CN114835798B

CN114835798B - Construction method and application of CD36 gene humanized non-human animal

Info

Publication number: CN114835798B
Application number: CN202210590458.1A
Authority: CN
Inventors: 刘畅; 尚诚彰; 李惠琳
Original assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Current assignee: Baccetus Beijing Pharmaceutical Technology Co ltd
Priority date: 2021-05-28
Filing date: 2022-05-27
Publication date: 2024-02-23
Anticipated expiration: 2042-05-27
Also published as: WO2022247936A1; CN114835798A

Abstract

The invention provides a humanized CD36 protein, a humanized CD36 gene, a targeting vector of the CD36 gene, a CD36 gene humanized non-human animal, a construction method thereof and application thereof in the field of biological medicine, and a nucleotide sequence for encoding the human CD36 protein is introduced into a genome of the non-human animal by utilizing a homologous recombination mode, so that the human or humanized CD36 protein can be normally expressed in the animal body, and the humanized CD36 protein can be used as an animal model for human CD36 signal mechanism research and tumor and autoimmune disease drug screening, and has important application value for developing new drugs of immune targets.

Description

Construction method and application of CD36 gene humanized non-human animal

Technical Field

The invention belongs to the fields of animal genetic engineering and genetic modification, and particularly relates to a construction method of a CD36 gene modified non-human animal model and application thereof in the field of biological medicine.

Background

CD36 (Cluster of differentiation 36) is also known as platelet glycoprotein 4, fatty Acid Translocase (FAT), scavenger receptor class B member 3 (SCARB 3), glycoprotein 88 (GP 88), GPIIB, GPIV, members of the cell surface protein class B scavenger receptor family. Mainly expressed on the surfaces of capillary endothelial cells, mammary gland secretion epithelial cells, differentiated fat cells, platelets, erythrocytes, monocytes, B cells, macrophages, skeletal muscle cells and the like. CD36 is also expressed on a variety of tumor cells and is associated with poor prognosis for breast, prostate, ovarian, colon, oral squamous cell carcinoma, and acute myelogenous leukemia. The ligand comprises: collagen, thrombospondin (TSP-1), erythrocytes parasitized by plasmodium falciparum, oxidized low density lipoprotein (oxLDL), natural lipoprotein, oxidized phospholipid, long chain Fatty Acid (FA), etc., CD36 functional diversity depends on binding of different ligands: a. as a lipid aminotransferase, CD36 recognizes and endocytoses denatured LDL (oxLDL and AcLDL), resulting in macrophage foaming, secretion of various inflammatory cytokines, and initiation of atherosclerosis; b. on macrophages, CD36 forms part of a non-opsonin receptor (CD 36/αvβ3 complex), involved in phagocytosis; c. CD36 on macrophages and DC cells can also bind the heat shock protein gp96, stimulating an immune response in the body by releasing cytokines, chemokines and nitrogen oxides; d. recognizing exogenous substances such as red blood cells infected with plasmodium falciparum and cell wall components of staphylococci and mycobacteria, exerting innate immune functions; e. recognizing endogenous ligands, CD36 on macrophages can bind to beta-amyloid peptide, playing a role in Alzheimer's disease; f. promoting aseptic inflammation by assembly of TLR2/6 or TLR4/6 heterodimers; g. in the tumor microenvironment, CD36 on tumor microvascular endothelial cells promotes vascular apoptosis by binding to TSP-1, while recruiting Src family members to mediate VEGFR2 dephosphorylation, inhibiting the VEGF pathway, resulting in anti-angiogenesis. In addition, CD36 activates Wnt/TGF-beta signals to promote tumor metastasis through EMT, and absorbs lipid molecules to enable lipid deposition in immune cells, so that aseptic inflammation and antigen presenting dysfunction in DC cells are caused, thereby inducing tumor immunosuppression and initiating growth, invasion and metastasis of tumor cells.

The experimental animal disease model is an indispensable research tool for researching the etiology and pathogenesis of human disease occurrence, developing control technology and developing medicines. However, due to the differences of physiological structures and metabolic systems of animals and humans, the traditional animal model cannot well reflect the real conditions of human bodies, and establishing a disease model in animals, which is closer to the physiological characteristics of humans, is an urgent need of the biomedical industry.

With the continuous development and maturation of genetic engineering techniques, the substitution or replacement of animal homologous genes with human genes has been achieved, and the development of a humanized experimental animal model in this way is a future development direction of animal models. Wherein the humanized animal model of the gene, namely, utilize the gene editing technology, replace the homologous gene of animal genome with the normal or mutated gene of human origin, can set up the normal or mutated gene animal model more similar to human physiology or disease characteristic. The humanized animal has important application value, such as the humanized animal model transplanted by cells or tissues can be improved and promoted by gene humanized, and more importantly, the humanized protein can be expressed or partially expressed in the animal body due to the insertion of human gene fragments, can be used as a target spot of a medicament capable of only recognizing human protein sequences, and provides possibility for screening anti-human antibodies and other medicaments at animal level. However, due to differences in physiology and pathology between animals and humans, coupled with the complexity of genes, e.g., 84% identity of human and mouse CD36 proteins, it remains a major challenge to construct "efficient" humanized animal models for new drug development.

In view of the complex mechanism of action of CD36 and the great application value in the field of tumor therapy, in order to further explore its related biological characteristics, the effectiveness of preclinical pharmacodynamic tests is improved, the success rate of research and development is improved, preclinical tests are more effective, and research and development failure is minimized, and there is an urgent need in the art to develop non-human animal models of CD36 related signaling pathways. In addition, the non-human animal obtained by the method can also be mated with other humanized non-human animals to obtain a polygenic humanized animal model, which is used for screening and evaluating the study of the drug effect of the human drug and the combined drug aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

In a first aspect of the invention, there is provided a humanized CD36 protein, said humanized CD36 protein comprising all or part of a human CD36 protein.

Preferably, the humanized CD36 protein comprises all or a portion of a transmembrane region, cytoplasmic region and/or extracellular region of a human CD36 protein, and more preferably, the humanized CD36 protein comprises all or a portion of an extracellular region of a human CD36 protein.

In a specific embodiment of the invention, the humanized CD36 protein comprises all or a portion of an extracellular region of a human CD36 protein, preferably, an extracellular region of a human CD36 protein comprising at least 50 consecutive amino acids, e.g., an extracellular region of a human CD36 protein comprising at least 50, 70, 100, 130, 150, 170, 200, 230, 250, 270, 300, 320, 350, 360, 370, 390, 400, 405, 406, 407, 408, 409, 410 consecutive amino acids, further preferably, an extracellular region of a human CD36 protein comprising 410 consecutive amino acids; more preferably, the amino acid sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 30-439 of SEQ ID NO. 2 or the amino acid sequence comprising positions 30-439 of SEQ ID NO. 2.

Preferably, the humanized CD36 protein further comprises a portion of a non-human animal CD36 protein, and more preferably, the portion of a non-human animal CD36 protein is all or part of the transmembrane and/or cytoplasmic regions of a non-human animal CD36 protein. Preferably comprising SEQ ID NO:1 from position 1 to position 29 and/or from position 440 to 472; alternatively, comprising a sequence identical to SEQ ID NO: amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% as shown at positions 1-29 and/or 440-472; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequences shown at positions 1-29 and/or positions 440-472 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:1 from position 1 to 29 and/or from position 440 to 472, comprising substitutions, deletions and/or insertions of one or more amino acid residues, or, preferably, comprising the amino acid sequence of SEQ ID NO:1, from positions 8 to 29, from positions 440 to 461, from positions 1 to 7 or from positions 462 to 472; alternatively, comprising a sequence identical to SEQ ID NO: amino acid sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% as shown at positions 8-29, 440-461, 1-7 or 462-472 of 1; alternatively, comprising a sequence identical to SEQ ID NO: the amino acid sequences shown at positions 1, 8-29, 440-461, 1-7 or 462-472 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; alternatively, comprising a sequence identical to SEQ ID NO:1, from positions 8 to 29, from positions 440 to 461, from positions 1 to 7, or from positions 462 to 472, comprising substitutions, deletions, and/or insertions of one or more amino acid residues.

Preferably, the humanized CD36 protein comprises all or a portion of the amino acid sequence encoded by exons 1 to 14 of a human CD36 gene. It is further preferred that all or part of the amino acid sequence encoded by a combination of any one, two, three or more, two or more consecutive exons from exon 1 to exon 14 is comprised. Still more preferably, all or part of the amino acid sequence encoded by exons 3 to 14 is included. Still further preferred, the portion comprising exon 3, all of exons 4 to 13 and exon 14, wherein the portion of exon 3 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp, further preferred, comprises a nucleotide sequence of 33 bp; preferably, the portion of exon 3 comprises a nucleotide sequence encoding an extracellular region of exon 3, and the portion of exon 14 comprises at least a 30bp nucleotide sequence, e.g., at least a 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 439, 440bp nucleotide sequence, further preferably, a 63bp nucleotide sequence; preferably, the portion of exon 14 comprises a nucleotide sequence encoding an extracellular region of exon 14. Most preferably, the humanized CD36 protein comprises a sequence identical to SEQ ID NO:5 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the amino acid sequence encoded by SEQ ID NO:5, and a coding amino acid sequence.

Preferably, the transmembrane and cytoplasmic regions of the humanized CD36 protein are derived from a non-human animal.

Preferably, the humanized CD36 protein further comprises all or part of an amino acid sequence encoded by a CD36 gene of a non-human animal, preferably all or part of an amino acid sequence encoded by exons 1-3 and/or exon 16 of a CD36 gene of a non-human animal, more preferably all or part of an amino acid sequence encoded by a part of exon 4 and/or a part of exon 15.

In one embodiment of the invention, the amino acid sequence of the humanized CD36 protein comprises one of the group consisting of:

a) Is SEQ ID NO:2 from amino acid position 30 to 439;

b) And SEQ ID NO:2 at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) And SEQ ID NO: the amino acid sequence shown at positions 30-439 of 2 differs by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or (b)

D) And SEQ ID NO:2, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a specific embodiment of the present invention, the amino acid sequence of the humanized CD36 protein is selected from one of the following groups:

i) Is SEQ ID NO:11 or a portion of the amino acid sequence;

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

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

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

Preferably, the non-human animal is selected from any non-human animal that can be genetically edited to produce a humanized gene, such as rodents, pigs, rabbits, monkeys, etc.

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 mouse.

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

In a second aspect of the invention, there is provided a nucleic acid encoding a humanized CD36 protein as described above.

In a third aspect of the invention, there is provided a humanized CD36 gene, said humanized CD36 gene comprising a portion of a human CD36 gene and a portion of a non-human animal CD36 gene.

Preferably, the humanized CD36 gene encodes a humanized CD36 protein as described above.

Preferably, the humanized CD36 gene comprises all or a portion of a nucleotide sequence encoding a transmembrane region, cytoplasmic region and/or extracellular region of a human CD36 protein, wherein the humanized CD36 gene comprises all or a portion of a nucleotide sequence encoding an extracellular region of a human CD36 protein, preferably, a nucleotide sequence encoding an extracellular region of a human CD36 protein of at least 50 consecutive amino acids, such as at least a nucleotide sequence encoding an extracellular region of a human CD36 protein of 50, 70, 100, 130, 150, 170, 200, 230, 250, 270, 300, 320, 350, 360, 370, 390, 400, 405, 406, 407, 408, 409, 410 consecutive amino acids, more preferably, a nucleotide sequence encoding an extracellular region of a human CD36 protein of 410 consecutive amino acids; more preferably, the nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequence encoding positions 30-439 of SEQ ID NO. 2 or the nucleotide sequence comprising positions 30-439 of SEQ ID NO. 2.

Preferably, the humanized CD36 gene comprises all or part of exons 1 to 14 of a human CD36 gene. It is further preferred that all or part of a combination comprising any one, two, three or more, two or more consecutive exons from exon 1 to exon 14. Still more preferably, all or part of exons 3 to 14 are included. Still further preferred, the portion comprising exon 3, all of exon 4 to exon 13 and exon 14, preferably further comprises intron 3-4 and/or intron 13-14, more preferred any of the introns 3-14, wherein the portion of exon 3 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp, further preferred a nucleotide sequence of 33 bp; preferably, the portion of exon 3 comprises a nucleotide sequence encoding an extracellular region of exon 3, and the portion of exon 14 comprises at least a 30bp nucleotide sequence, e.g., at least a 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 439, 440bp nucleotide sequence, further preferably, a 63bp nucleotide sequence; preferably, the portion of exon 14 comprises a nucleotide sequence encoding an extracellular region of exon 14.

Preferably, the part of the non-human animal CD36 gene is exons 1-3 and/or 16 of the non-human animal CD36 gene; further preferably also comprises a part of exon 4 and/or a part of exon 15 of the non-human animal.

Preferably, the humanized CD36 gene further comprises a nucleotide sequence encoding a transmembrane region and/or an intracellular region of a CD36 protein of a non-human animal.

In one embodiment of the invention, the humanized CD36 gene further comprises a nucleotide sequence encoding SEQ ID NO:1 from positions 1 to 29 and/or from positions 440 to 472; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO: nucleotide sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% at positions 1-29 and/or 440-472; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO: the nucleotide sequences at positions 1 to 29 and/or positions 440 to 472 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO:1 from position 1 to 29 and/or from position 440 to 472, comprising a nucleotide sequence comprising one or more substitutions, deletions and/or insertions of nucleotides, or the humanized CD36 gene further comprises a nucleotide sequence encoding SEQ ID NO:1, from positions 8 to 29, from 440 to 461, from 2 to 7 or from 462 to 472; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO: nucleotide sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% at positions 8-29, 440-461, 2-7 or 462-472; alternatively, it comprises a sequence identical to the sequence encoding SEQ ID NO: the nucleotide sequences at positions 1, 8-29, 440-461, 2-7 or 462-472 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; alternatively, it comprises a polypeptide having a sequence encoding SEQ ID NO:1, from positions 8-29, from 440-461, from 2-7, or from 462-472, including nucleotide sequences in which one or more nucleotides are substituted, deleted, and/or inserted.

In one embodiment of the invention, the humanized CD36 gene comprises a sequence identical to SEQ ID NO:6 and/or 7, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity, or comprising the nucleotide sequence of SEQ ID NO:6 and/or 7.

In one embodiment of the invention, the humanized CD36 gene comprises one of the following groups:

(A) Is SEQ ID NO:5 or a portion thereof;

(B) And SEQ ID NO:5 is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

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

(D) Has the sequence of SEQ ID NO:5, including substitution, deletion and/or insertion of one or more nucleotides.

In one embodiment of the invention, the mRNA transcribed from the humanized CD36 gene is selected from one of the following groups:

(i) Is SEQ ID NO:10 or a portion of the nucleotide sequence set forth in seq id no;

(ii) And SEQ ID NO:10 is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) And SEQ ID NO:10 does not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or not more than 1 nucleotide; or (b)

(iv) And SEQ ID NO:10, comprising substitutions, deletions and/or insertions of one or more nucleotides.

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

Preferably, the humanized CD36 gene further comprises a specific inducer or repressor. Further preferably, the specific inducer or repressor may be a substance that is conventionally inducible or repressible. In one embodiment of the invention, the specific inducer is selected from the group consisting of the tetracycline System (Tet-Off System/Tet-On System) and the Tamoxifen System (Tamoxifen System).

In a fourth aspect of the invention, there is provided a targeting vector comprising a donor nucleotide sequence comprising a portion of the human CD36 gene.

Preferably, the portion of the human CD36 gene comprises all or part of exons 1 to 14 of the human CD36 gene. Further preferred are combinations comprising any one, two, three or more, two or more consecutive exons from exon 1 to exon 14. Still further preferred, comprises all or part of exons 3 to 14. Even more preferably, the portion comprising exon 3, all of exons 4 to 13 and exon 14, wherein the portion of exon 3 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp, even more preferably, comprises a nucleotide sequence of 33 bp; preferably, the portion of exon 3 comprises a nucleotide sequence encoding an extracellular region of exon 3, and the portion of exon 14 comprises at least a 30bp nucleotide sequence, e.g., at least a 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 439, 440bp nucleotide sequence, further preferably, a 63bp nucleotide sequence; preferably, the portion of exon 14 comprises a nucleotide sequence encoding an extracellular region of exon 14. Most preferably, the targeting vector comprises a sequence identical to SEQ ID NO:5 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity or comprising SEQ ID NO: 5.

Preferably, the targeting vector comprises a nucleotide sequence encoding a human CD36 protein or a humanized CD36 protein as described above.

Preferably, the targeting vector comprises a nucleotide sequence encoding the extracellular region of human CD36 protein.

Preferably, the targeting vector comprises the nucleotide sequence of the humanized CD36 gene described above.

Preferably, the targeting vector further comprises a DNA fragment homologous to the 5' end of the switching region to be altered, i.e.a 5' arm (5 ' homology arm), selected from the group consisting of nucleotides of 100-10000 in length of genomic DNA of the CD36 gene of the non-human animal. Further preferred, the 5' arm has at least 90% homology to NCBI accession number NC_ 000071.7. Still further preferred, the 5' arm sequence hybridizes to SEQ ID NO:3, or a sequence as set forth in SEQ ID NO: 3. And/or, the targeting vector further comprises a DNA fragment homologous to the 3' -end of the transition region to be changed, i.e., a 3' -arm (3 ' -homology arm), selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the CD36 gene of the non-human animal. Further preferred, the 3' arm has at least 90% homology to NCBI accession number NC_ 000071.7. Still further preferred, the 3' arm sequence hybridizes to SEQ ID NO:4, or a sequence as set forth in SEQ ID NO: 4.

In one embodiment of the invention, the targeting vector comprises a sequence identical to SEQ ID NO: 6. 7, 8 and/or 9, or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity, or comprising SEQ ID NO: 6. 7, 8 and/or 9.

Preferably, the transition region to be altered is located at the non-human animal CD36 locus. Further preferably, it is located on exons 1 to 16 of the CD36 gene. Still more preferably, it is located on exons 4 to 15.

Preferably, the non-human animal is an immunodeficient non-human mammal. Further preferably, the immunodeficient non-human mammal is immuneEpidemic-deficient rodents, immunodeficient pigs, immunodeficient rabbits or immunodeficient monkeys. Still more preferably, the immunodeficient rodent is an immunodeficient mouse or rat. Most preferably, the immunodeficient mouse is NOD-Prkdc ^scid IL-2rγ ^null Mouse, NOD-Rag 1 ^-/- -IL2rg ^-/- (NRG) mice, rag 2 ^-/- -IL2rg ^-/- (RG) mice, NOD/SCID mice or nude mice.

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 the diphtheria toxin A subunit encoding gene (DTA).

In one embodiment of the invention, the targeting vector further comprises a resistance gene selected from positive clones. Further preferably, the resistance gene screened 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 (conventional LoxP recombination systems may also be selected). The specific recombination system is provided with two Frt recombination sites which are respectively connected with two sides of the resistance gene.

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

In a sixth aspect of the invention there is provided the use of a targeting vector as described above, or a cell as described above, in the modification of the CD36 gene, preferably said use including but not limited to knockdown, insertion or substitution.

In a seventh aspect of the invention there is provided a non-human animal humanized with the CD36 gene, said non-human animal expressing human or humanized CD36 protein in vivo.

Preferably, the non-human animal has reduced or absent expression of endogenous CD36 protein.

Preferably, the humanized CD36 protein is expressed in the non-human animal.

Preferably, a portion of the human CD36 gene or the nucleotide sequence of the humanized CD36 gene is operably linked to endogenous regulatory elements at an endogenous CD36 locus in at least one chromosome.

Preferably, the non-human animal body comprises a portion of the human CD36 gene, more preferably comprises the humanized CD36 gene described above.

Preferably, the genome of the non-human animal comprises a portion of the human CD36 gene. Further preferably, the genome of the non-human animal comprises all or part of exons 1 to 14 of the human CD36 gene. It is further preferred that all or part of a combination comprising any one, two, three or more, two or more consecutive exons from exon 1 to exon 14. Still more preferably, all or part of exons 3 to 14 are included. Still further preferred, the portion comprising exon 3, all of exon 4 to exon 13 and exon 14, preferably further comprises intron 3-4 and/or intron 13-14, more preferred any of the introns 3-14, wherein the portion of exon 3 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp, further preferred a nucleotide sequence of 33 bp; preferably, the portion of exon 3 comprises a nucleotide sequence encoding an extracellular region of exon 3, and the portion of exon 14 comprises at least a 30bp nucleotide sequence, e.g., at least a 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 439, 440bp nucleotide sequence, further preferably, a 63bp nucleotide sequence; preferably, the portion of exon 14 comprises a nucleotide sequence encoding an extracellular region of exon 14.

In a specific embodiment of the invention, the genome of the non-human animal comprises a nucleotide sequence encoding an extracellular region of a human CD36 protein, wherein the genome of the non-human animal comprises all or part of a nucleotide sequence encoding an extracellular region of a human CD36 protein, preferably comprises a nucleotide sequence encoding an extracellular region of a human CD36 protein of at least 50 consecutive amino acids, e.g. comprises at least a nucleotide sequence encoding an extracellular region of a human CD36 protein of 50, 70, 100, 130, 150, 170, 200, 230, 250, 270, 300, 320, 350, 360, 370, 390, 400, 405, 406, 407, 408, 409, 410 consecutive amino acids, further preferably comprises a nucleotide sequence encoding an extracellular region of a human CD36 protein of 410 consecutive amino acids; more preferably, the nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequence encoding positions 30-439 of SEQ ID NO. 2 or the nucleotide sequence comprising positions 30-439 of SEQ ID NO. 2.

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

According to some embodiments of the invention, the CD36 gene and/or the other gene is homozygous or heterozygous for the endogenous replaced locus.

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

In an eighth aspect of the present invention, there is provided a non-human animal having a deletion of all or part of exons 1 to 16 of an endogenous CD36 gene. Preferably, the non-human animal lacks all or part of exons 4 to 15 of the endogenous CD36 gene. More preferably, the non-human animal lacks part of exon 4, all of exons 5 to 14 and part of exon 15 of the endogenous CD36 gene.

In a ninth aspect of the present invention, a construction method of a non-human animal with a CD36 gene deletion is provided, the construction method comprising preparing the non-human animal using the targeting vector described above.

In a tenth aspect of the present invention, there is provided a method for constructing a non-human animal humanized with a CD36 gene, said method comprising introducing a CD36 locus of a non-human animal with a partial nucleotide sequence comprising a human CD36 gene, said non-human animal expressing a human or humanized CD36 protein in vivo.

Preferably, the humanized CD36 protein comprises a humanized CD36 protein as described above.

Preferably, the genome of the non-human animal further comprises a humanized CD36 gene, and the humanized CD36 gene comprises the humanized CD36 gene.

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

Preferably, the construction method comprises introducing a nucleotide sequence comprising a nucleotide sequence encoding a human CD36 protein or a humanized CD36 protein as described above into a CD36 locus of a non-human animal.

Preferably, the construction method comprises introducing a nucleotide sequence comprising an extracellular region encoding human CD36 protein into a CD36 locus of a non-human animal.

Preferably, the construction method comprises introducing a nucleotide sequence comprising the humanized CD36 gene described above into a CD36 locus of a non-human animal.

Preferably, the partial nucleotide sequence of the human CD36 gene comprises all or part of the nucleotide sequence of exons No. 1 to 14 of the human CD36 gene, more preferably, comprises all or part of any one, two, three or more, two or more consecutive combinations of three or more exons No. 1 to 14, still more preferably, comprises all or part of the nucleotide sequence of exons No. 3 to 14. Most preferably, the portion comprising exon 3, all of exon 4 to exon 13 and exon 14, preferably further comprises intron 3-4 and/or intron 13-14, more preferably any of the introns 3-14, wherein the portion of exon 3 comprises at least a nucleotide sequence of 10bp, e.g. at least a nucleotide sequence of 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp, further preferably a nucleotide sequence of 33 bp; preferably, the portion of exon 3 comprises a nucleotide sequence encoding an extracellular region of exon 3, and the portion of exon 14 comprises at least a 30bp nucleotide sequence, e.g., at least a 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 70, 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 439, 440bp nucleotide sequence, further preferably, a 63bp nucleotide sequence; preferably, the portion of exon 14 comprises a nucleotide sequence encoding an extracellular region of exon 14. In one embodiment of the invention, a polypeptide comprising a sequence identical to SEQ ID NO:5 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity or comprising SEQ ID NO: 5.

Preferably, the construction method comprises introducing a non-human animal CD36 locus with a nucleic acid sequence comprising all or part of a nucleotide sequence encoding a human CD36 protein. Further preferred, the construction method comprises introducing a non-human animal CD36 locus with all or part of a nucleotide sequence comprising a transmembrane region, cytoplasmic region and/or extracellular region encoding a human CD36 protein; preferably, the non-human animal CD36 locus is introduced with a nucleotide sequence comprising all or part of an extracellular region encoding a human CD36 protein, wherein said extracellular region comprises all or part of an extracellular region of a human CD36 protein, preferably an extracellular region of a human CD36 protein comprising at least 50 consecutive amino acids, e.g. an extracellular region of a human CD36 protein comprising at least 50, 70, 100, 130, 150, 170, 200, 230, 250, 270, 300, 320, 350, 360, 370, 390, 400, 405, 406, 407, 408, 409, 410 consecutive amino acids, further preferably an extracellular region of a human CD36 protein comprising 410 consecutive amino acids; more preferably, the nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 30-439 of SEQ ID NO. 2 or the amino acid sequence comprising positions 30-439 of SEQ ID NO. 2, and even more preferably, the nucleotide sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequence comprising positions 30-439 of SEQ ID NO. 2 or the nucleotide sequence comprising positions 30-439 of SEQ ID NO. 2 is used for introducing into the non-human animal CD36 gene locus.

In one embodiment of the invention, the non-human animal CD36 locus is introduced with a cDNA sequence comprising a sequence encoding a human CD36 protein.

In one embodiment of the invention, the non-human animal CD36 locus is introduced with a nucleotide sequence comprising a sequence encoding a humanized CD36 protein.

In one embodiment of the invention, the nucleotide sequence comprising the humanized CD36 gene is used to introduce the non-human animal CD36 locus.

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

Preferably, the introducing is a substitution or insertion, and in one embodiment of the invention, the non-human animal CD36 locus is a substitution of the corresponding region of the non-human animal, more preferably, all or part of exons 4 to 15 of the non-human animal CD36 gene is substituted, even more preferably, part of exons 4, 5 to 14 and part of exons 15 of the non-human animal CSF1 gene are substituted.

Preferably, the nucleotide sequence encoding the extracellular region of the endogenous CD36 protein in the genome of the non-human animal is replaced, and more preferably, the nucleotide sequence encoding positions 30-439 of SEQ ID NO. 1 in the genome of the non-human animal is replaced.

Preferably, the site of insertion or substitution is subsequent to the endogenous regulatory element of the CD36 gene.

Preferably, the insertion is performed by first disrupting the coding box of the endogenous CD36 gene of the non-human animal or disrupting the coding box of the endogenous CD36 gene after the insertion sequence. Or the step of inserting can not only cause frame shift mutation to the endogenous CD36 gene, but also realize the step of inserting human sequence.

It is further preferred that an auxiliary sequence (e.g., a stop codon or a sequence containing a stop function, etc.) or other means (e.g., a flip sequence, or a knockout sequence) can be added to the insertion sequence such that the non-human animal endogenous CD36 protein after the insertion site is not normally expressed.

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

Preferably, the regulatory elements include, but are not limited to, endogenous promoters.

In one embodiment of the invention, the endogenous regulatory element is derived from the non-human animal CD36 gene. The exogenous regulatory element is derived from the human CD36 gene.

Preferably, the human or humanized CD36 gene is homozygous or heterozygous for the endogenous replaced CD36 in the non-human animal.

Preferably, the humanized CD36 gene is contained on at least one chromosome in the genome of the non-human animal.

Preferably, at least one cell in said non-human animal expresses human or humanized CD36 protein.

Preferably, the construction of the non-human animal is performed using gene editing techniques including gene targeting techniques using embryonic stem cells, regular clustered interval short palindromic repeat (CRISPR/Cas 9) techniques, zinc Finger Nuclease (ZFN) techniques, transcription activator-like effector nuclease (TALEN) techniques, homing endonucleases (megabase megaribozymes) or other molecular biology techniques.

In one embodiment of the invention, the construction method comprises modifying the coding box of the non-human animal CD36 gene, inserting a nucleotide sequence encoding a human or humanized CD36 protein or a nucleotide sequence of a humanized CD36 gene into an endogenous regulatory element of the non-human animal CD36 gene. Wherein, the coding frame of the modified non-human animal CD36 gene can be a functional region of the non-human animal CD36 gene or a sequence inserted into the functional region, so that the non-human animal CD36 protein is not expressed or the protein with reduced expression or expression is not functional. Further preferably, the coding box of the modified non-human animal CD36 gene may be the entire or partial nucleotide sequence of exon 4 to exon 15 of the non-human animal CD36 gene.

In one embodiment of the invention, the construction method comprises inserting a nucleotide sequence encoding a human or humanized CD36 protein or a nucleotide sequence and/or helper sequence of a humanized CD36 gene into an endogenous regulatory element of a CD36 gene of a non-human animal. Preferably, the helper sequence may be a stop codon such that the CD36 gene is humanized to express human CD36 protein and not non-human animal CD36 protein in an animal model. Further preferably, the helper sequence is WPRE and/or polyA.

In one embodiment of the invention, the construction method comprises introducing all or part of the gene encoding human CD36 protein (preferably the extracellular region, signal peptide, intracellular region and/or cytoplasmic region of human CD36 protein, more preferably the extracellular region) into the corresponding region of the CD36 gene of the non-human animal (preferably the nucleotide sequence encoding the extracellular region of the CD36 protein of the non-human animal).

Preferably, the construction of the non-human animal is performed using the targeting vector described above.

In one embodiment of the present invention, the construction method comprises introducing the targeting vector into a non-human animal cell, culturing the cell (preferably an embryonic stem cell), transplanting the cultured cell into a female non-human animal oviduct, allowing the female non-human animal to develop, and identifying and screening the non-human animal to obtain the CD36 gene humanization.

According to some embodiments of the invention, the method of constructing further comprises: and mating the CD36 gene humanized non-human animal with other non-human animals modified by genes, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-gene modified non-human animal.

Preferably, the other gene is a non-human animal genetically modified with at least one of PD-1, PD-L1, TIGIT and CD 226.

Preferably, the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, TIGIT and CD226 proteins.

Preferably, each of the plurality of genes modified in the genome of the polygenously modified non-human animal is heterozygous for the endogenous replaced locus.

Preferably, each of the plurality of genes modified in the genome of the polygenously modified non-human animal is homozygous for the endogenous replaced locus.

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

providing the non-human animal and the non-human animal obtained by the construction method;

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

Preferably, the other genetically modified non-human animals include non-human animals humanized with one or a combination of two or more of the genes PD-1, PD-L1, TIGIT or CD 226.

Preferably, the polygene modified non-human animal is a double-gene humanized non-human animal, a three-gene humanized non-human animal, a four-gene humanized non-human animal or a five-gene humanized non-human animal.

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

In a twelfth aspect of the present invention, there is provided a non-human animal or progeny thereof obtained by the above construction method.

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

In a fourteenth aspect of the present invention, there is provided a method for constructing an animal model comprising the above-described method for constructing a non-human animal, a non-human animal or a progeny thereof, a genetically deleted animal or a polygenic modified non-human animal. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a fifteenth aspect of the present invention there is provided the use of a non-human animal derived from the above described non-human animal, the above described non-human animal obtained by the above described construction method, the above described non-human animal or progeny thereof or the above described constructed polygenically modified non-human animal in the preparation of an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a sixteenth aspect of the invention, there is provided a cell or cell line or primary cell culture derived from the above-described non-human animal, the non-human animal obtained by the above-described construction method, the above-described non-human animal or progeny thereof or the above-described animal model. Preferably, the cell or cell line or primary cell culture comprises a cell or cell line or primary cell culture that can develop into an animal subject or cannot develop into an animal subject.

In a seventeenth aspect of the present invention, there is provided a tissue or organ or a culture thereof derived from the above-described non-human animal, the above-described non-human animal obtained by the construction method, the above-described non-human animal or a progeny thereof or the above-described animal model. Preferably, the tissue or organ or culture thereof includes a tissue or organ or culture thereof that may or may not develop into an animal subject.

In an eighteenth aspect of the present invention, there is provided a tumor tissue after tumor-bearing, said tumor tissue being derived from the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny or the above-mentioned animal model. Preferably, the tumor-bearing tumor tissue includes tumor-bearing tumor tissue that can develop into an animal individual or cannot develop into an animal individual.

In a nineteenth aspect of the invention, there is provided a CD36 gene humanized cell that expresses a human or humanized CD36 protein.

Preferably, the cells express the humanized CD36 protein described above.

Preferably, the genome of the cell comprises a portion of the human CD36 gene. More preferably, the cell comprises the humanized CD36 gene described above. Preferably, the cells include cells that can develop into an animal subject or cannot develop into an animal subject.

In a twentieth aspect of the present invention, there is provided a construct expressing the above-described humanized CD36 protein or comprising the above-described humanized CD36 gene.

In a twenty-first aspect of the invention, there is provided a cell comprising the construct described above. Preferably, the cells include cells that can develop into an animal subject or cannot develop into an animal subject.

In a twenty-second aspect of the invention, there is provided a tissue comprising the above-described cells. Preferably, the tissue includes a tissue that may or may not develop into an animal subject.

In a twenty-third aspect of the present invention, there is provided the use of a polypeptide derived from the above-mentioned humanized CD36 protein, the above-mentioned nucleic acid, the above-mentioned humanized CD36 gene, the above-mentioned non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or a progeny thereof, the above-mentioned animal 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 tissue after tumor loading, the above-mentioned cell, the above-mentioned construct, the above-mentioned cell or the above-mentioned tissue, the above-mentioned use comprising:

A) Use in the product development of CD 36-associated immune processes involving human cells;

b) Use in model systems related to CD36 as pharmacological, immunological, microbiological and medical studies;

c) To the use of animal experimental disease models for the production and use in research of etiology associated with CD36 and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

d) Application in screening, drug effect detection, efficacy evaluation, verification or evaluation of human CD36 signal pathway modulators in vivo; or,

e) Research on CD36 gene function, research on medicine and medicine effect aiming at human CD36 target site, research on medicine for immune related diseases related to CD36 and application in anti-tumor medicine.

Preferably, the use includes therapeutic and diagnostic purposes for diseases or therapeutic and diagnostic purposes for non-diseases.

In a twenty-fourth aspect of the present invention, there is provided a method of screening for a human CD 36-specific modulator, said method comprising administering the modulator to an individual implanted with tumor cells, and detecting tumor suppression; wherein the individual is selected from the group consisting of the above-described non-human animal, the non-human animal obtained by the above-described construction method, the above-described non-human animal or its progeny, or the above-described animal model.

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

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

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

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

Preferably, the detecting comprises assessing an individual's weight, fat mass, activation pathway, neuroprotective activity, or metabolic change, including a change in food consumption or water consumption.

Preferably, the tumor cells are derived from a human or non-human animal.

Preferably, the method of screening for a human CD 36-specific modulator comprises a therapeutic or non-therapeutic method.

In one embodiment of the present application, the method is used to screen or evaluate drugs, detect and compare the efficacy of candidate drugs to determine which candidate drugs may be drugs and which may not be drugs, or compare the sensitivity of the efficacy of different drugs, i.e., the therapeutic effect is not necessarily, but is only one possibility.

In a twenty-fifth aspect of the present invention, there is provided a method of evaluating an intervention program, the method comprising implanting tumor cells into an individual, applying the intervention program to the individual implanted with the tumor cells, 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-described non-human animal, the non-human animal obtained by the above-described construction method, the above-described non-human animal or progeny thereof, or the above-described animal model.

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

Preferably, the tumor cells are derived from a human or non-human animal.

Preferably, the method of evaluation of the intervention regimen comprises a therapeutic or non-therapeutic method treatment regimen.

In one embodiment of the present application, the evaluation method detects and evaluates the effect of an intervention program to determine whether the intervention program has a therapeutic effect, i.e. the therapeutic effect is not necessarily, but is only one possibility.

In a twenty-sixth aspect of the present invention, there is provided a use of a non-human animal derived from the above-described non-human animal, the above-described non-human animal or progeny thereof, the above-described animal model in the preparation of a human CD 36-specific modulator.

In a twenty-seventh aspect, the present invention provides a use of a non-human animal derived from the above-described non-human animal, the above-described non-human animal obtained by the above-described construction method, the above-described non-human animal or progeny thereof, the above-described animal model, in the preparation of a medicament for treating a tumor or an immune-related disease.

The humanized non-human animal of the CD36 gene can normally express human or humanized CD36 protein in vivo, can be used for drug screening, drug effect evaluation, immune related diseases and tumor treatment aiming at target sites of human CD36 channels, and can accelerate the development process of new drugs, save time and cost.

The "immune-related diseases" described herein include, but are not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain or neurological disorders, and the like.

The "tumor" as described herein 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, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcomas. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the 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 sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In one embodiment of the invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

The invention relates to all or part of the whole, the whole is the whole, the part is the part of the whole or the whole individual.

The "humanized CD36 protein" of the present invention includes a portion derived from human CD36 protein and a portion derived from a non-human CD36 protein.

The "humanized CD36 protein" described herein comprises a portion derived from a human CD36 protein. Wherein, the "human CD36 protein" is identical to the "whole human CD36 protein", i.e. the amino acid sequence thereof is identical to the full-length amino acid sequence of the human CD36 protein. The "part of human CD36 protein" is continuous or interval 5-472 (preferably 10-410) amino acid sequences, which are identical to the amino acid sequence of human CD36 protein or have over 70% homology with the amino acid sequence of human CD36 protein.

The "humanized CD36 gene" of the present invention includes a portion derived from a human CD36 gene and a portion derived from a non-human CD36 gene. Wherein, the "human CD36 gene" is identical to the "whole human CD36 gene", i.e. the nucleotide sequence thereof is identical to the full-length nucleotide sequence of the human CD36 gene. The part of the human CD36 gene is 20-77071bp (preferably 20-27218bp or 20-1230 bp) nucleotide sequence which is continuous or interval, and is consistent with the human CD36 nucleotide sequence or has more than 70% of homology with the human CD36 nucleotide sequence.

The "xx-to-xxx exons" or the "all of xx-to-xxx exons" described herein include exons and nucleotide sequences of introns therebetween, e.g., the "3-to-4 exons" described herein include all nucleotide sequences of 3 exons, 3-4 introns, and 4 exons.

The "x-xx number intron" as used herein means an intron between the x-exon and the xx number exon. For example, "intron No. 3-4" means an intron between exon No. 3 and exon No. 4.

"part of an exon" as used herein means that several, tens or hundreds of nucleotide sequences are identical to all exon nucleotide sequences, either consecutively or at intervals. For example, the portion of exon 3 of the human CD36 gene comprises a contiguous or spaced 5-209bp, preferably 10-33bp nucleotide sequence that corresponds to the nucleotide sequence of exon 3 of the human CD36 gene. In one embodiment of the present invention, the "portion of exon 3" included in the "humanized CD36 gene" includes at least a nucleotide sequence encoding an extracellular region of exon 3.

The "locus" as used herein refers broadly to the location of a gene on a chromosome, and in a narrow sense to a DNA fragment on a gene, either a gene or a portion of a gene. For example, the "CD36 locus" means a DNA fragment of an optional stretch on exons 1 to 16 of the CD36 gene. Preferably any one or a combination of two or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16 or an intron during the period, or all or part of one or two or more, more preferably on exon 4 to exon 15 of the CD36 gene.

The "nucleotide sequence" as used herein includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Preferably DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, tRNA.

The term "three or more" as used herein includes, but is not limited to, three, four, five, six or seven, etc.

The term "three or more consecutive" as used herein includes, but is not limited to, three consecutive, four consecutive, five consecutive, six consecutive, seven consecutive, etc. Wherein "more than three consecutive exons from No. 1 to No. 6" includes three consecutive exons, four, five or six, etc., and also includes intronic nucleotide sequences of the period.

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a 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 signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

"homology" as used herein means that a person skilled in the art, while maintaining a structure or function similar to a known sequence, can adjust the sequence according to actual working requirements, using sequences having (including but not limited to) 1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, 99.99.3%, 99.99.7%, 99.99.99%, 99.9%, as compared with sequences obtained in the prior art.

One skilled in the art can determine and compare sequence elements or degrees of identity to distinguish 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 murine. In one embodiment, the genetically humanized non-human animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from a murine family. In one embodiment, the genetically modified animal is from a family selected from the group consisting of the hamsidae (e.g., hamster-like), hamsidae (e.g., hamster, new world rats and mice, voles), murine superfamily (true mice and rats, gerbils, spiny rats, coronary rats), equine island murine (mountain climbing mice, rock mice, tailed rats, motor gas rats and mice), spiny murine (e.g., spiny sleeping rats) and mole murine (e.g., mole rats, bamboo rats and zokors). In a particular embodiment, the genetically modified rodent is selected from the group consisting of a true mouse or rat (murine superfamily), a gerbil, a spiny mouse, and a coronary 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, which is a mouse selected from the group consisting of BALB/C, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola of the C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strain.

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,2ndEd., by Sambrook, fritschand Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning, volumes I and II (D.N.Glcovered., 1985); oligonucleotide Synthesis (m.j. Gaited., 1984); mullisetal, u.s.pat.no.4, 683, 195; nucleic Acid Hybridization (B.D.Hames & S.J.Higginseds.1984); transcription And Translation (B.D.Hames & S.J.Higginseds.1984); culture Of Animal Cells (R.I.Freshney, alanR.Liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); perbal, A Practical Guide To Molecular Cloning (1984); the services, methods In ENZYMOLOGY (j. Abelson and m. Simon, eds. Inch, academic Press, inc., new York), special, vols.154and 155 (wuetal. Eds.) and vol.185, "Gene Expression Technology" (d. Goeddel, ed.); gene Transfer Vectors For Mammalian 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 illustrative of some aspects of the present invention and is not, nor should it be construed as limiting the invention in any respect.

All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto 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 present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: schematic comparison of mouse CD36 locus and human CD36 locus (not to scale);

fig. 2: schematic representation (not to scale) of humanized modification of the mouse CD36 gene;

fig. 3: schematic (not to scale) of CD36 gene targeting strategy and targeting vector design;

fig. 4: cell Southern blot results after CD36 recombination, where WT is wild type control;

fig. 5: schematic (not to scale) of the process of FRT recombination in a CD36 gene humanized mouse;

fig. 6: CD36 gene humanized mouse F1 generation tail PCR identification result, wherein WT is wild type, H ₂ O is water control, PC is positive control;

Fig. 7: c57BL/6 wild-type mice (+/+) and CD36 Gene humanized homozygous mice (H/H) RT-PCR assay results, wherein H ₂ O is water control, GAPDH is glyceraldehyde-3-phosphate dehydrogenase reference;

fig. 8: flow detection result of leukocyte subpopulation ratio in spleen;

fig. 9: flow-through detection of T cell subpopulations in spleen;

fig. 10: flow detection results of the white blood cell subpopulation ratio in the peripheral blood;

fig. 11: flow detection results of T cell subpopulations in peripheral blood;

fig. 12: flow-through detection of leukocyte subpopulations in lymph nodes;

fig. 13: flow detection results of T cell subpopulations in lymph nodes;

fig. 14: implanting a mouse colon cancer cell MC38 into a humanized CD36 mouse, and carrying out an anti-tumor efficacy test on the result of the body weight of the mouse;

fig. 15: implanting a mouse colon cancer cell MC38 into a humanized CD36 mouse, and carrying out an anti-tumor efficacy test on the result of weight change of the mouse;

fig. 16: the tumor volume results of mice after the anti-tumor efficacy test are shown by implanting the mouse colon cancer cell MC38 into the humanized CD36 mice.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

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

ScaI and EcoNI enzymes were purchased from NEB under the product numbers R3122S, R0521S, respectively;

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

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

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) anti-body available from bioleged under the accession number 108426;

v450 Rat Anti-mouse CD11b is purchased from Biolegend, cat 8232657;

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

APC anti-mouse CD36 anti-body was purchased from Biolegend, cat# 102611;

PE anti-human CD36 anti-body is available from bioleged under the designation 336205;

mouse IgG2A PE-conjugated Antibody was purchased from RD, cat No. IC003P;

APC Armenian Hamster IgG Isotype Ctrl Antibody from Biolegend, cat 400912;

purified anti-mouse CD16/32 was purchased from Biolegend under accession number 101302.

EXAMPLE 1 CD36 Gene humanized mice

The alignment of the mouse CD36 Gene (NCBI Gene ID:12491,Primary source:MGI:107899,UniProt:Q08857, located at positions 17986688 to 18093957 of chromosome 5 NC-000071.7, based on transcript NM-001159558.1 and its encoded protein NP-001153030.1 (SEQ ID NO: 1)) and the human CD36 Gene (NCBI Gene ID:948,Primary source:HGNC:1663,UniProt ID:P16671-1, located at positions 80602207 to 80679277 of chromosome 7 NC-000007.14, based on transcript NM-000072.3 and its encoded protein NP-000063.2 (SEQ ID NO: 2)) is shown in FIG. 1.

For the purposes of the present invention, a nucleotide sequence encoding a human CD36 protein may be introduced at the endogenous CD36 locus of a mouse, such that the mouse expresses the human or humanized CD36 protein. Specifically, under the control of the regulatory element of the mouse CD36 gene, the humanized CD36 gene locus is obtained by replacing about 27kb of the partial sequence of the mouse exon 4 to about 43kb of the partial sequence of the exon 15 with about 27kb of the partial sequence of the exon 14 comprising the human CD36 gene under the control of the regulatory element of the mouse CD36 gene, and the schematic diagram of the humanized CD36 gene locus is shown in FIG. 2.

Targeting strategies were designed as shown in FIG. 3, which shows targeting vectors containing homologous arm sequences upstream and downstream of the mouse CD36 gene, as well as fragments A comprising the human CD36 sequence. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to nucleotide sequence 18033806 to 18038888 of NCBI accession No. NC_000071.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to nucleotide sequence 17986807 to 17990821 of NCBI accession No. NC_ 000071.7. The nucleotide sequence of human CD36 on fragment A (SEQ ID NO: 5) was compared with NCBIThe 80646828 th to 80674045 th nucleotide sequence of accession NC_000007.14 is identical; the linkage to mice upstream of the human CD36 sequence was designed as: (SEQ ID NO: 6) wherein the sequence "AGTC"middle" C "is the last nucleotide of the mouse, the sequence +.>The first "G" of (3) is the first nucleotide of a human. The connection of the human CD36 sequence downstream to the mouse is designed as +.> Wherein the sequence is "AAAC"C" in "is the last nucleotide of the human, sequence>The first "C" of (3) is the first nucleotide of the mouse sequence.

The targeting vector also comprises a resistance gene for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination systems Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo box (neocassette). Wherein the connection between the 5' end of the Neo box and the human gene is designed as follows Wherein the sequence is "GATC"middle" C "is the last nucleotide of the human, the sequence +.>Is the first nucleotide of the Neo cassette; the connection of the 3' -end of Neo cassette to human gene is designed as +.> Wherein the sequence is "AGCCThe last "C" of the "is the last nucleotide of the Neo cassette, sequence +.>In (2) is the first nucleotide of a human. In addition, a coding gene (coding gene for diphtheria toxin A subunit (DTA)) with a negative selection marker was also constructed downstream of the targeting vector 3' homology arm. The mRNA sequence of the modified humanized mouse CD36 is shown as SEQ ID NO:10, the expressed protein sequence is shown as SEQ ID NO: 11.

Whereas human CD36 has multiple subtypes or transcripts, the methods described herein are applicable to other subtypes or transcripts.

Targeting vector construction can be performed by conventional methods, such as enzyme digestion ligation, and the like. After the constructed targeting vector is subjected to primary verification through enzyme digestion, the targeting vector is sent to a sequencing company for sequencing verification. The sequencing and correct targeting vector is transfected into embryonic stem cells of a C57BL/6 mouse through electroporation, the obtained cells are screened by utilizing a positive clone screening marker gene, the integration condition of exogenous genes is confirmed through detection by utilizing PCR and Southern Blot technology, correct positive cloned cells are screened, clones which are identified as positive through PCR are detected, southern Blot (cell DNA is digested by ScaI or EcoNI enzyme respectively and hybridization is carried out by using 2 probes, the lengths of the probes and target fragments are shown in table 1), an exemplary result is shown in fig. 4, and the detection result shows that 12 clones which are identified as positive through PCR are further verified through sequencing, and 4 clones which are positive are found without random insertion are specifically numbered 2-B04, 2-D02, 2-D03 and 3-F05.

Table 1: specific probe and fragment length of interest

Restriction enzyme	Probe with a probe tip	Wild-type fragment	Recombinant sequence fragments
				ScaI	3’Probe	10.5kb	15.7kb
EcoNI	5’Probe	11.2kb	15.4kb

Wherein the PCR assay comprises the following primers:

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

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

southern Blot detection included the following probe primers:

5'probe (5' probe):

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

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

3 'probe (5' probe):

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

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

the correctly positive cloned cells (black mice) are introduced into the isolated blasts (white mice) according to the known technique in the art, and the obtained chimeric blasts are transferred to a culture solution for short culture and then transplanted into oviducts of recipient mice (white mice), so that F0 generation chimeric mice (black-white interphase) can be produced. And backcrossing the F0 generation chimeric mice and the wild mice to obtain F1 generation mice, and then mating the F1 generation heterozygous mice to obtain F2 generation homozygous mice. The positive mice and the Flp tool mice can also be mated to remove the positive clone screening marker gene (the process is schematically shown in figure 5), and then the CD36 gene humanized homozygote mice can be obtained through the mating. The genotype of somatic cells of the offspring mice can be identified by PCR (primers shown in Table 2), and the identification results of exemplary F1-generation mice (from which the Neo marker gene has been deleted) are shown in FIG. 6, wherein the mice numbered F1-01 and F1-02 2 are positive heterozygous mice. This shows that the method can be used for constructing the CD36 gene humanized mice which can be stably passaged and have no random insertion.

Table 2: primer name and specific sequence

The expression of the humanized CD36 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. Specifically, 1 female C57BL/6 wild-type mouse of 7 weeks old and 1 female CD36 gene humanized heterozygote mouse of 7 weeks old were taken respectively, bone marrow tissue was taken after neck-removed euthanasia, and anti-mouse CD36 Antibody APC anti-mouse CD36 Antibody (mCD 36), anti-human CD36 Antibody PE anti-human CD36 Antibody (hCD 36), mouse leukocyte recognition Antibody Brilliant Violet 510 were used respectively ^TM Anti-Mouse CD45 (mCD 45), perCP Anti-Mouse Ly-6G/Ly-6C (Gr-1) Anti-body (mGr-1), murine monocyte/macrophage specific recognition Antibody V450 Rat Anti-Mouse CD11b (mCD 11 b), murine macrophage specific recognition Antibody FITC Anti-Mouse F4/80 (mF 4/80), murine IgG2A Antibody MoIgG 2A PE-conjugated Antibody, murine Antibody APCArmenian Hamster IgG Isotype Ctrl Antibody, mouse CD16/32 antibody Purified anti-mouse CD16/32 and the like, and performing flow detection after identification staining.

The results showed that 53.0% of macrophages (characterized by mCD45+ mGr-1-mCD11+mF4/80+) in the bone marrow of C57BL/6 mice expressed the mouse CD36 protein (characterized by mCD45+ mGr-1-mCD11+mF4/80+mCD36+), and 0.33% expressed the humanized CD36 protein (characterized by mCD45+ mGr-1-mCD11+mF4/80+hCD36+). Macrophages 49.8% expressed mouse CD36 protein, 23.4% expressed humanized CD36 protein in CD36 humanized heterozygous mouse bone marrow. It was demonstrated that C57BL/6 mouse bone marrow cells did not substantially express the humanized CD36 protein, and that CD36 humanized mouse bone marrow cells were able to successfully express the humanized CD36 protein.

The expression of CD36 mRNA in mice was detected by RT-PCR. Specifically, 1 female C57BL/6 wild-type mouse of 10 weeks old and the humanized homozygous mouse of CD36 gene obtained in this example were taken, respectively, lung tissue was taken after neck-removed euthanasia, cellular RNA was extracted according to Trizol kit instructions, reverse transcription was performed to cDNA, and RT-PCR detection (primers see Table 3) was performed, and the detection results (shown in FIG. 7) showed that: only murine CD36 mRNA was detected in C57BL/6 wild-type mice, and no human CD36 mRNA was detected; only human CD36 mRNA was detected in the CD36 gene humanized homozygous mice, and no murine CD36 mRNA was detected.

TABLE 3 RT-PCR primer names and specific sequences

The expression of the humanized CD36 protein in the CD36 gene humanized homozygous mice can be confirmed using the same flow cytometry detection method as described above. Specifically, 1 female C57BL/6 wild type mouse and CD36 gene humanized homozygous mouse of 7 weeks old were taken respectively, and the peritoneal exudation macrophages were taken for detection after neck-off euthanasia. The results show that the humanized or humanized CD36 protein is not detected in the C57BL/6 mice, and the humanized CD36 protein is detected only in the CD36 humanized homozygote mice, which shows that the humanized CD36 protein can be successfully expressed in the CD36 gene humanized mice prepared by the implementation.

Further, by performing immunotyping detection on C57BL/6 wild-type mice and CD36 gene humanized homozygous mice (H/H, hCD 36) 3 mice each, spleen, peripheral blood and lymph node tissues of the mice after euthanasia (H/H, hCD 36), white blood subtype and T cell subtype detection results in the spleen are shown in FIG. 8 and FIG. 9 respectively, white blood subtype and T cell subtype detection results in the peripheral blood are shown in FIG. 10 and FIG. 11 respectively, and it can be seen from the figures that B Cells (B Cells), T Cells (T Cells), NK Cells (T Cells), CD4+ T Cells (CD 4+ T Cells), CD8+ T Cells (CD 8+ T Cells), granulocytes (Grancyte), DC Cells (Dendritics), macrophages (monoclonal) and Monocytes (monoclonal) in the peripheral blood are basically consistent with those in the wild-type mice (CD 8+ T Cells) and the wild-type mice (CD 8+ T Cells) such as CD36 gene humanized homozygous mice (B Cells), T Cells (T Cells), NK Cells (T Cells), CD4+ T Cells (CD 4+ T Cells), CD8+ T Cells (CD 8+ T Cells), granulocytes (Grandic Cells), macrophages (monoclonal Cells) and the like (CD 8+ T Cells) are basically consistent with those in the wild-type mice (CD 8+ cell type mice (CD 8+ CD 8) and CD8+ cell Cells).

The results of the detection of the leukocyte subtypes in lymph nodes and T cell subtypes are shown in FIG. 12 and FIG. 13, respectively, and it can be seen from the figures that the leukocyte subtypes such as B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells and the like in lymph nodes of the CD36 gene humanized homozygous mice are substantially identical to those of the C57BL/6 wild type mice (FIG. 12), and the T cell subtype percentages such as CD4+ T cells, CD8+ T cells and Tregs cells are substantially identical to those of the C57BL/6 wild type mice (FIG. 13).

The results indicate that the humanized modification of the CD36 gene did not affect the differentiation, development and distribution of leukocytes and T cells in mice.

EXAMPLE 2 efficacy verification

The CD36 gene humanized mice prepared by the invention are used for constructing tumor models, and can be used for testing the drug effect of drugs targeting human CD 36. Specifically, a CD36 gene humanized homozygote mouse (8-week-old female) prepared in example 1 was selected, and a mouse colon cancer cell MC38 (5×10) ⁵ And the volume of the tumor is about 100-150mm ³ Thereafter, the tumor volume was counted as a control group or a treatment group (n=6/group). The control group was injected with PBS,the treatment group used anti-CD 36 antibody 1G04 (for information on drugs see patent WO2021176424 A1). The administration mode is as follows: intraperitoneal injection (i.p.), the group was given 3 times per week for 6 times on the day of the group. Tumor volume was measured 2 times per week and the tumor volume of individual mice reached 3000mm after inoculation ³ Euthanasia is performed at that time. The specific groupings and dosing are shown in Table 4. The body weight and tumor volume measurements of the mice over the experimental period are shown in figures 14-16, respectively.

Table 4: grouping and administration conditions

The main data and analysis results of each experiment are shown in Table 5, and include tumor volume at the time of grouping, 11 days after grouping, 18 days after grouping, survival of mice, tumor-free mice, tumor (volume) inhibition rate (Tumor Growth Inhibition value, TGI) _TV ) And statistical differences in tumor volume (P-value) between treated and control mice.

Table 5: tumor volume, survival and tumor inhibition rate

As shown in fig. 14, 15 and table 5, the animal health status was good throughout each group of experiments, and at day 18, all treatment groups (G2, G3) showed a trend of increasing animal weight (fig. 14 and 15) compared to the control group (G1), and there was no significant difference (P)>0.05 The animal is well tolerated against the human CD36 antibody 1G04, no obvious toxic effect is generated on the animal, and the safety is good. As shown in fig. 16 and table 5, from the tumor volume results, the tumor volume of the treatment group was smaller than that of the control group at each period, and the tumor volumes of the G2 and G3 mice were 1782±172mm on day 18, respectively ³ And 1397+ -239 mm ³ Are smaller than 2555+/-432 mm of the control group ³ In particular, the G3 group had a significant difference from the control group (P<0.05). Experimental results show that the method of the inventionThe prepared CD36 humanized mice can be used for in vivo efficacy detection of drugs (e.g., antibody drugs) targeting human CD 36.

Example 3 preparation of double humanized or multiple double humanized mice

Double or multiple humanized mouse models can also be prepared using the present methods or resulting CD36 mice. For example, in the aforementioned example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other genetic modifications such as PD-1, PD-L1, TIGIT, CD226, or may be obtained by isolating mouse ES embryonic stem cells and using a genetic recombination targeting technique on the basis of humanized CD36 mice. The CD36 mouse homozygote or heterozygote obtained by the method can be mated with other genetically modified homozygote or heterozygote, the offspring thereof is screened, according to Mendelian genetic law, the humanized CD36 and other genetically modified double-gene or polygene modified heterozygote can be obtained with a certain probability, and the heterozygote can be mated with each other to obtain the double-gene or polygene modified homozygote, and the double-gene or polygene modified mice can be utilized to carry out in vivo pharmacodynamic verification of targeted human CD36 and other gene regulators.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Sequence listing

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ccttatcttt tcttggttct attggcatct gtgtagcgct cttggctatg ggcattttga 3720

tgttacacaa aactgttcta aagtacaaac cacaggtata caccacacca tgaagggtta 3780

aacaccatca taaaactgtc actgaagccc aacttcttct tcaagtattg atcaaaaaga 3840

cacgattcta tcagttttgt tacagcagga tcctgttgtt ctgtaaattg cttctttcca 3900

atgttatata atacccacac aaaataagat attgcaaatt aaactgcctg catggagtgc 3960

atagtagcaa tactactgta tttaagagca ttgacatgaa tctctacata ggaaaaaaat 4020

aatgctacta gaattggata cacaagggaa tctttaagaa taacaaaaaa ccaactataa 4080

tatatacata ttatatttgt atcttcaaat tctttagcat atgttttgtc aaaatcattg 4140

ataatgaaat cagaaaccat atacttttaa aagtttctgg aatgttttcc acactgtatg 4200

gggaaagttt caggcatgaa cataaaaact ggaaaggaag tggaattttc attaatgcta 4260

tgattatatt aaagaaaata gtagtattga ttttgtgata atgttttttc ctctgcattt 4320

tttttgttgt tgttttgaca ggtagtttac aaatgatttg aagtgctgat cctttcagag 4380

tctcaacaga ttatttctta aacagctcat acattgctgt ttatgcatga attaggtacg 4440

ttaccactac ggtcatgggt taatagacaa ggtatatcag tccagccaca tttgttttgc 4500

aggagaattc ctggcaaagc actcagggtt ctgtttgggt ggagaaacta gatagggcgt 4560

gaaggtataa aaaagcatgc cacactgtcg taaggagtat tgggatggga atatgtttat 4620

atggctcgcc ttagataaga ttatagtatg ttaatcacga agggattaat gtgcaaaaca 4680

gaaaagttta aaaaccaagc attctctctg ttactccttc tgcttctggt gagttttttt 4740

atgaacaaaa agaaggagta gaacctagaa atccagtgtg gtgaaacaaa caaaaagact 4800

tgtttgttag ttggcaaaat catctgtatc agtagcatga tttctttgta ctttgatctt 4860

tttgtgctaa tacttaagct tcttttttat gacctctttc taatagtaga accgggccac 4920

gtagaaaaca ctgtgattgt acctgggagt tggcgagaaa accagtgctc tcccttgatt 4980

ctgctgcacg aggagaatgg gctgtgatcg gaactgtggg ctcattgctg gagctgttat 5040

tggtgcagtc ctggctgtgt ttggaggcat tctcatgcca gtc 5083

<210> 4

<211> 4015

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctccttggca tggtagagat ggccttactt gggattggag tggtgatgtt tgttgctttt 60

atgatttcat attgtgcttg caaatccaag aatggaaaat aagtaagtgt tcttaagcaa 120

agtatgtatt atttaactat tttcatctta tcaaaatgtg attaaaaatg aaggcacaaa 180

tatttctgga catgtttaac cagctatgat ttcttaatat atatcccaat ttttgatgta 240

attgttgtaa attaagaaac aaatgattaa aatcatgacc ataggaaaat tagcacaatg 300

ctaacttctg tccaaattta gagtttctaa tttcttttat tttaaaattt attaaaatta 360

tactttatta aaaattataa ttataaaatt ttagcagatt gaacttcata agaacaagat 420

gaagatgtag aaatatataa attttgataa aacacaggtg taaaggagtt tatcatcata 480

aactgtggga ggatgcttgt agatgccagc catgcctttt tccaattagg gcttaatact 540

atagcatcct gatgatgaat taaaaatcac tgtctaagag ttagaatata aaattacaag 600

ataataatat aattaattaa ttaattaatt aactgagtca ctcagtttag agaaaaagta 660

catcattttg gataatactt aagagttttg gcaaaattgt acacgccttt ttatatatta 720

gtgaccactg tgtctagact ttaataccca tatttttcta catgaacaaa aatggaagct 780

taagctactt tctctggcct aaatggaagt aaactttgaa atagaagtct ccaatccctg 840

tcagaaaaat gaagtatgag tagaaaatgc ttggtttgaa aaatcacttc aaaataaata 900

aacttattct acttaagccc aaaattgacc ataatatgta cctttcagaa agttttattt 960

taattccaat aactgatatt gaacaaatag tgacaatgct aaacatttta catgaattat 1020

cacattatcc ctatgtatgt ttccaataac ctggagtaaa tactatggta tatgttctag 1080

aggctttcta gaaatttata acagatatca aactatccat attgaagtct aagataacca 1140

tgttaatgtt ttctaaagta gtgtggaata tagttatcat atttatttta tgggaactga 1200

gcatcttaat ggtgtagaaa atgcaactca atgttttgtt ctggatcatt tgtgtcaccc 1260

tatttaaatt tgtttgcact acatctgttt aaaaggctag caagacatat atacattata 1320

atgcttaaaa taagaggtgg agccatgaca ggtggctaag agcaccattg ctcttctaaa 1380

ggaccctttc ccaccaggtg gctgcttact ccagtaccac tagctgatgt cttctggcgt 1440

ctacagtctt tgcacacaca caagggtaca cagacgtgca ggcaaaatac ccacacatat 1500

aaaattaaaa cttttaaatc tattttgtga aagacattta cttttaataa aatcatgaga 1560

aaaatgtctc ctccaaagtt aaaatacttt aatatttctc attagcccag caagttataa 1620

cggaacaaat gtgaacatat gttgatatca tagattaaac agaatgctaa aaatgtatca 1680

aatcctgctc aaataaaaaa gcatgcctat acaaattatt attttatcta gttaaatgaa 1740

taactttatc attgtttaat gatccttgat tataagatta cttaggctca tagactacta 1800

attaataaca gaattaaaag ctcttagtgt ctacatcatt aagtgcctgt tttattcaaa 1860

ctttccacta ttttaaagaa aaataacacc ttttaaaggt gatgtttccg caaaacagat 1920

ttttccttgg caataagttg actttactag gctcagaatt attagaggca ggtttgctga 1980

aataaccatc ttataatatt ctaaaaatgt aagtctatgg gaaaatgtgt gcataatttt 2040

aaaagaggat aaatgtaaag aatgggataa ggtagcatgt tagggagaaa gtggcttgct 2100

gcttacagat tgtgaggata cactgagctc agttttcatg ttgggcatgt gcacttcttt 2160

atggaaaaca gtcttgagtg aatggtgatg cttggcagca aatttaatta actaccctcc 2220

atgttcttat tattatcaga tcttggaggt caggatgcag ctctctatgt gtcattcttg 2280

aataggaaca tgggtctcct ttgtaatgaa gctcttacat gacttctttc tttagcttga 2340

actgcagaac tgaaagtatc attatcttga catgatataa taaataagac atgatatatg 2400

tgatatgata tgactatttt gcctttttat tatctcactg ctttctgatt gataatgttt 2460

cttctatttt ataggtagtg gatgagccta catatacact ggctacatct ttggtaaagc 2520

cgatctacaa aatgaaaact taatatatgc ttcgttttta caaaacacac ctatctttag 2580

gagaagaaat ggtggtgtgt gctctctctc tctttctctt tctctcttat tgcagatata 2640

tatttatttg taaatatata tatatgcaat aagtcacagc atatttcaaa agattaatat 2700

gtcactatag gcaatatttt ttaataaaat cttgcacttt tattaaaagt ccatcatttg 2760

caactgagtg gacttcaatt tctgtagacc caattatcct gtttggttct gatttactga 2820

tttgttccat gttggcaaat ttcaagaatg tacattctga gaaatttttg ttttccctca 2880

ctggaggaaa ctgctatcat gactggggtg gcccctttgt ttatagcaaa tttggcttgc 2940

aactgtcagc acatggcata agtataacat cttgaaagac ttaagaatga aaaatgaaca 3000

attcacatgt gagccactgc ttatatatta agtctctccc tctctggagt tcttggctac 3060

agcaaggcca gatatcacat tggttttggt tttgttgttt ttgttttgtt tttgtttttt 3120

actctctgac acagagctta tgaaatggac tttttttttt ttttttagca taccttagct 3180

ctttgtattt taagtatgtc gtcatgttcc atgctgcata gctctttaaa aaacctgagt 3240

aggtttttct ctttctgctc agctgcaact aataacaacc ttggagagct gttatagtgt 3300

taaaagatgt aaatgataat aaaagaatta ttaaatggaa tcctacaaaa gcaacaatgg 3360

gctttaatat atatttgtgg taatatctcc tgctttcata atcacccaaa aaaaggactg 3420

gtttctaaca ttaaaagaag tcgttcctta aattcaacct ttttgtctag ttactattga 3480

atcacagtat gttatttatt gtattttatt ttgttttttg tagaagtgtt ctatctgaga 3540

ttctgtgtcc ttctgaatca tttaaccttt ctgctatgtc ttaatgcaat tgaacacaga 3600

tattttgtta taattttata gcagcagtgt gagactgaag gcaaatattt tccagggtaa 3660

gacacagtga taggttgttc tacttccttt agcagcataa tcaatttcac ttagtattag 3720

tcccaataaa ttctggtata agacacaaaa taaaaacaaa cattttctgt tctatacgta 3780

gaaaaataaa aggaaaggtg agacaactaa ctctcttgtc tagatataaa aataatcaaa 3840

actgcatggc agctttgggc aggagtgctg gattagtggt taggagtcat caatgttcct 3900

acagaagacc tgggcttggt acccagcttc tatacgtctg ctcacagcct catatgattc 3960

ctgtcagttg atactatgcc cgcctctcct gtgcaccctt ctgcactgca tgtgg 4015

<210> 5

<211> 27218

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ggagacctgc ttatccagaa gacaattaaa aaggtacaag tagtccaaag aatatgcctt 60

ctcattttga ttgattctaa cttctctttt tttgctttgt atttacctgc tttatatttc 120

atggtaactg ctaattttgt atctttgaca taaaggtaat tatgaaccac tgcaactcta 180

tatgatgcga ctttatgtga aatgttataa gtataatgta tatttaacat gactccattg 240

ctgtcttaaa tataaatacc aaattctatt aaaagctgtc tacaggtatg catgttagta 300

gaaataattg ttttaagtta tgtccaaaga gcatgttggc atgcttttga gtaggaaata 360

agtgagtata ttttgtaaaa gcacatttat aaaagaagtt gcactttagt taatactgag 420

aaaagtaaaa ctgtgtgtgt gtgtgtgtgt gtgtgtaatg tgtttaatat tgaaacataa 480

atccttatta aattgtaggt aaacttgttt ggtaatacac tgtttagtaa tccactattt 540

ttatatatgt gtaataatct catctcataa atattttcta tttgtgaagc ttcatattgg 600

aatcttagaa aatactttca gaaatatgca gaacatgtct tagtataaaa caaattgact 660

gtagtgtgaa aaaacagaat gattgaatag atgggctttg cacaacaacc tagaattcat 720

ctcccaccct agcttattcg aattagctac acactcactc atcagaaacg ttgattgata 780

gggggaagaa gagaataaag ggaagcagtt ctgctgaagt tctaaatcag ggatggcaaa 840

ttcaaatggc tgcaggagtt tggatacagg ggttaaacaa aatttaaggt gccaagaaca 900

agataaaatg aagaacagga actgctgtga actagactgt gtgtttactg cctgaaggca 960

taaatgttca ttttattaaa cataatactg gccaaataaa acgagttctg ccttcaactc 1020

tctacctggt taggatggca atgacctaga caaagggtta gtaagcatag tgcacgatgg 1080

agaacaggag gaattgaatt tttattaaat ttttattgct atattgttag tatttttaat 1140

atttttgatc cacagttggt tgaatctgca gatgtggaac ccatgaatac agagggctga 1200

ctgtattgtg tttattgcta acatattgca gaacttcagc ctcatgcatt taactgaaaa 1260

aacatatgta aattagtatc ccctgccttg gaattcttag ttttcttgat agtattttta 1320

atacaggcca atggaaacag acaggtaata gtgaggtgtg gggcttatct aatagtggga 1380

tggagtggcc attgatctga catccttcct actcataaac tatgttctca cccagatctt 1440

atgcagagaa agtacaagat cagtgtctgt gttaacgtac agactacaac atcatttgga 1500

aaagttttcc aaattcaaaa atcacaataa tcttccaatg cacagcgatt tagactactt 1560

ttttttgctg acacataatt attggtcaat aactgagtat tgatgattta atttttttct 1620

ttgtcatgca acaaaactgg tacatgtaga ttcttttgaa tagcatgtga ggtgctagca 1680

tttattttaa ttctttaata tttatcttta tgcttataat agttaaccat gagaaagtaa 1740

gttttctgac atcaaaatgt gcttttgtat aatgactaag agaataatat aatctcatct 1800

attgtaagct ataaccaggg gaagatatat tataataaaa aataccgaga cctatgagac 1860

tctagaattg aattggaaaa gtaaatgctg taatactttg aaagagaaat ctctcagagt 1920

attcaagaaa cttcaggaaa aaggtaggac ttgattcaga ttttaaagaa gtgtagaatt 1980

ttgaaagtct caaataactg cagtaaccat attaaaggac tgattgatag tatgaaaaac 2040

cctgtgaaaa tgctaccaaa tgcagtgaag atggaaggaa gaaggtagag aatgtgccca 2100

taatactgga ttaacagcag aaaagattag aagttaacat ggggttagag ttaggaagca 2160

agcttagaac atcaggcaga aaaatactta attgatgata caatacacaa tagtcgtcgg 2220

tatgtttttc agcaagtgta agatatgata aaacctgaat ttagagatta gatggctaac 2280

atttaattct tactaagagt ttcctatata ctagttcact taatcttcaa aaatatctgt 2340

gagaagttgc tgttatttta ctcatcttac aggcgcaagt ccttaaatca acttgccaac 2400

tcacctagcc aatagggaaa gaagtcattc tccaaatgta agcaatctgt aggaagagcc 2460

tagatgaaaa acctggcctt gtatactact ctacagcaga aaggttgcaa agtgaggata 2520

ccagagatag gaaaacaaac aaacaaaaaa ctgtaggata tgaccacttt caaggagaat 2580

ataagattcc cttttagaaa gatatttgca taatctgcac attatataac aaaaggcaca 2640

ctggtctggc atggttgagt atatagaaaa aaaaaactga tagataatag gatgaaatgg 2700

aagcttggag agacagatta aattagagga gtaaaagcat caaactcaag atgtccagtg 2760

agttattgat agaaggtatg tatatacctt acaaagtaaa acagaggttt tataagtgat 2820

gttttatgaa catcagtctg tgtttcaaag gtcagagttc aaatagattt gatcattgct 2880

ataaagatta aaaataaaac taaacagtag atttactttt agagagtttt aatggagaga 2940

ggaaaaagca gaagaattat tacagaattt gtaataattc agaggcagga agacaaaagt 3000

agaaaaggaa aagaatgatc ttatatgaag aaacaaacaa agagtacaat cctagaagtt 3060

aataggaggc cagaactcaa ggaaagaaag cataaaatgt tctcaggcag atgaaaatta 3120

agaaaataat gattagaaca tggtcaccaa taattttgaa agtatacttt gagtagggta 3180

ataaagaagg gggaattaaa tcaatggagt gcttttcctt cttgtttttg aaataagaat 3240

tatccgtgca atcttttaca aggaaaagag aagattcaga cttcctagtg tagattcaaa 3300

ctcaaggagg tggtacaaca gagactcata aaaactgtca aatgacagtg cacattgtaa 3360

gccccctaca tgtccataga ctaagactca tcatcagaat ccttatattg gtcaagatgt 3420

atggtacatt taaggctaag cttctttttg ctctcagatt cctacacaac catgttgacc 3480

ttgttaattt agtcatatga agtaggtgga atattgtttg aatagactgt ttcttttttc 3540

ttcagtttac agactatttt ttaaatgata ttggcgtatg tatatctttg gcatagagta 3600

ggagtcaaat atttgaattt tgtgtaaacc tagagagcca agtgtcttac tgctgcctca 3660

gaaatactta gagtaatgct atttcacaaa catatgggtt ttggtactgt tgacttctta 3720

tttgtgtacc attaaaactc atttaatatt actttgttta gctgactaat agcaaattaa 3780

gaaagctatt gtatacaagt gatatttgga aaaaaataat taaacttctt aattgagaat 3840

gatgagaaaa tcatttcata tttagtgtag agttgcagtg ctgtgatacc ttccaattct 3900

gaaagatgtc attaccattt gaagtagtct aaaacagttt agcaagctta tttgtacttt 3960

atttttgctc tttattttca agggatgtct ctggtatcct catgcttttt atggattata 4020

gctgcaatct ttcttaccag tatttttgac cgtgattctt aatagagttg tgtgcagcca 4080

gcaagtttat gcagtctttc aaaaaaaaaa aaaaaactaa aaatcaatag caagagctgc 4140

catgaaagta gaaactacca aagcactgtg gaaaagggtc aaaattctac caccatttac 4200

aaaagataat ttatcacttg acattattaa tgtcagttga taaaaagttt agtgaatatt 4260

gtataacata acataggtag ttgtatgcta agcagattaa tgcaggaaca gaaaaccaga 4320

taccacatgt tctcacttgt aagtaggagc taaatattga atacacgtgg atataaagaa 4380

gggaacaata gacacctggg gctaatcgag ggtgggaagg gggaggagag cggaggatca 4440

aaaaactacc tatcaggtac tatgcttatt acctgcgtga agaaataatc cgtacaccaa 4500

acctccatga cacacaattt acctatataa caaacttgca catgtacctc tgaccctaaa 4560

ataaaagttg gaagaaaaaa ataaaattta aataagttct atagttctaa ctatagaaca 4620

ccaaaaaagg aattataggt ttctttgttc ctgttagaga actaatatta aatatcatca 4680

ttcaattata ccatttttcc ttctcccatt cttgaaaaca atgatccttc tgtagcttgt 4740

aagaaatgcc ttcagacaaa ataaattgaa atcatgattc attaaaatgg ttatacatgg 4800

ctgggcatgg tggctcatgc ctataatcct agtacttcgg gaggctgaga cgggtggatc 4860

acttgagctc aggagtttga gaccagcctg ggcaacatgg caaaacccca tctctacgaa 4920

taatacaaaa aaattagctg gatatggtta cacatgcctg tagtcctagc ttttgtgggg 4980

ctgaagtggg aggatccctt gagcccagga ggttgaggct gcagtgagct aaggtcgcgc 5040

ctctgtactt cagcctgggt gacaaagtga gaccctgtct ccccccgcaa aaaattatgt 5100

ttgaattaat agtccatatt ttaaacatat aatttgttta tattaccttt ataatgtatg 5160

cttctagtat ttccagatat aataaaataa tttgtacttt ttcatagtat ctaaaagctc 5220

aaaaataaat gactaaaaat attaaagatg gtaaagtttg ttttatattt aataacgtaa 5280

gaacaaccca aaatatttaa attaaaatag cagtataaaa acgaattaaa cactatgtat 5340

atgtgcatat agctgtaata acaaatgtca cttaaagaca atgtttcaag aaatagacaa 5400

gcatatatgt ttcttctact agtccaattt aatatcaaca tagtttactt ttcatgccaa 5460

acacaacata gtttactttt cataccattt taggtactta cataaattct tggaggctaa 5520

tttctaggtt tcagaaacat attaaagctt actactccaa tacatttcac ctaattttat 5580

tctgggtttt agttgatgat gatgttctga aactaaagcc attttcagat tctctgcata 5640

actgtcacta caattctaat tcattctcaa cttacacaca cttgcataat gcaaaaacat 5700

tagaatttct tttaacatgg ggaaatgatt cgttttaggc ccaattacaa gcaaatggta 5760

gcagctagtg ggaaggatgg aagtatgctt ctggttttta agaaagtttt tactttacta 5820

gaaagagaaa tattaaaatg attaaataac agaaaaataa ttgtcacagg atattatata 5880

agttattggc acatgactgg cttagacagt aaatgctatg aaccaagcaa ttaaagcaga 5940

atggagaagt aaatggtatt agagttcttc ctctaaagga tggatagaat ttgtaaaata 6000

ctaatagctg acatttattg aatgtctggt ttgtactaac actgttctaa gtgttcacgt 6060

ttattgatcc ccataacaac ctatgaggat aaatatttaa gccacatgaa aatgccaaaa 6120

ttcaactttt tttgattttc aaaaatacta agtctatatg gttcaatcta acattactgt 6180

tataatttca cagataagga aagtgaggta caaagaaatt aaataatctg ctcaaacttc 6240

aatgatgatg attattatta agtgatcaaa ctcatagcat ctaatttcat tgttggccac 6300

ttcagtctat gctttaggga agaagccact ataccttgtt agggtgggga tagggtaaaa 6360

aaatctaact cgtagattaa aatctgctaa acatacttct gtctcttggt agaaatgtgt 6420

aaagtccttg aaaagtgaaa caaaatattt tctacatttg tactctactc catgcgtagc 6480

aaagtatgaa gggcatttta ataatgaaag ttgtcagtta tgtagatgat tattgctgtt 6540

gcatgaaaac agtattcaag tgagtgcatg actacatata ggatattaag gtcccaacaa 6600

gcatctgttt tcaaattaat gtagcagaat agaagtttca aggtcagtgc tttcaaccca 6660

gctatagaac atgctatgtg agagtcacca ggttgaaatc cagttgttgt tggggcttgg 6720

tagtctgcat gcttagagtc agggacaaat acacacatac acattagcca agtgagaaaa 6780

tcttatatca ctttttctgt gtacatactt accagctttg tacttcaaat ttagtaaaat 6840

atttgcaaaa tagtctagac agtccaaatt caatttaaaa tcaagttctt ttaaattcac 6900

aaaggacata acatcttttg tttcttgtga catatttagt cttggtattc aaggagtgac 6960

tacaatttaa tacatatagt agtggtagcg tcagaagcat attattacta cttcgactac 7020

ttcaatattt ttctctatgt ctctaaacaa atcatgatgc tatggaaact atataaaatt 7080

gaagtgaaag gacctgattt ttctattatc tgctagtaaa tttgtcacat aacattgagt 7140

cagacatctc ttttctctag aacccatttt ccacctctaa aaaaatggac ggggttagat 7200

agtcctgatt ctaacagttg ataattcaac acattcattt caaccctgtg tttcacgttt 7260

gccttatttg tcgctagaga tattttttaa gggaaaagtg atttgtggcc tttagaaaaa 7320

aaggtctcat acacgtacaa tgtcttttgg tgtttagatg gacactcatt gtagagttag 7380

gaaggcattc aatcctgtca agcacattct gtcacagact ctcttcaatt ctataagagt 7440

ttgttgagtt tctttcatta agagatatta ggtcggtaca aaagtaattg tgatttttgt 7500

cattaaaatc gcaaaaaccg taattacttg tgtacaaacc taatatgtac cttgagaggc 7560

acttgatgaa taacaggtat tgtttttgag cattaagatt ctaatagtgt gaggagaata 7620

atgtttgtgt ttgtgtgttc tcccttgtta agtctcttga aatggagaaa tcaaccgttt 7680

tcagcttcta tttagctttg agcttcaaca tcctcaccaa acatatttac atttagaaga 7740

atatccataa tgctttaagc aattttgtct taatattaga cttagtatgg aaataagcct 7800

ttttgggagc aaataaaaag tttttcaagg acaatttgtt gtaaaaagta ataaagattc 7860

catcactcta gcaggtccaa tgtaccccgt ataagtggat tacagcagcg taggattttg 7920

aaattccatt ttaacttggt gcccagttct tctctaagga attcgcttat tccctacatt 7980

aagcaataag taaaatatca tgaagatatg aaacttaatt tgacttctac caactaagtg 8040

cagagggtga cggaaaaaaa aaaaaaaaag acagtcatag catggtgtaa aggaggacta 8100

gtctaagggt catggatgag gatttttgtc atgaattcac tactgtttgc ctaggctgtc 8160

cttgggccag ccacctggtt tctcagagcc tcagtgtgat ccactggaaa atgtgatcat 8220

gttcaatgtc ctttcctaat ctgtcattct gtgaagtggt gcttccaggt caaggaaaca 8280

aataggtcag aatgttcctg tggtcattgt tctctttagg ctcattttcc atgaaatagt 8340

tgtattcctc ttcctgtagc acactcaggt taagttggag ctctttaacc tgggcattct 8400

tcccaatact tcattagaaa taccgcctgc ttcaaaatca cctttgtttg cactatctac 8460

tacagcgtaa tagacttctg gctcagagag tgaaagctgt ggaagaaaac tagaagaata 8520

tttatgttaa ctcccttatt ttacagctaa tgaagttgac cctagggagg tttgatgact 8580

ctccacccag tcccaaagct cctaaagcaa ggaatcgagt tcctaatgta ggatttttaa 8640

ttctcttatt ggtgtatttt ccacttgtct atctttacca aaggagcatc agtgattttt 8700

agtggatttc acaaaggagt aataggacag cttccttgtc ctgttttttt ctttaaaaac 8760

tacaacccaa taatgatgag tgaggctgtt gtaatttttg gaaacatgat aatgggttgt 8820

tgcaaatata ttgaaagtta ggggctgtgt gcagtggctc acacctgtaa ttccagcctt 8880

ttgggagtcc aggtcaggtg gattgcttca gctcaggagt tcaaggtcag cctgaccaat 8940

atggtgaaat cctgtttcta ctaaaactac aaaaactagg tgggcatggt ggcacaggtg 9000

agaggatcac ttgagcctgg gaggtcaagg ctgcaaggag ccaagattgt accactgtac 9060

tccagcctgg gtgacagagc aatatcttgt ctcaaaaaaa aaaaaaagaa aagaaaaaga 9120

aaaaggttag ggaagccaga ttgagcagga ttttgaagac acggtgtcaa atttgtgttc 9180

ttcatgtaga tatttttatt ttcttaatgt agtgagtgtt aagtaccatc ctctgaagtg 9240

tccaactctt gaaggaaaag aaaaagcttc caataccatt agatttttca ctcagttttt 9300

gtgttttgtt tcatcagtcc aacttagggg cagagaagaa gttcaactca gtaagaactt 9360

ttttgaactt cggtaaaatt tgcttaataa attatgttga ctgttgcaat attttcaaag 9420

cgtcactcta aagcttgccg aagggtcact ttaaagtttg ccttaaaatc aacagtcgtg 9480

tcttcagtat tacacactga ttctctttgt aaaaggctaa aaagactgct gtaataataa 9540

tttgttgaaa acatttctgc tgcaagtgta tggtaaggtt gcaaaggttc tcatgaatga 9600

ggtacttggg cttggtcctt ttattctggc tgactcaagg ctgcaaacaa tcttccagaa 9660

gtgcctgtac ttactacaaa gacataaccc aaacttattt tctttttcat agcaagttgt 9720

cctcgaagaa ggtacaattg cttttaaaaa ttgggttaaa acaggcacag aagtttacag 9780

acagttttgg atctttgatg tgcaaaatcc acaggaagtg atgatgaaca gcagcaacat 9840

tcaagttaag caaagaggtc cttatacgta caggtgagtg agtccccaca aatatgagac 9900

actcttacct tgaccatgta tttctgagaa gtcttctact tggcaaatgt cattgtattg 9960

aaatgtactt attattttct tgccaaaaat atactttaaa atatttttcc tgtctgtata 10020

gaatcctaat ctaagaattt aatgattata aggtatttat tttgaaaaaa gtggaagata 10080

tatacattat ccaaatattt attagacaat atatagaaaa gataaacaga ttttattata 10140

aacttaccag tatcaaataa cttccctatg tttatgaagt tagtgttttg ttattgtggc 10200

atatattctc aacaactctg aaatattcct gctgaggaaa aaaaaatcag ttttcacatc 10260

ttaaaattta aagcatattt taacagtggt tctcaaagtg tagctgggcc acagcaaagt 10320

gctgggccag ctgcatcagc attaacaggg aattgggaaa catgcaaatc cctccacccc 10380

atcccagacc aaatgaatga aagagttgag gcccactaaa gagttttaca aatcctgggt 10440

gatttgaata cacaattttg agaaccaccg tgctaaattc atttttccta actgcggtac 10500

ctactcaaga tttatattca aattaacttc tgggtcatat agatgtttac attgatctat 10560

tttagtttca ctcttatatt tcctctagtg ttgagacaat attttatatc tttcctctgc 10620

cttccctgat agtttaacac acttttgaac agaatcttag agtattagaa ctagaggaaa 10680

tgatcagcca gaatctgtca ttttgtgagt taaaaatgcc tgatccagtg gatgtgaggt 10740

cacttgtcca tgttcacaca gatgtaggta ctggtagctc ctagattttt gtataccttt 10800

tcttccattt caattataga cactctgtct ttttcactaa aaagggaaaa aagggggaga 10860

ttgaggtgcc aaaaacacta ccttaaccaa ggattaggaa tatatttatg gtatccatat 10920

ggaaacatag ttatgttctg ctatagaatt tgttacctaa atgtgtattt tccctcatgg 10980

ataatcacaa ctagttatac accatgatta gttctcatca caaagttttg tatttctaat 11040

gattttttat aatttgtagt tttagcatct tatctgtttt ggttatcgga tgattgtata 11100

attttccttc ttccccaaag cattactatg cttccagatt gaaaattcac acccaaaata 11160

aaatgaaata acaggcatgg tgagggtggt gcctactacc atagcacatg acatataagc 11220

cccagagggc cttggtgcat gtcctgtctt agcagtttta cccgaatcgg gtgtctctgt 11280

gcgcatctgt gacaattcat aattttgtca atgaagactt agtttttatg ggtacaaaaa 11340

catcttacct tagaattttc ttcatggtct taaagttttt atattattag caagagtcta 11400

tacaagtatc ttgttgcaaa gaatgttgtt aggtgtacca tatatatata cacacatata 11460

tatatatgta ttcataggtg tatatatact tatgaaaatc tagaattcac atatgaccta 11520

aggagagtat cagcataatt gtttaagaaa tagaacctaa gtgaactgag taagtgtgag 11580

gaaagaaaat agttgttaga atttgagaac tcatcatgat tttttttgtt tgtttggggg 11640

aagttatatt tttcacttct gtttttaatt ttgtttattt tttgaggcaa ggtcttgttc 11700

tgtcacccac tctagagtgc agtggcacga tcacagttca ctgcatcctc aacctcctag 11760

gctcaagtga tcctcccacc tcagcctcct gagtagctgg gaccacaggc gcatgccact 11820

atacccagct aatttttatg ggttttgttt tgtagacaag gtttcgccat gttgctcatg 11880

ctggcctcaa actcctgggc tcaagcaatc tatgtgcctt ggcctcgcaa agtgttggga 11940

ttacaggtgt gacccaccat gcccgaccat atttttcact tttaaatata ttatttggga 12000

tatgagaaaa catattattg aaatagttct cctctggtag ttgacagctg atctatattt 12060

tattcttaaa gcctttaacc tatctctcta agggacaaaa aggggatgtg ttagatatat 12120

ttggtttgct tagagaggca atggagcaaa gactggagaa gaacagttgc atttacagct 12180

aaattttaga atccataaat agctctttct gtaacaattt taaagggatt cctgattatc 12240

tgtaggtcat cttgtctcag catgtcacca aaatagtctt ttattgtttg cctgagtgcc 12300

ttaaataatg gaaaaacaac cagtaccttt tagaaaaaaa attaacactt tgatagtgca 12360

tgtgttgagc taaacatgct ttttcataac taattatacc ctaaatccat ctgacattgg 12420

aagtatgtga gaatgtccct cctcaaacag aaccacaggc tgtatttggc cattgtctgc 12480

taaagtaagc ttgattacac tttgacaaga tatgacctga atcaaagcac gaaattgctt 12540

gggttgagat cttatgtgtg tttctaccta caccatctct atgaagatgc tgtaattcct 12600

tcagaatatt cttagatctg taattcattt tttgagttac tttgctttgg aaagaatata 12660

aaaagtaatc ttgagaaaag cataaattcc ttcccttgac cttataaaca aacacgttcc 12720

aaaacttcca tacagtctaa cttttaaaca aaaaattcag taattatgtt ccctttttaa 12780

aacaacattc taaaggcagg gctgttttaa ggatttttct tctagtgact gccatctact 12840

ggtataatgt tgtcagtaca ctttaggtaa cgtgacaatt atgttgctat tgttttaata 12900

tatgggcata tttctgaaaa ttgaagtact ttgatttgaa atattagtaa tatgcacact 12960

aatttaaaat acaattgtcc tgagtgctgt ttcagatttt tatattcttt gtgtgtctgc 13020

agtacttcaa gaatagtagt tttaataaaa ttatattaaa aatatatttc tagccaactt 13080

tgaatcctct ttttaaagaa tctgagacat ttgtttagta aatacattta ctgagaactc 13140

attatatacc aggcatgatg cttagagata caaaacactc ataaacccct accctgtagg 13200

aatttacatt tttattataa tggaataaaa tgggttttta aaaaattcag tccaattgct 13260

taagtcttta ccttggctat ctactcactt cacaggcatt caaatatgac agtgcaatta 13320

tgatatatat cttatttctt ctcagtgaat atcttccact tgtgaaatgt atttttcctg 13380

taataatcac ccttcaccgc gttcctaaaa tgtagttggt actttgcagc ctagatatgt 13440

tagagtcagg agattagaac tagcaaaagt attcaatata cttgaggggc tggggaagtt 13500

agtctataac ccttaaaaat ggaaattata ttcttatctc taattctcat taaagaagtc 13560

ctgaaaaagg gaaaaagcag tgctggatag gccttgtatt ttgatcattc tttctaatat 13620

aagaaaatgt gctctaaata ttcccctgct gaagcccctg tactttttcc tccttatcct 13680

caggataaag tctcgtttga gtattagatg caactctttt atgtttgaga tcttacttgt 13740

ttcaataact ttatttccac tactctcctc acctcttact ctgtaatcca accacactca 13800

aatatctgca tttcccaaaa catattatgt tcgttcttat gcatgctacc atctgccgta 13860

ctttacctaa ctaaacttgt atttgttaca tagaccattt atgtagactt gtactaaaaa 13920

gtgttttaac cctcacccct accctgacca ttctaaatct ggatctggat ctgttttgtg 13980

ttttcccatg gagtctccat tataacaata ttccttctct ctcactattt taatttccag 14040

tttgccagtt taagacccct tctcgttagt ttgctagaga ccctggctga tttctcattt 14100

taacaccatt gcttacatac tatctatctg gcatattctg tgtgttcaaa aatcatttgt 14160

tgaatgaatg acatttgaga tctaatgttc acatatgaca aatgttttga attttgttta 14220

ctgctatttc tttagagttc gttttctagc caaggaaaat gtaacccagg acgctgagga 14280

caacacagtc tctttcctgc agcccaatgg tgccatcttc gaaccttcac tatcagttgg 14340

aacagaggct gacaacttca cagttctcaa tctggctgtg gcagtgagta gacaaacaac 14400

aaagttatct attttaaaat actctagaac tcatgtaatt aatcctatca ttagaattat 14460

taggttttac ttgtttttcc atttttatca aaacgtattc ctatatcatt attttgaatg 14520

gaggcatggt catttggaaa gtgacaaaat cataagaatc gactgcatta gaagaccact 14580

ttattttttt cattattttt tgtcgaaaca ttctctatat ttaatttcaa ttttataaca 14640

gattacagga agatgcttaa gaaacaagta caacatttgt ttcagtatgt ctttaaatga 14700

aagactttta agtatgtaag caactatata ataaaaggtt tccaaacgca gcctgtaaga 14760

aatcaggcaa attttactat aagcaataaa ccattccgag ctttccagac agtgtaccag 14820

tagctgtacc agtgggcaat aacctttaac caaacaaaaa caaacaaaca aacaaacaaa 14880

agcactttgc aatttgttgc tgcaaaatgg ggagaaaaaa agagtatata aacttgatgg 14940

aatcacaact gtcaatataa tttaagggaa aataaagtcg ataaggttga tggtgtctat 15000

tgtttggaaa gtcgaattcg gctatttgct tggggctcta gagaccacac cactgaataa 15060

acaaaactct gcagagtcta gctatccgcc aacagggggt gccgtccaaa ttcatggcaa 15120

ataaagggca tttggtgctc accattcaca acaggcgggc atttatgtgg atgaagtaca 15180

atttcttcag caagctcagc aaaaactctt aaggggcaaa tatgaactct gcattttaag 15240

aaaaatagaa aacggaaaca caaaatccta aaaagtatat gaaggctctg cattagcaca 15300

ctggcatcaa accacacatc cacagcacat cctaattcta tgggaaacaa tccttgctta 15360

tcggaggtgc atatctaaat tcaatagctt accaatatat tcttgggagt aggccaaaag 15420

gaaacagaaa acccacatta aaaaaagaag tttcttttcc taaacatttt cctgaagctg 15480

aaattgaagt ggagaggaag tcagttgtcc tcgtcgaaat cgtagtcctc ctcatcctcc 15540

ccaacctggg acaccggggt cttcaccctg gagatgctgt actgagacct gttggagctt 15600

gtggccagca tttcatccgc accattggtc aggtcactgg cagagagcct cgtgccgtta 15660

gacgtggaac ctgccgttgt gatgaacacg cctgcaacaa ttgtctgcgc catttctgtc 15720

acgtgtggct ccagcgcctt tgggaccaga cttatggctt tttttttttt taagttctgg 15780

gatacatgtg ctgaatgtgc aggattgtta cataggtata catgtgccat ggtgctttgc 15840

tgcacctatc aaaccatcat ctaggtttta agccccgtat gccttaatgc attagatatt 15900

tgagaagacc actttaagtg tatcgttaaa taaatagagc ttaacttgga atgtcgtctt 15960

cttgtggctg gcactgaggc aaagaaatgt aatcatctag gaattagacg aattgcattt 16020

tgagttttgg caggatctgg cagtaatttt aaagataagc tttaaaaagt tttgtattaa 16080

gctcaatatt agcatttaat ccatttattt gttaaaatct aatattgtat tcttgtctta 16140

aacagtgact ttgtttttgt aggctgcatc ccatatctat caaaatcaat ttgttcaaat 16200

gatcctcaat tcacttatta acaagtcaaa atcttctatg ttccaagtca gaactttgag 16260

agaactgtta tggggctata gggatccatt tttgagtttg gttccgtacc ctgttactac 16320

cacagttggt ctgttttatc ctgtaagtac caaatatgaa tggcaatatt attacatttt 16380

aatttaatta attcaatggc attggcaagg cataatttta taatttagct cattagtctt 16440

attgctgatc tggagacata tatcctaact ttttaaaaag tccacttctc attatagctt 16500

cagctttcct agttgggaaa ttcatctgaa tttaacaatt aaatttaaac ctgaagaata 16560

gatttaataa ggtttctact catttataaa tacacaattt tttttaaatt agccggtaag 16620

ctagtctgta aatctttgag cactgttttt ggcttttatt tccctattca cataatcaag 16680

tttaatacca tatttttatt tgttttaaat atactcctca ttcctccttt tctagacctc 16740

ttacaatttt aatttatatt taaaagttag cctaatgttc acatctcaat actgataagg 16800

taatagactt cattttaatg ggatttgtaa ataagaattt ttagtagtcc ataatgtcat 16860

gaaatggcag cttgaagatt aaggaaaatg tgaacttgat ggtgtacttg attaccgctt 16920

aatgttttga attacaaata ggataagcta actactgaat tggaagttgg actatgactt 16980

catttggcac tatatgtgaa tattatgttc tctagttcat tgttttactt ttagatactg 17040

ttaggattac aaggttatat atcaattata aatgaatgta gaaagccata atgaatcaaa 17100

ttcattctga ttttaactca atactcatag ctctttcttt ggctaatgct ttaacttttg 17160

gatgtctaat ttttatcatt ttagtaacca cttattatca ttttagtaac cacttattaa 17220

tgactgtaac atttagaata cccctagaaa tcattgttct tataggtttg ttcaatgtcc 17280

ctcacctcaa catagtaaga atagtgatca aaatgccctc attggcttaa ttatgacaga 17340

gtgctagagt tcacatcatg tcagcttctg atatgtatct tctttgtcac agcatctagc 17400

acttatttct aggcaccttt cacaattttt aaggccaata atttaaaaaa atgtattgca 17460

gatgtatttc aagtcatttg agtaaccagt gattgagaaa tgtgaaagtg agttatgtat 17520

tgtacaactt tgaaaaaatg acttgtagaa gtaacatttt cccatacata tatttcagta 17580

caacaatact gcagatggag tttataaagt tttcaatgga aaagataaca taagtaaagt 17640

tgccataatc gacacatata aaggtaaaag gtaagtattc tggtaaaatg tgcatgtatg 17700

ttactagggt actcttaagc aggaatagta ttcatttaac atctcataag acataggcat 17760

caacctatag aacagacctg gttataattc agctctggaa actcctgttc tgctaggtat 17820

taactcttta gttgtggtaa ctggtgagtt cacaccagtg catagctgct gactatcagc 17880

tccactttaa ggtttggttc acctttctgc acaggttatg gttgtgttac ataaatcccc 17940

aaagggacta ttttttcatc tctgctactt atccagcatt acagtataat tattcttaca 18000

attagataac cataaatgaa aaggtaaaaa aaaaaaaaac aacacatcaa ctgattgtgt 18060

agtagatgga aacttttttt ttacttttta aatcgagcat atcgaattcc atattccagt 18120

ggcatgacct aaatgtgtct ataaagatgg aagcttaatg aatccaggca actgctttca 18180

tgaccttccc cctgcaaata gtctttaata attttccata ttgataactc agctttttta 18240

actttatcaa tgcaaaaata gaatgaatat ttcaagtgca gttctacaat gtaaatacaa 18300

aatgtgaaaa tgaagacttt gccaacttta aagtggtaaa ataacaaatc agcttcctaa 18360

gccattattt cctttttttt tttctagctc cagcctattc acctaaagaa tttataattt 18420

atcatatatg taaactagga agaaccctaa taaatatcag aggaacaagc tttcttctcc 18480

ataaaattaa caattgtgtg tttgactagt ttttttctga aaaagaataa agtgatcaaa 18540

accaaaagag atgaaatgtt tttattaata taaagtaacc cctacctcaa actgagtcta 18600

tctaatgatt gcttttaaca gctaaatatt actagtggag tactttttct tctaaagagt 18660

ccacagttac atatttttat agaaaaagtc agtagaggga aaaaaacact tctaagtatt 18720

cacttaaaag gaaatcacag caatttttta tattgagaaa taacgagcat ttcactctaa 18780

tattacagag agatgtggag gggagttgca aagcactcct agttagagta agaatttcac 18840

atcattttaa gattgtaagg ttgatttaac tcatggcaag actgaacatg attaaccact 18900

tattttgttt aagcaatggc ttgtcttgac atgtcactgt caggagtgga aaaaaagttt 18960

ttccatcacc aaaaagtgag gatgcaggga aactattact attgttttta tcacttccag 19020

atatatatag cttatttaga cagcaaagta taagttagta aactctttcc tactttaacg 19080

ccagtccatg ggagaattaa agggaggaag gggcaagaat ataaattaat catctacatg 19140

ttttgaatgt ttgctgatcc aaacatctgc ttctttcctc ttccctgctt ctctcttctg 19200

cctgccttgt acttagctta tatcagttac ccacttaaca ctttccttct attcattctg 19260

aacacttata ctactgagta attcatgtat cccattggaa aaaaaaaaat ttcccctaag 19320

tagagatcta agtttctgat aaatgttttt aatttttttc ttattccaag atgtttctaa 19380

ttaacacctg gcagttttta ttttatgatc tggctaccta atggcatcag gtacattgca 19440

ataagataaa aggttcaaac aaaacataaa cagaattgaa catttcttaa acttagtact 19500

tgtcacattt aaatgcatca tattaacaga agtattgaat tataatagaa aaagtaatgt 19560

aagaaaggta ttctttaaat aagaatgttt attcattgtc tttttctatt cctaggaatc 19620

tgtcctattg ggaaagtcac tgcgacatga ttaatggtac aggtaagaat atttgttttg 19680

tggtcatcac agttaatcca cctccctttc ccacaaatcc accgttgtac tgacagtgtt 19740

ctgaaagttg agggtgtgtg tttacttgcc tttatatccc cacaacaaaa ttcagagtca 19800

ctatttgtat ataggctaaa ggtctgttca atgtgatggg atttgtaaag attaatcata 19860

aaaaattagc ccttactgca tgattcagag aaatgtttgc tttgtaaaaa ctttactgcc 19920

atcctggcaa cagaagtaaa tggtaaaaaa caaacaacaa cacaaaacac tttagttaca 19980

tttcaaatat tttataaata gtatacagat aagttagaat tgaaagaatt aaaaaaagct 20040

aattacaaaa tagaaaacat atctaagcaa ggcttatagc tgagatagaa taaagttgca 20100

agacagaacc atccattttt ggataaggtg gtcagaatga gagagaaaac aataacaaag 20160

tcagatgtct tcttaactgt gaaaaatata tgtaaaaaat gagcattcat aaacttagat 20220

ttaaggtatg gttagatcag gcttattctc cttcaggaaa agcaaagtac agatatgcct 20280

attagtttaa gaattaagat ttttaaaaag caatgggagg ctggtcacag tggctcatgt 20340

ctataatccc agcactttgg gagaccaagg caggtagact acttgtgccc cggaggttga 20400

gaccagcctg ggcaacatag caaaatccca tctctacaaa aaaaatacaa atattagctg 20460

ggtgtgctgg cacacgcttt tagttccggc tacttgggag gctgaggcag gaggatcact 20520

tgattccagg aggtggaggc tgcagtgagc tataattgtg cctctgcact ctagcctggg 20580

caatagtgtg agaccctgtc tcaaaaaaaa aaaaaaaaaa aaaagtggtg ggaatcgaat 20640

accatgaagt aattaatatc cagaggcaag ctttcaacta ctaggaaaca aaagcatgaa 20700

gtttagaaaa ttagggaata tcccatagga aaatgatata acaaagataa agacacttaa 20760

gtaaaaatga agtacagaga atgtttactt tgccaaaggt atgattattg acttcgtatg 20820

cgtacttgat tctatcattg ccttgtagga aatgataaac aaaaaaagca aacaaaaaaa 20880

acagcaaaaa cactgttgga tttgcagggg ttttcttttg tttttttttt tttttttttt 20940

tgagacatag tctggctctg ttgccaggct ggagtgcagt ggtgcgatct cggctcactg 21000

caacctctgc ctcctggggt tcaagtgatt ctcctacctc aatctcatga gtagttggga 21060

ctacaggcat gtgccaccac acccagctaa tttttgtatt tttagtagag acggtttcac 21120

catgttggcc aggatggtct tgatctcttg accttgtgat ccgcccccct cggcctccca 21180

aagtgctggg attacaggct tgagccacca cacctggcca aggctttgca gggttttgag 21240

ccataggagt gggcaaataa gctattttct aagtaaagca tttctggaaa taaattttca 21300

agtcctcaat actaccaatg ctagtagttg tataagcgga atacttagtc cttagatgca 21360

gagataaata tactgtgtta atcctagtaa aggattcttt aaggagaaaa atctttaaaa 21420

agaagattca ttgacaataa tagaaatgca tgagcatcag gcatgatgtt aaaaatatct 21480

atggggaaaa tagggctgat acaaactagg aaaagaggga atttggacag agcaggggat 21540

gatccctgtg agagagattc ttgcttatgg cactagcgaa aagaaagtaa taaaacagta 21600

aggttctaat gtgttttcat cgtaatttag aagtgcatcc tctttacctt cttcagtaag 21660

tttgaaaaga cttaaaatga aagtattggt atacatttag aatgtttctt ttgggaggac 21720

agaaaaaaag ctctgcaaat atcaactatg ttgcagtcat tgtccatggt agttgctggg 21780

tataattagg acataatctt caatgatatt ttagataatg atcttaaaag cttttcaatg 21840

catttttctt taattattaa ggaaatggaa aagcagttgt tgtcaatgtc catatggggg 21900

cttatatttt acttgtcaca agagtgtaaa ctaatgaaaa caaggtcaca aggttgacac 21960

acttgccaga gaaagaacct acaatgcata atcaaagatg agacttacca tttaaaactc 22020

attcaatgac ggctcgtatt agtgatattc atgtgagaag tacatggagg agttaactat 22080

ttcttcatat atccaatgtt tgccatggat catagctgta gcaaaaagta aattcaataa 22140

atgacttttt tatacacttt caaagcattt gaagttctgt ttagtgagag aactttagta 22200

aatattttta gaagtagtaa tcagccatta ggacaaatga gaaaaaaaat cactacaaat 22260

aaatgtggac atggcaggag atccaaatga acttcactgg aagaaaagtg ccactctact 22320

ggtggggtag ggcatttcaa aaaacaaaca caatgttagc cttaacattt catgtttaag 22380

tttcttttat tttgtaccat taaatatgta tagtatgtag atttgttgtt gacaatagca 22440

gccgccagcc atatgtaact gttaaggact caaaatctgg ctagtatgat ttgaaatgtg 22500

ctgtaaatat aaaatgcaca gtagattttg agactttaag aatttaaaat attttataat 22560

ggttacatgt taaaatattt tggatattaa taagttaaat atagttttgt tttgtttgag 22620

acaaagtttt ccacttgtcg cccaggctgg agtgcaatgg catgatcttg gctcactgca 22680

gtatccgcct cctgggtgca agcaattatc ctgcctcagg atagctggga ttacgagcgc 22740

ctatctcagg atagctgagg caggcctatc ctgcctcagg atagctgcct cccaagtagc 22800

tgggattaca agcgcctacc accacaccca gctaattttg tgtattttta gtagagatgg 22860

ggtttcacca tgtaggccag gctggtctca aacccctgac ctcaggtgat ccacccgcct 22920

tggccttcca aagtgctggg cttgcaggca tgagccagca tgcctggccg gttatttctt 22980

tttacctttt aaaaatgtgg ctgctagata aattacttag gcagtttgca ttacatttct 23040

atggactaca ctggaggaga gatttctagg tttttttcta gaacacacat tacatctaat 23100

catttgccac tcgattttta aacagatgca gcctcatttc caccttttgt tgagaaaagc 23160

caggtattgc agttcttttc ttctgatatt tgcaggtaag acagatactg aagtataagt 23220

atgtctgagt cagaccccag gtgacaaaat gcagaccaag aaacttaaac acagcatagg 23280

aaattcatca tgtttattaa ctaactcttt gcaaaatgtt cttctgcatc ttccaatttt 23340

taatagtaca aatttttttt ttttttgcca tttctatcta agcaagaacc attttgcctt 23400

ttaaaaacta aactagtagt ctattaacga tcaagtccag aagggcgtgc ccaatctttc 23460

taaagacata cagagaagat gatgcaaatg taacagaact gtgttaatgt gctctgctca 23520

gatttgtggg gcccagtaga taacacacaa gcactggagc ctttaccact acccttgagc 23580

ccaagctctg tccctaaaga ctgtatgctt gtgggtaggc atttaacatc tctgaatttt 23640

tttcttatct gtacaattta agacagcagt atttcttcat atacgtgtat ctggggataa 23700

gaaaaatatg tgtattgaac cctatgatag acacttggta aacgatggaa atgtaccagg 23760

tatatatcca gcatgtatat gcatacactc cagtgagtgg tctttctttc caggattaat 23820

taagccgtga aagaaactat ttcatttaaa ctgatcacaa ataaagtatt tgaaggaagt 23880

cctataaata tttactctat tggataaatt gcctgtgaga agtaacttga gtataaataa 23940

acatggtact tcacaaacaa gaatagttca tgcttggcta ttgagtttta gtatgtgtta 24000

aaatttccca atcacttttt ttctaagaat gaaacaagaa tttaaaagag tatatgatgt 24060

ttctaagtta aaacaagaat aagaaaaaat gaatctccag aatgtaagtt caggttcctg 24120

gaatgcagct cttttttctc tgtatttagg tcaatctatg ctgtatttga atccgacgtt 24180

aatctgaaag gaatccctgt gtatagattt gttcttccat ccaaggcctt tgcctctcca 24240

gttgaaaacc cagacaacta ttgtttctgc acagaaaaaa ttatctcaaa aaattgtaca 24300

tcatatggtg tgctagacat cagcaaatgc aaagaaggtg agtaaataac ctcagtagca 24360

cagtccatac cataatttgt gatattcttt aagatgagaa ctttaccata atcctttagc 24420

aaccaaaatt taaaatatat cataatttgt gatattcttt aaaatgagaa ctttaccata 24480

atcctttagc aaccaaaatt taaaattaaa gtaagaaagt aattagggca gaagaaagaa 24540

tggtggcaga aaattttagt gctgattttg tattttggga agatcccact tgtgtttcag 24600

tattacaaaa tttagttaaa accacaccag tatttccttg tggctgcttt tagatttagg 24660

gtgaaatgaa aataattccg agaacacatt aaacatcctg ttattcatct gtcctaactt 24720

ttttcactag aaaatggtac aggtaaatgt attttcagta tgtatctaaa gctagagtta 24780

aacataaaat ttggagacta gcttatcctg tacatattta tcatactaac gtgggtgtgg 24840

aagaagaaag aaaaaactag tgttaaataa attcttagtc catagacata ttactgcctg 24900

aaagctttac atattgaaaa ttaatactga aggagtttat agtagaaatc aactgacata 24960

attcttcccc acccatgtta aaaaccatgt attttttaat gcaagaagct ttagttttgt 25020

ggaaatattt tttgagttat atgtgaaatg aaggaagtta ttaattccaa ttgactctta 25080

aaacttgtct tcagggagac ctgtgtacat ttcacttcct cattttctgt atgcaagtcc 25140

tgatgtttca gaacctattg atggattaaa cccaaatgaa gaagaacata ggacatactt 25200

ggatattgaa cctgtaagaa aacaccttat tgatctgatt tggttgatat ttttaaaaat 25260

acaattgaaa taaaaataat cttgtcgatg attatttatt caataaataa tcatatttat 25320

tgaatcacat tcttgaaagt tactgaaact taggtcgatt tcttcctatg gtttatgaag 25380

tgattctaat tggcttaaaa taattttata taaatattta tgtttagatg aggagttatt 25440

gtatattatg caaaagtcaa agagtatatg tgagtttgac tatacttatt gtcaaaatct 25500

ataaaattgt tgtagcgcaa cagttttaat catctttatt tttgtatttt ccttttcaaa 25560

aagtaaatga aattctagga ttttagtagt tatgttttag tttaaacaat gacacatgga 25620

ttctaactga atatatattt gaccaggaat tatctgagat cttatatttt gttgctgatt 25680

tttgattttt taaaaaccat ttcaagtaac tcacaaatct aactaactaa aaccttgaca 25740

ttcgattggg caaataaatt gtgtgtatct atatggatgc atgtgtatat aactataact 25800

atatatgcag ttttaaaagt ttcaattagt cctgtttaac cttaagttac taccttctct 25860

tctgctgtaa gaaaaataag ttttgaatag tataaaataa tgtttttaaa agttggtaat 25920

tatttagttg ttctcttttt agataactgg attcacttta caatttgcaa aacggctgca 25980

ggtcaaccta ttggtcaagc catcagaaaa aattcagtga gtctcttgaa aatggttatt 26040

ttgatatgat ctgtagtatc gtagtatctt cttgtaagaa catgagtaaa tctatgtaag 26100

taagtggaaa taacatctga tatcaactta tctttagctt aatgtcacca atcattatta 26160

aatgcttatg actaatttca cagattttgg aatggtttta tggttttatt tgagcatttg 26220

atagcatctc ttattttgtt agctgcccaa atatttctat gacaataatt aatttttgga 26280

attcatattt cagttccccg agaatttatt gaaaggaaaa atccacactt gtgaaaaaaa 26340

atcaatgtga ttagaagaca tataagagca aaggaagtca aaaacaacta tattaaaatt 26400

taaatgagtc attacaggaa caaaatcaaa ttagcaacag caactaattt atgaacattt 26460

attttaaagt ttgttatata taaatattag tttatatgtt cataattatt ttcaacgtat 26520

attacagagt attaaagaat ctgaagagga actatattgt gcctattctt tggcttaatg 26580

aggtttgtat ttgcagctgt tagtcattaa aaacaacctt ctttgtatat aaacaagctc 26640

ttgatgtttc aaaagaatgt atagtattta aagctatatg tatttccatt acccatatgg 26700

atgagtatac atttatttaa cctatttgag atgatccaat tgaacaaaaa catttcctat 26760

catttaagat tttcttcaaa aatgcatcta ttaaacacat tttcttgttg taacatttgt 26820

cttctattgc ctgacaaggt atttttacta taaatccatg cattgatagc tataaaaata 26880

ggaaaaacat tgaataagtc tttggagcaa atgaaactgt tgaccctttg atagttctga 26940

agagcaaatg aatcctagta cattgaagag taccgtactc tatctggcac ttaattgcct 27000

ttcttgactt gcaaaaggaa ttccattaac ttgccttata gatactgatg actaacacca 27060

atagaggtgt tagaaaaaag ggtgataggc aattgaaggg tttattttgt tttactaacg 27120

tacccaaata atgttgatta ttaacttgat tacagactgg gaccattggt gatgagaagg 27180

caaacatgtt cagaagtcaa gtaactggaa aaataaac 27218

<210> 6

<211> 62

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tgtgtttgga ggcattctca tgccagtcgg agacctgctt atccagaaga caattaaaaa 60

gg 62

<210> 7

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aagtcaagta actggaaaaa taaacctcct tggcatggta gagatggc 48

<210> 8

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tgtggacatg gcaggagatc ggatccccta ggagtactat catctg 46

<210> 9

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tagattacca caactccgag cccaaatgaa cttcactgga agaaaag 47

<210> 10

<211> 3566

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcagagacct atatttatct cctagtaggc gtgggtctga aggactggaa tgctcaggat 60

gtcaatggct gtcaggcgtc aggataacct agaagataga tgaatggttg agaccccgtg 120

cctcctccca gaattctcag ctgctccgct gtggaaatgg tgtgtttact ttctgtgtct 180

ccttcaacag tctccctgaa gccaatctta agatcctgtg actgggcaag caagctgttc 240

tagaagttgc tgccttctga aatgtgtgga gcaactggtg gatggtttcc tagcctttca 300

aaggtagttt acaaatgatt tgaagtgctg atcctttcag agtctcaaca gattatttct 360

taaacagctc atacattgct gtttatgcat gaattagtag aaccgggcca cgtagaaaac 420

actgtgattg tacctgggag ttggcgagaa aaccagtgct ctcccttgat tctgctgcac 480

gaggagaatg ggctgtgatc ggaactgtgg gctcattgct ggagctgtta ttggtgcagt 540

cctggctgtg tttggaggca ttctcatgcc agtcggagac ctgcttatcc agaagacaat 600

taaaaagcaa gttgtcctcg aagaaggtac aattgctttt aaaaattggg ttaaaacagg 660

cacagaagtt tacagacagt tttggatctt tgatgtgcaa aatccacagg aagtgatgat 720

gaacagcagc aacattcaag ttaagcaaag aggtccttat acgtacagag ttcgttttct 780

agccaaggaa aatgtaaccc aggacgctga ggacaacaca gtctctttcc tgcagcccaa 840

tggtgccatc ttcgaacctt cactatcagt tggaacagag gctgacaact tcacagttct 900

caatctggct gtggcagctg catcccatat ctatcaaaat caatttgttc aaatgatcct 960

caattcactt attaacaagt caaaatcttc tatgttccaa gtcagaactt tgagagaact 1020

gttatggggc tatagggatc catttttgag tttggttccg taccctgtta ctaccacagt 1080

tggtctgttt tatccttaca acaatactgc agatggagtt tataaagttt tcaatggaaa 1140

agataacata agtaaagttg ccataatcga cacatataaa ggtaaaagga atctgtccta 1200

ttgggaaagt cactgcgaca tgattaatgg tacagatgca gcctcatttc caccttttgt 1260

tgagaaaagc caggtattgc agttcttttc ttctgatatt tgcaggtcaa tctatgctgt 1320

atttgaatcc gacgttaatc tgaaaggaat ccctgtgtat agatttgttc ttccatccaa 1380

ggcctttgcc tctccagttg aaaacccaga caactattgt ttctgcacag aaaaaattat 1440

ctcaaaaaat tgtacatcat atggtgtgct agacatcagc aaatgcaaag aagggagacc 1500

tgtgtacatt tcacttcctc attttctgta tgcaagtcct gatgtttcag aacctattga 1560

tggattaaac ccaaatgaag aagaacatag gacatacttg gatattgaac ctataactgg 1620

attcacttta caatttgcaa aacggctgca ggtcaaccta ttggtcaagc catcagaaaa 1680

aattcaagta ttaaagaatc tgaagaggaa ctatattgtg cctattcttt ggcttaatga 1740

gactgggacc attggtgatg agaaggcaaa catgttcaga agtcaagtaa ctggaaaaat 1800

aaacctcctt ggcatggtag agatggcctt acttgggatt ggagtggtga tgtttgttgc 1860

ttttatgatt tcatattgtg cttgcaaatc caagaatgga aaataagtag tggatgagcc 1920

tacatataca ctggctacat ctttggtaaa gccgatctac aaaatgaaaa cttaatatat 1980

gcttcgtttt tacaaaacac acctatcttt aggagaagaa atggtggtgt gtgctctctc 2040

tctctttctc tttctctctt attgcagata tatatttatt tgtaaatata tatatatgca 2100

ataagtcaca gcatatttca aaagattaat atgtcactat aggcaatatt ttttaataaa 2160

atcttgcact tttattaaaa gtccatcatt tgcaactgag tggacttcaa tttctgtaga 2220

cccaattatc ctgtttggtt ctgatttact gatttgttcc atgttggcaa atttcaagaa 2280

tgtacattct gagaaatttt tgttttccct cactggagga aactgctatc atgactgggg 2340

tggccccttt gtttatagca aatttggctt gcaactgtca gcacatggca taagtataac 2400

atcttgaaag acttaagaat gaaaaatgaa caattcacat gtgagccact gcttatatat 2460

taagtctctc cctctctgga gttcttggct acagcaaggc cagatatcac attggttttg 2520

gttttgttgt ttttgttttg tttttgtttt ttactctctg acacagagct tatgaaatgg 2580

actttttttt ttttttttag cataccttag ctctttgtat tttaagtatg tcgtcatgtt 2640

ccatgctgca tagctcttta aaaaacctga gtaggttttt ctctttctgc tcagctgcaa 2700

ctaataacaa ccttggagag ctgttatagt gttaaaagat gtaaatgata ataaaagaat 2760

tattaaatgg aatcctacaa aagcaacaat gggctttaat atatatttgt ggtaatatct 2820

cctgctttca taatcaccca aaaaaaggac tggtttctaa cattaaaaga agtcgttcct 2880

taaattcaac ctttttgtct agttactatt gaatcacagt atgttattta ttgtatttta 2940

ttttgttttt tgtagaagtg ttctatctga gattctgtgt ccttctgaat catttaacct 3000

ttctgctatg tcttaatgca attgaacaca gatattttgt tataatttta tagcagcagt 3060

gtgagactga aggcaaatat tttccagggt aagacacagt gataggttgt tctacttcct 3120

ttagcagcat aatcaatttc acttagtatt agtcccaata aattctggta taagacacaa 3180

aataaaaaca aacattttct gttctatacg tagaaaaata aaaggaaagg tgagacaact 3240

aactctcttg tctagatata aaaataatca aaactgcatg gcagctttgg gcaggagtgc 3300

tggattagtg gttaggagtc atcaatgttc ctacagaaga cctgggcttg gtacccagct 3360

tctatacgtc tgctcacagc ctcatatgat tcctgtcagt tgatactatg cccgcctctc 3420

ctgtgcaccc ttctgcactg catgtggtga tcatttatac actcaggtat gcatgcacac 3480

atggaattta aagaggctgg aagagtgtat gagaaggttg tagaggagaa aggagtgtgg 3540

gaaacgatgt cattaaaatc agattt 3566

<210> 11

<211> 472

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Met Gly Cys Asp Arg Asn Cys Gly Leu Ile Ala Gly Ala Val Ile Gly

1 5 10 15

Ala Val Leu Ala Val Phe Gly Gly Ile Leu Met Pro Val Gly Asp Leu

20 25 30

Leu Ile Gln Lys Thr Ile Lys Lys Gln Val Val Leu Glu Glu Gly Thr

35 40 45

Ile Ala Phe Lys Asn Trp Val Lys Thr Gly Thr Glu Val Tyr Arg Gln

50 55 60

Phe Trp Ile Phe Asp Val Gln Asn Pro Gln Glu Val Met Met Asn Ser

65 70 75 80

Ser Asn Ile Gln Val Lys Gln Arg Gly Pro Tyr Thr Tyr Arg Val Arg

85 90 95

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

100 105 110

Ser Phe Leu Gln Pro Asn Gly Ala Ile Phe Glu Pro Ser Leu Ser Val

115 120 125

Gly Thr Glu Ala Asp Asn Phe Thr Val Leu Asn Leu Ala Val Ala Ala

130 135 140

Ala Ser His Ile Tyr Gln Asn Gln Phe Val Gln Met Ile Leu Asn Ser

145 150 155 160

Leu Ile Asn Lys Ser Lys Ser Ser Met Phe Gln Val Arg Thr Leu Arg

165 170 175

Glu Leu Leu Trp Gly Tyr Arg Asp Pro Phe Leu Ser Leu Val Pro Tyr

180 185 190

Pro Val Thr Thr Thr Val Gly Leu Phe Tyr Pro Tyr Asn Asn Thr Ala

195 200 205

Asp Gly Val Tyr Lys Val Phe Asn Gly Lys Asp Asn Ile Ser Lys Val

210 215 220

Ala Ile Ile Asp Thr Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp Glu

225 230 235 240

Ser His Cys Asp Met Ile Asn Gly Thr Asp Ala Ala Ser Phe Pro Pro

245 250 255

Phe Val Glu Lys Ser Gln Val Leu Gln Phe Phe Ser Ser Asp Ile Cys

260 265 270

Arg Ser Ile Tyr Ala Val Phe Glu Ser Asp Val Asn Leu Lys Gly Ile

275 280 285

Pro Val Tyr Arg Phe Val Leu Pro Ser Lys Ala Phe Ala Ser Pro Val

290 295 300

Glu Asn Pro Asp Asn Tyr Cys Phe Cys Thr Glu Lys Ile Ile Ser Lys

305 310 315 320

Asn Cys Thr Ser Tyr Gly Val Leu Asp Ile Ser Lys Cys Lys Glu Gly

325 330 335

Arg Pro Val Tyr Ile Ser Leu Pro His Phe Leu Tyr Ala Ser Pro Asp

340 345 350

Val Ser Glu Pro Ile Asp Gly Leu Asn Pro Asn Glu Glu Glu His Arg

355 360 365

Thr Tyr Leu Asp Ile Glu Pro Ile Thr Gly Phe Thr Leu Gln Phe Ala

370 375 380

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

385 390 395 400

Val Leu Lys Asn Leu Lys Arg Asn Tyr Ile Val Pro Ile Leu Trp Leu

405 410 415

Asn Glu Thr Gly Thr Ile Gly Asp Glu Lys Ala Asn Met Phe Arg Ser

420 425 430

Gln Val Thr Gly Lys Ile Asn Leu Leu Gly Met Val Glu Met Ala Leu

435 440 445

Leu Gly Ile Gly Val Val Met Phe Val Ala Phe Met Ile Ser Tyr Cys

450 455 460

Ala Cys Lys Ser Lys Asn Gly Lys

465 470

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

aaccagtgct ctcccttgat tctg 24

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gtcgcatcat atagagttgc agtgg 25

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agataacagg ctggcctgga gaaaa 25

<210> 15

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gttgcccaat accataattt ggaaaagt 28

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ctttagcacc aggtgtgcgt cagta 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ggtcatgcac caacatccac acaac 25

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gaaggccatg ccagtgagct t 21

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

aaccagtgct ctcccttgat tctg 24

<210> 20

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

aaagcatgcc agtctactcc aga 23

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gtcgcatcat atagagttgc agtgg 25

<210> 22

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ttgccatgga tcatagctgt agc 23

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

agttacatat ggctggcggc tgct 24

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gacaagcgtt agtaggcaca tatac 25

<210> 25

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gctccaattt cccacaacat tagt 24

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

aaacccagat gacgtggcaa 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ttcagatccg aacacagcgt 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

aatgtaaccc aggacgctga 20

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gtggaaatga ggctgcatct g 21

<210> 30

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

tcaccatctt ccaggagcga ga 22

Claims

1. A method for constructing a humanized non-human animal of a CD36 gene, wherein the humanized CD36 protein is expressed in the non-human animal, and/or wherein the genome of the non-human animal comprises the humanized CD36 gene; the humanized CD36 gene encodes the humanized CD36 protein;

The construction method comprises the steps of introducing part of human CD36 gene into a non-human animal CD36 gene locus;

the amino acid sequence of the humanized CD36 protein is shown in SEQ ID NO: 11;

the non-human animal has reduced or absent expression of endogenous CD36 protein.

2. The method of claim 1, wherein the portion of the human CD36 gene comprises a portion of exon 3, all of exons 4 to 13, and a portion of exon 14 of the human CD36 gene.

3. The method of claim 1, wherein the portion of the human CD36 gene comprises the amino acid sequence of SEQ ID NO:5, and a nucleotide sequence shown in SEQ ID NO.

4. The method of claim 1, wherein the humanized CD36 gene comprises a portion of exon 4 and/or a portion of exon 15 of the non-human animal CD36 gene.

5. The method according to claim 4, wherein the humanized CD36 gene further comprises all or part of exons 1 to 3 and/or exons 16 of a non-human animal CD36 gene.

6. The method according to claim 1, wherein the humanized CD36 gene transcribed mRNA is represented by SEQ ID NO: shown at 10.

7. The method of claim 6, wherein the humanized CD36 gene is regulated in a non-human animal by endogenous regulatory elements.

8. The construction method according to claim 1, wherein the non-human animal CD36 gene locus is replaced with a non-human animal corresponding region, and the non-human animal CD36 gene has a portion of exon 4, all of exons 5 to 14 and a portion of exon 15 replaced.

9. The method of claim 1, wherein the method of constructing comprises constructing a non-human animal using a targeting vector; the targeting vector comprises a donor nucleotide sequence comprising a portion of the human CD36 gene.

10. The method of claim 9, wherein the portion of the human CD36 gene comprises a portion of exon 3, all of exons 4 to 13, and a portion of exon 14 of the human CD36 gene.

11. The method of claim 10, wherein the portion of the human CD36 gene comprises the amino acid sequence of SEQ ID NO:5, and a nucleotide sequence shown in SEQ ID NO.

12. The method of claim 9, wherein the targeting vector further comprises a 5' arm and/or a 3' arm, and wherein the 5' arm has the sequence set forth in SEQ ID NO:3 is shown in the figure; the 3' arm sequence is shown in SEQ ID NO: 4.

13. The construction method according to claim 1, wherein the construction method comprises mating a CD36 gene humanized non-human animal with other genetically modified non-human animals, in vitro fertilization or direct gene editing, and screening to obtain a polygenic modified non-human animal.

14. The method of claim 13, wherein the additional gene is selected from at least one of PD-1, PD-L1, TIGIT, and CD 226.

15. The method of any one of claims 1-14, wherein the non-human animal is a rat or a mouse.

16. A humanized CD36 protein comprising all or a portion of an extracellular domain of a human CD36 protein and all or a portion of a transmembrane and/or cytoplasmic domain of a non-human animal CD36 protein;

the amino acid sequence of the humanized CD36 protein is shown in SEQ ID NO: 11.

17. The humanized CD36 protein of claim 16 wherein the non-human animal is a rat or a mouse.

18. A humanized CD36 gene, wherein the mRNA transcribed from the humanized CD36 gene is as set forth in SEQ ID NO: shown at 10.

19. A targeting vector comprising the sequence set forth in SEQ ID NO:5, and a nucleotide sequence shown in SEQ ID NO.

20. The targeting vector according to claim 19, wherein said targeting vector further comprises a 5' arm and/or a 3' arm, said 5' arm having the sequence set forth in SEQ ID NO:3 is shown in the figure; the 3' arm sequence is shown in SEQ ID NO: 4.

21. A cell, tissue or organ which expresses the humanized CD36 protein of claim 16 or 17, or which comprises the humanized CD36 gene of claim 18, or which is derived from a non-human animal obtained by the construction method of any one of claims 1 to 15, which is incapable of developing into an animal individual.

22. Use of a humanized CD36 protein according to claim 16 or 17, a humanized CD36 gene according to claim 18, a non-human animal obtained by a construction method according to any of claims 1-15 or a cell, tissue or organ according to claim 21, characterized in that said use comprises:

c) To the use of animal experimental disease models for the production and use in research of etiology associated with CD 36;

e) In researching the functions of CD36 gene and researching the application of drugs and drug effects aiming at human CD36 target sites,

the application is for the diagnosis and treatment of non-diseases.

23. The use according to claim 22, wherein said drugs against human CD36 target sites in E) comprise immune-related disease drugs and anti-tumor drugs.