CN113603765A

CN113603765A - IL17F gene humanized non-human animal and construction method and application thereof

Info

Publication number: CN113603765A
Application number: CN202110803051.8A
Authority: CN
Inventors: 沈月雷; 张美玲; 黄蕤; 白阳; 郭朝设; 赵磊
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd; Beijing Biocytogen Co Ltd
Priority date: 2020-07-15
Filing date: 2021-07-15
Publication date: 2021-11-05
Also published as: WO2022012636A1

Abstract

The invention provides a non-human animal humanized by an IL17F gene and a construction method and application thereof. The invention also provides a construction method of the IL17A and IL17F gene modified non-human animal or multi-gene modified non-human animal, and application of the prepared IL17A gene and/or IL17F gene humanized non-human animal or multi-gene modified non-human animal in screening and drug effect evaluation of the targeted human IL17 signal path antibody.

Description

IL17F 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 non-human animal humanized by IL17A and/or IL17F genes, a construction method thereof and application thereof in the field of biomedicine.

Background

The Interleukin (IL) 17 cytokine (abbreviated as IL17) family is a characteristic cytokine, which is secreted mainly by activated Th17 cells. The IL17 family has now found 6 members, IL17A, IL17B, IL17C, IL17D, IL17E (also known as IL25) and IL 17F. The IL17 receptor (abbreviated as IL17R) also forms a unique family, and 5 homologous subunits are found at present, namely IL17RA, IL17RB, IL17RC, IL17RD and IL17 RE. IL17 stimulates various cells to produce inflammatory mediators by binding to receptors, initiating their downstream signaling pathways (including the MAP kinase pathway, NF-kB pathway, mRNA stabilization signaling pathway, ERK signaling pathway, and JAK/STAT signaling pathway, etc.), has become a key contributor to immune and inflammatory diseases, and can lead to organ-specific or systemic autoimmune diseases such as psoriasis, systemic lupus erythematosus, ulcerative colitis, ankylosing spondylitis, etc.

ILl7A and ILl7F have strong homology and have similar regulation, signal path and function as main effector factors secreted by Th17 cells. Activated Th17 cells can secrete homodimers of IL17A and IL17F, express heterodimers consisting of IL17A and IL17F (IL17A/IL17F), and the heterodimers are combined with heterodimer receptor complexes consisting of IL17RC and IL17RA of IL17 receptor family, so that downstream cell signal transduction pathways are started, and a series of proinflammatory cytokines, chemochemokines, Matrix Metalloproteinases (MMPs) and the like are induced to promote tissue inflammation injury. Three currently approved targeted human IL-17/IL-17R-related antibodies by the FDA, including IL-17A monoclonal antibodies Secukinumab, Ixekizumab and IL-17RA monoclonal antibody Brodalumab, are mainly indicated for moderate-severe plaque psoriasis. However, these drugs have significant side effects such as infection, diarrhea, etc., and Brodalumab is associated with a patient's suicidal tendency. Considering that most of the existing medicines for treating autoimmune diseases can only improve the disease symptoms and can not fully meet the clinical requirements, more medicines targeting IL-17/IL-17R still need to be developed.

Because the amino acid sequence of human IL17 has significant differences from the corresponding protein in rodents, for example, the sequence identity between human IL17A and mouse IL17A protein is only 60%, and the sequence identity between human IL17F and mouse IL17F protein is only 56%, therefore, antibodies recognizing human IL17A protein and IL17F protein generally cannot recognize mouse IL17A and IL17F, i.e., ordinary mice cannot be used to screen and evaluate the efficacy of drugs targeting IL17/IL17R signaling pathway.

In view of the broad involvement of IL17/IL17R in the disease development process and the great utility value of targeting this signaling pathway, there remains an urgent need in the art to develop non-human animal models related to the humanized IL17/IL17R signaling pathway in order to make preclinical trials more effective and minimize development failures. Therefore, the application provides an experimental animal disease model which is used for researching the etiology and pathogenesis of human diseases and developing prevention and treatment technologies and medicines.

Disclosure of Invention

In a first aspect of the invention there is provided a humanized IL17F protein, said humanized IL17F protein comprising all or part of a human IL17F protein. Preferably, no IL17F protein signal peptide is included.

Preferably, the humanized IL17F protein further comprises a portion of a non-human animal IL17F protein, preferably a signal peptide of a non-human animal IL17F protein.

Preferably, the humanized IL17F protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17F gene. Further preferred is a polypeptide comprising the amino acid sequence encoded in whole or in part by exons 2 to 3. Further preferably, it does not contain an amino acid sequence encoding a signal peptide. Still further preferred, the polypeptide comprising SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

Preferably, the humanized IL17F protein further comprises an amino acid sequence encoded by all or part of exons 1 to 2 of a non-human animal IL17F gene. Further preferred is an amino acid sequence encoded by all of exon 1 and part of exon 2 of the non-human animal IL17F gene. Wherein the portion of exon 2 comprises a nucleotide sequence encoding a signal peptide.

In one embodiment of the present invention, the amino acid sequence of the humanized IL17F protein comprises one of the following groups:

A) SEQ ID NO: 8, all or part of the amino acid sequence from position 31 to 163;

B) and SEQ ID NO: 8, 31-163 amino acid sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 8, positions 31-163, differ 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: 8, positions 31-163, including substitution, deletion and/or insertion of one or more amino acid residues.

In one embodiment of the invention, the humanized IL17F protein comprises a sequence identical to SEQ ID NO: 7, or an amino acid sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 7, 1-28.

I) SEQ ID NO: 16 amino acid sequence, in whole or in part;

II) and SEQ ID NO: 16 amino acid sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

III) and SEQ ID NO: 16 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or

IV) and SEQ ID NO: 16, comprising substitution, deletion and/or insertion of one or more amino acid residues.

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

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

In one embodiment of the present invention, the non-human animal is a non-human animal humanized with IL17A gene.

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, said immunodeficiency virus is administered to a mammalThe dental animal is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc^scid IL-2rγ^nullMouse, NOD-Rag 1^-/--IL2rg^-/-(NRG) mice, Rag 2^-/--IL2rg^-/-(RG) mice, NOD/SCID mice or nude mice.

In a second aspect of the invention, there is provided a humanized IL17F gene, said humanized IL17F gene comprising a portion of the human IL17F gene.

Preferably, the humanized IL17F gene comprises all or part of exons 1 to 3 of human IL17F gene. Further preferably, it comprises all or part of exon 2 to exon 3 of the human IL17F gene. Even more preferably, the part of exon 2 and part of exon 3, and preferably all of introns 2-3 are included, wherein the part of exon 2 at least comprises a nucleotide sequence of 20bp, for example, at least comprises a nucleotide sequence of 20, 50, 70, 100, 150, 160, 161, 162, 163, 164, 165, 170, 200, 220, 221bp, and even more preferably, comprises a nucleotide sequence of 164 bp; the portion of exon 2 preferably comprises a sequence encoding SEQ ID NO: 8, 31-85, and a part of exon 3 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 100, 150, 200, 220, 230, 231, 232, 233, 234, 235, 238, 250, 270, 300, 350, 400, 450, 480, 488bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon.

Preferably, the humanized IL17F gene comprises a nucleotide sequence encoding human IL17F protein. Further preferably, the polypeptide does not contain a nucleotide sequence encoding a signal peptide of human IL17F protein. Even more preferably comprises a nucleotide sequence encoding SEQ ID NO: 8 from 31 to 163.

Preferably, the humanized IL17F gene comprises a cDNA encoding human IL17F protein.

Preferably, the humanized IL17F gene further comprises a non-coding region for human IL 17F.

Preferably, the humanized IL17F gene further comprises a portion of a non-human animal IL17F gene. Further preferably, the non-human animal IL17F gene comprises all or part of exons 1 to 2. Still further preferably comprises all of exon 1 and/or a portion of exon 2 of the non-human animal IL17F gene, preferably further comprises all of introns 1-2, wherein the portion of exon 2 preferably comprises a nucleotide sequence encoding a signal peptide.

In one embodiment of the present invention, the humanized IL17F gene comprises a nucleotide sequence of human IL17F gene comprising one of the following group:

(A) 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%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

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

In one embodiment of the present invention, the humanized IL17F gene comprises mRNA transcribed from the human IL17F gene comprising one of the following group:

(i) SEQ ID NO: 55, or a portion or all of a nucleotide sequence set forth in seq id no;

(ii) and SEQ ID NO: 55 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) and SEQ ID NO: 55 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

(iv) And SEQ ID NO: 55, and comprises a nucleotide sequence with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment of the invention, the humanized IL17F gene comprises a nucleotide sequence identical to SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 56, or a nucleotide sequence comprising at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14 or SEQ ID NO: 56.

In one embodiment of the present invention, the mRNA transcribed from the humanized IL17F gene comprises one of the following groups:

(i) SEQ ID NO: 15, all or part of a nucleotide sequence set forth in seq id no;

(ii) and SEQ ID NO: 15 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

(iii) and SEQ ID NO: 15 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

(iv) And SEQ ID NO: 15, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

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

Preferably, the humanized IL17F gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a conventionally induced or repressed substance.

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 invention, there is provided a targeting vector comprising a portion of the human IL17F gene.

Preferably, the targeting vector comprises all or part of exons 1 to 3 of the human IL17F gene. Further preferably, it comprises all or part of exon 2 to exon 3 of the human IL17F gene. Even more preferably, the part of exon 2 and part of exon 3, and preferably all of introns 2-3 are included, wherein the part of exon 2 at least comprises a nucleotide sequence of 20bp, for example, at least comprises a nucleotide sequence of 20, 50, 70, 100, 150, 160, 161, 162, 163, 164, 165, 170, 200, 220, 221bp, and even more preferably, comprises a nucleotide sequence of 164 bp; the portion of exon 2 preferably comprises a sequence encoding SEQ ID NO: 8, 31-85, and a part of exon 3 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 100, 150, 200, 220, 230, 231, 232, 233, 234, 235, 238, 250, 270, 300, 350, 400, 450, 480, 488bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon. Most preferably comprises a sequence identical to SEQ ID NO: 11 or a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 11 are identical.

Preferably, the targeting vector further comprises a5 'arm (5' homology arm) selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the non-human animal IL17F gene. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000067.6. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 9 or 24, or as shown in SEQ ID NO: 9 or 24. And/or, the targeting vector also comprises a3 'arm (3' homologous arm), which is selected from 100-10000 nucleotides in length of the genomic DNA of the non-human animal IL17F gene. Preferably, the 3' arm has at least 90% homology with NCBI accession number NC _ 000067.6. Further preferably, the 3' arm sequence is identical to SEQ ID NO: 10 or 25 has at least 90% homology, or as shown in SEQ ID NO: 10 or 25.

Preferably, the targeting vector further comprises SEQ ID NO: 12. 13, 14 and/or 56.

Preferably, the 5 'arm is a DNA fragment homologous to the 5' end of the transition region to be altered.

Preferably, the 3 'arm is a DNA fragment homologous to the 3' end of the transition region to be altered.

Preferably, the transition region to be altered is located at the IL17F locus of the non-human animal. Further preferably, it is located on exon 1 to exon 3 of the IL17F gene. Even more preferably, it is located on exon 2 to exon 3 of the IL17F gene.

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

Preferably, the targeting vector further comprises a marker gene. 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 fourth aspect of the invention, there is provided a sgRNA that targets a non-human animal IL17F gene, while the sequence of the sgRNA is on a target sequence on the IL17F gene to be altered.

Preferably, the target site of the sgRNA is located on exon 1 to exon 3 sequences of the IL17F gene.

Preferably, the target site of the sgRNA is located on exon 2 to exon 3 sequences of the IL17F gene.

Preferably, the target site of the sgRNA is located on exon 2 and/or exon 3 sequences of the IL17F gene.

Preferably, the sgRNA targets a target site sequence at the 5' end as set forth in SEQ ID NO: 26-33. Further preferred is SEQ ID NO: 29.

preferably, the sgRNA targets a target site sequence at the 3' end as set forth in SEQ ID NO: 34-41. Further preferred is SEQ ID NO: 34.

in a specific embodiment of the invention, the sgRNA target site sequence is selected from SEQ ID NO: 29 and 34.

In a fifth aspect of the invention, a DNA molecule encoding the sgRNA described above 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.

In one embodiment of the present invention, the double-stranded nucleotide sequence of the DNA molecule is as shown in SEQ ID NO: 42 and 44, SEQ ID NO: 46 and 48, SEQ ID NO: 43 and 45, SEQ ID NO: 47, and 49.

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

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

In an eighth aspect, the present invention provides a use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector, or the cell for modifying IL17F gene. Preferably, said use includes, but is not limited to, knock-out, insertion or substitution.

In a ninth aspect of the invention, there is provided a non-human animal humanized with an IL17F gene, said non-human animal expressing a human or humanized IL17F protein.

Preferably, the non-human animal has reduced or absent expression of endogenous IL17F protein.

Preferably, the non-human animal in vivo expression of the humanized IL17F protein.

Preferably, the non-human animal body contains a part of human IL17F gene, more preferably the humanized IL17F gene.

Preferably, the nucleotide sequence of the human IL17F gene or the humanized IL17F gene is operably linked to a non-human animal endogenous regulatory element.

Preferably, the non-human animal body expresses a human or humanized IL17A protein.

Further preferably, the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17A gene.

In one embodiment of the invention, the non-human animal expresses fully human IL17A protein. The fully human IL17A protein may be the full length IL17A protein comprising a leader signal peptide or the mature IL17A protein after cleavage of the leader signal peptide.

In one embodiment of the invention, the non-human animal humanized with the IL17A gene expresses the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Preferably, the non-human animal body contains a human or humanized IL17A gene. Further preferably, the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene. Still more preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and part of exon 3 of IL17A gene, preferably further comprises intron 1-2 and/or intron 2-3, wherein part of exon 1 comprises at least 10bp of nucleotide sequence, for example at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

In one embodiment of the invention, the non-human animal humanized with the IL17A gene comprises SEQ ID NO: 5 human IL17A gene.

In one embodiment of the invention, the humanized IL17A gene comprises SEQ ID NO: 5.

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

In a tenth aspect of the present invention, there is provided a method of constructing the above non-human animal expressing human or humanized IL17F protein.

Preferably, the humanized IL17F protein is the humanized IL17F protein described above.

Preferably, the genome of the non-human animal further comprises a human or humanized IL17F gene, and the humanized IL17F gene is the humanized IL17F gene.

Preferably, the construction method comprises introducing a nucleotide sequence encoding human IL17F protein into the non-human animal IL17F locus. Further preferably, the nucleotide sequence encoding human IL17F protein does not comprise a nucleotide sequence encoding a signal peptide. Still further preferred, comprises the use of a polypeptide comprising a sequence encoding an amino acid sequence substantially as shown in SEQ ID NO: 8, position 31-163, or at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, or at least 99% identical to SEQ ID NO: 8 from position 31 to 163 into the IL17F locus of a non-human animal.

Preferably, the method comprises introducing the nucleotide sequence encoding humanized IL17F into the non-human animal IL17F locus.

Preferably, the method comprises introducing a cDNA sequence encoding human IL17F into the non-human animal IL17F locus.

Preferably, the construction method comprises introducing a portion comprising the human IL17F gene into the non-human animal IL17F locus. Further preferred, it comprises introducing into the non-human animal IL17F locus all or part of an exon 1 to 3 comprising the human IL17F gene. Even more preferably, the method comprises introducing all or part of exon 2 to exon 3 of a gene comprising human IL17F into the locus of IL17F of a non-human animal. Still further preferred, comprising introducing into the non-human animal IL17F locus a nucleotide sequence comprising part of exon 2 and part of exon 3, preferably also comprising all of introns 2-3, wherein part of exon 2 comprises at least a nucleotide sequence of 20bp, for example at least a nucleotide sequence of 20, 50, 70, 100, 150, 160, 161, 162, 163, 164, 165, 170, 200, 220, 221bp, further preferred a nucleotide sequence of 164 bp; the portion of exon 2 preferably comprises a sequence encoding SEQ ID NO: 8, 31-85, and a part of exon 3 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 100, 150, 200, 220, 230, 231, 232, 233, 234, 235, 238, 250, 270, 300, 350, 400, 450, 480, 488bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon. Even more preferably, the method comprises the step of contacting the polypeptide with a polypeptide comprising an amino acid sequence substantially identical to that of SEQ ID NO: 11 or a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 11 into the IL17F locus of a non-human animal.

Preferably, the method comprises introducing a nucleotide sequence comprising a humanized IL17F gene into the non-human animal IL17F locus.

Preferably, the method comprises introducing the CDS sequence comprising the human IL17F gene into the non-human animal IL17F locus.

Preferably, the construction method comprises introducing a portion of the human IL17F gene into exons 2 to 3 of a non-human animal.

Preferably, the construction method comprises introducing part of the human IL17F gene into part of exon 2 and part of exon 3 of the non-human animal, wherein the part of exon 2 comprises at least 20bp nucleotide sequence, such as at least 20, 50, 70, 100, 150, 160, 161, 162, 163, 164, 165, 170, 200, 220, 221bp nucleotide sequence, more preferably 164bp nucleotide sequence; the portion of exon 2 preferably comprises a sequence encoding SEQ ID NO: 7, 29-83, and a part of exon 3 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 800, 850, 859bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon.

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

Preferably, the human IL17F gene, the nucleotide sequence encoding human IL17F protein, the humanized IL17F or the nucleotide sequence encoding humanized IL17F protein are regulated by endogenous regulatory elements.

Preferably, the site of insertion or substitution is after the endogenous regulatory element of the IL17F gene.

Preferably, the insertion is performed by firstly destroying the coding frame of the endogenous IL17F gene of the non-human animal or the coding frame of the endogenous IL17F gene after the insertion sequence is destroyed, and then performing the insertion operation. Or the insertion step can cause frame shift mutation to the endogenous IL17F gene and realize the step of inserting the human sequence.

Further preferably, an auxiliary sequence (e.g., a stop codon or a sequence having a function of termination, etc.) or other methods (e.g., a sequence for inversion, or a sequence for knock-out) may be added to the inserted sequence so that the endogenous IL17F protein in the non-human animal after the insertion site is not normally expressed.

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

Preferably, the genome of the non-human animal comprises a humanized IL17F gene on at least one chromosome.

Preferably, at least one cell in the non-human animal expresses a human or humanized IL17F protein.

Preferably, the non-human animal is constructed using gene editing techniques including gene targeting using embryonic stem cells, regular clustered spacer short palindromic repeats (CRISPR/Cas9) techniques, Zinc Finger Nucleases (ZFNs) techniques, transcription activator-like effector nucleases (TALENs) techniques, homing endonucleases (megabase megaribozymes), or other molecular biology techniques.

Preferably, the construction of a non-human animal is performed using a targeting vector, wherein said targeting vector comprises a portion of the human IL17F gene. Further preferably, the recombinant human IL17F comprises all or part of exon 1 to exon 3 of the human IL17F gene. Still further preferably, the recombinant human IL17F comprises all or part of exons 2 to 3 of the gene. Even more preferably, the part of exon 2 and part of exon 3, and preferably all of introns 2-3 are included, wherein the part of exon 2 at least comprises a nucleotide sequence of 20bp, such as at least a nucleotide sequence of 20, 50, 70, 100, 150, 160, 161, 162, 163, 164, 165, 170, 200, 220, 221bp, and even more preferably, a nucleotide sequence of 164 bp; the portion of exon 2 preferably comprises a sequence encoding SEQ ID NO: 8, 31-85, and a part of exon 3 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 100, 150, 200, 220, 230, 231, 232, 233, 234, 235, 238, 250, 270, 300, 350, 400, 450, 480, 488bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon. Most preferably comprises a sequence identical to SEQ ID NO: 11 or a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 11 are identical.

Preferably, the targeting vector further comprises a 5' arm selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the IL17F gene of a non-human animal. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000067.6. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 9 or 24, or as shown in SEQ ID NO: 9 or 24. And/or, the targeting vector further comprises a 3' arm selected from 100-10000 nucleotides in length of the genomic DNA of the IL17F gene of the non-human animal. Preferably, the 3' arm has at least 90% homology with NCBI accession number NC _ 000067.6. Further preferably, the 3' arm sequence is identical to SEQ ID NO: 10 or 25 has at least 90% homology, or as shown in SEQ ID NO: 10 or 25.

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector into a cell of a non-human animal, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal humanized with the IL17F gene.

Preferably, to improve recombination efficiency, sgRNA can also be used together with the targeting vector to construct a non-human animal. Wherein the sgRNA targets the non-human animal IL17F gene, while the sequence of the sgRNA is on the target sequence on the IL17F gene to be altered.

In a specific embodiment of the invention, the construction method comprises introducing the targeting vector, the sgRNA and the Cas9 into a non-human animal cell, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into an oviduct of a female non-human animal, allowing the female non-human animal to develop, and identifying and screening the non-human animal humanized with the IL17F gene.

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

Preferably, the non-human mammal further expresses at least one of human or humanized IL17A, IL12, IL23, IL4R and IL6 proteins.

Preferably, the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17A gene.

Preferably, the non-human animal body contains a human or humanized IL17A gene, preferably, the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene, further preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and exon 3 of human IL17A gene, wherein the part of exon 1 comprises at least 10bp nucleotide sequence, such as at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp nucleotide sequence, further preferably 27bp nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the constructing method comprises introducing all or part of exon 1 to exon 3 of the human IL17A gene into the IL17A locus of the non-human animal, preferably comprises introducing part of exon 1, exon 2 and part of exon 3 of the human IL17A gene into the IL17A locus of the non-human animal, wherein the part of exon 1 comprises at least 10bp of nucleotide sequence, such as at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, and more preferably comprises 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

In the eleventh aspect of the present invention, there is provided a non-human animal in which the IL17F gene is deleted, wherein the non-human animal is deleted of all or part of the nucleotide sequence of the IL17F gene.

Preferably, the non-human animal lacks all or part of exons 1 to 3 of the IL17F gene. It is further preferred that all or part of exons 2 to 3 are deleted.

In one embodiment of the invention, the part of exon 2 and the part of exon 3, preferably also comprising all of introns 2 to 3, wherein the part of exon 2 deleted comprises the sequence encoding SEQ ID NO: 8, positions 31-85, the portion of exon 3 deleted comprising the coding region.

In one embodiment of the present invention, a non-human animal with a deletion of IL17F gene is prepared using the sgRNA described above.

In the twelfth aspect of the present invention, there is provided a method for constructing a non-human animal genetically modified with IL17A and IL17F, comprising the steps of:

providing a non-human animal humanized with an IL17A gene expressing human or humanized IL17A protein;

and (II) modifying the non-human animal gene provided in the step (I) by adopting a construction method of a non-human animal humanized by the IL17F gene to obtain the non-human animal modified by the IL17A and IL17F genes.

In one embodiment of the invention, the IL17A gene is humanized and a non-human animal expresses a fully human IL17A protein in vivo. The fully human IL17A protein may be the full length IL17A protein comprising a leader signal peptide or the mature IL17A protein after cleavage of the leader signal peptide.

Preferably, the humanized non-human animal of IL17A gene comprises human or humanized IL17A gene. Further preferably, the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene. Still more preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and exon 3 of human IL17A gene, preferably further comprises intron 1-2 and/or intron 2-3, wherein the part of exon 1 comprises at least 10bp of nucleotide sequence, such as at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the method for constructing a non-human animal humanized with IL17A gene in step (one) comprises introducing into the non-human animal IL17A gene locus a sequence comprising all or part of the nucleotide sequence encoding human IL17A protein.

Preferably, the method for constructing a non-human animal humanized with IL17A gene in step (a) comprises introducing all or part of the nucleotide sequence comprising exons 1 to 3 of human IL17A gene into the IL17A locus of a non-human animal. Further preferably comprises introducing the part of the nucleotide sequence comprising exon 1, exon 2 and part of the nucleotide sequence comprising exon 3 of the human IL17A gene into the IL17A locus of a non-human animal, preferably further comprising intron 1-2 and/or intron 2-3, wherein the part of exon 1 comprises at least a nucleotide sequence of 10bp, such as at least a nucleotide sequence of 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp, further preferably a nucleotide sequence of 27 bp; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the construction of the non-human animal humanized with the IL17A gene is performed using a targeting vector of the IL17A gene, said targeting vector comprising part of the human IL17A gene, preferably comprising all or part of the nucleotide sequence of exons 1 to 3, further preferably comprising part of the exon 1, exon 2 and part of the nucleotide sequence of exon 3 of the human IL17A gene, preferably further comprising introns 1-2 and/or introns 2-3, wherein part of the exon 1 comprises at least 10bp of nucleotide sequence, for example at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

In one embodiment of the present invention, the targeting vector of IL17A gene comprises SEQ ID NO: 5.

Preferably, the targeting vector for the IL17A gene further comprises a5 'and/or 3' arm.

In one embodiment of the invention, the 5' arm is identical to SEQ ID NO: 3 or as shown in SEQ ID NO: 3, respectively.

In one embodiment of the invention, the 3' arm is identical to SEQ ID NO: 4 or as shown in SEQ ID NO: 4, respectively.

In a thirteenth aspect of the present invention, there is provided a method for constructing a non-human animal genetically modified with IL17A and IL17F, comprising the steps of:

(I) providing the above non-human animal humanized with IL17F gene or a non-human animal obtained by the method for constructing the same;

and (II) carrying out gene modification on the non-human animal provided in the step (I) so as to enable the non-human animal to express human or humanized IL17A protein, thereby obtaining the non-human animal with the gene modified IL17A and IL 17F.

Preferably, the genetic modification in step (two) comprises insertion or substitution into the non-human animal IL17A locus with a nucleotide sequence comprising all or part of the nucleotide sequence encoding human IL17A protein.

Preferably, the genetic modification in step (two) comprises introducing into the non-human animal IL17A locus all or part of a nucleotide sequence comprising exons 1 to 3 of human IL17A gene. Further preferably comprises introducing the part of the nucleotide sequence comprising exon 1, exon 2 and part of the nucleotide sequence comprising exon 3 of the human IL17A gene into the IL17A locus of a non-human animal, preferably further comprising intron 1-2 and/or intron 2-3, wherein the part of exon 1 comprises at least a nucleotide sequence of 10bp, such as at least a nucleotide sequence of 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp, further preferably a nucleotide sequence of 27 bp; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the construction of the non-human animal is performed by using a targeting vector of IL17A gene, which comprises a part of human IL17A gene. Preferably, the targeting vector comprises all or part of the nucleotide sequence of the exon 1 to 3 of the human IL17A gene, preferably comprises part of the exon 1, exon 2 and part of the nucleotide sequence of the exon 3 of the human IL17A gene, preferably further comprises the intron 1-2 and/or the intron 2-3, wherein the part of the exon 1 comprises at least 10bp of nucleotide sequence, for example at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, and further preferably comprises 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

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

providing a non-human animal humanized with IL17F gene or obtained by the construction method;

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

Preferably, the other genetically modified non-human animal comprises a non-human animal modified by one or a combination of two or more of genes IL12, IL23, IL4R or IL 6.

Preferably, the modification may be a knockout, a mutation, an insertion or a substitution, and the like, or a combination thereof.

Preferably, the polygenetically modified non-human animal is a three-genetically modified non-human animal, a four-genetically modified non-human animal, a five-genetically modified non-human animal, a six-genetically modified non-human animal, a seven-genetically modified non-human animal, an eight-genetically modified non-human animal, or a nine-genetically modified 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 fifteenth aspect of the present invention, there is provided a non-human animal or its progeny obtained by the above-described construction method.

In a sixteenth aspect of the invention, there is provided a non-human animal genetically modified with IL17A and IL17F, said non-human animal expressing a human or humanized IL17F protein and a human or humanized IL17A protein.

Preferably, the humanized IL17F protein is selected from the humanized IL17F protein of the invention.

Preferably, the non-human animal body contains a human or humanized IL17F gene and a human or humanized IL17A gene.

Preferably, the humanized IL17F gene is selected from the humanized IL17F gene of the invention.

Preferably, the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene. Further preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and part of exon 3 of IL17A gene, preferably further comprises intron 1-2 and/or intron 2-3, wherein the part of exon 1 comprises at least 10bp of nucleotide sequence, such as at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

The seventeenth aspect of the invention provides an animal tumor-bearing or inflammation model, wherein the tumor-bearing or inflammation model is derived from the non-human animal or the offspring thereof and the non-human animal obtained by the construction method.

In an eighteenth aspect of the present invention, there is provided a method for producing a tumor-bearing or inflammatory model in an animal, comprising any one of the methods for constructing a non-human animal of the present invention.

In a nineteenth aspect of the present invention, an application of the above non-human animal or its progeny, the non-human animal obtained by the above construction method, or the above tumor-bearing or inflammation model in the preparation of a disease animal model is provided.

Preferably, the animal model of the disease is selected from multiple sclerosis, psoriasis, asthma, and allergic animal models. Further preferred are psoriasis models.

In a twentieth aspect of the present invention, there is provided an application of the above non-human animal or its progeny, the non-human animal obtained by the above construction method, or the above tumor-bearing or inflammation model in the preparation of a medicament for treating immune-related diseases, tumors and/or inflammations.

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

In a twenty-fourth aspect of the invention, there is provided a cell humanised with the IL17F gene, said cell expressing a human or humanised IL17F protein. Preferably, the cell expresses the above-described humanized IL17F protein.

Preferably, the cell expresses a human or humanized IL17A protein. Further preferably, the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17A gene.

Preferably, the genome of said cell comprises part of the human IL17F gene. Further preferably, the cell comprises the above-mentioned humanized IL17F gene.

Preferably, the genome of said cell comprises part of the human IL17A gene. Further preferably, it comprises all or part of exon 1 to exon 3 of the human IL17A gene. Still more preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and part of exon 3 of IL17A gene, preferably further comprises intron 1-2 and/or intron 2-3, wherein part of exon 1 comprises at least 10bp of nucleotide sequence, for example at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the cells are not capable of developing into an individual animal.

In a twenty-fifth aspect of the present invention, there is provided a cell deleted of IL17F gene, said cell being deleted of all or part of the nucleotide sequence of IL17F gene.

Preferably, the cell lacks all or part of exon 1 to exon 3 of the IL17F gene. It is further preferred that all or part of exons 2 to 3 are deleted.

In one embodiment of the present invention, cells in which the IL17F gene is deleted are prepared using the sgrnas described above.

Preferably, the cells are not capable of developing into an individual animal.

In a twenty-sixth aspect of the invention, there is provided a construct expressing the humanized IL17F protein described above.

Preferably, the construct expresses a human or humanized IL17A protein. Further preferably, the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17A gene.

Preferably, the construct comprises a portion of the human IL17F gene. Further preferably, said construct comprises the humanized IL17F gene of the present invention.

Preferably, the construct expresses a human or humanized IL17F protein, and the humanized IL17F protein is the humanized IL17F protein.

Preferably, the genome of said construct comprises part of the human IL17A gene. Further preferably, it comprises all or part of exon 1 to exon 3 of the human IL17A gene. Still more preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and part of exon 3 of IL17A gene, preferably further comprises intron 1-2 and/or intron 2-3, wherein part of exon 1 comprises at least 10bp of nucleotide sequence, for example at least 10, 20, 25, 26, 27, 28, 29, 30, 50, 70, 80, 84bp of nucleotide sequence, further preferably 27bp of nucleotide sequence; part of exon 1 from the start codon to the last nucleotide of exon 1, and part of exon 3 comprising at least 100bp of nucleotide sequence, for example at least 100, 150, 200, 220, 230, 235, 236, 237, 238, 239, 240, 250, 300, 500, 700, 900, 1000, 1300, 1500, 1584bp of nucleotide sequence, and more preferably 238bp of nucleotide sequence; the portion of exon 3 extends from the first nucleotide of exon 3 to the stop codon.

Preferably, the construct also expresses human IL17A protein.

In a twenty-seventh aspect of the invention, there is provided a cell comprising the above construct. Preferably, the cells are not capable of developing into an individual animal.

In a twenty-eighth aspect of the invention, there is provided a tissue comprising the above-described cells. Preferably, the tissue is incapable of developing into an individual animal.

A twenty-ninth aspect of the present invention provides a use of the protein derived from the above-mentioned humanized IL17F, the above-mentioned humanized IL17F gene, the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, 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 culture thereof, the above-mentioned tumor-bearing tissue, the above-mentioned cell, the above-mentioned construct, the above-mentioned cell or the above-mentioned tissue for product development requiring an immune process involving human cells, for antibody production, 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 IL17F channel function, human IL17F channel signal mechanism, human-targeting antibody, human-targeting drug, drug effect, inflammation drug, immune-related disease drug and anti-tumor 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 thirtieth aspect of the present invention, there is provided a method for screening for a modulator specific for human IL17F, 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 above non-human animal, the non-human animal obtained by the above construction method, and the above 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 method of screening for a modulator specific for human IL17F 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 thirty-first 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 applying 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-second aspect of the present invention, there is provided a use of a non-human animal derived from the above-mentioned non-human animal, a non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, the above-mentioned tumor-bearing or inflammation model for producing a human IL 17F-specific modulator.

The IL-17F gene humanized non-human animal prepared by the invention can normally express human or humanized IL-17F protein in vivo, and the expressed human or humanized IL-17F protein can specifically bind to a human IL-17F antibody. The IL-17F and IL-17A gene double humanized non-human animal prepared by the invention can normally express human or humanized IL-17F protein and human or humanized IL-17A protein in vivo, and the expressed protein can be specifically combined with IL-17F or IL-17A antibody. The non-human animals prepared herein can be induced to prepare a variety of disease models, such as asthma, allergy, psoriasis, or multiple sclerosis, among others. And can be used for drug screening and drug effect evaluation aiming at human IL-17F pathway target sites, and screening or evaluating drugs for treating immune-related diseases, inflammations or tumors. Meanwhile, the prior art shows that: IL17A and IL-17F are expressed in skin lesions and inflammatory synovium of patients with psoriatic arthritis, and compared with the simple blocking of IL-17A, the dual neutralization of IL-17A and IL-17F leads to the reduction of the expression level of inflammation-related genes and cytokines and the enhancement of the inhibition effect on the expression of disease-related genes. In addition, both IL-17A and IL-17F have been shown to co-stimulate the production of key pro-inflammatory cytokines and amplify tissue inflammation in preclinical models with TNF. The IL-17F and IL-17A gene double humanized non-human animal prepared by the application has considerable advantages in preparing disease animal models and screening and evaluating antibodies for treating immune-related diseases, inflammations or tumors.

The subject of the present invention, cells or cell lines or primary cell cultures, tissues or organs or cultures thereof, cannot develop into animals, wherein said cells are not stem cells or fertilized egg cells. The cells may be somatic cells, lymphocytes (preferably T cells or B cells), or tumor cells, among others. The tissue may be spleen, lymph node, bone marrow, tumor or its culture, etc.

The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, psoriasis, 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. In one embodiment of the invention, psoriasis may be treated.

The "inflammation" according to the present invention may be inflammation of various tissues or organs, including acute and chronic. Preferably degenerative inflammation, exudative inflammation (e.g. serous inflammation, cellulolytic inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation, proliferative inflammation, specific inflammation (e.g. tuberculosis, syphilis, leprosy, lymphogranuloma, etc.) in one embodiment of the invention, ulcerative colitis or ankylosing spondylitis, etc. may be mentioned.

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

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 IL17F protein comprises a part derived from human IL17F protein and a part derived from non-human IL17F protein.

Wherein, the humanized IL17F protein comprises 5-163 continuous or alternate amino acid sequences consistent with the amino acid sequence of the human IL17F protein, preferably 10-133 continuous or alternate amino acid sequences, and more preferably 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 or 163 continuous amino acid sequences consistent with the amino acid sequence of the human IL17F protein.

The humanized IL17A protein comprises a part derived from human IL17A protein and a part derived from non-human IL17A protein.

Wherein, the humanized IL17A protein comprises 5-155 amino acid sequences which are continuous or spaced and are consistent with the amino acid sequence of the human IL17A protein, preferably 10-155 amino acid sequences which are continuous or spaced, and more preferably 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 155 amino acid sequences which are continuous and are consistent with the amino acid sequence of the human IL17A protein.

The "humanized IL17F gene" of the present invention comprises a part derived from human IL17F gene and a part derived from non-human IL17F gene.

Wherein, the humanized IL17F gene comprises a continuous or alternate 20bp-9000bp nucleotide sequence which is consistent with the nucleotide sequence of the human IL17F gene, preferably a continuous or alternate 20-1963 nucleotide sequence, and more preferably 20-402 nucleotide sequences, 50, 100, 200, 300, 400, 402, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 1963, 2000, 2500, 3000, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500 or 9000bp nucleotide sequence which is consistent with the nucleotide sequence of the human IL17F gene.

The "humanized IL17A gene" of the present invention comprises a part derived from human IL17A gene and a part derived from non-human IL17A gene.

Wherein, the humanized IL17A gene comprises a continuous or alternate 20bp-3591bp nucleotide sequence which is consistent with the nucleotide sequence of the human IL17A gene, preferably a continuous or alternate 20-2861 nucleotide sequence, and more preferably a 20-468 nucleotide sequence, 50, 100, 200, 300, 400, 468, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 2861, 3000 or 3591bp nucleotide sequence which is consistent with the nucleotide sequence of the human IL17A gene.

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

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 "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 "IL 17F locus" refers to a DNA fragment of any of exons 1 to 3 of IL17F gene. In one embodiment of the invention, the inserted or replaced IL17F locus may be a DNA fragment of an optional stretch of exon 1 to exon 3 of the IL17F gene. In one embodiment of the invention, the IL17F locus to be replaced may be a DNA fragment of an optional stretch of exon 2 to exon 3 of the IL17F 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 "treating" (or "treatment") as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treatment" or the like refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

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

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

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal to which the gene is humanized is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the crimyspascimyscimysciaenopsis (for example of the crimysciaeidae (for example of the hamsters, the new world rats and the new world rats, the rats and the rats, the. In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily 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 strain of C57BL, C58, a/Br, CBA/Ca, CBA/J, CBA/CBA/mouse selected from BALB/C, a/He, a/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10 sn, C57BL/10Cr and C57 BL/Ola.

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

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

All patents and publications mentioned in this specification are herein incorporated 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 human and mouse IL17A loci (not to scale).

FIG. 2: schematic representation (not to scale) of the humanized IL17A locus.

FIG. 3: schematic representation (not to scale) of IL17A targeting strategy.

FIG. 4: the humanized IL17A mice disclosed herein were used to induce the EAE model, the body weight profile of each group of mice.

FIG. 5: the humanized IL17A mice disclosed herein were used to induce clinical symptoms in the EAE model, groups of mice.

FIG. 6: the humanized IL17A mouse disclosed in the invention is used to induce EAE model, and female mouse HE staining is used to observe histopathological changes.

FIG. 7: the humanized IL17A mouse disclosed in the present invention was used to induce the EAE model, female mice were stained for histopathological changes by IHC.

FIG. 8: the humanized IL17A mouse disclosed by the invention is used for inducing an EAE model, and the percentage results of hIL17+ CD3+ CD4+ T cells and IFN gamma + T cells in CD3+ CD4+ T cells of a mouse lymph node after MOG immunization are obtained.

FIG. 9: schematic comparison of human and mouse IL17F loci (not to scale).

FIG. 10: schematic representation (not to scale) of the humanized IL17F locus.

FIG. 11: IL17F gene targeting strategy and targeting vector design scheme one (not to scale).

FIG. 12: the genotype detection result of the humanized mouse IL17F in the F1 generation, wherein M is Marker, WT is wild type control, H₂O is water control, F1-01, F1-02, F1-03 and F1-04 are mouse numbers.

FIG. 13: the genotype detection result of the F1 generation IL17A/IL17F double-gene humanized mouse, wherein M is Marker, WT is wild-type control, PC is positive control, H₂O is water control, F1-01, F1-02 are mouse numbers.

FIG. 14: IL17F gene targeting strategy and targeting vector design scheme two (not to scale).

FIG. 15: and (3) detecting the activity of sgRNA1-sgRNA16, wherein Con is a negative control, and PC is a positive control.

FIG. 16: f0 mouse genotype identification results, wherein M is Marker, WT is wild typeControl, H₂O is water control, PC1 and PC2 are positive controls, and F0-01, F0-02 and F0-03 are mouse numbers.

FIG. 17: f1 mouse tail genotype identification results, wherein M is Marker, WT is wild type control, H₂O is water control; PC, PC1 and PC2 are positive controls, and F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08 and F1-09 are mouse numbers.

FIG. 18: and F1 Southern Blot detection results, wherein WT is wild type control, and F1-01, F1-02, F1-03, F1-04, F1-05, F1-06, F1-07, F1-08 and F1-09 are mouse numbers.

FIG. 19: and (3) detecting the expression detection result of IL17A/IL17F double-gene humanized mouse IL17A protein, wherein the B-hIL17A/hIL17F mic is an IL17A/IL17F double-gene humanized heterozygote mouse.

FIG. 20: and (3) detecting the expression detection result of IL17A/IL17F double-gene humanized mouse IL17F protein, wherein the B-hIL17A/hIL17F mic is an IL17A/IL17F double-gene humanized heterozygote mouse.

FIG. 21: percentage of leukocyte subtypes in the spleen of wild-type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice.

FIG. 22: percentage of T cell subtypes in the spleen of wild type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice.

FIG. 23: percentage of leukocyte subtypes in lymph nodes of wild-type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice.

FIG. 24: percentage of T cell subtypes in lymph nodes of wild type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice.

FIG. 25: the experimental design of anti-human IL17A/IL17F antibody Ab in IMQ-induced psoriasis model was evaluated using IL17A/IL17F double-gene humanized homozygote mice.

FIG. 26: statistical body weight plots of control (G1), model (G2) and anti-human IL17A/IL17F antibody dosing (G3-G4) mice in an imiquimod-induced psoriasis model using IL17AIL17F double-gene humanized homozygote mice.

FIG. 27 is a schematic view showing: statistical plots of erythema scores at psoriatic lesions of control (G1), model (G2) and anti-human IL17A/IL17F antibody dosing (G3-G4) mice in an imiquimod-induced psoriasis model using IL17AIL17F double-gene humanized homozygote mice.

FIG. 28: statistical plots of psoriasis-like squamous score of mice of control (G1), model (G2) and anti-human IL17A/IL17F antibody dosing (G3-G4) at different concentrations in an imiquimod-induced psoriasis model using IL17AIL17F double-gene humanized homozygote mice.

FIG. 29: PASI composite score statistics for control (G1), model (G2) and anti-human IL17A/IL17F antibody dosing (G3-G4) mice in an imiquimod-induced psoriasis model using IL17AIL17F double-gene humanized homozygote mice.

FIG. 30: HE staining results of dorsal tissue sections of mice on day 8 post-induction using IL17A/IL17F double-gene humanized homozygote mice in imiquimod-induced psoriasis model control group (G1), model group (G2), and anti-human IL17A/IL17F antibody administration group (G3-G4) at different concentrations.

FIG. 31: statistical plots of dorsal tissue epidermal thickness in mice of imiquimod-induced psoriasis model control (G1), model (G2) and anti-human IL17A/IL17F antibody administration (G3-G4) at various concentrations using IL17A/IL17F double-gene humanized homozygote mice.

FIG. 32: statistical plots of pathological scores using IL17A/IL17F double-gene humanized homozygote mice in dorsal tissue sections of imiquimod-induced psoriasis model control (G1), model (G2) and anti-human IL17A/IL17F antibody administration (G3-G4) mice at various concentrations.

FIG. 33: the dorsal skin condition of mice on day 6 post-induction in an imiquimod-induced psoriasis model control group (G1), a model group (G2) and a group administered with anti-human IL17A/IL17F antibody at different concentrations (G3-G4) using IL17A/IL17F double-gene humanized homozygote mice;

FIG. 34: the detection results of RT-PCR of wild-type and IL17A/IL17F double-gene humanized homozygote mice, wherein, the graph A is the detection result of IL17A, the graph B is the detection result of IL17F, the wild type is +/-and the H/H is IL17A/IL17F double-gene humanized homozygote mice;

FIG. 35: percentage of leukocyte subtypes in thymus of wild-type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice;

FIG. 36: percentage of T cell subtypes in thymus of wild type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice;

FIG. 37: the results of conventional detection of blood of wild type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice;

FIG. 38: the results of biochemical tests of blood of wild type C57BL/6 mice and IL17A/IL17F double-gene humanized homozygote mice.

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:

BbsI, EcoRI, BamHI, NcoI, ScaI enzymes were purchased from NEB under the respective accession numbers R0539S, R0101M, R0136M, R0193M, R3122M;

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

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

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

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

the HumanIL17A ELISA kit was purchased from Biolegend under cat No. 433917;

mouse IL17A ELISA kit was purchased from Biolegend under cat No. 432507;

the HumanIL17F ELISA kit was purchased from Biolegend under cat No. 435707;

mouse IL17F ELISA kit was purchased from Biolegend under cat No. 436107;

PE anti-human IL17A antibody (anti-human IL17A PE) was purchased from Biolegend under code No. 512305;

APC anti-mouse IFN- γ antibodies (anti-mouse IFN- γ APC) purchased from Biolegend under cat # 505809;

Alexa

488 anti-mouse CD3 antibody was purchased from Biolegend, cat # 100212;

Brilliant Violet 421^TManti-mouse CD 4antibody was purchased from Biolegend, cat No. 100443;

Brilliant Violet 510^TManti-mouse CD45 antibody was purchased from Biolegend, cat No. 103137;

eBioscience^TMfoxp 3/transformation Factor stabilizing Buffer Set, available from ThermoFisher under a cat number of 00-5523-00;

in VivoMAb anti-mouse CD3 antibody was purchased from BioXCell, cat # BE 0001-1;

in VivoMAb anti-mouse CD28 antibody was purchased from BioXCell, cat # BE 0015-5;

Ultra-LEAF^TMpurified anti-mouse IFN-. gamma.antibody was purchased from Biolegend, cat # 505834;

recombinant Mouse TGF-beta1 Protein (mTGF beta) was purchased from R & D under cat number 7666-MB-005;

CD4+ T Cell Isolation Kit mouse available from Miltenyi Biotec, cat # 130-;

Ultra-LEAF^TMpurified anti-mouse IL-4Antibody, available from Biolegend under cat # 504121;

recombinant Mouse IL-6(carrier-free) (mIL6) was purchased from Biolegend, cat # 575702;

PMA was purchased from Sigma, cat # P1585;

ionomycin was purchased from Sigma, cat # 407952;

imiquimod cream (idangle) purchased from 3M Health Care Limited, specification 250mg:12.5mg, approved article No. H20160079;

MOG35-55 was purchased from ProSpec, Specification 100 mg;

pertussis Toxin (PTX) was purchased from millipore, cat # 516560.

Example 1 preparation of IL17A Gene-humanized mouse

A comparison scheme between the mouse IL17A Gene (NCBI Gene ID: 16171, Primary source: MGI: 107364, UniProt ID: Q62386, from position 20730905 to 20734496 on chromosome 1 NC-000067.6, based on transcript NM-010552.3 and its encoded protein NP-034682.1 (SEQ ID NO: 1)) and the human IL17A Gene (NCBI Gene ID: 3605, Primary source: HGNC:5981, UniProt ID: Q16552, from position 52186387 to 52190638 on chromosome 6 NC-000006.12, based on transcript NM-002190.3 and its encoded protein NP-002181.1 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purpose of the present invention, a gene sequence encoding human IL17A protein may be introduced at the endogenous IL17A locus of a mouse, so that the mouse expresses human IL17A protein. Specifically, the mouse IL17A gene can be humanized by replacing a specific mouse IL17A gene sequence with a sequence of the human IL17A gene at the mouse endogenous IL17A locus by gene editing techniques, such as replacing a sequence of about 2.9kb including at least the start codon ATG to the stop codon TAA of the mouse IL17A gene with a corresponding human DNA sequence to obtain a humanized IL17A locus (schematic diagram shown in fig. 2).

The targeting strategy is further schematically shown in FIG. 3, which shows a targeting vector comprising a5 'homology arm, a 3' homology arm, and an A fragment comprising a human DNA fragment (IL 17A). Wherein the 5' homology arm (SEQ ID NO: 3) is identical to the NCBI accession number with nucleotide sequence 20727254-20730961 of NC-000067.6; the 3' homology arm (SEQ ID NO: 4) is identical to the nucleotide sequence at positions 20735137 and 20739901 of NC-000067.6 for the NCBI accession number; the human DNA fragment (SEQ ID NO: 5) is identical to the nucleotide sequence at positions 52186432 and 52189292 of NCBI accession No. NC-000006.12. The mRNA sequence and the protein sequence of the humanized mouse IL17A after being transformed are respectively shown as SEQ ID NO: 6 and SEQ ID NO: 2, respectively.

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 method comprises the steps of transfecting a targeting vector with correct sequencing verification into embryonic stem cells of a C57BL/6 mouse by electroporation, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technologies, screening correct positive clone cells, introducing the screened correct positive clone cells (black mice) into separated blastocysts (white mice) according to the technology known in the field, transferring the obtained chimeric blastocysts into a culture solution, carrying out short-time culture, and transplanting the chimeric blastocysts into an oviduct of a recipient mother mouse (white mouse) to produce an F0 generation chimeric mouse (black and white alternating). 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.

The humanized mouse disclosed by the invention can be used for inducing and preparing various human disease models, including models of multiple sclerosis, asthma, allergy and the like, and can be used for testing the in-vivo efficacy of a human specific antibody. Taking the preparation of an Experimental Autoimmune Encephalomyelitis (EAE) model as an example, IL17A gene-humanized mice (10 weeks old) prepared in the present invention were selected, immunized 1 time with MOG (day 0, injected subcutaneously with 200 μ g/mouse), and given intraperitoneally with Pertussis Toxin (PTX) twice (day 0 and day 1, at a dose of 400 μ g/mouse). After the first immunization, the mice are weighed and continuously observed every day, and are grouped after the mice are attacked, and the administration is carried out through various ways such as gavage, intraperitoneal injection or rat tail intravenous injection. The in vivo efficacy of different human medicines can be evaluated by multiple detection indexes such as behavioral scoring, brain/spinal cord IHC (immunological histochemist) pathology, HE pathology examination, Th17 type multi-cytokine detection of serum/brain homogenate, and flow cytometry of CNS, spleen and lymph node. The experimental groups are shown in table 1.

Table 1: experiment grouping

Group of	Immunization	The week of the year	Number of mice	Sex	Genotype(s)
						G1 (control group)	PBS	10	4	Female	IL17A(h/h)
G2 (building block set)	MOG	10	5	Female	IL17A(h/h)
						G3 (control group)	PBS	10	5	Male sex	IL17A(h/h)
G4 (building block set)	MOG	10	5	Male sex	IL17A(h/h)

After induction by the method, none of the PBS control groups (G1, G3) developed disease, and only the mice were found to be diseased in the model building groups (G2, G4), and clinical symptoms were manifested as listlessness, weight loss, loss of rat tail tension, hind limb or limb paralysis, incontinence of urine and feces, and individual mice were manifested as ataxia. The two building groups had total 10 mice with onset at 10-12 days after first immunization, and the mice in groups G1, G2, and G3 had weight loss. The number of cases is gradually increased along with the increase of days after sensitization, clinical symptoms reach a peak 3-5 days after the onset of the disease, and then the weight is gradually increased in a remission stage, so that the trend of disease onset-remission is presented.

The disease incidence of the female and male mice of the model group was compared, and the animal body weights were measured daily and neurological indices were evaluated according to a 4-point scoring system (clinical score): 0 is normal; 1 ═ tail weakness; 2 ═ part of hind limb paralysis; 3 ═ total hind limb paralysis; 4-tetraplegia. It is found that the incidence, the onset time, the time to peak and the severity of symptoms of the amphoteric mice are not obviously different in the modeling process, but the body weight and the clinical symptoms of the female mice are better recovered automatically (see fig. 4and fig. 5), and at the end of the experiment (day 45), spinal cord tissues of the female mice are taken to be fixed by paraformaldehyde, and HE and IHC staining are carried out to observe histopathological changes after paraffin embedding and sectioning. When the longitudinal section of the lumbar enlargement white matter of the spinal cord is stained, as shown by the results in fig. 6 and fig. 7, the spinal cord of the MOG immunized mice (building module) is infiltrated by a large amount of inflammatory cells, myelin proteins are greatly reduced, and the spinal cord of the control group of mice is not abnormal.

In the EAE model, IL17A was mainly produced by CD4+ Th17 cells during disease progression. To detect the production of human IL17 in mice, MOG-immunized IL17A humanized mouse homozygote (female, n-5) lymph node cells were isolated and stimulated with PMA and ionomycin in the presence of brefeldin a for 6 hours. Cells producing IL17 and IFN γ were analyzed by FACS. Fig. 8 shows exemplary flow cytometry results. The results show that the percentages of hIL17+ CD3+ CD4+ T cells and IFN gamma + T cells in CD3+ CD4+ T cells of mouse lymph nodes are increased after MOG immunization, and the building success of an EAE model is proved from a molecular level.

The above results indicate that the gene-humanized mice prepared using the method of the present invention can be used to establish a stable EAE model.

Example 2 preparation of IL17F Gene-humanized mouse

A comparative scheme of the mouse IL17F Gene (NCBI Gene ID: 257630, Primary source: MGI: 2676631, UniProt: Q7TNI7, from positions 20777146 to 20785274 on chromosome 1 NC-000067.6, based on transcript NM-145856.2 and its encoded protein NP-665855.2 (SEQ ID NO: 7)) and the human IL17F Gene (NCBI Gene ID: 112744, Primary source: HGNC: 16404, UniProt ID: Q96PD4, from positions 52236681 to 52245689 on chromosome 6 NC-000006.12, based on transcript NM-052872.4 and its encoded protein NP-443104.1 (SEQ ID NO: 8)) is shown in FIG. 9.

To achieve the object of the present invention, a nucleotide sequence partially encoding human IL17F protein may be introduced at the endogenous IL17F locus in a mouse, so that the mouse expresses humanized IL17F protein. Specifically, the partial sequence of mouse IL17F gene exons 2 to 3 can be replaced by the corresponding DNA sequence of human IL17F by gene editing technology to obtain the humanized IL17F gene sequence (schematic diagram is shown in fig. 10), thereby realizing the humanized modification of mouse IL17F gene.

The targeting strategy is schematically designed as shown in FIG. 11, which shows the homology arm sequences containing the upstream and downstream of the mouse IL17F gene on the targeting vector, and the A1 fragment containing the DNA sequence of human IL 17F. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 9) is identical to the nucleotide sequence at 20782346 and 20779455 of NCBI accession No. NC-000067.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 10) is identical to the nucleotide sequence at 20776845 and 20772788 of NCBI accession No. NC-000067.6; the DNA sequence of human IL17F (SEQ ID NO: 11) and the nucleotide sequence at positions 52238893-52236931 of NCBI accession No. NC-000006.12Has 99 percent of homology and is different in that the 'G' at the 52237842 th site is replaced by the 'A'; the mRNA sequence transcribed from the human IL17F sequence in fragment A1 is SEQ ID NO: 55; the connection of the upstream of the human IL17F DNA sequence in the A1 fragment with mice was designed

Wherein the sequence "cagct"t" in "is the last nucleotide, sequence, of the mouse

"c" in (a) is the first nucleotide of a human; the connection of the downstream of the human IL17F DNA sequence to the mouse was designed

Wherein the sequence "agtaaThe last "a" in "is the last nucleotide, sequence, of a human

The first "c" in (a) is the first nucleotide in the 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 is designed as

Therein, itMiddle sequence "caaag"g" 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 is designed as

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

The first "c" of (a) is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse IL17F after being transformed is shown as SEQ ID NO: 15, the expressed protein sequence is shown as SEQ ID NO: shown at 16.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. And (3) performing electroporation transfection on the targeting vector which is verified to be correct by sequencing into the embryonic stem cells of the wild mouse, screening the obtained cells by using a positive clone screening marker gene, and detecting the integration condition of the exogenous gene by using PCR (polymerase chain reaction) to screen out the correct positive clone cells. 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 wild type mice, and the F1 generation heterozygous mice are mutually mated to obtain F2 generation homozygous son mice. The positive mice and Flp tool mice can be mated to remove the positive clone screening marker gene, and then the mice are mated with each other to obtain the IL17F gene humanized homozygote mice. The genotype of the somatic cells of the progeny mice can be identified by PCR. The results of identification of the exemplary F1 mouse (with the Neo marker gene removed) are shown in FIG. 12, in which the mice numbered F1-01, F1-02, F1-03, and F1-04 are positive heterozygous mice.

The PCR detection primer sequences are as follows:

L-F1-F(SEQ ID NO：17)：5’-CCGAACTATAGTGACTTTCAGTCTTGCT-3’

L-F1-R(SEQ ID NO：18)：5’-ATTTATCCTGCCAGCTTGCCATTGT-3’

example 3 two-Gene humanized mouse and Multi-Gene humanized mouse

A two-gene humanized or polygenic humanized mouse containing IL17A and/or IL17F can also be prepared using IL17A obtained in example 1 and/or IL17F humanized mice obtained in example 2. For example, in example 2, a double-gene humanized mouse in which IL17A and IL17F genes were humanized can be obtained by selecting IL17A gene-humanized positive clone cells obtained in example 1 from ES cells used for electroporation. The homozygous or heterozygote of IL17A and/or IL17F mouse obtained by the method can also be mated with other gene modified homozygous or heterozygote mouse, the offspring is screened, IL17A and/or IL17F humanized and other gene modified double-gene or multi-gene modified heterozygous mouse can be obtained with certain probability according to Mendel genetic rule, and then the heterozygotes are mated with each other to obtain double-gene or multi-gene modified homozygote.

Take IL17A/IL17F double-gene humanized mouse as an example. Since the IL17A and IL17F genes of mice are located on chromosome 1, after IL17A humanized positive ES cells are obtained, secondary targeting is performed according to the method of example 2, and then the positive progeny mice are screened to finally obtain the IL17A/IL17F double-gene humanized mice. The somatic cell genotype of the offspring mice can be identified by PCR, and the identification result of an exemplary F1 generation mouse (with the Neo marker gene removed) is shown in FIG. 13 (the PCR detection primer sequences and the length of the target fragment are shown in Table 2), wherein the mice with numbers F1-01 and F1-02 are positive heterozygous mice.

Table 2: PCR detection primer sequence and target fragment length

Wherein, the position of the primer L-F1-F is positioned at the left side of the 5 'homologous arm of IL17F, R-F1-R is positioned at the right side of the 3' homologous arm of IL17F, and L-F1-R and R-F1-F are both positioned on the human sequence of IL 17F; primer WT-F is located on the 5' homology arm of IL17A, Mut-R is located on the human sequence of IL17A, and WT-R is located on the mouse intron 1-2 sequence.

In addition, CRISPR/Cas system can be introduced for gene editing to design a targeting strategy as shown in FIG. 14, wherein a targeting vector contains homologous arm sequences of the upstream and downstream of mouse IL17F gene and a human IL17F DNA sequence, wherein the upstream homologous arm sequence (5 'homologous arm, SEQ ID NO: 24) is the same as the nucleotide sequence of 20781021 and 20779455 of NCBI accession number NC-000067.6, and the downstream homologous arm sequence (3' homologous arm, SEQ ID NO: 25) is the same as the nucleotide sequence of 20777766 and 20776366 of NCBI accession number NC-000067.6; the human IL17F DNA sequence is identical to the human IL17F DNA sequence of fragment A1 in FIG. 11. The mRNA sequence and the protein sequence of the humanized mouse IL17F after being transformed are respectively equal to the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 are identical.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. 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.

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

exons

2 and 3 of the IL17F gene, and the target site sequence of each sgRNA on IL17F is as follows:

sgRNA1 target site sequence (SEQ ID NO: 26): 5'-AGCGGTTCTGGAATTCACGTGGG-3'

sgRNA2 target site sequence (SEQ ID NO: 27): 5'-GCTCGGAAGAACCCCAAAGCAGG-3'

sgRNA3 target site sequence (SEQ ID NO: 28): 5'-CGAATCTTCAACCAAAACCAGGG-3'

sgRNA4 target site sequence (SEQ ID NO: 29): 5'-ATGGGGAACTGGAGCGGTTCTGG-3'

sgRNA5 target site sequence (SEQ ID NO: 30): 5'-ACAGTGTTATCCTCCAGGGGAGG-3'

sgRNA6 target site sequence (SEQ ID NO: 31): 5'-CTCTCACAGTGTTATCCTCCAGG-3'

sgRNA7 target site sequence (SEQ ID NO: 32): 5'-TGGGAACTGTCCTCCCCTGGAGG-3'

sgRNA8 target site sequence (SEQ ID NO: 33): 5'-TTCCCAGCCTTCTGCAAGGCAGG-3'

sgRNA9 target site sequence (SEQ ID NO: 34): 5'-AGCGTTGTCAGGCCGCTTGGTGG-3'

sgRNA10 target site sequence (SEQ ID NO: 35): 5'-TGCAGCGTTGTCAGGCCGCTTGG-3'

sgRNA11 target site sequence (SEQ ID NO: 36): 5'-CAGGCCGCTTGGTGGACAATGGG-3'

sgRNA12 target site sequence (SEQ ID NO: 37): 5'-TCAGGCCGCTTGGTGGACAATGG-3'

sgRNA13 target site sequence (SEQ ID NO: 38): 5'-GTGGACAATGGGCTTGACACAGG-3'

sgRNA14 target site sequence (SEQ ID NO: 39): 5'-AGGGCTGTTCTAATTCCTTCAGG-3'

sgRNA15 target site sequence (SEQ ID NO: 40): 5'-GAAGGAATTAGAACAGCCCTGGG-3'

sgRNA16 target site sequence (SEQ ID NO: 41): 5'-GAGAAGATGCTCCTAAAAGTTGG-3'

Table 3: relative activity detection result of sgRNA

The activity of multiple sgrnas is detected by using a UCA kit, and the results show that the sgrnas have different activities, wherein although the activities of the

sgrnas

6 and 8 are relatively low, which may be caused by the specificity of a target site sequence, according to our experiments, the values of the

sgrnas

6 and 8 are still significantly higher than those of a control group, and the sgrnas 6 and 8 can still be judged to be active, so that the activities meet the requirements of gene targeting experiments, and the specific detection results are shown in table 3 and fig. 15. From these sgrnas 4and 9 were randomly selected 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 (see table 4), and after annealing, the annealed products are respectively connected to pT7-sgRNA plasmids (the plasmids are firstly linearized by BbsI), so as to obtain expression vectors pT7-IL17F-4 and pT7-IL 17F-9.

Table 4: sgRNA 4and sgRNA9 positive and negative oligonucleotide sequences

pT7-sgRNA vector was synthesized by plasmid synthesis company as a fragment DNA (SEQ ID NO: 50) 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.

The mouse germ cell at prokaryotic stage obtained from the IL17A humanized mouse obtained in example 1 was taken, and the in vitro transcription products of pT7-IL17F-4 and pT7-IL17F-9 plasmids (using Ambion in vitro transcription kit, transcription was performed according to the method described in the specification), the targeting vector and Cas9mRNA were premixed and injected into the cytoplasm or nucleus of the mouse germ cell. 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 fertilized eggs after injection 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 IL17A/IL17F double-gene mutant mouse strain.

The genotype of somatic cells of F0 mouse can be identified by the same PCR method as that described in the example (the primer sequences are the same), and the identification results of some F0 mice are shown in FIG. 16, in which 3 mice numbered F0-01, F0-02 and F0-03 are positive mice, and the 3 mice are further verified to be positive mice by sequencing without random insertion.

The IL17A/IL17F double-gene humanized mouse identified as positive by F0 was mated with the IL17A humanized mouse to give F1 generation mice. The same PCR method can be used to genotype F1 generation mice, and exemplary results are shown in FIG. 17, where 9 mice numbered F1-01 through F1-09 are all positive in FIG. 17. The 9 mice identified as positive by F1 PCR were subjected to Southern blot analysis to confirm the presence of random insertions. Extracting genome DNA from the mouse tail, digesting the genome with BspHI enzyme or EcoNI enzyme, transferring the membrane, and hybridizing. Probes 5 'Probe and 3' Probe are located on the left side of the 5 'homology arm and on the 3' homology arm, respectively, and the lengths of the probes and the target fragment are shown in Table 5.

Table 5: length of specific probes and target fragments

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				BspHI	5’Probe	——	5.6kb
EcoNI	3’Probe	20.0kb	11.4kb

The probe synthesis primers were as follows:

5’Probe-F(SEQ ID NO：51)：5’-GCATCATCAATGAAAACCAGCGCGT-3’

5’Probe-R(SEQ ID NO：52)：5’-AGAACCCTCTCTTCCAACACAGGAA-3’

3’Probe-F(SEQ ID NO：53)：5’-CCTATCTGGGAGTTGGTTTGGGGTC-3’

3’Probe-R(SEQ ID NO：54)：5’-GAACTCGGAGCCTGCAGATCCAATC-3’

the Southern blot assay results are shown in FIG. 18. The results of 5 'Probe and 3' Probe were combined and further verified by sequencing that none of 5 mice numbered F1-02, F1-03, F1-04, F1-08, F1-09 were randomly inserted, confirming that these 5 mice were positively heterozygous and that there was no random insertion. This shows that the method can construct the IL17A/IL17F double-gene humanized gene engineering mouse which can be stably passaged and has no random insertion.

The expression of the humanized IL17AmRNA and the humanized IL17F mRNA in the humanized mouse of the IL17A/IL17F gene can be detected by RT-PCR. 3 wild type C57BL/6 mice and humanized IL17A/IL17F homozygote mice at 7 weeks of age were selected, spleen tissues were taken after cervical euthanasia, total RNA of cells was extracted, and PCR amplification was performed after reverse transcription into cDNA using a reverse transcription kit, and the primer sequences were shown in Table 6.

TABLE 6 RT-PCR detection primer sequences and target fragment lengths

Partial results of the assay showed (see FIG. 34) that murine IL17F and Il17A mRNA expression was detectable in wild type C57BL/6 mouse cells and humanized IL17F and Il17A mRNA expression was not detected; humanized IL17F and humanized IL17A mRNA expression was detected in humanized IL17A/IL17F homozygous mouse cells, and murine IL17F and IL17A mRNA expression was not detected. The expression of human IL17A protein and humanized IL17F protein in the obtained positive mice can be confirmed by conventional detection methods, for example, ELISA methods are used. Selecting 3 female wild-type C57BL/6 mice and IL17A/IL17F double-gene humanized heterozygote mice respectively, injecting 7.5 mu g of anti-mouse CD3 antibody (mCD3) and 4 mu g of anti-mouse CD28 antibody (mCD28) into each mouse abdominal cavity, taking serum after 2h to detect the expression condition of the human IL17A protein, and detecting results (shown in figure 19) show that the expression of the mouse IL17A protein is detected in the wild-type C57BL/6 mice, and the expression of the human IL17A protein is not detected; in IL17A/IL17F double gene humanized heterozygote mice in vivo detected in both mouse IL17A protein expression, and human IL17A protein expression. The expression of humanized IL17F protein in mice was also determined by ELISA. Respectively taking splenocytes of a female wild type C57BL/6 mouse and an IL17A/IL17F double-gene humanized heterozygote mouse, sorting CD4+ T cells, adding a 96-well plate pre-coated with 2ug/mL anti-mouse CD3 antibody and 5ug/mL anti-mouse CD28 antibody, adding 3ng/mL mTGF beta, 20ng/mL mIL6, 10ug/mL anti-mouse eIFN-gamma and 10ug/mL anti-mouse IL-4antibody, culturing for 72h, adding 50ng/mL PMA and 1ug/mL Ionomycin (Ionomycin) 5h before the culture, inducing the CD4+ T cells to differentiate into Th17 cells, and centrifuging the supernatant after the culture is finished to carry out ELISA detection. The results of the assay (see FIG. 20) showed that the expression of murine IL17F protein was detected in wild-type C57BL/6 mice, and the expression of humanized IL17F protein was not detected; expression of murine IL17F protein, as well as humanized IL17F protein, was detected in IL17A/IL17F double-gene humanized heterozygote mice.

Wild type C57BL/6 mice and IL17A/IL17F double gene humanized homozygote mice were further analyzed for immune cell subtypes in vivo by flow cytometry. FIGS. 21 and 22 are the leukocyte subtype and T-cell subtype percentages in spleen, FIGS. 23 and 24 are the leukocyte subtype and T-cell subtype percentages in lymph nodes, respectively, FIGS. 35 and 36 are the leukocyte subtype and T-cell subtype percentages in thymocytes, respectively, and it can be seen from the graphs that the expression profile of the leukocyte subpopulations in IL17A/IL17F two-gene humanized homozygote mice is similar to that of C57BL/6 mice, and the humanization of IL17A and IL17F does not affect the differentiation of T Cells (T Cells), B Cells (B Cells), NK Cells (NK Cells), granulocytes (Granulaces), Monocytes (Monocytes), DC Cells (Dentic Cells), and Macrophages (Macrophages), nor the DN Cells, DP Cells, CD4 in T Cells⁺T cell, CD8⁺Differentiation of T cells, Tregs cells.

Selecting 1 mouse of wild type C57BL/6 and humanized IL17A/IL17F homozygote respectively at 8 weeks of age, taking peripheral blood, and performing blood routine and blood biochemical index detection, wherein the blood routine comprises white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HB), Hematocrit (HCT), Mean Corpuscular Volume (MCV), mean corpuscular hemoglobin content (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), platelet count (PLT), lymphocyte count (LY), monocyte count (MO), neutrophil count (NEUT), red blood cell distribution width (RDW) and Mean Platelet Volume (MPV); the blood biochemical index detection comprises Albumin (ALB), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), total Cholesterol (CHOL), Creatinine (CR), blood sugar (GlU), Triglyceride (TG) and UREA (UREA), the blood routine index detection result is shown in figure 37, and compared with a wild-type mouse, the blood routine parameters of a humanized IL17A/IL17F homozygote mouse are consistent, which indicates that the method provided by the embodiment of the invention carries out humanization on IL17A and IL17F genes of the mouse, and the blood cell composition and the blood cell form are not changed; blood biochemical indicators test results as shown in fig. 38, the humanized IL17A/IL17F homozygous mice had no statistical difference in serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels compared to wild type mice, indicating that the methods of the present example humanized the IL17A and IL17F genes in mice without altering the ALT and AST levels or liver health in mice.

Example 4 in vivo efficacy validation of animal models

The humanized mice with IL17A and/or IL17F can induce and prepare various human disease models, including psoriasis, multiple sclerosis, asthma, allergy and other models, for testing the in vivo efficacy of the human specific antibody. A psoriasis model was established by Imiquimod (IMQ) induction with IL17A/IL17F double-gene humanized mouse homozygotes prepared in example 3. IL17A/IL17F double-gene humanized homozygote mice were divided into a control group (G1: vaseline), a model group (G2: IMQ + vaseline), a low dose group (G3: 1mg/kg Ab) and a high dose group (G4: 3mg/kg Ab) by weight, and each group had 5 mice. The mice were shaved 3 days prior to the experiment using a shaver to expose a 2cm x 4cm area of skin. After 3 days (D0), 10mg/cm was used for mice in the model group and the administration group²The IMQ cream is applied to the back skin area every day for 6 days; control mice were coated with petrolatum. Mice were given 2 doses of anti-human IL17A/IL17F antibody (Ab, obtained by immunizing mice using a conventional method, see Janeway's immunology (9th edition)) intraperitoneally at D0, D3. The total experimental period was 8 days, and the specific experimental protocol is shown in fig. 25.

Mice were weighed daily starting from D0, photographed and observed for dorsal status, and clinically scored for disease. The scoring items include erythema and scaling of the skin lesions of the mice. Each was classified by severity into 0-4 points, and the PASI scoring criteria were as follows: 0-none; 1-mild; 2-moderate; 3-severe; 4-very severe. The scores of each mouse and the total scores of the two mice in each group are averaged and compared. At the end of the experiment (D8), mouse dorsal skin specimens were sectioned and stained with Hematoxylin and Eosin (HE). The severity of dorsal erosion, spinous process appearance, parakeratosis and inflammatory mixed cell infiltration of each group of mice was scored (0.5-2 points): 0.5-mild, 1-mild, 1.5-moderate, 2-severe; stromal cell proliferation was scored (0.5-2 points): 0.5 is 2-4 layers, 1 is 4-6 layers, 1.5 is 6-8 layers, 2 is 8-10 layers; crust appearance: 0.5 min. Results statistics and inter-group pathology analysis scores were performed and epidermal thickness was measured.

From the change of the body weight of the mice with time (fig. 26), the body weight of the control group was stable throughout the whole experimental period; the body weight trends of the model group and the administration group are consistent, the body weight trends of the model group and the administration group are firstly reduced from D0, about D3 is reduced to the lowest, and then slowly increased, the body weight differences of the two groups in the experimental process are not large, and the body weights of all the mice in all the groups at the experimental end point are close and have no obvious difference. The results of the dorsal cutaneous erythema, scaling and combined PASI scores shown in figures 27-29 indicate that none of the control groups developed disease, while the model and administered groups exhibited varying degrees of disease. Compared with the model group and the administration group, the mice in the administration group (G3, G4) have the skin PASI scores which are obviously lower than that in the model group (G2), and the mice in the 3mg/kg treatment group (G4) have the skin PASI scores which are lower than that in the 1mg/kg treatment group (G3), which shows that the treatment of the administration of the anti-human IL17A/IL17F antibody to the mice in the administration group has the treatment effect on psoriasis, the treatment effect of the psoriasis is different in the treatment group of the mice with different administration doses, and the treatment effect of the 3mg/kg treatment group (G4) is better than that of the treatment group with 1mg/kg (G3) and presents a dose-dependent trend. HE staining results (fig. 30) of the dorsal tissue sections, statistical results (fig. 31) of the epidermal thickness of the dorsal tissue sections, and statistical results (fig. 32) of the pathology scores of the dorsal tissue sections of the mice showed that the degree of lesion of the dorsal skin in terms of stromal cell proliferation and epidermal thickening was lower in the administration group than in the model group. In addition, crusts were observed on the dorsal skin of some mice in the model group, while no such lesions were observed in the administered group (fig. 33), suggesting that the skin of animals in the model group was too ulcerated or eroded, and the severity of lesions was higher than that of the administered group.

The above results demonstrate that the humanized mice of the invention can be used to establish a psoriasis model to evaluate the in vivo efficacy of a medicament against human IL17A/IL 17F.

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 Diagram (Beijing) pharmaceutical science and technology Co., Ltd

<120> IL17F gene humanized non-human animal and construction method and application thereof

<130> 1

<150> CN202010681755.8

<151> 2020-07-15

<150> CN202011586262.2

<151> 2020-12-28

<160> 72

<170> SIPOSequenceListing 1.0

<210> 1

<211> 158

<212> PRT

<213> Mouse (Mouse)

<400> 1

Met Ser Pro Gly Arg Ala Ser Ser Val Ser Leu Met Leu Leu Leu Leu

1 5 10 15

Leu Ser Leu Ala Ala Thr Val Lys Ala Ala Ala Ile Ile Pro Gln Ser

20 25 30

Ser Ala Cys Pro Asn Thr Glu Ala Lys Asp Phe Leu Gln Asn Val Lys

35 40 45

Val Asn Leu Lys Val Phe Asn Ser Leu Gly Ala Lys Val Ser Ser Arg

50 55 60

Arg Pro Ser Asp Tyr Leu Asn Arg Ser Thr Ser Pro Trp Thr Leu His

65 70 75 80

Arg Asn Glu Asp Pro Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala Gln

85 90 95

Cys Arg His Gln Arg Cys Val Asn Ala Glu Gly Lys Leu Asp His His

100 105 110

Met Asn Ser Val Leu Ile Gln Gln Glu Ile Leu Val Leu Lys Arg Glu

115 120 125

Pro Glu Ser Cys Pro Phe Thr Phe Arg Val Glu Lys Met Leu Val Gly

130 135 140

Val Gly Cys Thr Cys Val Ala Ser Ile Val Arg Gln Ala Ala

145 150 155

<210> 2

<211> 155

<212> PRT

<213> human (human)

<400> 2

Met Thr Pro Gly Lys Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser

1 5 10 15

Leu Glu Ala Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly

20 25 30

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

35 40 45

Leu Asn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser

50 55 60

Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg Asn Glu

65 70 75 80

Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg His

85 90 95

Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser

100 105 110

Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Pro His

115 120 125

Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser Val Gly Cys

130 135 140

Thr Cys Val Thr Pro Ile Val His His Val Ala

145 150 155

<210> 3

<211> 3708

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tagtactcat tactgggact tcccatcata gaattgttca gagtggtcct cagaagcaca 60

aattataggt aagactgagt taaaatgtag atctggataa gatcctagct ttaccaattc 120

cataaggcct cccatgtggt cattattgca aataccctcc taccttaaca acaaatcttt 180

cctttaagtg tcaatttggg atttttttcc agtccaggtt gggcatcttc atacagtagc 240

tcctacattt aggacctaag gacactttct ctagaactgt tctctattaa acaaaaccaa 300

tggagaagga gccttatgca atactgtaca atgcattaaa ctaaatggca cctacctgtt 360

caaacagcca ttgtcttctt taaattagga tgcatgttaa ccactttaaa ctgtgaaatc 420

cctccttttt acagaccact gctgttattg ctttttcctg aaaagggcca tagaatacaa 480

attagtgaaa taatagattc tcaatggtag ccctgccatc tgagcagaaa acaaaactga 540

tttttatgtt tttagtaacg tcttaatttt ccaccacact gagacaaaag atattttatc 600

ctgtttacaa aagtaagaat tttcccttat ttttggtgtt atcctctctt ataggtagaa 660

aataatactg tataatggcc taatgatttc aagaagcccc aaagtatctt ttcccttaaa 720

acatcacttg actacaagca tcctcctgtt tcataccctc ccacacacag ccctggtcct 780

taaactgtag ctgcccagtc ttgtagctgc tgttttgaat ctgcagagaa gcaagatgga 840

tacacttgaa ggccactcct gagcaaaatc atcattccta acctactgct tttgtttatt 900

cattgctatt ttccttctgc aaaaagtcaa ccacaatgaa caggtgagac aaaacctcat 960

tttataaaac agaagagcat gtctacaaaa aagatgaagt aggtctcaga aaggcaaagg 1020

cacaacgaaa gtgaagatgc caatctcttg gtaccctcag cacaccaggt ctcactatgg 1080

ccttccttct gcctctgaag gctaagagcc aatcctgaat aacagcaagc acaaaagaaa 1140

cctgaccttc tgtgcacagc ccctgtgttc ccaattgctc ctccaaggac aaggctttca 1200

gatggaggga gtctctatgg ctgggaaatc cgttcttctc aaagccaggc acagccagct 1260

ccatgtctaa atcttcacac tgttgtgaaa actgatcttg tctctccccc ttccctttat 1320

ttttagacaa agattgtcac ctctcaaagg cccatacata ggctgagttt tacgactgtg 1380

ggacagtgga ctggggagat tattagatct tcctcttcat catcagttga cagatttggc 1440

aggaaacctc ttcctctcct gccagatagc aacaaggagt gtttattcct tggagttcag 1500

cataggctac aacatagtca tacacttttc aaaggcataa ggatttagag ccaagggata 1560

agacaaccca ggaatgtctt agactgcagc tgagttctaa aaagaaacaa ggtcatgaat 1620

ttctgacata atgtttcata ttgccaatca ataacaacaa caacaacaaa agttcagctg 1680

ttattttcta aaccctatgc agttggtaca aagattaaca aaatagctac aggtgttcaa 1740

gtaacttaca atatagaaag taaatcagta agcaaacaat atacaggtca agagaaacag 1800

acagccacat accaaagaga caaatgagtc taaagtaatc catggagctg gagagatggc 1860

tcagcagtta agagtactga ctgctcttcc agaggtcttg agttcaattc ccagaaacca 1920

catggtggct cacaaccatt tgtaatggga tctgatacct tcttctggtg tgtctgaaga 1980

tagcatcagt gtattcacat atattaaata aataaatatt ttttaaaaag taatccatga 2040

agacctatgt tgtataaaaa gttatattat gtacaacaaa atttaagaaa atgagtaaca 2100

cattagccca taaagaagcc aatgttgtgg tatgtttatt ccagtgatgt tattccccga 2160

tagaataact atttgaatag gatcctacaa gtgtgctgat aaacagggtg aaagaggaca 2220

ttgcctaggc tgtattttaa cagaaggtat gtaatttgta gaaggatgac acgtagaaag 2280

ccaggacctg gtgaacccta acatggagca tagtaacatg gaggctagaa gggatgttaa 2340

ttcaaactgc aatactaatt caaggagttc atgcttctca ttcaggaagc tagagaatgg 2400

gaaaagacct ggcaatcaga ggtgtgtgtg agcattatcc cagggataat gccaagggta 2460

ttatcccaag ggtatcccaa gaagtgtcag aaaagcaaac atgatccaaa caggtgaaag 2520

tcagagttac cagccagcaa aagacctaga aagaagagca aggtgtgagg tgctgcaact 2580

tctgagaaca cggtgatcat gaacagaatc cagcaatcct accagacatg ccatctattg 2640

aacaggagct atcggtccac ctcatgctgc atgtcagaca aaagctgaag agctgggacc 2700

taatgacccc catattcacc atcttgtcct catatctgct attcctgaag aaaaagactt 2760

ctcaaagaca taaaggcaaa ggtcatctca tggagaggag agaacatgag agagctgttt 2820

ccatcttccc ttctcatccc tcatctcctc ctgttagtag tctccacccg gcagtgcctc 2880

agtgtctcca ctgtctttca gccttcatct tgatttctaa ttctttcttc gatttatcca 2940

atcagtccct tattctttca cttcatttcc ttcctcctta aaagaaaggc ttgataccga 3000

acctcaaaac agcaaatatt aacaggtttc ttgataacat gcaaccgtaa tgacttcact 3060

agtaaacctc atgtctctcg ctactcctta ataactaact agcctttgtg attgtttctt 3120

gcagagaata gacattcaag gaaaaacagt tgcggtactc agttaaatag aacgtgttcc 3180

gttggtgtta aattatttat tttgtatgtc tgtttacata ctaagacatt gagtgggttt 3240

ctttgggcaa gggatgctct ctagccaggg aatttggtag aaaagtgaga aagatcaagt 3300

caaaattcaa agtgtgtgtc actaggagac tgtcaagaga ctcacaaacc attactatgg 3360

agcccagctc tgcagcagct tcagatatgt ccatacacac atgatactga atcacagcaa 3420

agcatctctg ttcagctccc aagaagtcat gcttctttgc atagtgaact tctgcccttc 3480

ccatctacct tcgagacaga tgttgcccgt cataaagggg tggttctgtg ctgacctcat 3540

ttgaggatgg aatctttact caaatggtgt caccccccaa cccactcttg acgtaagtga 3600

ccacagaggt agtaaaaccg tataaaaaga gagaaaggag cactactctt catccacctc 3660

acacgaggca caagtgcacc cagcaccagc tgatcaggac gcgcaaac 3708

<210> 4

<211> 4765

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atctgtagct cggggaacat catgagagag gagcagaaag attgtaagag cagaacaaca 60

ggaagtctgc tgggaaacag tctcctagaa atgtcttcat gaacaagcca agacaatatc 120

aatggatata ttaacatgga aggggtaatt tcctcaggtc ctacccctaa acaaagaact 180

acaggcatcc attgacttct gggagaatgt aaaagcctct accagagatg agccccttat 240

tgtttgccta atgcagagca gtcagccctg aaaccatatg cacaacaaca acaaaaatgg 300

actcagcagg ttgtgagtgc atgtgtgtgt gtgtgtatgt gtatgcaaac acatacatgt 360

gcatatacat gtatgtggca ataataaaga aaaatactat catttaagag tggaggtata 420

tggaaggggt tcaaggaaga gtgggtctgg aggaagaaag ggaagtggaa aatgacatga 480

ttctatctgt accaaaaaaa acactttttt aaaatgtaat atgttgagtc ccccaggaag 540

tagggaggtc tggtggggtg ggaggaatgc aatgaagaac tgtcagaagg tagaccagaa 600

gggggataat gaccagactg taaaaaagag attaaagaaa ataaatttta taaataaata 660

aattaattaa ttgattgatt gattccaaca aaaatagtat attttacacg attataaatt 720

tgtggtaatt gtacatgaat aatagaaatt tgtgactgca caaatttgga tttcaatcct 780

ggctttcctt attcgctgct ccctgagaaa tatttctaag cctcttccct cttaatccat 840

gaaatagaaa actgtgagag taaaacataa ataaataaag aatggcgtag tctctgatag 900

gtgtcagtag agaaggacac atacagcctt ctcccctgtc agtcttccct aaaacaatgc 960

ggcagtgcag atgtttgcac agggtcacat tctgtgagat actgtaagta ttacagatga 1020

gtttcagcac aatggaagga tggagggagg ttacatgcaa acaccactat cttatgcaag 1080

gagatgagcg tctgcagact ttgatatgtg ggagggactg tcctgttact cacctcaaac 1140

actctgaagg tcttttgaac ttcagcccct tcagctctcc cctgttggga aaaatatgcc 1200

tcagagtaac aatgcccttg aaccacccaa ctccacattt ccaatttcat attgccttta 1260

agacaatgag gacaaggctc ttcccgtacc aggcaatgct atgagcagaa gcattctgca 1320

ataaattaag caaaaagaac agtggcccta ggaaaagaca catcgctgtt agagaccggg 1380

gtgggggggg ggggggcgtc ctaaaactat aacctgatat ttgacataaa agaaattctt 1440

caaggaaact ctgttttcaa aataggagtc cttgctgtga tgaccaaggg ccaccagaga 1500

tcaggattcc ctctcagagc ttaggtctgc cctcagtctc tcatccagtg cacccaagca 1560

ttcagaaggg aagcggcaag aaagagtcag cctcaagaat aaaagttgtt caacacaagc 1620

aaagtcaaaa ctgagtcata tttgccaaca ggtcagtttc aagctttgaa tcatagccat 1680

cccagtcccc acgcccgagc ctgttattat gcccagttca gggatccttc tgaatggttc 1740

tccagccatt cccacgggct ctctgcttct tcttttaatt aatttctttg ggaagaggaa 1800

gcaggcacct ttagctgctg agctgtctca ttagccttca ctctttactc tctagcaggt 1860

ccatcaccac ccagaggtct acagtacagc ttccctctcc ttcattctaa tcccaaagcc 1920

aggaggcaac ttccccagtt cctcttgtca ctagcacctt gtgctctttc agcttggtac 1980

tgagagccaa cgcgtcacaa gcacacacgc acacacacag ccactgtagt ggtttgaatg 2040

tgcctggccc agtgagtggc actattagga gatgtggcct tgttggagtg ggtgtggcct 2100

tgttagagaa agtgtgtcac tgtgggcatg agctttaaga ccctcatccc agcttcattg 2160

gaagacagtc ttctagcggc cttcagatga agatgtagaa ctcgcagctc ctccagcatt 2220

atgccatgct cccaccttga tgataataga ctgaatctct gaacctgtaa gccagtccca 2280

attaaacgtt gtccttacaa gagttgcctt ggtcatgtgt ctgttcacag cagtaaaatt 2340

ctaagacaca cacacatatg cgcatggggg gggaggggag agagagagag agcattcaca 2400

ggtgctatct taatgtttta taatcctgct gctcatcatt tctttgggct tcgcagcatg 2460

gaagctttag gagcaccact aaggaagagc acacatgaac ctgtagtccc agaccgttat 2520

ttcatattag ttttactatt attggaggct aggaaaaagc cttctgtctt aggatttttg 2580

ttgttgttgt tgttgttgtt tgtttgtttg tttgtttgtt tttttagtgt tgttataact 2640

gtaacaaaaa ttttgaattc tgtttattaa gaaaagagat ttatttcact gacagttcta 2700

gagttgatgg ccattgcacc tgcatcctgc atcagtgtag cttctggtca aggcttcatg 2760

gcaaatgtca tccacataaa gaaacatgtc ctaaagtggc atagagagga ggtaccaggc 2820

tcttctagaa cagcccatct gtcaggacag tcccatgaga cctggtctgc tctctaatga 2880

cagtactgat ctctcccaac agtgattcct tctacaacta aattaccttg cactaggtct 2940

aactctttaa agctgtaagc acttcaaacc caccacaatg gagaccgagt atccagcagt 3000

ggatatttag agaccttatt ggatactttt ttgttttttc tttttctagg aattcaatgg 3060

tgtgttattt tagtgtactt tatttattca ctttacatcc caatcacagc ccccccccaa 3120

cctgtcatcc cccctcctcc ctgcttctct tctcctctag aaggatggag gggtcccctg 3180

ggcatccact ccccaccctg gcacatcaag tctctgaaag gctaggtgct tcctctccca 3240

ctgaggccag acaaaacagc ccagctagaa gaacatatcc cacagacagc caactgcttt 3300

gaggatagct cccgttccag ttgttcagga cccacatgaa gaccaagctg cacatctgct 3360

acatatgaac agggaggcct aggtccagtc catgtatgct tttttgttgg tagttcagtc 3420

cctgagaccc tccaagggtc caggttaatt gactgtgttg gtcttcctgt ggagttccta 3480

tccccttcag gctgaaatcc tttcttctgt tcttctataa gagcccagga gctccatcta 3540

ctgtttgtct gaagctccac aacctctcca aacagtgctg taagctagtc attaaatgct 3600

taaagatgca cctacagggg acagttcaca gaacaatctt gcgcatctct cattccctga 3660

tacacatcac ttggattcta actttgtctt cccaactctc accattgctc tggatggttt 3720

ccatgcgcct gcagatgcta cctacacaca aagtcaaagg ccacaaagta ctgtctggtt 3780

tacctcacgc cactttctgc ataggtatgt tctaagtgca gtgaggttca cagcctgtgc 3840

actggggctc ctctgctgga atggcttaac ctttctttcc tagttgttcc tagttgctcc 3900

tctttttttt cacagattta tttatttatt tatttattta tttatttatt tattgtacgt 3960

gagtacactg tcactctatt cagagacacc agaaattaca ttacagatgt ctgtgagcca 4020

ccatgtggtt gctgggattt gaactcggga cctctggtta agagctactg ctcttaaccg 4080

ctgagctatc tctccagccc tgctagctct tctttatatt catagccttc ctttccttcc 4140

tgctatacct taaccctatt ttgtgatgaa agcatgtggc aaaggaaaca aaatgagaga 4200

gaataagagt ctgaggtcgc agccccttca agggtctgcc cacaaaagcc ttactgcctt 4260

ctattaggtc tcatctgcta aaggttccac ctcctcccac tatcactaaa ggctaggaac 4320

caaaccccta acatgcagcc cttcagaaaa cactggccaa aacaacatca tgcctggatt 4380

tgttcattct tacccactca tttcaagctc aatgctttgc cttatacaac aaaactcttc 4440

ccttcatatt cacaagggct tcacatcctt aggatccatg ttttgtgtct tttgctacct 4500

ttctcaagat gctctgtgga gacagggagt gattagaaga cctcatagat gggcccagac 4560

gaccaagagt ggttgctaat gcccctatct cagagtatcc atgatggagc aggatttaat 4620

gagaggcatt gaagaattgc cttcacaagc tgtgtaacta ggtttttctc cactgaagca 4680

aacccaactg accaaatgca gtttatgtcc tttctcagca cagagccaca gagtatggat 4740

ccaatgtgac agaaaccggt cagtg 4765

<210> 5

<211> 2861

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgactcctg ggaagacctc attggtggtg agtcctgcac taacgtgcga tgctcttgct 60

gatttggacc agatagtatt tctggaccgt gggcatgaaa cgctgggttc tgactatgga 120

gatccaggaa tactgtatat gtaggatagg aaatgaaagc tttggtaggt atttaagtca 180

ttgtgcagca ttttcaagaa ctgatacaca gcagtttgaa agataagatt aaaactgaaa 240

gatagctata ttggggctaa accacacaag aagtgtcaca tgatgctgtg cagtaagaaa 300

gaaaatttat tgaaagtctg tttttctgag tacaaaggat ttaatataat tctcccacgg 360

catttttctt taaaatgggt cactatcctt gagattttga aagccgtagc agcaacaacc 420

tttgtttcca ttatctcgta ccatattctc agtacattga aactatgtat tctaactaaa 480

cataggtata actgtgtttt agaataagtg gggtttatat tttttaaata tttaacttca 540

agtatctttt ttgaaatctg attttattac agaatcaata catgttaaat ttagaacaac 600

tggaaaatat acctaagaaa acatgaagga gatcgagttt ttagttggat gcctgccagt 660

agcaccaaca gcacttctag catgaatatt gataccacat agattttcta tagctctttc 720

ttccaatgtg aatgtttgac ttcacgatga gtttcacaga atatgggact gagaacaatg 780

gtgcaggagg atatttctac ctagaaaatc aaggttatta ttccttccca gacctgacaa 840

tgatgcatgt gctgataggc taatgacatg ccatgacttg acatttttat taaaattatt 900

gccaaccaat ggataacatg tctttcctaa gtcaaaagga gaatgttgaa actagttttt 960

ttaaaaaaat tttaaagcca tggtgttaac attatgttgg tcatctacct agatttttct 1020

ctagctgatc tgaaaaatgt agtatagatt gtcctggaac attgtgtgtt ctctatgatt 1080

agcaatgcat catcatcaca attaatttgt caaaaagaac cacatagtaa tctaatctcc 1140

aacctctctc tcctttccca ttcaattcta gtcactgcta ctgctgctga gcctggaggc 1200

catagtgaag gcaggaatca caatcccacg aaatccagga tgcccaaatt ctgaggacaa 1260

gaacttcccc cggactgtga tggtcaacct gaacatccat aaccggaata ccaataccaa 1320

tcccaaaagg tcctcagatt actacaaccg atccacctca ccttggaatc tccagtacgt 1380

aaagcttcca gataaaaatg ctatattctt catccctctt atgcatcaga ctgccagtta 1440

aatctccctg aggatgattt tattcattta gaattaccag tcaaacctgg aaggaccact 1500

gtgaagagca attctcaaac tttctacaga tttctttaac caagcacagg acagcctcca 1560

ataatcccta tcctgttaga tctaattgtc actgacacca ataatcaacc caaattaatt 1620

ataatcatta ttctaatatt tatgagaccc caagtctatt ctttatttat tcaaagaata 1680

gacatttatc aaagaggatt aatgctttta ttatcttaac cagagctgcc attgagaaga 1740

tttattgcaa ataattaata attagggttt tttactttta ttcttttgct tatttttgtt 1800

tttgaatccc agtggaataa gtatcactgg ggtatttcta cccctttgtg tgttaaatag 1860

tcttgatcta cttcctaaca tacctatgct tgctgtatcc ttagtatacc cagtatttag 1920

accccatcaa gggttaaata ccaaatgtat tttgatcatt tgacttcata caaataagtc 1980

tctgttctgt ggagcctaca gattggtctg attgtaggat ttcttctctt cttcccatta 2040

ctaggaagag tcaaaataaa tcaattcaaa aatgcaagca aatcattcac tgatctaaaa 2100

gagagaggga agagaaggtc atagagacac ttaacctttt gtttccagcc ctttatctca 2160

gctctgggct ctgtcccacg aatgtgatct cagataaaat tttgatgtat tccctcttca 2220

aagacagact tcatcaagtc aaataaacag ctatcttatt ctagatggtt ccaagtctac 2280

tcttcctttg gtcttcttct gtctgtcaaa tgtaccctaa aaaagctatc atttgtgtca 2340

aacttaaatt ttttctgtgg cctcagtcta tcttatttta ttcattcttc aaataaattg 2400

gagaaaaact gatcactgtc ttcttttcta taacaattca cgtgcttgaa aaaaaaatcc 2460

aatttgtccc caaagttctt cttcaaacta acatcattta aagaatttgc aatgcctata 2520

atttgtcatc ctgtgaactt gcctctcttc atgtattcct gttttatttc tttcccactt 2580

taccaggaat tcactttcct cctgattttt ctcccctctg cagccgcaat gaggaccctg 2640

agagatatcc ctctgtgatc tgggaggcaa agtgccgcca cttgggctgc atcaacgctg 2700

atgggaacgt ggactaccac atgaactctg tccccatcca gcaagagatc ctggtcctgc 2760

gcagggagcc tccacactgc cccaactcct tccggctgga gaagatactg gtgtccgtgg 2820

gctgcacctg tgtcaccccg attgtccacc atgtggccta a 2861

<210> 6

<211> 1162

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atccacctca cacgaggcac aagtgcaccc agcaccagct gatcaggacg cgcaaacatg 60

actcctggga agacctcatt ggtgtcactg ctactgctgc tgagcctgga ggccatagtg 120

aaggcaggaa tcacaatccc acgaaatcca ggatgcccaa attctgagga caagaacttc 180

ccccggactg tgatggtcaa cctgaacatc cataaccgga ataccaatac caatcccaaa 240

aggtcctcag attactacaa ccgatccacc tcaccttgga atctccaccg caatgaggac 300

cctgagagat atccctctgt gatctgggag gcaaagtgcc gccacttggg ctgcatcaac 360

gctgatggga acgtggacta ccacatgaac tctgtcccca tccagcaaga gatcctggtc 420

ctgcgcaggg agcctccaca ctgccccaac tccttccggc tggagaagat actggtgtcc 480

gtgggctgca cctgtgtcac cccgattgtc caccatgtgg cctaaacaga gacccgcggc 540

tgacccctaa gaaaccccca cgtttctcag caaacttact tgcattttta aaacagttcg 600

tgctattgat tttcagcaag gaatgtggat tcagaggcag attcagaatt gtctgccctc 660

cacaatgaaa agaaggtgta aaggggtccc aaactgcttc gtgtttgttt ttctgtggac 720

tttaaattat ttgtgtattt acaatatccc aagatagctt tgaagcgtaa cttattttaa 780

tgaagtatct acattattat tatgtttctt tctgaagaag acaaaattca agactcagaa 840

attttattat ttaaaaggta aagcctatat ttatatgagc tatttatgaa tctatttatt 900

tttcttcagt atttgaagta ttaagaacat gattttcaga tctacctagg gaagtcctaa 960

gtaagattaa atattaatgg aaatttcagc tttactattt gtttatttaa ggttctctcc 1020

tctgaatggg gtgaaaacca aacttagttt tatgtttaat aactttttaa attattgaag 1080

attcaaaaaa ttggataatt tagctcccta ctctgtttta aaaaaaaatt gtaacaatat 1140

cactgtaata ataaagtttt gg 1162

<210> 7

<211> 161

<212> PRT

<213> Mouse (Mouse)

<400> 7

Met Lys Cys Thr Arg Glu Thr Ala Met Val Lys Ser Leu Leu Leu Leu

1 5 10 15

Met Leu Gly Leu Ala Ile Leu Arg Glu Val Ala Ala Arg Lys Asn Pro

20 25 30

Lys Ala Gly Val Pro Ala Leu Gln Lys Ala Gly Asn Cys Pro Pro Leu

35 40 45

Glu Asp Asn Thr Val Arg Val Asp Ile Arg Ile Phe Asn Gln Asn Gln

50 55 60

Gly Ile Ser Val Pro Arg Glu Phe Gln Asn Arg Ser Ser Ser Pro Trp

65 70 75 80

Asp Tyr Asn Ile Thr Arg Asp Pro His Arg Phe Pro Ser Glu Ile Ala

85 90 95

Glu Ala Gln Cys Arg His Ser Gly Cys Ile Asn Ala Gln Gly Gln Glu

100 105 110

Asp Ser Thr Met Asn Ser Val Ala Ile Gln Gln Glu Ile Leu Val Leu

115 120 125

Arg Arg Glu Pro Gln Gly Cys Ser Asn Ser Phe Arg Leu Glu Lys Met

130 135 140

Leu Leu Lys Val Gly Cys Thr Cys Val Lys Pro Ile Val His Gln Ala

145 150 155 160

Ala

<210> 8

<211> 163

<212> PRT

<213> human (human)

<400> 8

Met Thr Val Lys Thr Leu His Gly Pro Ala Met Val Lys Tyr Leu Leu

1 5 10 15

Leu Ser Ile Leu Gly Leu Ala Phe Leu Ser Glu Ala Ala Ala Arg Lys

20 25 30

Ile Pro Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro

35 40 45

Pro Val Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu

50 55 60

Asn Gln Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser

65 70 75 80

Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu

85 90 95

Val Val Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly

100 105 110

Lys Glu Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu

115 120 125

Val Val Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln Leu Glu

130 135 140

Lys Val Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val Ile His

145 150 155 160

His Val Gln

<210> 9

<211> 2892

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aattgaggga aatttggatg gagcttggag ctgaccagtc tctgtcaccc attcctgtgt 60

agggaggaaa gtgtaggggc aagtgtgcag atgttctgcc accagcgagc acacaagagt 120

aatgtacaaa gctaagctgg gaagattcac actctatgac tgaacacctt cagtttcaat 180

attctcctag ctgtttccca tcctacagga tttgaatcac acctatcaat cccctttaaa 240

ggtggaattt aaacctgtaa aatctcccga ctcccctgag aagatggatt gtttctattg 300

tttttccata ctcagcacca gccttatcag agctgcaaaa caccaaaaaa caaacaaaca 360

aacaaacaaa accccaaaaa cgcataaagc aaacagtggt ttgtggcttt caatacttct 420

gtcttactga tgcagtgcag agagtgttta gaattcgtgg ctcttgaaat tagacacagt 480

gaagactaat gtgaagaata gtaatgacct tcagtcgctg tggccaagcc ttgccataga 540

aggtggatcc tatagattgt cacgatgagg ctggggaacc ctgccgggtt gtttattgcc 600

tctctgacat aagtcctttg agagctgatt agatagttag gatgagtttc atctatgagt 660

tagctgttca aatcttgtgt gaaaacattt tctaaaatac tccaagaatg tctagctatt 720

aataaagaaa cataatgagc tgttagggac atgatcgttg tcttagttat ttttcgattg 780

ctgtgataaa acactgtaac catggcaaca tataaaagga agtgtttaat ttggggctta 840

cagttgccaa gggttaagct gcttgatcat catggctgga ggcaggaagg taggcatggc 900

gctggggaag ttgctgggag ttcacatctt aagacataac catgaggcag aaagaactaa 960

ctgggaatga tataagactt gaaatttcga agcccacctc agtgacacac ctcctccaac 1020

aagaccacac ctcctactcc ttcccaaata attccaccaa ctggggacca agcatccaca 1080

taaataagcc tatgcaggtt gcttcgcagt tcaaccacca caacaactta cattaatcac 1140

ataaccccga actatagtga ctttcagtct tgctagtcaa aatgagaagt gagacaataa 1200

acatgacata taagaagaaa gagaagattc tggaggagaa atttacttaa gaaatctaaa 1260

gaagtggttg atagatgtgt gactatgttt tctaaatttc aaagcacctg tgaagactgc 1320

attttacatc catcccatgc acagggtaac atcaatacac tgccaaccgt tgttgccttg 1380

tggaactact gcatgcttac agggatcttt gggatttatg aggatgtagg aaaccaagtc 1440

attgtgcggt gagtcctata aatgaattat ggactccaca cagttaactc tagagataat 1500

gcctgaaacc acaacactgt ggcctcgagg attagctact cagtgaacga atgacttcag 1560

aggcgaaggc atatggctgc tgattatctg gagacaaatc tgtttcatcc tgcagcttgc 1620

tttcaatgta ttatcagtga tcgtagtcca gcaggctgac tccatgacct ctttgtttat 1680

tctgcatatt tcttgcctcc aggatgttga aaagaacagg caaatcttta aatccaacat 1740

gcacagtcac tttccctacc tacacagtgt gagcctgaac aacacggagc ccagaaatga 1800

aaagctcagc cttagctctt ctgccttacc atgggctttg ttcttcccca ggaaggtgtg 1860

agcagggacg gcaaaggaca tttgcaccca aggggagctg ttgaggtttt agctcttctt 1920

gtgtttagag ctccactgag cactttgtgt gcgctagtaa cagcagcgaa actgatttca 1980

gtgttagtac agaaaagata gattctagag ctaaaagaaa tgggttctgc actctgagtg 2040

atgccaggcg tcatcagtgt ccctgcttct ttcaattagc atggataaat gaaattttgc 2100

tgtgcttagt ttcccataat ccttttactc aatgagcgtg gcataactat actggttaat 2160

ttaactgggc tgccctgttc attcacttcc ccactctctc attcattcag aaagttttca 2220

tggaccaact atgctctcgc aggaagttaa ctctgagtct ggtatgaaca cagatatgac 2280

atctttagag aacagaaaaa atacttcttg agtgatacat gctaaaatct ttattattta 2340

atgttgataa atatactttt aaaaaaatat ttattgtatg tataagagtt cactgtttct 2400

gcctttagac acaccagaag agggcattgg atgccattac agatggttgt gagccactat 2460

gtggttgctg ggaactgaac tcaggacctc tagaagagca atcagtgctc ttagctgtgg 2520

agccattcct ccatcccaat aaatatactt ctaattgaat tacatatggg accttgacag 2580

tagggtgagg tttggtggaa tgtcccagag aaaggttttg tggactataa ggccggagat 2640

aaactccaga gagctttact cctgaccctt tctgctttgt atcctgataa cattaaacac 2700

ggcagttctc attgctagtg aaccatgaaa aaaaaaaaaa aaaagaaacc ttgatcctgt 2760

aaagtgaccc tatacagagt tcaagtacat tgcccaccac cagggctcta acctgtggta 2820

atctctattt tccaggtcaa gtctttgcta ctgttgatgt tgggacttgc cattctgagg 2880

gaggtagcag ct 2892

<210> 10

<211> 4058

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cttctctcca tgatggaact tgtaaacgta acaattccat tcccctctaa gttgtttttg 60

ttcatggtgt ttaacatggc agcacagaaa gcagctagac aagtatttgc ttctgccttc 120

agcttgaact ccaagacttt acaaccctca acattttgaa catctgtata tcatttgttt 180

ttccaaattt cctcctcaga gctcccatgt tagagttatt gttattaatg gaactgaggt 240

cccgttacac ttagaaagag agttaaggga aatggtgcaa atggaattca agtgtggggc 300

cagcatgtta cacttccagc cttctggcca catcttgtta caaagcaaac acttactgtc 360

cactttgcta gcttctggag ttatgatgtg gagaaagatg tacgtagaag agccactgta 420

ttggcagtag ccacccccag aaccctgtca ttagggccac atggttcctt tgccttcttc 480

tccctatgac ttctaggggt caccataaat cttttatcag actcccatgc tacttgccgg 540

ccaatacaca gtatgcagac cctaggctcc agggaaacgt gtgtctcccc tctgtctccc 600

ttgcctggat acagtgcacc acatctctct ttcactgcta ctatgctttg agagcatcta 660

tattgcagct acattccagg tttctaaaat ctctatgctc cagaggaagt agaaagtgag 720

gtctttctcc tatctgggag ttggtttggg gtcaggcagg gagcaggaac ttggctccct 780

tgtctaactt gtctatgata ggcaggatgt tgggcaggct ccctttagtc atttacaagt 840

gaatcctgct gcagacaaca gtagggttca ttgcctaatg accccaccca tggttagctc 900

tgggtgcagg actgcaggag tggcagaggc ctggcaattc cccaagataa ctcgggtgat 960

gtcaaacaga cctgaattct gaggatcaat taaaggttgg agagaaagag attcctcttg 1020

atggtagtct tccatgatta aggtcaaata gaacactcta gtccctgatt tacttctctc 1080

atttatgaat gtagcctgga ggaattggct ggcttgctca gtgtcactca agaagttact 1140

ggcaggattg gatctgcagg ctccgagttc aagaccttta cattttggtt ttctagcatt 1200

gccaaaaaat ataaaagatg gatggactag tattagctgt gggagaaaat acagaagtac 1260

cttatcctac ctctcccaat actttcattg ctcacaaagc cccccagagg ctaagccaag 1320

cttggcttcc tcactcgtgg ctaatgctgg ctgtcatcag gaaacagcac agcctgttcg 1380

attcacaagg tggcacatgg aacccacaca ttcttggatt ccttagcaga gagagaagag 1440

gaagaaatgg ctgctttatg gggacacctg tttgctcagg ggatgtgtgc aactgtggag 1500

cagagtgagc atgctgcact catcagcttt gccaagctgt ctgagaggca gaagtccccc 1560

attttgcctg aagatacaga cgaagttgca tgctttacat ttttgaatag agggtagcat 1620

cgtagtacgt ggagagattt ccacataaaa acttgaaaac acatggctat ttcctttctt 1680

tcctttcctc tgtccctccc tctctttgct tgttggctga cccctcttta atgcctcata 1740

tagatcaagc tgtcctggag cttgaacctc taatcctcct gcacctacct cctgagtgct 1800

gggattacag gtatgtacca tgacaaccag gtttatgggg attcctgcat gctagttatt 1860

taatttttta atatttagtt atttatgaag gtattgggtg aaagggaaac taatattgat 1920

tctcctctat cccatgccct tgtcttaata atgacaaatg acaaatgcaa gcacacccct 1980

tgtacccaac atcaaagttc tttgtaatct gctctgttgt gggccatcca gtggctctcg 2040

aggaacaggg tatggctgaa gcggcagact caggttgaaa gagagggaac tagatggatg 2100

ggagaaagac cagagctagg acaagattct gatcaaagct caattttagt attcggaaca 2160

cggggttata aagaagggag aggggcccat tcctgccaaa tcatctttgg agtccagctg 2220

caggtgacca catgagtact ctggaacagc taggtggcag atagcagcag tgggagtggc 2280

agaacaatag tgtggcagac tccacccctc gatgatctcc tagtgataac agtcaagcct 2340

gatcagccag atttcaggca ggggagggag ggggaggtta catctcctcc ctgttgttaa 2400

tataaaaaag aaaggctggg ctgaggcata aaatttattt atgctctata gttctgcctg 2460

aattcttagg gtctaatgaa gaaacctttc tcagtgggat tccctaactt ttctcatggt 2520

aaacccttgt ttggttaaga ctgtcctttc tgtcctagta aacattttgc agactagccc 2580

tgagctgttg gatctgtctt ttgtaatgct aattagttac tgaataggta acctccttgc 2640

tgaattccta ctgaattcca agtagtcagc tacatgctct ttctaggaca ttggaacact 2700

ggtgaagact taactgtatt aggataaaat tttaaaaggc actatagtaa gataacgcta 2760

aaggagagta tgtggacctc cttaacagca ggcaggctgt taaggagggt gacaagcagg 2820

gaacaaagag gagggggtga tgaccggctc cttagcacaa ggccatttgg cttgttagga 2880

tgggaggctg tgaagggctt gccttttcac ggtgtggtct ccaacctctc tccccccttt 2940

caattaaatt taataaggcc acacttaact gaggtgatca aatgatcttc tcatctttgg 3000

atgagtgggc attaaccgtg gctgggggag cattgacccg attcctcccg tgggtgctgt 3060

catgacccac aatcatggct cttggtatct tttggggagg ggaggcagag ccttagaaaa 3120

atacttttgg ttgtaactgg aactagatac caacctccat ccaaatcggc tgtgtctttc 3180

agggaactca gaaatggtca agttggacct tcccctgcag tgcttagttt cgtacccatg 3240

ctggtgccca tactggattt gtattatcac gaaccagcat gcacagttct gtgtactgtg 3300

cagctcccaa gggagaattg tcaacctcag attcagcaag acctctacaa gaattatgcc 3360

acttgtaata ccacaatttt tggctcttag gctaatttag gagctggagg tcaccctcct 3420

catccctaat atttccacag cctgaggaac caaggggacc ttgcatttgc agcaagggtg 3480

gaaaactgga gaccagctaa ggttctaagc ccgtcttatc agaataaaaa caacattttt 3540

ctgatatctg aaatttctgc ctttctagcc ctcatagcaa accttaatcc caaatcagac 3600

caaaggccac aatatgtgct gggatgcttt tagaatgaaa ataagtacct gccccctcat 3660

gctcttgggg gcgggatgca cgcctgctgc agtcaggctg aagggtgttg attgacctgc 3720

ttgaaaaaca aaatccaggc tggtgagatg gctcagtggg taagagcacc cgactgctct 3780

tccaaaggtc cggattcttc cagtgataat catctgggct actgcttcat gatatggcaa 3840

gatattacat ttaggagaaa tcctcgaatg tatgcttgct ccttagtgat cttatgttgg 3900

agccttaagg tatggctaga gagatgaaac ttttcatgat cacgtttggc caaagcaaca 3960

ggatccgaaa ttaagtcttc tcctattaga gctctgtcaa tgcaatcatt gccttcagct 4020

aaaggtccag gaagaccaga gtgagattgt atgtggcc 4058

<210> 11

<211> 1963

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cggaaaatcc ccaaagtagg acatactttt ttccaaaagc ctgagagttg cccgcctgtg 60

ccaggaggta gtatgaagct tgacattggc atcatcaatg aaaaccagcg cgtttccatg 120

tcacgtaaca tcgagagccg ctccacctcc ccctggaatt acacgtaagt gcaaatgaga 180

ctgattttca cattctttgg aaaaatatgt taattcattc taatcataaa gtagaaaatg 240

tagaatacat agaaaaaata aaagggaaaa tcctcttaac aatgaaaatt tcactttacc 300

tgggagctca tatctttctt gttttggact aggttcacct ttgtggttga tggtcctaat 360

attttgcttc cacgttctca tagactttgt gcctttttct gcttcatgta gttctaggat 420

agaattcaca aattttcctg tgttggaaga gagggttctt taaagcagat tgcataatta 480

tctcctcatg aggataaatg ggaaacagaa aagtcggttt aaggagattc actcagatta 540

gacattatca tttaaggagt gggaatgagt ggaggagaaa ggccagtctg ggaaatggga 600

tgttaggtgg agaccttcag ggtacagtct tgtaatttgg cattaagata caggttcttg 660

gaagaccaag cactccctca tactctccaa atgtaagtat caaagtagat tccagggaca 720

attgaagtgc ttctggggtt gtcacatagc ttctcatgtg agtgtgttat gagaaaacct 780

tgggacggta gtgccccctg cctcgaactg ggaagatatg gagagatcaa agaatgactt 840

ggacaagtcc agcttagcga gtggatgagt ttattaagac ttactatggg gcgccctagg 900

gccacagcag gacagatcta gagatccgct ctgcctccca tctctaagct gctcttaagc 960

gaattttctg gctctttgcc tactgtgtgt gtccaatggg actgttttcc ttggtaggtt 1020

ctcagatact ctctgagatg tttgggttct cagtggcctg ctccttggct gtgcatggtg 1080

gccttggctc actgcccgtc cttcagggtt cagacagtgg acacacaccc tcaagtaacc 1140

tggtgggggt cctgtcacac tacaagtgga atatgtacaa tggcaagctg gcaggataaa 1200

tgttgtggtc tttatgcctc caggcagagg ggcctattaa aggagaaatg gagagaaggt 1260

ctgagattca cagacctctg gggctgtggc tttcctttcc aagcagcagg ctctgtagac 1320

cgactagtga tggaagactg actagtgacg ttcaatctcc aggcagagaa taaagaattg 1380

taataataat aataataatg ccaatgaaag atctaatgtt aattgaatgc taacatttgt 1440

acagtggggt attttatatg tacttgtatc actgagtatt ctcaacagtt gcatgagaaa 1500

atggtatatg aaaatcccca tcttatagtt gaggaaactg aggcttagaa aggtaagcca 1560

ctgccagagg ctggtgtatg cttccaggct gtgtggctcc agaacctgtg ccacagccct 1620

gcagaactgc ctccctgcag gtgaacactc agtgctgtgg ggctctcatt cgctgtcttc 1680

ctcccccatg cctcaccatt gtgctttggc ttgctcctcc tgcagtgtca cttgggaccc 1740

caaccggtac ccctcggaag ttgtacaggc ccagtgtagg aacttgggct gcatcaatgc 1800

tcaaggaaag gaagacatct ccatgaattc cgttcccatc cagcaagaga ccctggtcgt 1860

ccggaggaag caccaaggct gctctgtttc tttccagttg gagaaggtgc tggtgactgt 1920

tggctgcacc tgcgtcaccc ctgtcatcca ccatgtgcag taa 1963

<210> 12

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ctgcacctgc gtcacccctg tcatccacca tgtgcagtaa caacgctgca tacaaaaatc 60

agttgaagac ttccactgag 80

<210> 13

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tcatatctgc tttaggtcct gccttaagtt cctgccaaag gatatcgaat tccgaagttc 60

ctattctcta gaaagtatag 80

<210> 14

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtataggaac ttcatcagtc aggtacataa tggtggatcc cttctctcca tgatggaact 60

tgtaaacgta acaattccat 80

<210> 15

<211> 1178

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gacataccca ggaagacata cttagaagaa agtacttcct gagggaagaa gcagccattg 60

gagaaaccag catgaagtgc acccgtgaaa cagccatggt caagtctttg ctactgttga 120

tgttgggact tgccattctg agggaggtag cagctcggaa aatccccaaa gtaggacata 180

cttttttcca aaagcctgag agttgcccgc ctgtgccagg aggtagtatg aagcttgaca 240

ttggcatcat caatgaaaac cagcgcgttt ccatgtcacg taacatcgag agccgctcca 300

cctccccctg gaattacact gtcacttggg accccaaccg gtacccctcg gaagttgtac 360

aggcccagtg taggaacttg ggctgcatca atgctcaagg aaaggaagac atctccatga 420

attccgttcc catccagcaa gagaccctgg tcgtccggag gaagcaccaa ggctgctctg 480

tttctttcca gttggagaag gtgctggtga ctgttggctg cacctgcgtc acccctgtca 540

tccaccatgt gcagtaacaa cgctgcatac aaaaatcagt tgaagacttc cactgagaaa 600

aagcctcctt ttatccagag ctctgtgtga agcccgatct ccaagtcttt atgctttcta 660

ggactctcag taaggtgtgc atggcattct tgcagctctg cagtagatat agcttgaact 720

ttctggcttg ctttagaatt gttaccagtc ctggtgtgtt cccaatgcct cacttaaact 780

cctaaaaaca aaggtaaatg aaggcaccct tggcccagtc tctttgtgtt agatgatgac 840

cacagttctt gtctgtcaag actgtcactt tgatgcagta ccattcttgt agagttcaat 900

gaaatgctca tatttgtgat ggaaagaaaa caaacataat cagaatgcat gggaggaggc 960

ctgtgtagca ggggctggac tggcaaggaa gaagcacaca tggtatgtgt gtttttagag 1020

aaaaatttgg taaatataaa tgaagttggc ataaactatt atatatttat gttggtaggg 1080

gaactataca tatatttatg acctattgat tatatatcaa tttagtagaa aattaatata 1140

tccattaatt atataataaa atatttttaa ttcaaaaa 1178

<210> 16

<211> 161

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Lys Cys Thr Arg Glu Thr Ala Met Val Lys Ser Leu Leu Leu Leu

1 5 10 15

Met Leu Gly Leu Ala Ile Leu Arg Glu Val Ala Ala Arg Lys Ile Pro

20 25 30

Lys Val Gly His Thr Phe Phe Gln Lys Pro Glu Ser Cys Pro Pro Val

35 40 45

Pro Gly Gly Ser Met Lys Leu Asp Ile Gly Ile Ile Asn Glu Asn Gln

50 55 60

Arg Val Ser Met Ser Arg Asn Ile Glu Ser Arg Ser Thr Ser Pro Trp

65 70 75 80

Asn Tyr Thr Val Thr Trp Asp Pro Asn Arg Tyr Pro Ser Glu Val Val

85 90 95

Gln Ala Gln Cys Arg Asn Leu Gly Cys Ile Asn Ala Gln Gly Lys Glu

100 105 110

Asp Ile Ser Met Asn Ser Val Pro Ile Gln Gln Glu Thr Leu Val Val

115 120 125

Arg Arg Lys His Gln Gly Cys Ser Val Ser Phe Gln Leu Glu Lys Val

130 135 140

Leu Val Thr Val Gly Cys Thr Cys Val Thr Pro Val Ile His His Val

145 150 155 160

Gln

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccgaactata gtgactttca gtcttgct 28

<210> 18

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atttatcctg ccagcttgcc attgt 25

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gagaaaacct tgggacggta gtgcc 25

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ttagacaagg gagccaagtt cctgc 25

<210> 21

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tctctgttca gctcccaaga agtca 25

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgcccacgg tccagaaata ctat 24

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctcattgcat agcgtcatgt gaca 24

<210> 24

<211> 1567

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

acatccatcc catgcacagg gtaacatcaa tacactgcca accgttgttg ccttgtggaa 60

ctactgcatg cttacaggga tctttgggat ttatgaggat gtaggaaacc aagtcattgt 120

gcggtgagtc ctataaatga attatggact ccacacagtt aactctagag ataatgcctg 180

aaaccacaac actgtggcct cgaggattag ctactcagtg aacgaatgac ttcagaggcg 240

aaggcatatg gctgctgatt atctggagac aaatctgttt catcctgcag cttgctttca 300

atgtattatc agtgatcgta gtccagcagg ctgactccat gacctctttg tttattctgc 360

atatttcttg cctccaggat gttgaaaaga acaggcaaat ctttaaatcc aacatgcaca 420

gtcactttcc ctacctacac agtgtgagcc tgaacaacac ggagcccaga aatgaaaagc 480

tcagccttag ctcttctgcc ttaccatggg ctttgttctt ccccaggaag gtgtgagcag 540

ggacggcaaa ggacatttgc acccaagggg agctgttgag gttttagctc ttcttgtgtt 600

tagagctcca ctgagcactt tgtgtgcgct agtaacagca gcgaaactga tttcagtgtt 660

agtacagaaa agatagattc tagagctaaa agaaatgggt tctgcactct gagtgatgcc 720

aggcgtcatc agtgtccctg cttctttcaa ttagcatgga taaatgaaat tttgctgtgc 780

ttagtttccc ataatccttt tactcaatga gcgtggcata actatactgg ttaatttaac 840

tgggctgccc tgttcattca cttccccact ctctcattca ttcagaaagt tttcatggac 900

caactatgct ctcgcaggaa gttaactctg agtctggtat gaacacagat atgacatctt 960

tagagaacag aaaaaatact tcttgagtga tacatgctaa aatctttatt atttaatgtt 1020

gataaatata cttttaaaaa aatatttatt gtatgtataa gagttcactg tttctgcctt 1080

tagacacacc agaagagggc attggatgcc attacagatg gttgtgagcc actatgtggt 1140

tgctgggaac tgaactcagg acctctagaa gagcaatcag tgctcttagc tgtggagcca 1200

ttcctccatc ccaataaata tacttctaat tgaattacat atgggacctt gacagtaggg 1260

tgaggtttgg tggaatgtcc cagagaaagg ttttgtggac tataaggccg gagataaact 1320

ccagagagct ttactcctga ccctttctgc tttgtatcct gataacatta aacacggcag 1380

ttctcattgc tagtgaacca tgaaaaaaaa aaaaaaaaag aaaccttgat cctgtaaagt 1440

gaccctatac agagttcaag tacattgccc accaccaggg ctctaacctg tggtaatctc 1500

tattttccag gtcaagtctt tgctactgtt gatgttggga cttgccattc tgagggaggt 1560

agcagct 1567

<210> 25

<211> 1401

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

caacgctgca tacaaaaatc agttgaagac ttccactgag aaaaagcctc cttttatcca 60

gagctctgtg tgaagcccga tctccaagtc tttatgcttt ctaggactct cagtaaggtg 120

tgcatggcat tcttgcagct ctgcagtaga tatagcttga actttctggc ttgctttaga 180

attgttacca gtcctggtgt gttcccaatg cctcacttaa actcctaaaa acaaaggtaa 240

atgaaggcac ccttggccca gtctctttgt gttagatgat gaccacagtt cttgtctgtc 300

aagactgtca ctttgatgca gtaccattct tgtagagttc aatgaaatgc tcatatttgt 360

gatggaaaga aaacaaacat aatcagaatg catgggagga ggcctgtgta gcaggggctg 420

gactggcaag gaagaagcac acatggtatg tgtgttttta gagaaaaatt tggtaaatat 480

aaatgaagtt ggcataaact attatatatt tatgttggta ggggaactat acatatattt 540

atgacctatt gattatatat caatttagta gaaaattaat atatccatta attatataat 600

aaaatatttt taattcaaaa atatttgaag tctgtgctgg ttagtttttg ttaatgtggt 660

atatacttga gccacctggc aagaggaaag cttagttggg gaattgcccc tatcagattg 720

gtctctgggc atgtctgggg ggcattttct tggttaatgg ttgtttcggg agggcctagc 780

ccattgtggg tggtgcctgt tgtggggagg tggtcctgag ttatataaga aaacaagctg 840

ggcagaccat ggagaggaag ctactgagca gcactctgtc atcatatctg ctttaggtcc 900

tgccttaagt tcctgccaaa gcttctctcc atgatggaac ttgtaaacgt aacaattcca 960

ttcccctcta agttgttttt gttcatggtg tttaacatgg cagcacagaa agcagctaga 1020

caagtatttg cttctgcctt cagcttgaac tccaagactt tacaaccctc aacattttga 1080

acatctgtat atcatttgtt tttccaaatt tcctcctcag agctcccatg ttagagttat 1140

tgttattaat ggaactgagg tcccgttaca cttagaaaga gagttaaggg aaatggtgca 1200

aatggaattc aagtgtgggg ccagcatgtt acacttccag ccttctggcc acatcttgtt 1260

acaaagcaaa cacttactgt ccactttgct agcttctgga gttatgatgt ggagaaagat 1320

gtacgtagaa gagccactgt attggcagta gccaccccca gaaccctgtc attagggcca 1380

catggttcct ttgccttctt c 1401

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agcggttctg gaattcacgt ggg 23

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gctcggaaga accccaaagc agg 23

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cgaatcttca accaaaacca ggg 23

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atggggaact ggagcggttc tgg 23

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

acagtgttat cctccagggg agg 23

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ctctcacagt gttatcctcc agg 23

<210> 32

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tgggaactgt cctcccctgg agg 23

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ttcccagcct tctgcaaggc agg 23

<210> 34

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

agcgttgtca ggccgcttgg tgg 23

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tgcagcgttg tcaggccgct tgg 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

caggccgctt ggtggacaat ggg 23

<210> 37

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tcaggccgct tggtggacaa tgg 23

<210> 38

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gtggacaatg ggcttgacac agg 23

<210> 39

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

agggctgttc taattccttc agg 23

<210> 40

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gaaggaatta gaacagccct ggg 23

<210> 41

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gagaagatgc tcctaaaagt tgg 23

<210> 42

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

atggggaact ggagcggttc tgg 23

<210> 43

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

taggatgggg aactggagcg gttctgg 27

<210> 44

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

ccagaaccgc tccagttcca t 21

<210> 45

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

aaacccagaa ccgctccagt tccat 25

<210> 46

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

agcgttgtca ggccgcttgg tgg 23

<210> 47

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

taggagcgtt gtcaggccgc ttggtgg 27

<210> 48

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ccaccaagcg gcctgacaac gct 23

<210> 49

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

aaacccacca agcggcctga caacgct 27

<210> 50

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 51

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

gcatcatcaa tgaaaaccag cgcgt 25

<210> 52

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

agaaccctct cttccaacac aggaa 25

<210> 53

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

cctatctggg agttggtttg gggtc 25

<210> 54

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

gaactcggag cctgcagatc caatc 25

<210> 55

<211> 402

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

cggaaaatcc ccaaagtagg acatactttt ttccaaaagc ctgagagttg cccgcctgtg 60

ccaggaggta gtatgaagct tgacattggc atcatcaatg aaaaccagcg cgtttccatg 120

tcacgtaaca tcgagagccg ctccacctcc ccctggaatt acactgtcac ttgggacccc 180

aaccggtacc cctcggaagt tgtacaggcc cagtgtagga acttgggctg catcaatgct 240

caaggaaagg aagacatctc catgaattcc gttcccatcc agcaagagac cctggtcgtc 300

cggaggaagc accaaggctg ctctgtttct ttccagttgg agaaggtgct ggtgactgtt 360

ggctgcacct gcgtcacccc tgtcatccac catgtgcagt aa 402

<210> 56

<211> 102

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

gtcaagtctt tgctactgtt gatgttggga cttgccattc tgagggaggt agcagctcgg 60

aaaatcccca aagtaggaca tacttttttc caaaagcctg ag 102

<210> 57

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

actgttgatg ttgggacttg ccatt 25

<210> 58

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

tactgcacat ggtggatgac aggg 24

<210> 59

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

acccgtgaaa cagccatggt caa 23

<210> 60

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

agtcaccagc accttctcca actg 24

<210> 61

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

tcaggccgct tggtggacaa tg 22

<210> 62

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

acacaggtgc agccaacttt tagg 24

<210> 63

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

acctcattgg tgtcactgct actgc 25

<210> 64

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

attgcggtgg agattccaag gtgag 25

<210> 65

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

aggcaggaat cacaatccca cgaaa 25

<210> 66

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

tcctcattgc ggtggagatt ccaag 25

<210> 67

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

tccagggaga gcttcatctg tgtct 25

<210> 68

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

gctcactttt gcgccaaggg agtta 25

<210> 69

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

tccaaacact gaggccaagg acttc 25

<210> 70

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

accagcatct tctcgaccct gaaag 25

<210> 71

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

tcaccatctt ccaggagcga ga 22

<210> 72

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

gaaggccatg ccagtgagct t 21

Claims

1. A humanized IL17F protein, wherein the humanized IL17F protein comprises all or part of a human IL17F protein.

2. The humanized IL17F protein of claim 1, wherein the portion of humanized IL17F protein comprises an amino acid sequence encoded by all or part of exons 2 to 3 of the human IL17F gene.

3. The humanized IL17F protein of any one of claims 1-2, wherein the amino acid sequence of the humanized IL17F protein comprises one of the following group:

4. The humanized IL17F protein of any one of claims 1 to 3, wherein the amino acid sequence of the humanized IL17F protein comprises one of the following group:

I) SEQ ID NO: 16 amino acid sequence, in whole or in part;

5. A humanized IL17F gene, wherein said humanized IL17F gene comprises a portion of the human IL17F gene.

6. The humanized IL17F gene according to claim 5, characterized in that the humanized IL17F gene comprises all or part of human IL17F gene exon 1 to exon 3, preferably comprises all or part of human IL17F gene exon 2 to exon 3, further preferably comprises part of exon 2 and part of exon 3, wherein part of exon 2 comprises at least a 20bp nucleotide sequence and part of exon 2 preferably comprises a nucleotide sequence encoding SEQ ID NO: 8, 31-85, the part of exon 3 at least comprises 50bp of nucleotide sequence, and the part of exon 3 is from the 1 st nucleotide of exon 3 to the stop codon.

7. The humanized IL17F gene according to any of claims 5-6, wherein the humanized IL17F gene further comprises a portion of the non-human animal IL17F gene, preferably comprises all or part of exon 1 to exon 2 of the non-human animal IL17F gene, further preferably comprises all, part of exon 2 of exon 1 of the non-human animal IL17F gene, wherein part of exon 2 preferably comprises a nucleotide sequence encoding a signal peptide.

8. The humanized IL17F gene of any one of claims 5 to 7, wherein the humanized IL17F gene comprises a nucleotide sequence of the human IL17F gene comprising one of the following group:

9. The humanized IL17F gene of any one of claims 5 to 8, wherein the humanized IL17F gene comprises mRNA transcribed from the human IL17F gene comprising one of the following group:

10. The humanized IL17F gene of any one of claims 5-9, wherein the mRNA transcribed from the humanized IL17F gene comprises one of the following group:

(i) SEQ ID NO: 15, all or part of a nucleotide sequence set forth in seq id no;

11. A targeting vector, wherein said targeting vector comprises a portion of the human IL17F gene, preferably said targeting vector comprises all or part of exon 1 to exon 3 of the human IL17F gene, preferably comprises all or part of exon 2 to exon 3 of the human IL17F gene, further preferably comprises part of exon 2 and part of exon 3, wherein part of exon 2 comprises at least a 20bp nucleotide sequence, and part of exon 2 preferably comprises a nucleotide sequence encoding SEQ ID NO: 8, 31-85, the part of exon 3 comprises at least 50bp of nucleotide sequence, and the part of exon 3 extends from nucleotide 1 of exon 3 to the stop codon, and more preferably comprises nucleotide sequence similar to SEQ ID NO: 11 or a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 11 are identical.

12. The targeting vector according to claim 11, wherein said targeting vector further comprises a 5' arm selected from the group consisting of 100-10000 nucleotides in length of genomic DNA of the IL17F gene of a non-human animal; preferably, said 5' arm has at least 90% homology to NCBI accession No. NC _ 000067.6; further preferably, the 5' arm sequence is identical to SEQ ID NO: 9 or 24, or as shown in SEQ ID NO: 9 or 24; and/or, the targeting vector further comprises a 3' arm selected from 100-10000 nucleotides in length of the genomic DNA of the IL17F gene of the non-human animal; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000067.6; further preferably, the 3' arm sequence is identical to SEQ ID NO: 10 or 25 has at least 90% homology, or as shown in SEQ ID NO: 10 or 25.

13. A method for constructing a genetically humanized non-human animal, wherein said non-human animal expresses human or humanized IL17F protein.

14. The method of claim 13, wherein the humanized IL17F protein is the humanized IL17F protein of any one of claims 1-4.

15. The method of claim 13 or 14, wherein the genome of the non-human animal comprises a humanized IL17F gene, and the humanized IL17F gene comprises the humanized IL17F gene of any one of claims 5-10.

16. The method of constructing according to any one of claims 13-15, comprising introducing a portion of a human IL17F gene into the non-human animal IL17F locus, preferably said introduced portion of a human IL17F gene comprises all or part of exons 1 to 3 of the human IL17F gene, further preferably comprises all or part of exons 2 to 3 of the human IL17F gene, more preferably comprises part of exons 2 and part of exons 3, wherein said part of exons 2 comprises at least 20bp of nucleotide sequence, and said part of exons 2 preferably comprises a nucleotide sequence encoding SEQ ID NO: 8, 31-85, said portion of exon 3 comprising at least 50bp of the nucleotide sequence, preferably said portion of exon 3 extends from nucleotide 1 of exon 3 to the stop codon, more preferably said portion of the introduced human IL17F gene comprises a nucleotide sequence identical to SEQ ID NO: 11 or a nucleotide sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95% or at least 99% identity to SEQ ID NO: 11, or a nucleotide sequence identical to the nucleotide sequence shown in figure 11.

17. The construct of any of claims 13-16, which comprises introducing a portion of the human IL17F gene into exons 2 to 3 of a non-human animal.

18. The construct of any of claims 13-17, wherein the human IL17F gene or the nucleotide sequence encoding human IL17F protein is regulated by endogenous regulatory elements.

19. The method of any one of claims 13 to 18, wherein the targeting vector of any one of claims 11 to 12 is used to construct a non-human animal.

20. The construct of any of claims 13-19, wherein 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.

21. The method of constructing according to claim 20, wherein the non-human mammal further expresses at least one of human or humanized IL17A, IL12, IL23, IL4R and IL6 proteins.

22. The method of claim 21, wherein the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of human IL17A gene.

23. The method of claim 21 or 22, wherein the non-human animal comprises a human or humanized IL17A gene, preferably wherein the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene, more preferably wherein the humanized IL17A gene comprises part of exon 1, exon 2 and exon 3 of human IL17A gene, wherein the part of exon 1 comprises at least 10bp of nucleotide sequence, the part of exon 1 comprises from the start codon to the last nucleotide of exon 1, and the part of exon 3 comprises at least 100bp of nucleotide sequence, preferably the part of exon 3 comprises from the first nucleotide of exon 3 to the stop codon.

24. The construct of any of claims 21-23, wherein the construct comprises introducing into the non-human animal IL17A locus all or part of exon 1 to exon 3 of the human IL17A gene, preferably comprises introducing into the non-human animal IL17A locus all or part of exon 1 to exon 3 of the human IL17A gene, wherein said part of exon 1 comprises at least 10bp of nucleotide sequence, preferably said part of exon 1 comprises at least 100bp of nucleotide sequence from the start codon to the last nucleotide of exon 1, and said part of exon 3 comprises at least 100bp of nucleotide sequence, preferably said part of exon 3 comprises from the first nucleotide of exon 3 to the stop codon.

25. A cell, tissue or organ that is humanized by a gene, wherein the genome of said cell, tissue or organ comprises the humanized IL17F gene and/or the humanized IL17A gene of any one of claims 5 to 10, and said cell, tissue or organ expresses the humanized IL17F protein and/or the humanized IL17A protein of claims 1 to 4.

26. The cell, tissue or organ of claim 25, wherein the humanized IL17A protein comprises an amino acid sequence encoded by all or part of exons 1 to 3 of the human IL17A gene.

27. The cell, tissue or organ of claim 25 or 26, wherein the humanized IL17A gene comprises all or part of exons 1 to 3 of human IL17A gene, and further preferably, the humanized IL17A gene comprises part of exon 1, exon 2 and exon 3 of human IL17A gene, wherein the part of exon 1 comprises at least 10bp of nucleotide sequence, the part of exon 1 comprises the last nucleotide of the exon 1, and the part of exon 3 comprises at least 100bp of nucleotide sequence, and preferably, the part of exon 3 comprises the first nucleotide of exon 3 comprises the stop codon.

28. Use of a cell, tissue or organ derived from a humanized IL17F protein according to any one of claims 1 to 4, a humanized IL17F gene according to any one of claims 5 to 10, a non-human animal obtained by the construction process according to any one of claims 13 to 24, or a cell, tissue or organ according to any one of claims 25 to 27 in product development requiring an immune process involving human cells, in the manufacture of antibodies, or as a model system for pharmacological, immunological, microbiological or 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 IL17F channel function, human IL17F channel signal mechanism, human-targeting antibody, human-targeting drug, drug effect, inflammation drug, immune-related disease drug and anti-tumor drug, screening and evaluating human drug and drug effect research.