780

aattccccca catattcaag caaccattta agaagaccac tggagcagct caagaggaag 840

atgcttgtag ctgccgatgt ccacaggaag aagaaggagg aggaggaggc tatgagctgt 900

gatgtactat cctaggagat gtgtgggccg aaaccgagaa gcactaggac cccaccatcc 960

tgtggaacag cacaagcaac cccaccaccc tgttcttaca catcatccta gatgatgtgt 1020

gggcgcgcac ctcatccaag tctcttctaa cgctaacata tttgtcttta ccttttttaa 1080

atcttttttt aaatttaaat tttatgtgtg tgagtgtttt gcctgcctgt atgcacacgt 1140

gtgtgtgtgt gtgtgtgtga cactcctgat gcctgaggag gtcagaagag aaagggttgg 1200

ttccataaga actggagtta tggatggctg tgagcctttg tgtgggtgct aggaatcaaa 1260

cctgggtcct ctacaagggc agccagtgct cttaaccact gagtcagctt tccagccctg 1320

ccctggacag tttttaaaat ttaacttaat tttttttttt ttttacttaa gcccttaaca 1380

tttttaatag gactgtggga agatcaattt ctagattctc cttaacaata tacatcatat 1440

acatatacac atacatatac atatacatat atattctgag aaaatgacag tttcagttgg 1500

atctcataga ccaatggtcc agttaaaata actgtaaaat cagtgtgtgt gtgtgtgtgt 1560

gtgtgtgtgt gcaaatatga tgcatgacaa tagccataag atgcagtata ttaccctatc 1620

ccattttcct ttggtttctc actcactata ataacacctc cactgtctga agggggagac 1680

ccatgcatcc ctgtctggag gaagcctgac agattttgag gggaatcttc agagcagttc 1740

aagggcctgc ttctcctgtt tcctctgtgt caggcttttc aataaaaagg ccgtttagga 1800

aagggacaaa gcactgtgag gtggggaaca cctgtgaact cacagtagga acgcggcctt 1860

ccagtccacc atgggcagac atggctgccg cttgcctctg catgactcct ggacagctca 1920

agagctgaga atggttgtta cttttcttta ttttgagaca gcacctccct ctgtagtgct 1980

ggctggcctg gaagtcacag aaatcctcct gcctctgctt ccagagtact gggtcttaaa 2040

ctgttcacca cgtctcccct ccccaacccc caaactagcc tttccatttt taaaagccag 2100

ctaaaaatat taaagttgtg ctcttacaaa agtc 2134

<210> 52

<211> 256

<212> PRT

<213> Mouse (Mouse)

<400> 52

Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

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

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 53

<211> 6001

<212> DNA/RNA

<213> human (human)

<400> 53

caaggaggga tcccacagat gtcacagggc tgtcacagag ctgtggtggg aatttcccat 60

gagaccccgc ccctggctga gtcaccgcac tcctgtgttt gacctgaagt cctctcgagc 120

tgcagaagcc tgaagaccaa ggagtggaaa gttctccggc agccctgaga tctcaagagt 180

gacatttgtg agaccagcta atttgattaa aattctcttg gaatcagctt tgctagtatc 240

atacctgtgc cagatttcat catgggaaac agctgttaca acatagtagc cactctgttg 300

ctggtcctca actttgagag gacaagatca ttgcaggatc cttgtagtaa ctgcccagct 360

ggtacattct gtgataataa caggaatcag atttgcagtc cctgtcctcc aaatagtttc 420

tccagcgcag gtggacaaag gacctgtgac atatgcaggc agtgtaaagg tgttttcagg 480

accaggaagg agtgttcctc caccagcaat gcagagtgtg actgcactcc agggtttcac 540

tgcctggggg caggatgcag catgtgtgaa caggattgta aacaaggtca agaactgaca 600

aaaaaaggtt gtaaagactg ttgctttggg acatttaacg atcagaaacg tggcatctgt 660

cgaccctgga caaactgttc tttggatgga aagtctgtgc ttgtgaatgg gacgaaggag 720

agggacgtgg tctgtggacc atctccagcc gacctctctc cgggagcatc ctctgtgacc 780

ccgcctgccc ctgcgagaga gccaggacac tctccgcaga tcatctcctt ctttcttgcg 840

ctgacgtcga ctgcgttgct cttcctgctg ttcttcctca cgctccgttt ctctgttgtt 900

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 960

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1020

gaactgtgaa atggaagtca atagggctgt tgggactttc ttgaaaagaa gcaaggaaat 1080

atgagtcatc cgctatcaca gctttcaaaa gcaagaacac catcctacat aatacccagg 1140

attcccccaa cacacgttct tttctaaatg ccaatgagtt ggcctttaaa aatgcaccac 1200

tttttttttt tttttgacag ggtctcactc tgtcacccag gctggagtgc agtggcacca 1260

ccatggctct ctgcagcctt gacctctggg agctcaagtg atcctcctgc ctcagtctcc 1320

tgagtagctg gaactacaag gaagggccac cacacctgac taactttttt gttttttgtt 1380

tggtaaagat ggcatttcac catgttgtac aggctggtct caaactccta ggttcacttt 1440

ggcctcccaa agtgctggga ttacagacat gaactgccag gcccggccaa aataatgcac 1500

cacttttaac agaacagaca gatgaggaca gagctggtga taaaaaaaaa aaaaaaaaag 1560

cattttctag ataccactta acaggtttga gctagttttt ttgaaatcca aagaaaatta 1620

tagtttaaat tcaattacat agtccagtgg tccaactata attataatca aaatcaatgc 1680

aggtttgttt tttggtgcta atatgacata tgacaataag ccacgaggtg cagtaagtac 1740

ccgactaaag tttccgtggg ttctgtcatg taacacgaca tgctccaccg tcagggggga 1800

gtatgagcag agtgcctgag tttagggtca aggacaaaaa acctcaggcc tggaggaagt 1860

tttggaaaga gttcaagtgt ctgtatatcc tatggtcttc tccatcctca caccttctgc 1920

ctttgtcctg ctccctttta agccaggtta cattctaaaa attcttaact tttaacataa 1980

tattttatac caaagccaat aaatgaactg catatgatag gtatgaagta cagtgagaaa 2040

attaacacct gtgagctcat tgtcctacca cagcactaga gtgggggccg ccaaactccc 2100

atggccaaac ctggtgcacc atttgccttt gtttgtctgt tggtttgctt gagacagtct 2160

tgctctgttg cccaggctgg aatggagtgg ctattcacag gcacaatcat agcacacttt 2220

agccttaaac tcctgggctc aagtgatcca cccgcctcag tctcccaagt agctgggatt 2280

acaggtgcaa acctggcatg cctgccattg tttggcttat gatctaagga tagcttttta 2340

aattttattc attttatttt tttttgagac agtgtctcac tctgtctccc aggctggagt 2400

acagtggtac aatcttggat caccgcctcc cagtttcaag tgatctccct gcctcagcct 2460

cctaagtagc tgggactaca ggtatgtgcc accacgcctg gctaattttt atatttttag 2520

tagagacggg gtttcaccat gttgtccagg ctggtctcaa actcctgacc tcaggtgatc 2580

tgcccacctc tgcctcccaa agtgctggga ttacaggcat gagccaccat gcctggccat 2640

ttcttacact tttgtatgac atgcctattg caagcttgcg tgcctctgtc ccatgttatt 2700

ttactctggg atttaggtgg agggagcagc ttctatttgg aacattggcc atcgcatggc 2760

aaatgggtat ctgtcacttc tgctcctatt tagttggttc tactataacc tttagagcaa 2820

atcctgcagc caagccaggc atcaataggg cagaaaagta tattctgtaa ataggggtga 2880

ggagaagata tttctgaaca atagtctact gcagtaccaa attgcttttc aaagtggctg 2940

ttctaatgta ctcccgtcag tcatataagt gtcatgtaag tatcccattg atccacatcc 3000

ttgctaccct ctggtactat caggtgccct taattttgcc aagccagtgg gtatagaatg 3060

agatctcact gtggtcttag tttgcatttg cttggttact gatgagcacc ttgtcaaata 3120

tttatatacc atttgtgttt atttttttaa ataaaatgct tgctcatgct tttttgccca 3180

tttgcaaaaa aacttggggc cgggtgcagt ggctcatgcc tgtagtccca gctctttggg 3240

aggccaaggt gggcagatcg cttgagccca ggagttcgag accagccttg gcaacatggc 3300

gaaaccctgt ctttacaaaa aatacaaaaa ttagccgggt gtggtggtgt gcacctgaag 3360

tcccagctac tcagtaggtt cgctttgagc ctgggaggca gaggttgcag tgagctggga 3420

ccgcatcact acacttcagc ctgggcaaca gagaaaaacc ttttctcaga aacaaacaaa 3480

cccaaatgtg gttgtttgtc ctgattccta aaaggtcttt atgtattcta gataataatc 3540

tttggtcagt tatatgtgtt aaaaaatatc ttctttgtgg ccaggcacgg tagctcacac 3600

ctgtaatccc agcactttgc ggggctgagg tgggtggatc atctgaggtc aagagttcaa 3660

gatcagcctg gccaacacag tgaaacccca tctctactaa acatgtacaa aacttagctg 3720

ggtatggtgg cgggtgcctg taaccccagc tgctccagag gctgtggcag aagaatcgct 3780

tgaacccagg aggcagaggt tgcagcgagc caagattgtg ccattgcact ccagactggg 3840

tgacaagagt gaaattctgc ctatctatct atctatctat ctatatctat atatatatat 3900

atatatatcc tttgtaattt atttttccct ttttaaaatt ttttataaaa ttctttttta 3960

tttttatttt tagcagaggt gaggtttctg aggtttcatt atgttgccca ggctggtctt 4020

gaactcctga gctcaagtga tcctcccacc tcagccttcc aaagtgctgg aattgcagac 4080

atgagccacc gcgcccctcc tgtttttctc taattaatgg tgtctttctt tgtctttctg 4140

gtaataagca aaaagttctt catttgattt ggttaaattt ataactgttt tctcatatgg 4200

ttaacatttt ttcttgcctg gctaaagaaa tccttttctg cccaatacta taaagaggtt 4260

tgcccacatt ttattccaaa agttttaagt tttgtctttc atcttgaagt ctaatgtatc 4320

aggaactggc ttttgtgcct gttgggaggt agtgatccaa ttccatgtct tgcatgtagg 4380

taaccactgg tccctgcgcc atgtattcaa tacgtcgtct ttctcctgcg ggtctgcaat 4440

ctcacctacc atccatcaag tttccatagg gccatgggtc tgcttctggg ctccctgttc 4500

tgttccattg tcaatttgtc tatcctgtgc cagtatcaca ctgtgtttat tacaatagct 4560

ttgtaacagc tctcgatatc cggtaggaca tctccctcca ccttcttttt ctacttcaga 4620

agtgtcttag ctaggtcagg cacggtggct cacgcctgta atcccagcac tttgggaggc 4680

cgacgcggat ggatcacctg aggtcaggag ttttgagaca gcctggccaa catggtgaaa 4740

ccccatctct actaaaaaat acaaaaatta gtcaggcatg gtggcatgtg cctgtaatcc 4800

cagctatttg ggaggctgag gccggagaat tgcttgaacc cggggggcgg aggttgcagt 4860

gagccgagat cgtaccattg cactccagcc tgggtgacag agcgaaactc tgtctcagga 4920

aaaaaaagaa aagagatgtc ttggttattc ttggttcttt attattcaat ataaatttta 4980

gaagctgaat ttgaaaagat ttggattgga atttcattaa atctacaggt caatttaggg 5040

agagttgata attttacaga attgagtcat ctggtgttcc aataagaata agagaacaat 5100

tattggctgt acaattcttg ccaaatagta ggcaaagcaa agcttaggaa gtatactggt 5160

gccatttcag gaacaaagct aggtgcgaat atttttgtct ttctgaatca tgatgctgta 5220

agttctaaag tgatttctcc tcttggcttt ggacacatgg tgtttaatta cctactgctg 5280

actatccaca aacagaaaga gactggtcat gccccacagg gttggggtat ccaagataat 5340

ggagcgaggc tctcatgtgt cctaggttac acaccgaaaa tccacagttt attctgtgaa 5400

gaaaggaggc tatgtttatg atacagactg tgatattttt atcatagcct attctggtat 5460

catgtgcaaa agctataaat gaaaaacaca ggaacttggc atgtgagtca ttgctccccc 5520

taaatgacaa ttaataagga aggaacattg agacagaata aaatgatccc cttctgggtt 5580

taatttagaa agttccataa ttaggtttaa tagaaataaa tgtaaatttc tatgattaaa 5640

aataaattag cacatttagg gatacacaaa ttataaatca ttttctaaat gctaaaaaca 5700

agctcaggtt tttttcagaa gaaagtttta attttttttc tttagtggaa gatatcactc 5760

tgacggaaag ttttgatgtg aggggcggat gactataaag tgggcatctt cccccacagg 5820

aagatgtttc catctgtggg tgagaggtgc ccaccgcagc tagggcaggt tacatgtgcc 5880

ctgtgtgtgg taggacttgg agagtgatct ttatcaacgt ttttatttaa aagactatct 5940

aataaaacac aaaactatga tgttcacagg aaaaaaagaa taagaaaaaa agaaaaaaaa 6000

a 6001

<210> 54

<211> 255

<212> PRT

<213> human (human)

<400> 54

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 55

<211> 2137

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

ttcctgaaat tcaggtgctg caggcagccc tcagcacaga gagctgacag ggaccctggg 60

tcaggggttc tgagttccag ctgccactat tcttcttcac ctttggtgtc ctgtgcatgt 120

gacatttcgc catgggaaac agctgttaca acatagtagc cactctgttg ctggtcctca 180

actttgagag gacaagatca ttgcaggatc cttgtagtaa ctgcccagct ggtacattct 240

gtgataataa caggaatcag atttgcagtc cctgtcctcc aaatagtttc tccagcgcag 300

gtggacaaag gacctgtgac atatgcaggc agtgtaaagg tgttttcagg accaggaagg 360

agtgttcctc caccagcaat gcagagtgtg actgcactcc agggtttcac tgcctggggg 420

caggatgcag catgtgtgaa caggattgta aacaaggtca agaactgaca aaaaaaggtt 480

gtaaagactg ttgctttggg acatttaacg atcagaaacg tggcatctgt cgaccctgga 540

caaactgttc tttggatgga aagtctgtgc ttgtgaatgg gacgaaggag agggacgtgg 600

tctgtggacc atctccagcc gacctctctc cgggagcatc ctctgtgacc ccgcctgccc 660

ctgcgagaga gccaggacac tctttgcagg tccttacctt gttcctggcg ctgacatcgg 720

ctttgctgct ggccctgatc ttcattactc tcctgttctc tgtgctcaaa tggatcagga 780

aaaaattccc ccacatattc aagcaaccat ttaagaagac cactggagca gctcaagagg 840

aagatgcttg tagctgccga tgtccacagg aagaagaagg aggaggagga ggctatgagc 900

tgtgatgtac tatcctagga gatgtgtggg ccgaaaccga gaagcactag gaccccacca 960

tcctgtggaa cagcacaagc aaccccacca ccctgttctt acacatcatc ctagatgatg 1020

tgtgggcgcg cacctcatcc aagtctcttc taacgctaac atatttgtct ttaccttttt 1080

taaatctttt tttaaattta aattttatgt gtgtgagtgt tttgcctgcc tgtatgcaca 1140

cgtgtgtgtg tgtgtgtgtg tgacactcct gatgcctgag gaggtcagaa gagaaagggt 1200

tggttccata agaactggag ttatggatgg ctgtgagcct ttgtgtgggt gctaggaatc 1260

aaacctgggt cctctacaag ggcagccagt gctcttaacc actgagtcag ctttccagcc 1320

ctgccctgga cagtttttaa aatttaactt aatttttttt tttttttact taagccctta 1380

acatttttaa taggactgtg ggaagatcaa tttctagatt ctccttaaca atatacatca 1440

tatacatata cacatacata tacatataca tatatattct gagaaaatga cagtttcagt 1500

tggatctcat agaccaatgg tccagttaaa ataactgtaa aatcagtgtg tgtgtgtgtg 1560

tgtgtgtgtg tgtgcaaata tgatgcatga caatagccat aagatgcagt atattaccct 1620

atcccatttt cctttggttt ctcactcact ataataacac ctccactgtc tgaaggggga 1680

gacccatgca tccctgtctg gaggaagcct gacagatttt gaggggaatc ttcagagcag 1740

ttcaagggcc tgcttctcct gtttcctctg tgtcaggctt ttcaataaaa aggccgttta 1800

ggaaagggac aaagcactgt gaggtgggga acacctgtga actcacagta ggaacgcggc 1860

cttccagtcc accatgggca gacatggctg ccgcttgcct ctgcatgact cctggacagc 1920

tcaagagctg agaatggttg ttacttttct ttattttgag acagcacctc cctctgtagt 1980

gctggctggc ctggaagtca cagaaatcct cctgcctctg cttccagagt actgggtctt 2040

aaactgttca ccacgtctcc cctccccaac ccccaaacta gcctttccat ttttaaaagc 2100

cagctaaaaa tattaaagtt gtgctcttac aaaagtc 2137

<210> 56

<211> 257

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

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

180 185 190

Ala Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu

195 200 205

Phe Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys

210 215 220

Gln Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys

225 230 235 240

Ser Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu

245 250 255

Leu

<210> 57

<211> 552

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

atgggaaaca gctgttacaa catagtagcc actctgttgc tggtcctcaa ctttgagagg 60

acaagatcat tgcaggatcc ttgtagtaac tgcccagctg gtacattctg tgataataac 120

aggaatcaga tttgcagtcc ctgtcctcca aatagtttct ccagcgcagg tggacaaagg 180

acctgtgaca tatgcaggca gtgtaaaggt gttttcagga ccaggaagga gtgttcctcc 240

accagcaatg cagagtgtga ctgcactcca gggtttcact gcctgggggc aggatgcagc 300

atgtgtgaac aggattgtaa acaaggtcaa gaactgacaa aaaaaggttg taaagactgt 360

tgctttggga catttaacga tcagaaacgt ggcatctgtc gaccctggac aaactgttct 420

ttggatggaa agtctgtgct tgtgaatggg acgaaggaga gggacgtggt ctgtggacca 480

tctccagccg acctctctcc gggagcatcc tctgtgaccc cgcctgcccc tgcgagagag 540

ccaggacact ct 552

Claims

1. A humanized TNFSF9 protein, wherein said humanized TNFSF9 protein comprises all or a portion of a human TNFSF9 protein; preferably, the humanized TNFSF9 protein comprises all or part of the transmembrane, extracellular and/or cytoplasmic domain of human TNFSF9 protein; preferably, the humanized TNFSF9 protein comprises a transmembrane region and/or an extracellular region of a human TNFSF9 protein; preferably, said humanized TNFSF9 protein further comprises a portion of the cytoplasmic region of human TNFSF9 protein; preferably, the portion of the cytoplasmic domain of the human TNFSF9 protein comprises SEQ ID NO: 4, 26 th to 28 th amino acid sequence; preferably, said humanized TNFSF9 protein further comprises a portion of a non-human animal TNFSF9 protein, preferably a cytoplasmic region of a non-human animal TNFSF9 protein; preferably, said humanized TNFSF9 protein comprises all or part of the amino acid sequence encoded by exon 1 to exon 3 of the human TNFSF9 gene; preferably, the humanized TNFSF9 protein comprises all or part of an amino acid sequence encoded by one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of the human TNFSF9 gene; preferably, the humanized TNFSF9 protein comprises a portion of exon 1, an amino acid sequence encoded by exons 2 to 3 of the human TNFSF9 gene, wherein the portion of exon 1 preferably comprises a nucleotide sequence encoding a transmembrane region and/or an extracellular region, and further preferably further comprises a nucleotide sequence encoding SEQ ID NO: 4, nucleotide sequence 26-28; preferably, said humanized TNFSF9 protein further comprises a portion of the amino acid sequence encoded by exon 1 of a non-human animal TNFSF9 gene, preferably said portion of the amino acid sequence encoded by exon 1 of a non-human animal TNFSF9 gene that hybridizes to SEQ ID NO: 2 or at least 90% identical to the amino acid sequence at positions 1-82 of SEQ ID NO: 2, 1-82 amino acid sequence is consistent; preferably, the non-human animal is a non-human mammal; preferably, the non-human mammal is a rodent; further preferably, the rodent is a mouse or a rat; preferably, the partial amino acid sequence of the human TNFSF9 protein contained in the humanized TNFSF9 protein comprises one of the following groups:

D) And SEQ ID NO: 4, amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues, as shown at positions 26-254;

preferably, the amino acid sequence of the humanized TNFSF9 protein is selected from one of the following groups:

a) is SEQ ID NO: 12 amino acid sequence, in whole or in part;

d) And SEQ ID NO: 12, comprising substitution, deletion and/or insertion of one or more amino acid residues;

2. A humanized TNFSF9 gene, wherein said humanized TNFSF9 gene comprises at least a portion of a human TNFSF9 gene; preferably, said humanized TNFSF9 gene comprises all or part of a nucleotide sequence encoding a transmembrane, cytoplasmic, and/or extracellular region of human TNFSF9 protein; preferably, said humanized TNFSF9 gene comprises all or part of a nucleotide sequence encoding a transmembrane region and/or an extracellular region of human TNFSF9 protein; preferably, said humanized TNFSF9 gene further comprises a partial nucleotide sequence encoding a cytoplasmic region of human TNFSF9 protein; preferably, the humanized TNFSF9 gene further comprises a portion of a non-human animal TNFSF9 gene, preferably all or a portion of the nucleotide sequence of the cytoplasmic region encoding a non-human animal TNFSF9 protein, and more preferably a nucleotide sequence encoding SEQ ID NO: 2 or at least 90% identical to the amino acid sequence at positions 1-82 of SEQ ID NO: 2, 1-82 amino acid sequence is consistent; preferably, said humanized TNFSF9 gene encodes a humanized TNFSF9 protein according to any one of claims 1-10; preferably, said humanized TNFSF9 gene comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene; preferably, the humanized TNFSF9 gene comprises all or part of the nucleotide sequence of a combination of one, two, three or two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene, and further preferably, the humanized TNFSF9 gene comprises part of exon 1, all of exon 2 and part of the nucleotide sequence of exon 3 of human TNFSF9 gene, preferably further comprises intron 1-2 and/or intron 2-3; preferably, the humanized TNFSF9 gene comprises a partial nucleotide sequence of a human TNFSF9 gene comprising one of the following groups:

(D) Has the sequence shown in SEQ ID NO: 7, including substitution, deletion and/or insertion of one or more nucleotides;

preferably, said humanized TNFSF9 gene further comprises a portion of exon 1 and a portion of exon 3 of a non-human animal TNFSF9 gene; preferably, the part of the exon 1 is a nucleotide sequence coding for a cytoplasmic region of a non-human animal TNFSF9 gene, and the part of the exon 3 is a non-coding region of an exon 3 of a non-human animal TNFSF9 gene; preferably, the non-human animal is a non-human mammal; preferably, the non-human mammal is a rodent, and more preferably, the rodent is a mouse or a rat; preferably, the nucleotide sequence of the humanized TNFSF9 gene comprises a nucleotide sequence identical to SEQ ID NO: 8, or a nucleotide sequence comprising at least 60%, 70%, 80%, 90%, or at least 95% identity to SEQ ID NO: 8; preferably, the mRNA transcribed by the nucleotide sequence of the humanized TNFSF9 gene is selected from one of the following groups:

(d) And SEQ ID NO: 11, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted; preferably, the humanized TNFSF9 gene encodes a protein capable of recognizing and/or binding at least a portion of human 4-1BB protein, further wherein the human 4-1BB protein is encoded by a nucleotide sequence comprising one, two, three, two or more consecutive exons from exon 1 to exon 9 of the human 4-1BB gene; preferably, the human 4-1BB protein is encoded by a nucleotide sequence comprising all or part of exon 3 to exon 8 of human 4-1BB gene; preferably, the human 4-1BB protein consists of a nucleotide sequence comprising a signal peptide encoded by exon 3 and an extracellular domain of the human 4-1BB gene, all of exons 4 to 7, and an amino acid sequence encoded by exon 8 of SEQ ID NO: 54 at position 182-184.

3. A method of constructing a humanized non-human animal, wherein said non-human animal expresses human or humanized TNFSF9 protein; preferably, the constructing method comprises introducing a partial nucleotide sequence comprising the human TNFSF9 gene into the non-human animal TNFSF9 locus; preferably, the nucleotide sequence comprising all or part of exons 1 to 3 of the human TNFSF9 gene is introduced into the TNFSF9 locus of a non-human animal, more preferably, all or part of the nucleotide sequence comprising a combination of one, two, three or two consecutive exons 1 to 3 of the human TNFSF9 gene is introduced into the TNFSF9 locus of a non-human animal, even more preferably, part of the nucleotide sequence comprising part of exon 1, all of exon 2 and part of exon 3 of the human TNFSF9 gene is substituted into the TNFSF9 locus of a non-human animal, even more preferably, part of the nucleotide sequence comprising exon 1 of the human TNFSF9 gene, (ii) a partial nucleotide sequence of intron No. 1-2, all of exon No. 2, intron No. 2-3 and exon No. 3 is substituted at the non-human animal TNFSF9 locus to form a humanized TNFSF9 gene; optionally, the introduction is insertion or substitution; preferably, the method of construction comprises replacing at the non-human animal TNFSF9 locus with a nucleotide sequence comprising all or part of a transmembrane, cytoplasmic, and/or extracellular region encoding a human TNFSF9 protein; preferably, the nucleotide sequence comprising a transmembrane region and/or an extracellular region encoding human TNFSF9 protein is substituted at the non-human animal TNFSF9 locus, and more preferably, the nucleotide sequence comprises SEQ ID NO: 4 following replacement of an endogenous regulatory element at the TNFSF9 locus, optionally at least a portion of exon 1 through exon 3 of said TNFSF9 locus, preferably a transmembrane region and extracellular region of exon 1 through exon 3 of said TNFSF9 locus; preferably, the construction method uses a targeting vector to construct the non-human animal, wherein the targeting vector comprises a donor DNA sequence, and the donor DNA sequence comprises a part of the human TNFSF9 gene; preferably, the donor DNA sequence comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene; further preferably, the donor DNA sequence comprises all or part of a nucleotide sequence of one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of human TNFSF9 gene; preferably, the donor DNA sequence comprises a partial nucleotide sequence of exon 1, all of exon 2, and a partial nucleotide sequence of exon 3 of human TNFSF9 gene; preferably, the donor DNA sequence further comprises the nucleotide sequence of intron 1-2, intron 2-3 of the human TNFSF9 gene; preferably, the targeting vector further comprises a 5' arm selected from the group consisting of nucleotides of 100-10000 in length of genomic DNA of the TNFSF9 gene of a non-human animal; preferably, said 5' arm has at least 90% homology to NCBI accession No. NC _ 000083.6; further preferably, the 5' arm sequence is identical to SEQ ID NO: 5 or SEQ ID NO: 50 or as shown in SEQ ID NO: 5 or SEQ ID NO: 50 is shown; and/or, the targeting vector further comprises a 3' arm selected from 100-10000 nucleotides in length of the genomic DNA of the TNFSF9 gene of the non-human animal; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000083.6; preferably, the 3' arm sequence is identical to SEQ ID NO: 6 or SEQ ID NO: 13 or as shown in SEQ ID NO: 6 or SEQ ID NO: 13 is shown in the figure; and/or, the targeting vector further comprises a non-human animal 3' UTR, preferably, further comprises 4-1BB, PD-1, PD-L1 or OX40 gene humanization; preferably, said humanization of the 4-1BB gene comprises expression of a human or humanized 4-1BB protein in said non-human animal body; preferably, the humanized 4-1BB protein comprises all or part of a human 4-1BB protein, further preferably all or part of an extracellular region of a human 4-1BB protein, further preferably a signal peptide, and even more preferably a sequence identical to SEQ ID NO: 54, 1-184 or an amino acid sequence having 90% homology to SEQ ID NO: 54, 1 st to 184 th amino acid sequence; preferably, said gene editing of a non-human animal humanized with a 41BB gene comprises replacing a part of the nucleotide sequence of the human 4-1BB gene at the non-human animal 4-1BB locus, further preferably, replacing the nucleotide sequence of an exon comprising a combination of one, two, three, two or more consecutive exons 1 to 9 of the human 4-1BB gene at the non-human animal 4-1BB locus, further preferably, replacing all or a part of the nucleotide sequence of an exon comprising a combination of 3 to 8 of the human 4-1BB gene at the non-human animal 4-1BB locus, more preferably, coding the signal peptide and the nucleotide sequence of the extracellular region by using exon 3 containing human 4-1BB gene, all of exons 4-7 and exon 8 coding SEQ ID NO: 54 at position 182-184, to the non-human animal 4-1BB locus.

4. A targeting vector, wherein said targeting vector comprises a donor DNA sequence, said donor DNA sequence comprising a portion of the human TNFSF9 gene; preferably, the donor DNA sequence comprises all or part of the nucleotide sequence of exons 1 to 3 of human TNFSF9 gene; preferably, the donor DNA sequence comprises all or part of a nucleotide sequence of one, two, three or a combination of two consecutive exons from exon 1 to exon 3 of the human TNFSF9 gene; preferably, the donor DNA sequence comprises a partial nucleotide sequence of exon 1, all of exon 2, and a partial nucleotide sequence of exon 3 of human TNFSF9 gene; preferably, the donor DNA sequence further comprises nucleotide sequences of intron 1-2 and intron 2-3 of the human TNFSF9 gene; preferably, the targeting vector further comprises a 5' arm selected from the group consisting of nucleotides of 100-10000 in length of genomic DNA of the TNFSF9 gene of a non-human animal; preferably, said 5 'arm has at least 90% homology to NCBI accession No. NC _000083.6, and further preferably, said 5' arm sequence has at least 90% homology to SEQ ID NO: 5 or SEQ ID NO: 50 or as shown in SEQ ID NO: 5 or SEQ ID NO: 50 is shown; and/or, the targeting vector further comprises a 3' arm selected from 100-10000 nucleotides in length of the genomic DNA of the TNFSF9 gene of the non-human animal; preferably, said 3 'arm has at least 90% homology to NCBI accession No. NC _000083.6, and further preferably, said 3' arm sequence is identical to SEQ ID NO: 6 or SEQ ID NO: 13 or as shown in SEQ ID NO: 6 or SEQ ID NO: 13 is shown in the figure; and/or, the targeting vector further comprises a non-human animal 3' UTR; preferably, the non-human animal is a non-human mammal; preferably, the non-human mammal is a rodent; further preferably, the rodent is a mouse or a rat; preferably, the targeting vector further comprises a marker gene.

5. A cell comprising the targeting vector of claim 4.

6. The targeting vector of claim 4, or the use of the cell of claim 5 for the modification of the TNFSF9 gene.

7. A cell or cell line or primary cell culture derived from the non-human animal obtained by the construction method of claim 3.

8. A tissue or organ or culture thereof, wherein the tissue or organ or culture thereof is derived from the non-human animal obtained by the construction method according to claim 3.

9. A tumor tissue obtained by the method according to claim 3, wherein the tumor tissue is derived from a non-human animal.

10. A cell humanized with TNFSF9 gene, said cell expressing human or humanized TNFSF9 protein; preferably, the humanized TNFSF9 protein is the humanized TNFSF9 protein of claim 1; preferably, the genome of said cell comprises all or part of the human TNFSF9 gene; preferably, the cell comprises the humanized TNFSF9 gene of claim 2.

11. A TNFSF9 gene knock-out cell, wherein the cell lacks all or part of the nucleotide sequence of the TNFSF9 gene; preferably, the cell lacks all or part of exons 1 to 3 of the TNFSF9 gene, further preferably lacks all or part of a nucleotide sequence of a combination of one, two, three or two consecutive exons from exons 1 to 3, further preferably lacks all or part of an exon 1, all of an exon 2 and part of an exon 3, and further preferably lacks part of an exon 1, all of an intron 1 to 2, all of an exon 2, part of a nucleotide sequence of an intron 2 to 3 and part of an exon 3.

12. A construct expressing the humanized TNFSF9 protein of claim 1 or comprising the humanized TNFSF9 gene of claim 2.

13. A cell comprising the construct of claim 12.

14. A tissue comprising the cell of claim 13.

15. Use of a cell or cell line or primary cell culture derived from the humanized TNFSF9 protein of claim 1, the humanized TNFSF9 gene of claim 2, the non-human animal obtained by the construction method of claim 3, the cell or cell line or primary cell culture of claim 6, the tissue or organ or culture thereof of claim 7, the tumor-bearing tumor tissue of claim 8, the cell of any one of claims 10 or 11, the construct of claim 12, the cell of claim 13, or the tissue of claim 14 in product development requiring an immune process involving human cells, in the manufacture of antibodies, or as a model system for pharmacological, immunological, microbiological, medical research; or in the production and use of animal experimental disease models for the development of new diagnostic and/or therapeutic strategies; or screening, verifying, evaluating or researching TNFSF9 function, TNFSF9 signal mechanism, human-targeting antibody, human-targeting drug, drug effect, immune-related disease drug and anti-tumor or anti-inflammatory drug, screening and evaluating human drug and drug effect research.

16. A method of screening for a modulator specific for human TNFSF9, said method comprising administering the modulator to an individual implanted with tumor cells and detecting tumor suppression; wherein the individual is selected from the group consisting of the non-human animals obtained by the construction method according to claim 3; preferably, the modulator is selected from CAR-T, a drug; preferably, the drug is an antibody.