CN114835798A

CN114835798A - Construction method and application of CD36 gene humanized non-human animal

Info

Publication number: CN114835798A
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: 2022-08-02
Anticipated expiration: 2042-05-27
Also published as: WO2022247936A1; CN114835798B

Abstract

The invention provides a humanized CD36 protein, a humanized CD36 gene, a targeting vector of a CD36 gene, a humanized non-human animal of a CD36 gene, a construction method thereof and application thereof in the field of biomedicine, wherein a nucleotide sequence for coding the human CD36 protein is introduced into the genome of the non-human animal by utilizing a homologous recombination mode, the animal can normally express the human or humanized CD36 protein, can be used as an animal model for researching the signal mechanism of the human CD36 and screening drugs of tumors and autoimmune diseases, and has important application value for the research and development of new drugs of immune targets.

Description

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

Technical Field

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

Background

CD36(Cluster of differentiation 36), also known as platelet glycoprotein 4, Fatty Acid Translocase (FAT), scavenger receptor class B member 3(SCARB3), glycoprotein 88(GP88), GPIIIB, GPIV, members of the cell surface protein class B scavenger receptor family. Mainly expressed on the surfaces of capillary endothelial cells, mammary secretory epithelial cells, differentiated adipocytes, 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 in breast, prostate, ovarian, colon, oral squamous cell carcinoma and acute myeloid leukemia. The ligand comprises: collagen, thrombospondin (TSP-1), erythrocyte parasitized by plasmodium falciparum, oxidized low density lipoprotein (oxLDL), natural lipoprotein, oxidized phospholipid and long chain Fatty Acid (FA), etc., and the functional diversity of CD36 depends on the combination of different ligands: a. as a lipid transaminase, CD36 can recognize and endocytose denatured LDL (oxLDL and AcLDL), leading to foaming of macrophages, secretion of various inflammatory cytokines, initiation of atherogenesis; b. on macrophages, CD36 forms part of the non-opsonin receptor (CD36/α V β 3 complex) and is involved in phagocytosis; c. CD36 on macrophages and DC cells can also bind to heat shock protein gp96, stimulating immune responses in the body by releasing cytokines, chemokines and nitric oxide; d. recognizing exogenous substances, such as red blood cells infected with plasmodium falciparum and cell wall components of staphylococcus and mycobacterium, exerting innate immune function; e. recognizing endogenous ligands, CD36 on macrophages can bind to beta-amyloid peptide, and has a role in alzheimer's disease; f. promoting sterile inflammation through the assembly of TLR2/6 or TLR4/6 heterodimers; g. CD36 on tumor microvascular endothelial cells promotes vascular apoptosis by binding TSP-1 in the tumor microenvironment, while recruiting Src family members to mediate VEGFR2 dephosphorylation, inhibiting the VEGF pathway, leading to anti-angiogenesis. In addition, CD36 activates Wnt/TGF- β signaling to promote tumor metastasis by EMT, and also absorbs lipid molecules to deposit lipids in immune cells, resulting in sterile inflammation and dysfunction of antigen presentation in DC cells, 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 diseases, developing prevention and treatment technologies and developing medicines. However, due to the differences between the physiological structures and metabolic systems of animals and humans, the traditional animal models cannot reflect the real conditions of human bodies well, and the establishment of disease models closer to the physiological characteristics of human bodies in animal bodies is an urgent need of the biomedical industry.

With the continuous development and maturation of genetic engineering technology, the replacement or substitution of homologous genes of animals by human genes has been realized, and the development of humanized experimental animal models in this way is the future development direction of animal models. The gene humanized animal model is one animal model with normal or mutant gene replaced with homologous gene in animal genome and similar physiological or disease characteristics. The gene humanized animal not only has important application value, for example, the humanized animal model of cell or tissue transplantation can be improved and promoted by gene humanization, but also more importantly, the human protein can be expressed or partially expressed in the animal body due to the insertion of the human gene segment, and the gene humanized animal can be used as a target of a drug which can only recognize the human protein sequence, thereby providing possibility for screening anti-human antibodies and other drugs at the animal level. However, due to differences in physiology and pathology between animals and humans, coupled with the complexity of genes, for example, the identity of human and mouse CD36 protein is 84%, how to construct an "efficient" humanized animal model for new drug development remains the greatest challenge.

In view of the complex mechanism of action of CD36 and the huge application value in the field of tumor therapy, there is an urgent need in the art to develop a non-human animal model of CD 36-related signaling pathway in order to further explore its relevant biological properties, improve the effectiveness of preclinical drug efficacy tests, improve the success rate of research and development, make preclinical tests more effective and minimize the research and development failure. In addition, the non-human animal obtained by the method can be mated with other gene humanized non-human animals to obtain a multi-gene humanized animal model which is used for screening and evaluating the drug effect research of human drugs and combined drugs aiming at the signal path. The invention has wide application prospect in academic and clinical research.

Disclosure of Invention

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

Preferably, the humanized CD36 protein comprises all or part of the transmembrane region, cytoplasmic region and/or extracellular region of the human CD36 protein, and further preferably, the humanized CD36 protein comprises all or part of the extracellular region of the human CD36 protein.

In one embodiment of the present invention, the humanized CD36 protein comprises all or part of the extracellular domain of human CD36 protein, preferably, the extracellular domain of human CD36 protein comprising at least 50 consecutive amino acids, for example, the extracellular domain of 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, and further preferably, the extracellular domain of human CD36 protein comprising 410 consecutive amino acids; more preferably, it comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 30-439 of SEQ ID NO. 2 or comprises the amino acid sequence shown in 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 the non-human animal CD36 protein is all or part of the transmembrane region and/or cytoplasmic region of the non-human animal CD36 protein. Preferably comprises SEQ ID NO:1, 1-29 and/or 440-472; or, comprising a nucleotide sequence identical to SEQ ID NO:1 at positions 1-29 and/or 440-472 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, positions 1-29 and/or 440-472 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, and/or 440-472, including substitution, deletion and/or insertion of one or more amino acid residues, or, preferably, an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:1, 8-29, 440-461, 1-7 or 462-472; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 8-29, 440-461, 1-7 or 462-472 position by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 8-29, 440-461, 1-7 or 462-472 amino acid sequence of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:1, 8-29, 440-461, 1-7 or 462-472, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized CD36 protein comprises all or part of an amino acid sequence encoded by exons 1 to 14 of human CD36 gene. Further preferably, the polypeptide comprises all or part of an amino acid sequence encoded by any one, two, three or more, two or more consecutive exons from exon 1 to exon 14 in combination. Even more preferably, it comprises all or part of the amino acid sequence encoded by exons 3 to 14. Still further preferably, the kit comprises a part of exon 3, all of exons 4 to 13 and a part of exon 14, wherein the part of exon 3 comprises at least 10bp of nucleotide sequence, for example at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp of nucleotide sequence, and further preferably 33bp of nucleotide sequence; preferably, part of exon 3 comprises the nucleotide sequence of exon 3 coding for the extracellular region, and part of exon 14 comprises at least 30bp of nucleotide sequence, such as at least 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 of nucleotide sequence, and further preferably comprises 63bp of nucleotide sequence; preferably, the portion of exon 14 comprises the nucleotide sequence of exon 14 that encodes the extracellular domain. 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 an amino acid sequence encoded by SEQ ID NO: 5, or a pharmaceutically acceptable salt thereof.

Preferably, the transmembrane region and cytoplasmic region 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 the non-human animal CD36 gene, preferably all or part of an amino acid sequence encoded by exons 1 to 3 and/or 16 of the non-human animal CD36 gene, and further preferably comprises all or part of an amino acid sequence encoded by exons 4 and/or part of exons 15.

In one embodiment of the present invention, the amino acid sequence of the humanized CD36 protein comprises one of the following groups:

A) is SEQ ID NO:2, all or part of the amino acid sequence from 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% amino acid sequence identity between positions 30-439;

C) and SEQ ID NO:2 from position 30 to position 439 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or

D) And SEQ ID NO:2, and (b) an amino acid sequence comprising substitution, deletion and/or insertion of one or more amino acid residues, as shown in positions 30 to 439 of the above formula.

In one 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, all or part of an amino acid sequence;

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

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

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

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

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

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

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

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

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

Preferably, the humanized CD36 gene comprises all or part of a nucleotide sequence encoding a transmembrane region, cytoplasmic region and/or extracellular region of human CD36 protein, wherein the humanized CD36 gene comprises all or part of a nucleotide sequence encoding an extracellular region of human CD36 protein, preferably comprises a nucleotide sequence encoding an extracellular region of human CD36 protein of at least 50 contiguous amino acids, such as at least a nucleotide sequence encoding an extracellular region of 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 contiguous amino acids, further preferably comprises a nucleotide sequence encoding an extracellular region of human CD36 protein of 410 contiguous amino acids; more preferably, it comprises a nucleotide sequence having 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 comprises a nucleotide sequence encoding positions 30-439 of SEQ ID NO. 2.

Preferably, the humanized CD36 gene comprises all or part of exons 1 to 14 of human CD36 gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 14 is contained. Even more preferably, all or part of exons 3 to 14 are included. Still further preferably, a part of exon 3, all of exon 4 to exon 13 and a part of exon 14, preferably further intron 3-4 and/or intron 13-14, more preferably further any intron between exons 3-14, wherein the part of exon 3 comprises at least 10bp of nucleotide sequence, such as at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp of nucleotide sequence, further preferably 33bp of nucleotide sequence; preferably, part of exon 3 comprises the nucleotide sequence of exon 3 coding for the extracellular region, and part of exon 14 comprises at least 30bp of nucleotide sequence, such as at least 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 of nucleotide sequence, and more preferably 63bp of nucleotide sequence; preferably, the portion of exon 14 comprises the nucleotide sequence of exon 14 that encodes the extracellular domain.

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

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

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

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

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

(A) is SEQ ID NO: 5, all or part of a nucleotide sequence set forth in seq id no;

(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 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or

(D) Has the sequence shown in SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment of the present 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 or all of a 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 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; or

(iv) And SEQ ID NO: 10, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

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 tetracycline System (Tet-Off System/Tet-On System) or Tamoxifen System (Tamoxifen System).

In a fourth aspect of the invention, there is provided a targeting vector comprising a donor nucleotide sequence, said donor nucleotide sequence comprising part 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 preferably, any one, two, three or more, two or more consecutive exons among exons 1 to 14 are contained in combination. Still further preferably, all or part of exons 3 to 14 are comprised. Even more preferably, the part of exon 3, all of exon 4 to exon 13 and part of exon 14 are included, wherein the part of exon 3 at least comprises 10bp nucleotide sequence, such as at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp nucleotide sequence, and more preferably, 33bp nucleotide sequence; preferably, part of exon 3 comprises the nucleotide sequence of exon 3 coding for the extracellular region, and part of exon 14 comprises at least 30bp of nucleotide sequence, such as at least 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 of nucleotide sequence, and more preferably 63bp of nucleotide sequence; preferably, the portion of exon 14 comprises the nucleotide sequence of exon 14 that encodes the extracellular domain. 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 to a polypeptide comprising SEQ ID NO: 5.

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

Preferably, the targeting vector comprises a nucleotide sequence encoding an 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 transition region to be altered, i.e.the 5 ' arm (5 ' homology arm), which is selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD36 gene. Further preferred are nucleotides having at least 90% homology in the 5' arm with NCBI accession No. NC _ 000071.7. Still further preferably, the 5' arm sequence is identical to SEQ ID NO: 3 or as shown in SEQ ID NO: 3, respectively. And/or, the targeting vector further comprises a DNA fragment homologous to the 3 ' end of the transition region to be altered, i.e.the 3 ' arm (3 ' homology arm), which is selected from the group consisting of 100-10000 nucleotides in length of the genomic DNA of the non-human animal CD36 gene. Further preferred are nucleotides having at least 90% homology in the 3' arm with NCBI accession No. NC _ 000071.7. Still more preferably, the 3' arm sequence is identical to SEQ ID NO: 4 or as shown in SEQ ID NO: 4, respectively.

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

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

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

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

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

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

In a sixth aspect of the invention, there is provided the use of the above targeting vector, or the above cell, in the modification of the CD36 gene, preferably, said use includes but is not limited to knock-out, insertion or substitution.

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

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

Preferably, the non-human animal expresses the humanized CD36 protein described above.

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

Preferably, the non-human animal body contains a part of a human CD36 gene, and more preferably contains the humanized CD36 gene.

Preferably, the genome of the non-human animal comprises a portion of the human CD36 gene. Further preferably, the genome of said non-human animal comprises all or part of exons 1 to 14 of human CD36 gene. Further preferably, all or part of a combination of any one, two, three or more, two or three or more consecutive exons from exon 1 to exon 14 is contained. Even more preferably, all or part of exons 3 to 14 are included. Still further preferably, a part of exon 3, all of exon 4 to exon 13 and a part of exon 14, preferably further intron 3-4 and/or intron 13-14, more preferably further any intron between exons 3-14, wherein the part of exon 3 comprises at least 10bp of nucleotide sequence, such as at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp of nucleotide sequence, further preferably 33bp of nucleotide sequence; preferably, part of exon 3 comprises the nucleotide sequence of exon 3 coding for the extracellular region, and part of exon 14 comprises at least 30bp of nucleotide sequence, such as at least 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 of nucleotide sequence, and more preferably 63bp of nucleotide sequence; preferably, the portion of exon 14 comprises the nucleotide sequence of exon 14 that encodes the extracellular domain.

In one embodiment of the present invention, the genome of the non-human animal comprises a nucleotide sequence of an extracellular region encoding human CD36 protein, wherein the genome of the non-human animal comprises all or part of the nucleotide sequence of an extracellular region encoding human CD36 protein, preferably comprises a nucleotide sequence of an extracellular region encoding human CD36 protein with at least 50 consecutive amino acids, for example, comprises at least a nucleotide sequence of an extracellular region encoding human CD36 protein with 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, and further preferably comprises a nucleotide sequence of an extracellular region encoding human CD36 protein with 410 consecutive amino acids; more preferably, it comprises an amino acid sequence having 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 comprises a nucleotide sequence encoding positions 30-439 of SEQ ID NO. 2.

According to some embodiments of the invention, the non-human animal further comprises additional genetic modifications, the additional genes 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.

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

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

The ninth aspect of the invention provides a method for constructing a non-human animal with a CD36 gene deletion, which comprises the step of preparing the non-human animal by using the targeting vector.

In the tenth aspect of the invention, a method for constructing a non-human animal humanized with a CD36 gene is provided, the method comprises introducing a partial nucleotide sequence comprising a human CD36 gene into the CD36 locus of the non-human animal, and the non-human animal expresses a human or humanized CD36 protein.

Preferably, the humanized CD36 protein comprises the humanized CD36 protein 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.

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

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

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

Preferably, the partial nucleotide sequence of the human CD36 gene comprises all or partial nucleotide sequences of exons 1 to 14 of the human CD36 gene, more preferably comprises all or partial nucleotide sequences of any one, two, three or more, two or more continuous exons or combinations of three or more continuous exons of the exons 1 to 14, and still more preferably comprises all or partial nucleotide sequences of exons 3 to 14. Most preferably, part of exon 3, all of exon 4 to exon 13 and part of exon 14, preferably further comprises intron 3-4 and/or intron 13-14, more preferably comprises any intron between exons 3-14, wherein part of exon 3 comprises at least 10bp of nucleotide sequence, for example at least 10, 15, 20, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 180, 190, 200, 208, 209bp of nucleotide sequence, and even more preferably 33bp of nucleotide sequence; preferably, part of exon 3 comprises the nucleotide sequence of exon 3 coding for the extracellular region, and part of exon 14 comprises at least 30bp of nucleotide sequence, such as at least 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 of nucleotide sequence, and more preferably 63bp of nucleotide sequence; preferably, the portion of exon 14 comprises the nucleotide sequence of exon 14 that encodes the extracellular domain. In one embodiment of the invention, the polypeptide 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 to a polypeptide comprising SEQ ID NO: 5.

Preferably, the construction method comprises introducing the nucleotide sequence comprising all or part of the nucleotide sequence encoding human CD36 protein into the CD36 locus of the non-human animal. Further preferably, the constructing method comprises introducing into the non-human animal CD36 locus a nucleotide sequence comprising all or part of a transmembrane region, cytoplasmic region and/or extracellular region encoding human CD36 protein; preferably, the non-human animal CD36 gene 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, for example, 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 CD36 gene locus is introduced into a non-human animal by a gene comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to positions 30-439 of SEQ ID NO. 2 or by a gene comprising an amino acid sequence as shown in positions 30-439 of SEQ ID NO. 2, even more preferably by a gene comprising a nucleotide sequence encoding at least 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity to the nucleotide sequence of positions 30-439 of SEQ ID NO. 2 or by a gene comprising a nucleotide sequence encoding positions 30-439 of SEQ ID NO. 2.

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

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

In a specific embodiment of the invention, the nucleotide sequence comprising the humanized CD36 gene is introduced into 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 replacing or inserting, and in a specific embodiment of the invention, the introducing is to replace a corresponding region of the non-human animal CD36 gene locus, further preferably, all or part of exons 4 to 15 of the non-human animal CD36 gene is replaced, and further preferably, all or part of exons 4, 5 to 14, and 15 of the non-human animal CSF1 gene is replaced.

Preferably, the nucleotide sequence encoding the extracellular domain 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 after the endogenous regulatory elements of the CD36 gene.

Preferably, the insertion is performed by first disrupting the coding frame of the endogenous CD36 gene of the non-human animal or disrupting the coding frame of the endogenous CD36 gene following the insertion sequence, followed by insertion. Or the insertion step can cause frame shift mutation to the endogenous CD36 gene and can realize the step of inserting the human sequence.

Further preferably, an auxiliary sequence (e.g., a stop codon or a sequence having a function of termination, etc.) or other methods (e.g., a turn-over sequence, or a knock-out sequence) may be added to the inserted sequence so that the endogenous CD36 protein in the non-human animal after the insertion site is not normally expressed.

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

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

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

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

Preferably, the genome of the non-human animal comprises a humanized CD36 gene on at least one chromosome.

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

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

In one embodiment of the invention, the construction method comprises modifying the coding frame of the non-human animal CD36 gene, and inserting the nucleotide sequence of the coding human or humanized CD36 protein or the nucleotide sequence of the humanized CD36 gene into the endogenous regulatory elements of the non-human animal CD36 gene. Wherein, the coding frame of the modified non-human animal CD36 gene can adopt a functional region knocked out of a non-human animal CD36 gene or a sequence inserted so that the non-human animal CD36 protein is not expressed or the expression is reduced or the expressed protein is not functional. Further preferably, the coding frame of the modified non-human animal CD36 gene can be all or part of the nucleotide sequence from exon 4 to exon 15 of the knocked-out non-human animal CD36 gene.

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

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

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

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

According to some embodiments of the invention, the building method further comprises: the CD36 gene-humanized non-human animal is mated with another genetically modified non-human animal, fertilized in vitro or directly subjected to gene editing, and then screened to obtain a polygene-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 protein.

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

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

In the eleventh aspect of the present invention, there is provided a method for constructing a polygene-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 genetically modified non-human animals, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-gene modified non-human animal.

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized by a combination of one or more of the genes PD-1, PD-L1, TIGIT or CD 226.

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

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

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

In a thirteenth aspect of the present invention, an animal model is provided, wherein the animal model is derived from the above non-human animal, the non-human animal obtained by the above construction method, or the above non-human animal or its progeny. 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-mentioned method for constructing a non-human animal, a non-human animal or its offspring, a gene-deleted animal or a polygene-modified non-human animal. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

In a fifteenth aspect, the present invention provides the use of the non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, or the above constructed polygene-modified non-human animal for the preparation of an animal model. Preferably, the animal model is a tumor-bearing or inflammatory animal model.

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

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

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

In a nineteenth aspect of the invention, there is provided a cell humanized with the CD36 gene, said cell expressing human or humanized CD36 protein.

Preferably, the cell expresses the humanized CD36 protein described above.

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

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

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

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

In a twenty-third aspect, the present invention provides a use of the above-mentioned humanized CD36 protein, the above-mentioned nucleic acid, the above-mentioned humanized CD36 gene, the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the above-mentioned non-human animal or its progeny, the above-mentioned 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-bearing tissue, the above-mentioned cell, the above-mentioned construct, the above-mentioned cell or the above-mentioned tissue, the use comprising:

A) use in the development of products involving CD 36-related immune processes in human cells;

B) use as a model system in pharmacological, immunological, microbiological and medical research associated with CD 36;

C) to the production and use of animal experimental disease models for the research of CD 36-related etiology and/or for the development of diagnostic strategies and/or for the development of therapeutic strategies;

D) the application of the human CD36 signal channel regulator in screening, drug effect detection, curative effect evaluation, verification or evaluation is studied in vivo; alternatively, the first and second electrodes may be,

E) the functions of the CD36 gene are researched, the medicine and the drug effect aiming at the target site of the human CD36 are researched, and the application in the aspects of the medicine for the immune-related diseases related to the CD36 and the anti-tumor medicine is researched.

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

In a twenty-fourth aspect of the present invention, there is provided a method of screening for a modulator specific for human CD36, said method comprising administering the modulator to an individual implanted with tumor cells and detecting tumor suppression; wherein the individual is selected from the non-human animal, the non-human animal obtained by the construction method, the non-human animal or its offspring or the animal model.

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

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

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

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

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

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

Preferably, the screening method for a human 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, test and compare the potency of candidate drugs to determine which candidate drugs can act as drugs and which cannot act as drugs, or to compare the potency sensitivity of different drugs, i.e., the therapeutic effect is not necessarily but only a possibility.

According to a twenty-fifth aspect of the invention, an evaluation method of an intervention scheme is provided, the evaluation method comprises implanting tumor cells into an individual, applying the intervention scheme to the individual implanted with the tumor cells, and detecting and evaluating the tumor inhibition effect of the individual after applying the intervention scheme; wherein the individual is selected from the non-human animal, the non-human animal obtained by the construction method, the non-human animal or its offspring, or the animal model.

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

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

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

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

In a twenty-sixth aspect of the present invention, there is provided a use of the non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the above animal model in the preparation of a human CD 36-specific modulator.

The twenty-seventh aspect of the invention provides a non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above non-human animal or its progeny, and the use of the above animal model in the preparation of a medicament for treating tumor or immune-related diseases.

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

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

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

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

The humanized CD36 protein comprises a part derived from human CD36 protein and a part of non-human CD36 protein.

The humanized CD36 protein of the present invention includes part of human CD36 protein. Wherein, the "human CD36 protein" is the same as the whole "human CD36 protein", namely, the amino acid sequence of the "human CD36 protein" is consistent with the full-length amino acid sequence of the human CD36 protein. The "part of human CD36 protein" is continuous or alternate 5-472 (preferably 10-410) amino acid sequences which are consistent with the amino acid sequence of human CD36 protein or have more than 70% homology with the amino acid sequence of human CD36 protein.

The "humanized CD36 gene" of the present invention includes part derived from human CD36 gene and part of non-human CD36 gene. Wherein, the "human CD36 gene" is the same as the whole "human CD36 gene", namely, the nucleotide sequence of the "human CD36 gene" is consistent with the full-length nucleotide sequence of the human CD36 gene. The 'part of the human CD36 gene' is a continuous or spaced 20-77071bp (preferably 20-27218bp or 20-1230bp) nucleotide sequence which is consistent with the nucleotide sequence of the human CD36 or has more than 70 percent of homology with the nucleotide sequence of the human CD 36.

The "xx to xxx exons" or all of the "xx to xxx exons" in the present invention include nucleotide sequences of exons and introns therebetween, for example, the "3 to 4 exons" include all nucleotide sequences of exon 3, intron 3 to 4, and exon 4.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, "intron 3-4" means an intron between exon 3 and exon 4.

"part of an exon" as referred to herein means that the nucleotide sequence is identical to all exon nucleotide sequences in a sequence of several, several tens or several hundreds of nucleotides in succession or at intervals. For example, the portion of exon 3 of human CD36 gene comprises consecutive or spaced nucleotide sequences of 5-209bp, preferably 10-33bp, identical to the nucleotide sequence of exon 3 of human CD36 gene. In a specific embodiment of the present invention, the "part of exon 3" contained in the "humanized CD36 gene" at least includes a nucleotide sequence encoding an extracellular region in exon 3.

The "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense and refers to a DNA fragment of a certain gene in a narrow sense, and the gene may be a single gene or a part of a single gene. For example, the "CD 36 locus" refers to a DNA fragment of any one of exons 1 to 16 of the CD36 gene. Preferably 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 any combination of two or more thereof, or all or part of one or two or more thereof, more preferably exon 4 to exon 15 of the CD36 gene.

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

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

The term "three or more in succession" in the present invention includes, but is not limited to, three in succession, four in succession, five in succession, six in succession, seven in succession, and the like. Wherein "three or more consecutive exons 1 to 6" includes three, four, five or six, etc. consecutive exons, and also includes intron nucleotide sequences in between.

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

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

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

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

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

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

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

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

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

Drawings

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

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

FIG. 2: schematic representation of humanization of mouse CD36 gene (not to scale);

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

FIG. 4: CD36 post-recombination cellular Southern blot results, where WT is wild type control;

FIG. 5: schematic representation (not to scale) of FRT recombination process for humanized mouse of CD36 gene;

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

FIG. 7: RT-PCR detection results of C57BL/6 wild type mice (+/+) and CD36 gene humanized homozygote mice (H/H), wherein H is ₂ O is water control, GAPDH is glyceraldehyde-3-phosphate deprivationInternal reference of catalase;

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

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

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

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

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

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

FIG. 14: implanting mouse colon cancer cell MC38 into humanized CD36 mouse, and performing anti-tumor effect test to obtain the weight result of the mouse;

FIG. 15: implanting mouse colon cancer cell MC38 into humanized CD36 mouse, and performing antitumor effect test to obtain the weight change result of the mouse;

FIG. 16: the results of the tumor volume of mice after the antitumor effect test was performed by implanting mouse colon cancer cell MC38 into humanized CD36 mice.

Detailed Description

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

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

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

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

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

PerCP anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody, available from Biolegend under cat # 108426;

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

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

APC anti-mouse CD36 Antibody was purchased from Biolegend under Cat 102611;

PE anti-human CD36 Antibody was purchased from Biolegend, cat # 336205;

mouse IgG2A PE-conjugated Antibody was purchased from RD, cat # IC 003P;

APC Armenian Hamster IgG Isotype Ctrl Antibody was purchased from Biolegend under Cat 400912;

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

Example 1 humanized mouse with CD36 Gene

A comparison scheme between 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 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.

To achieve the object of the present invention, a nucleotide sequence encoding a human CD36 protein may be introduced at the endogenous CD36 locus of a mouse, so that the mouse expresses a human or humanized CD36 protein. Specifically, by using a gene editing technology, under the control of mouse CD36 gene regulatory elements, a part of the sequence from mouse exon 4 to a part of the sequence from mouse exon 15 is replaced by a part of the sequence from exon 3 to exon 14 of human CD36, which is about 27kb, so as to obtain a humanized CD36 locus diagram, which is shown in FIG. 2, thereby realizing the humanized modification of the mouse CD36 gene.

Design ofThe targeting strategy is shown in FIG. 3, which shows the homology arm sequences on the targeting vector containing the upstream and downstream of the mouse CD36 gene, as well as an A fragment containing the sequence of human CD 36. Wherein, the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence from position 18033806 to position 18038888 of NCBI accession No. NC-000071.7, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence from position 17986807 to position 17990821 of NCBI accession No. NC-000071.7. The nucleotide sequence of human CD36 on fragment A (SEQ ID NO: 5) is identical to the nucleotide sequence 80646828 to 80674045 of NCBI accession No. NC-000007.14; the ligation of the upstream human CD36 sequence to mice was designed as:

(SEQ ID NO: 6), wherein sequence "AGTC"middle" C "is the last nucleotide, sequence, of mouse

The first "G" in (A) is the first human nucleotide. The connection of the downstream of the human CD36 sequence and the mouse is designed

Wherein the sequence "AAAC"C" in "is the last nucleotide, sequence, of human

The first "C" in (a) is the first nucleotide of the mouse sequence.

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

Wherein the sequence "GATC"middle" C "is the last nucleotide, sequence, of human

The first "G" of (a) is the first nucleotide of the Neo cassette; the connection between the 3' end of the Neo cassette and the human gene is designed as

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

The "C" in (A) is the first nucleotide in humans. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector. The mRNA sequence of the humanized mouse CD36 after being transformed is shown as SEQ ID NO: 10, and the expressed protein sequence is shown as SEQ ID NO: shown at 11.

Given that human CD36 has multiple subtypes or transcripts, the methods described herein can be applied to other subtypes or transcripts.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The method comprises the steps of performing electroporation transfection on a targeting vector which is verified to be correct by sequencing into embryonic stem cells of a C57BL/6 mouse, screening the obtained cells by using a positive clone screening marker gene, detecting and confirming the integration condition of an exogenous gene by using PCR and Southern Blot technologies, screening out correct positive clone cells, detecting clones which are identified to be positive by PCR, performing Southern Blot (digesting cell DNA by using ScaI or EcoNI enzyme respectively and hybridizing by using 2 probes, wherein the lengths of the probes and target fragments are shown in Table 1), and detecting exemplary results are shown in FIG. 4, wherein the detection results show 12 clones which are verified to be positive by PCR, and 4 clones which are verified to be positive by sequencing are further verified to be free of random insertion and are specifically numbered as 2-B04, 2-D02, 2-D03 and 3-F05.

Table 1: specific probes and target fragment lengths

Restriction enzyme	Probe needle	Wild type fragment	Recombinant sequence fragment
				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)；

the Southern Blot detection comprises 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 selected correctly positive cloned cells (black mice) are introduced into the separated blastocysts (white mice) according to the known technology in the field, the obtained chimeric blastocysts are transferred into a culture solution for short-term culture and then transplanted into the oviduct of a recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white alternate) can be produced. The F0 generation chimeric mice and the wild mice are backcrossed to obtain F1 generation mice, and the F1 generation heterozygous mice are mutually mated to obtain F2 generation homozygous son mice. Alternatively, positive mice may be mated with Flp tool mice to remove the positive clone selection marker gene (see FIG. 5 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous mice for the CD36 gene. The somatic genotypes of the progeny mice were identified by PCR (primers shown in Table 2), and the results of identification of exemplary F1 mice (with the Neo marker gene removed) are shown in FIG. 6, in which both F1-01 and F1-022 mice were positive heterozygous mice. This shows that using this method, humanized mice of the CD36 gene can be constructed which can be stably passaged and have no random insertion.

Table 2: name and specific sequence of primer

The expression of humanized CD36 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry, and the like. Specifically, 1 mouse of 7-week-old female C57BL/6 wild-type mice and 7-week-old female CD36 gene humanized heterozygote mice were each harvested, bone marrow tissue was extracted after cervical dislocation, and anti-cervical bone marrow tissue was used separatelyMurine CD36 Antibody APC anti-mouse CD36 Antibody (mCD36), anti-human CD36 Antibody PE anti-human CD36 Antibody (hCD36), murine leukocyte recognition Antibody Brilliant Violet 510 ^TM Anti-Mouse CD45(mCD45), PerCP Anti-Mouse Ly-6G/Ly-6C (Gr-1) Anti-body (mGr-1), murine monocyte/macrophage specific recognition Antibody V450 Rat Anti-Mouse CD11b (mCD11b), murine macrophage specific recognition Antibody FITC Anti-Mouse F4/80(mF4/80), murine IgG2A Antibody Mouse IgG2A PE-conjugated Anti-body, murine Antibody APC Armenian Hamster IgG Isotype Ctrl Anti-body, murine CD16/32 Antibody Purified Anti-Mouse CD16/32, etc. after recognition staining, flow detection was performed.

The results show that 53.0% of macrophages (characterized by mCD45+ mGr-1-mCD11+ mF4/80+) in C57BL/6 mouse bone marrow express mouse CD36 protein (characterized by mCD45+ mGr-1-mCD11+ mF4/80+ mCD36+), and 0.33% express humanized CD36 protein (characterized by mCD45+ mGr-1-mCD11+ mF4/80+ hCD36 +). Macrophage cells expressed mouse CD36 protein at 49.8% and humanized CD36 protein at 23.4% in CD36 humanized hybrid mouse bone marrow. Therefore, the C57BL/6 mouse bone marrow cells basically do not express the humanized CD36 protein, and the CD36 humanized mouse bone marrow cells can successfully express the humanized CD36 protein.

The expression of CD36 mRNA was detected in mice by RT-PCR. Specifically, 1 mouse of 10-week-old female C57BL/6 wild-type mice and 1 mouse of humanized homozygote of CD36 gene obtained in this example were taken, lung tissue was harvested after cervical euthanasia, cellular RNA was extracted according to the instructions of Trizol kit, and after reverse transcription into cDNA, RT-PCR assay was performed (primers are shown in table 3), and the assay results (shown in fig. 7) were shown: only murine CD36 mRNA, no human CD36 mRNA was detected in C57BL/6 wild type mice; only human CD36 mRNA, and no murine CD36 mRNA were detected in humanized homozygous mice for the CD36 gene.

TABLE 3 RT-PCR primer names and specific sequences

The expression of humanized CD36 protein in humanized homozygote mice of the CD36 gene was confirmed by the same flow cytometry assay as described above. Specifically, 1 mouse of 7-week-old female C57BL/6 wild-type mice and 1 mouse of humanized homozygote of CD36 gene were taken, and the cells exuded from the abdominal cavity were examined after cervical dislocation. The results show that no human or humanized CD36 protein is detected in C57BL/6 mice, and only humanized CD36 protein is detected in CD36 humanized homozygote mice, which indicates that the humanized CD36 protein can be successfully expressed in the humanized mice of the CD36 gene prepared by the method.

Further, 3 mice each of C57BL/6 wild type mice and CD36 gene humanized homozygote mice (H/H, hCD36) were subjected to immunotyping using flow cytometry detection, spleen, peripheral blood and lymph node tissue from mice (H/H, hCD36) were taken after euthanasia, the results of detecting leukocyte subtypes and T-cell subtypes in spleen were shown in FIGS. 8 and 9, respectively, and the results of detecting leukocyte subtypes and T-cell subtypes in peripheral blood were shown in FIGS. 10 and 11, respectively, from which it can be seen that B Cells (B Cells), T Cells (T Cells), NK Cells (NK Cells), CD4+ T Cells (CD4+ T Cells), CD8+ T Cells (CD8+ T Cells), Granulocytes (Granulocytes), DC Cells (Dentic Cells), monoclonal Cells (macrophage Cells), macrophage Cells (macrophage Cells), etc. were consistent with those from wild type mouse spleen, macrophage, etc. (Mac 58), the percentage of T cell subtypes such as CD4+ T cells (CD4+ T cells), CD8+ T cells (CD8+ T cells), and Tregs cells (Tregs) was substantially identical to that of C57BL/6 wild-type mice (fig. 9 and 11).

The results of the detection of the leukocyte subtypes and the T-cell subtypes in the lymph nodes are shown in FIGS. 12 and 13, respectively, and it can be seen from the graphs that the leukocyte subtypes such as B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells and the like in the lymph nodes of the humanized homozygote mouse with the CD36 gene are substantially consistent with those of the wild-type C57BL/6 mouse (FIG. 12), and the percentages of the T-cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells are substantially consistent with those of the wild-type C57BL/6 mouse (FIG. 13).

The results show that the humanized modification of the CD36 gene does not affect the differentiation, development and distribution of white blood cells and T cells in mice.

Example 2 drug efficacy verification

The humanized mouse with the CD36 gene prepared by the invention is used for constructing a tumor model and can be used for testing the drug effect of a drug targeting human CD 36. Specifically, humanized homozygote mice (8-week-old, female) of the CD36 gene prepared in example 1 were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5X 10) ⁵ One), the tumor volume is about 100- ³ Then, the tumor volume was counted as control group or treatment group (n-6/group). Control group was injected with PBS, and treatment group was administered with anti-CD 36 antibody 1G04 (for drug information see patent WO2021176424a 1). The administration mode comprises the following steps: intraperitoneal (i.p.) injection, the administration is started on the same day, and 3 times per week and 6 times in total. Tumor volume was measured 2 times per week, and after inoculation, tumor volume of a single mouse reached 3000mm ³ And performing euthanasia. Specific groups and dosing are shown in table 4. The results of the measurement of the body weight and the tumor volume of the mice in the experimental period are shown in FIGS. 14 to 16, respectively.

Table 4: grouping and administration of drugs

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

Table 5: tumor volume, survival and tumor inhibition rate

As shown in FIGS. 14 and 15 and Table 5, the health status of the animals was good in the experimental process of each group, and there was no significant difference (P) in the weight gain (FIGS. 14 and 15) between all the treatment groups (G2, G3) and the control group (G1) on day 18 (P)>0.05), indicating that the animal is against humansThe CD36 antibody 1G04 has good tolerance, does not generate obvious toxic effect on animals, and has better safety. As shown in FIG. 16 and Table 5, from the tumor volume results, the tumor volumes of the treated groups were smaller than those of the control group at each stage, and the tumor volumes of the mice in the groups G2 and G3 were 1782. + -. 172mm on day 18, respectively ³ 1397. + -. 239mm ³ Are all smaller than 2555 +/-432 mm of a control group ³ In particular, the G3 group had a significant difference (P) from the control group<0.05). The experimental result shows that the humanized mouse of CD36 prepared by the method can be used for in vivo drug effect detection of drugs (such as antibody drugs) targeting human CD 36.

Example 3 preparation of double humanized or multiple double humanized mice

A double-humanized or multi-humanized mouse model can be prepared by using the method or the prepared CD36 mouse. For example, in example 1, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other gene modifications such as PD-1, PD-L1, TIGIT, CD226, etc., or may be humanized CD36 mice and subjected to gene recombination targeting techniques to obtain a two-gene or multi-gene modified mouse model of CD36 with other gene modifications. The homozygote or heterozygote of the CD36 mouse obtained by the method can also be mated with homozygote or heterozygote of other gene modification, the offspring of the homozygote or heterozygote is screened, the homozygote or heterozygote of humanized CD36 and heterozygote of double gene or multiple gene modification of other gene modification can be obtained with a certain probability according to Mendel genetic rule, the heterozygote is mated with each other to obtain homozygote of double gene or multiple gene modification, and the in vivo efficacy verification of targeted human CD36 and other gene regulators can be carried out by utilizing the mouse of double gene or multiple gene modification.

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

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

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

Sequence listing

<110> Baiosai Diagram (Beijing) pharmaceutical science and technology Co., Ltd

Construction method and application of <120> CD36 gene humanized non-human animal

<130> 1

<150> CN202110593226.7

<151> 2021-05-28

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caactgaatc cttaccatct tctgcctttt agcccttctc tgagcttata tttaacactg 60

aatcatatgt gctatttaac cagagagttc tatttctaca cataatttta taacaatttt 120

cctaggtctt ttgtttcagc atggtcaact actaccaaat agctgaacat cttcctatag 180

aggaatttta atctagcaac atggctgctc tagcaaatga tcatatccaa ggttctccta 240

ggtctatgtg atgtggctgg gatgtagaca catctttaat cctaggagac aagcagctct 300

cttgggttta agcagttcaa gaccatccag ggataaagca agtttagtgt aatgaaaagc 360

ttagctccat gcatagtggt acgtgtcttt taatcccagc actcaggaga cagaggcatg 420

tggacctctg agttctagtc agtcagttcc gttgaatcaa tgagttgaga ggcattgcag 480

tggagaggag ttcttggcaa ttcacttcat agagtcagct ttaccaggat agttttaaag 540

agatgggttg taggtgaaga cagaaggagc caaagaatga caaagagaca gaagattaga 600

atagattgac agagttagtt tgaggctaag cagagaaatt cagctataag ctgagagaag 660

ccagtttgag tcagtcagtc tggaaaagaa ttggggccag aaaagctgag ttgaatcaag 720

cagccagaat tcagaaagaa ctagaaaagg taagtgtatt cagcagtagg cctctgagat 780

gacaattgca tctggcaaat aaaattacat ttacaccttc ctgttaatct atgcttaaat 840

gtatatattg tctctataga atcatggctt tataaatacc aatagacagg gtggagggta 900

aatgtataaa aacacataat aaatttttta aaaatcctgt gttcatattt aacctgttat 960

tcattaaatt acctcaagtt gtatttccat attaggttat catgggtagg tgctgttctt 1020

ttcttgggaa aggaagggtc tataggaaaa gatgacagtt tttagagttt tatggcctgg 1080

tttcctatgt taagaataag aatatttagc tgttggtaga tggaggaaaa gagaatccat 1140

gtgccagact gattctggaa cattgtcatc ttcactgttc acatctcagc atgcgttacc 1200

tgtgggccat gtcttttcta ggtggagttt ctgagggaac agagtttgaa acacctgtgg 1260

gaataattaa gagattcctc ccgggaattt gctctacact ttggcctatt taccagggag 1320

tctgcttata gttcatccta cctcagactt tgcccaatct attgggttct actaggtttc 1380

tcatttagta aaagggccct gtgtttcaaa gttcataata gacaatcaga caacaccagg 1440

aaaaaaaata ataataccac ttctcctgct tcttgaattc actctgaagt tctgaaaatg 1500

caatctaaat gtactttgta cagtggataa gaaatagcag ctgacagatt tgtaactagt 1560

gatattttaa aacatggcat cctattccct cttctccttt agattagtcc aaacagttct 1620

ctggtcctga ttattctttg tcttttaaag ttcaatactt taagcaggaa acagtttgtt 1680

gtaaataaat atttgcacta tagaattaat gttagcaaaa ttaatgctaa gttagaaaat 1740

aaataacaat gtaaaatatg agcaggtatg ctattgagga aatgtaattt tctcagatcc 1800

aaattttgga gtccttaata tctaaattga cagaaaatag ttaaaaagac tatatgtttc 1860

tttgaagata agtatggatg gccaaggtgc tggctagaat gtgctaactt gagcatttat 1920

ctcttgaggt caataaaaca ccttcagttt tatgtcatgt actatgagaa agaaaagcac 1980

gaacacacta taaacccctc cttcctcgaa atgttaaggg actaaatgat aatcagtaag 2040

ccctagtgaa aaaaggatct ttcctatttt tagggcaatc ccagaaactt tattatgaca 2100

tgatgtaatg ataataaatc aatcattgaa aatatgaacc agtggataag ctaacttctt 2160

ccactctgtc aaatatcagc acatggttga aagaatgtta tataaatgct tatattaagg 2220

tttatcataa taatctaact aaatatattt attgataccc tcgttttcta agaagtctga 2280

ctgtacatca gtgggacaca tgcatgttct cacttccgtt tgaacagaca taactcagac 2340

catacctacc tctacctact gattattagt caggggtaca tgtcctgtga ttgatttctt 2400

ggcaatggaa aacaggttct gttttttgga tgcttcagta gatgctgtta taacttttca 2460

tctcttgtag aaaccttttg cttttaataa tagaaatact tcaataagaa aaatgtggaa 2520

atggaatact cacaattcat atttatggta atatagatcc aaacaaggga caaatatgtg 2580

cactcctagt tcaaaagcaa taatagtcca attttaaggt cattttatac tctacttgtc 2640

agaattgtaa gtgattttac taaataattt ctagctttta tttataaaat ttgtgtgtgt 2700

gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atgtgtgtgt gtattaaact ttcagggttt 2760

aattatctct ttctgcctta tagatgccaa tcaatatatc aggcttggca gcaagtgtac 2820

ttatgcactg tgccatctcc ctgccccaaa taaataattt taatgatgtt catatggaca 2880

ttaaataact ttccaagtaa agtaactcag taggtgcttt tacttagatc taagtaattt 2940

atattcacca aactcctata acacacagga tttcctgtcc cagttctaag atcatgggcc 3000

acatttcctt ttttttgggg gggggggagg gggcacattt tcttaaagag gctcttttaa 3060

cttttctaag aagagttagg cagatatgaa gtgtgtcagg aacaaagatc tctgggttgt 3120

caccttgctg tgtaatcata gttgccatac cttttggttt cccaggaaac aacttggaaa 3180

ccatccaaaa ctgggtgaat atcatttcgg ctactaacat aggaaattgc cactttcact 3240

ttagatccta ttttgaaaca ttagaaaagt caaaatatag agagccagta tttctttatt 3300

gcctcttaga aggaggcaga aaggtcgggt ttatttcatc tccaaccttt tatttgtgct 3360

gagtgctgga tccctctgct ctcatttctg ccttacaggt atgattcttc ctttacagtt 3420

ggcagaatcc agccttcaca ccagttctgt gagtcagttc tgtgagtgct tgtaagattg 3480

tatcaaggaa aagtctcaga ccttccaact gcatgaaagg agtagggcta aacaaagaga 3540

agctcggggg ttacagaggg agtacagttg ccaggaaagg gtctgggcgg tttgtgcttc 3600

tccatctttg agaaaggctt atggttgtac tcagagtatt ttataaatgc tctctgtgtg 3660

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 humanized CD36 protein, wherein said humanized CD36 protein comprises all or a portion of a human CD36 protein.

2. The humanized CD36 protein of claim 1, wherein the humanized CD36 protein comprises all or part of the extracellular, transmembrane and/or cytoplasmic region of a human CD36 protein, preferably wherein the humanized CD36 protein comprises all or part of the extracellular region of a human CD36 protein, further preferably wherein the extracellular region of a human CD36 protein comprises at least 50 contiguous amino acids, more preferably comprises the amino acid sequence of SEQ ID NO:2, positions 30-439; or, comprising a nucleotide sequence identical to SEQ ID NO:2 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical in amino acid sequence from position 30 to 439; or, comprising a nucleotide sequence identical to SEQ ID NO:2, positions 30-439, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or, comprising a nucleotide sequence identical to SEQ ID NO:2, and including substitution, deletion and/or insertion of one or more amino acid residues, and preferably, the humanized CD36 protein further comprises all or part of the transmembrane region and/or cytoplasmic region of a non-human animal CD36 protein.

3. The humanized CD36 protein of claim 1 or 2, wherein the amino acid sequence of the humanized CD36 protein comprises any one of the following group:

A) SEQ ID NO: 11;

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

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

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

4. A nucleic acid encoding the humanized CD36 protein of any one of claims 1-3.

5. A humanized CD36 gene, wherein the humanized CD36 gene comprises a portion of the human CD36 gene.

6. The humanized CD36 gene according to claim 5, wherein the humanized CD36 gene comprises all or part of exons 1 to 14, preferably all or part of exons 3 to 14, more preferably part of exons 3, 4 to 13 and 14, wherein part of exons 3 comprises at least 10bp of nucleotide sequence and part of exons 14 comprises at least 30bp of nucleotide sequence of human CD36 gene.

7. The humanized CD36 gene of claim 5 or 6, wherein the humanized CD36 gene comprises the amino acid sequence of SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

8. The humanized CD36 gene according to any one of claims 5 to 7, wherein the humanized CD36 gene further comprises a non-human animal CD36 gene portion, preferably comprises all or part of exon 1-3 and/or exon 16 of a non-human animal CD36 gene, further preferably further comprises part of exon 4 and/or part of the nucleotide sequence of exon 15.

9. The humanized CD36 gene according to any one of claims 5 to 8, wherein the nucleotide sequence of the humanized CD36 gene comprises any one of the following group:

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

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

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

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

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

10. A targeting vector, wherein said targeting vector comprises one of the group consisting of:

A) a nucleotide sequence encoding a human or humanized CD36 protein;

B) all or part of a nucleotide sequence encoding an extracellular region, a transmembrane region, a cytoplasmic region and/or a signal peptide of the human CD36 protein, preferably all or part of a nucleotide sequence encoding an extracellular region of the human CD36 protein;

C) a nucleotide sequence of a human or humanized CD36 gene; or the like, or a combination thereof,

D) all or part of exons 1 to 14 of human CD36 gene, preferably all or part of exons 3 to 14 of human CD36 gene, further preferably part of exons 3, 4 to 13 and 14 of human CD36 gene, wherein part of exon 3 comprises at least 10bp of nucleotide sequence, part of exon 14 comprises at least 30bp of nucleotide sequence, further preferably comprises SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; alternatively, a polypeptide comprising a sequence having SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

11. The targeting vector according to claim 10, wherein said targeting vector further comprises a 5 'arm and/or a 3' arm, preferably wherein said 5 'arm has at least 90% homology to NCBI accession No. NC _000071.7, further preferably wherein said 5' arm sequence is as set forth in SEQ ID NO: 3 is shown in the specification; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000071.7; further preferably, the 3' arm sequence is as set forth in SEQ ID NO: 4, respectively.

12. A method for constructing a non-human animal humanized with a CD36 gene, wherein the non-human animal expresses a human or humanized CD36 protein.

13. The method of constructing a recombinant human CD36 protein of claim 12, wherein the humanized CD36 protein is the humanized CD36 protein of any one of claims 1 to 3.

14. The method of claim 12, wherein the genome of the non-human animal comprises the nucleic acid of claim 4 or the humanized CD36 gene of any one of claims 5-9.

15. The construct of any of claims 12-14, comprising introducing a donor nucleotide sequence into the non-human animal CD36 locus, preferably wherein the donor nucleotide sequence comprises one of the following groups:

A) a nucleotide sequence encoding a human or humanized CD36 protein;

C) a nucleotide sequence of a humanized CD36 gene; or the like, or, alternatively,

D) exons 1 to 14 of human CD36 gene, preferably, all or part of exons 3 to 14 of human CD36 gene, and more preferably, part of exon 3, all of exons 4 to 13, and part of exon 14 of human CD36 gene, wherein part of exon 3 comprises at least 10bp of nucleotide sequence, and part of exon 14 comprises at least 30bp of nucleotide sequence, preferably comprising SEQ ID NO: 5; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%; or, comprising a nucleotide sequence identical to SEQ ID NO: 5 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or, comprises a polypeptide having the sequence of SEQ ID NO: 5, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

16. The method of claim 15, wherein the donor nucleotide sequence is regulated in the non-human animal by a regulatory element that is endogenous to the regulatory element.

17. The method of any one of claims 15-16, wherein said introducing is a substitution or insertion, optionally said introducing into the non-human animal CD36 locus is a substitution of a corresponding region of a non-human animal, preferably exon 4 through exon 15 of the non-human animal CD36 gene, more preferably a part of exon 4, all of exon 5 through exon 14, and a part of exon 15 of a non-human animal.

18. The method of construction according to any one of claims 12 to 17, wherein the targeting vector according to any one of claims 10 to 11 is used for construction of a non-human animal.

19. The method of constructing a recombinant human antibody according to any one of claims 12-18, wherein said method comprises mating a non-human animal humanized with the CD36 gene with another genetically modified non-human animal, in vitro fertilization or direct gene editing, and screening to obtain a polygenetically modified non-human animal, preferably wherein said another gene is at least one selected from the group consisting of PD-1, PD-L1, TIGIT and CD 226.

20. A cell, tissue or organ which expresses the humanized CD36 protein of any one of claims 1 to 3 or which comprises the nucleic acid of claim 4 or the humanized CD36 gene of any one of claims 5 to 9 or which is derived from a non-human animal obtained by the construction method of any one of claims 12 to 19.

21. Use of the humanized CD36 protein of any one of claims 1 to 3, the nucleic acid of claim 4, the humanized CD36 gene of any one of claims 5 to 9, the non-human animal obtained by the construction method of any one of claims 12 to 19, or the cell, tissue or organ of claim 20, wherein the use comprises:

22. The humanized CD36 gene according to any one of claims 5 to 9, the method of construction according to any one of claims 12 to 19 or the cell, tissue or organ according to claim 20, wherein the non-human animal is a non-human mammal; preferably, the non-human mammal is a rodent; further preferably, the rodent is a mouse or a rat.