CN112048522A

CN112048522A - Construction method and application of TMEM173 gene humanized and modified animal model

Info

Publication number: CN112048522A
Application number: CN202010911839.6A
Authority: CN
Inventors: 沈月雷; 白阳; 黄蕤; 周小飞; 张美玲; 姚佳维; 郭朝设; 郭雅南
Original assignee: Beijing Biocytogen Co Ltd
Current assignee: Beijing Biocytogen Co Ltd
Priority date: 2020-09-02
Filing date: 2020-09-02
Publication date: 2020-12-08

Abstract

The invention relates to a construction method of an animal model of TMEM173 gene humanized modification, in particular to a rodent of TMEM173 gene humanized modification, especially a mouse of gene humanized modification, and the animal model expresses humanized TMEM173 protein. In some examples, the animal model expressing the human or humanized TMEM173 protein also expresses at least one of a human immune checkpoint such as PD-1. The invention also provides application of the animal model obtained by the construction method in the field of biomedicine.

Description

Construction method and application of TMEM173 gene humanized and modified animal model

Technical Field

The application relates to a construction method and application of a humanized gene modified animal model, in particular to a construction method of a TMEM173 gene humanized modified animal model and application thereof in the field of biomedicine.

Background

The experimental animal disease model is an indispensable research tool for researching 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 with human genes has been realized, and the development of humanized experimental animal models (humanized animal models) in this way is the future development direction of animal models. The gene humanized animal model, that is, the gene editing technology is used to replace the homologous gene in animal genome with human normal or mutant gene, so as to establish normal or mutant gene animal model with physiological or disease characteristics similar to that of human. 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 (i.e., genetic factors), how to construct "efficient" humanized animal models for new Drug development remains the greatest challenge (Scheer N, Snaith M, Wolf CR, Seibler J. Generation and compliance of genetic humanized models, Drug Discov Today; 18(23-24):1200-11, 2013).

Immunotherapy, which attacks and kills cancer cells by activating the immune system, is an important area of tumor research and has been used in clinical therapy for the last decade. Despite the remarkable effects of both CTLA-4 and PD-1 classes of immune checkpoint blockers and the compelling success of cellular immunotherapy, the overall efficacy of such drugs and therapies is only about 20-30%, with a large unmet clinical need. In addition to the development of relevant monoclonal antibodies, the search for convenient small molecule compounds for targeted inhibition of immune checkpoints is also a leading area of tumor immunotherapy.

The interferon gene-stimulating protein sting (stimulator of interferon genes), also known as MITA, MPYS, ERIS and TMEM173, is a four-transmembrane protein that is mainly distributed in immune-related tissue cells and highly expressed in thymus, spleen and peripheral blood cells, is mainly localized on the Endoplasmic Reticulum (ER) and mitochondria, and is a major component of the innate immune system (lnnate immune system). DNA (whether the DNA is from a virus, a bacterium or an organism) in cytoplasm can be combined with enzyme of cyclic GMP-AMP synthase (cGAS) to form cGAMP (cyclic dinucleotide, CDN for short), dimeric TMEM173 is combined with the enzyme to generate conformational change, downstream transcription factor TBK1(STAT-6, NF-kb) is activated, transcription factor IRF3 is recruited and phosphorylated, IFNs (mainly type I IFN) are induced to express to kill tumor cells and viruses, and therefore a cGAS-cGAMP-TMEM173 signal channel is formed, and further researches find that small molecular compounds can also be combined with the TMEM173 to activate or inhibit the signal channel. Having recognized the TMEM173 signaling pathway, small molecules of similar structure were designed that would be expected to activate the TMEM173 signaling pathway against tumors and viruses while providing the basis for T cell activation and proliferation; or inhibiting the TMEM173 signaling pathway to treat autoimmune diseases.

The similarity (posives) of the human and murine TMEM173 proteins is as high as 81%, the former gene encodes 379 amino acids, and the latter gene encodes 378 amino acids, but due to species differences, it has been confirmed that the compound DMXAA (vadimezan) targeting TMEM173 binds only to murine TMEM173 and fails to activate the human TMEM173 protein, and thus despite its excellent efficacy in mouse anti-tumor models, DMXAA preclinical studies failed in phase III clinics. In recent years, international pharmaceutical companies such as BMS, nova and the like are increasingly searching for drugs capable of activating TMEM173, and it is expected that more and more domestic and foreign pharmaceutical companies participate in drug development targeting TMEM173 protein in the future. Therefore, developing more animal models that can mimic human drug development would help reduce the risk of development failure.

Disclosure of Invention

In view of the fact that the TMEM173 gene has great application value in the fields of tumor and immunotherapy, in order to further research the relevant biological characteristics of the TMEM173, improve the effectiveness of a preclinical drug effect test and improve the success rate of research and development, the invention provides a method for establishing a TMEM173 gene humanized modified animal model worldwide and obtains a TMEM173 gene humanized animal. Specifically, the invention aims to prepare a non-human animal model, the TMEM173 protein can be normally expressed in the animal body, the expressed TMEM173 protein can be identified and combined with a regulator targeting human TMEM173, and the method has wide application prospects in the aspects of drug screening, effectiveness verification and the like. In addition, the non-human animal obtained by the method can be mated with other humanized non-human animals with immune check points, such as B-hPD-1 mice to obtain a TMEM173 and PD-1 double-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 method comprises the following specific steps:

the invention relates to a first aspect of the invention, which relates to a construction method of an animal model of TMEM173 gene humanized modification, wherein the genome of the animal model comprises exons 5 to 8 of the TMEM173 gene.

Preferably, the genome of the animal model comprises a part of exon 5, intron 5-6, exon 6 to exon 7, intron 7-8 and a part of exon 8 of the human TMEM173 gene, wherein the part of exon 5 comprises a nucleotide sequence of exon 5 excluding the first 1-5 (e.g. 1, 2, 3, 4, 5) amino acids, and the part of exon 8 comprises the whole coding region.

Further preferably, the genome of said animal model comprises a nucleic acid sequence encoding SEQ ID NO: 4 from 139 th to 379 th.

Further preferably, the genome of said animal model comprises SEQ ID NO: 7.

Preferably, the construction method comprises replacing (preferably in situ replacement) the non-human animal TMEM173 locus with a nucleotide sequence comprising exon 5 to exon 8 of the human TMEM173 gene.

Preferably, the construction method comprises replacing (preferably in situ replacement) to the non-human animal TMEM173 locus a nucleotide sequence comprising part of exon 5, intron 5-6, exon 6 to all of exon 7, intron 7-8 and part of exon 8 of the human TMEM173 gene, wherein said part of exon 5 comprises a nucleotide sequence of exon 5 excluding the amino acids encoding the first 1-5 (e.g., 1, 2, 3, 4, 5) amino acids, and said part of exon 8 comprises the entire coding region.

Preferably, the method of construction comprises the step of using a polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 4 or a nucleotide sequence comprising SEQ ID NO: 7 (preferably in situ) to the non-human animal TMEM173 locus.

In one embodiment of the invention, the method of construction comprises contacting the nucleic acid sequence comprising the nucleic acid sequence encoding SEQ ID NO: 4 or a nucleotide sequence comprising SEQ ID NO: 7 (preferably in situ) to the corresponding region of the non-human animal TMEM173 gene.

Preferably, the method of construction comprises insertion or substitution into the non-human animal TMEM173 locus with a cDNA sequence comprising a gene encoding human TMEM 173. Wherein the insertion site is located after an endogenous regulatory element of the TMEM173 gene.

Preferably, the construction method comprises replacing (preferably in situ replacement) the region from exon 5 to exon 8 of the non-human animal TMEM173 gene with a nucleotide sequence comprising part of exon 5, intron 5-6, exon 6 to exon 7, intron 7-8 and part of exon 8 of the human TMEM173 gene, wherein said part of exon 5 of the human TMEM173 gene comprises a nucleotide sequence excluding exon 5 encoding the first 1-5 (e.g. 1, 2, 3, 4, 5) amino acids and said part of exon 8 of the human TMEM173 gene comprises the entire coding region.

Preferably, the human TMEM173 gene is regulated by TMEM173 regulatory elements. Further preferably, said human TMEM173 gene is regulated by endogenous TMEM173 regulatory elements.

Preferably, the animal model expresses human or humanized TMEM173 protein in vivo with reduced or absent expression of endogenous TMEM173 protein.

The invention uses gene editing technology to construct the TMEM173 gene humanized and modified animal model, wherein the gene editing technology comprises gene targeting technology by utilizing embryonic stem cells, CRISPR/Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology.

The non-human animal of the invention is a rodent; preferably, the rodent is a rat or a mouse.

In one embodiment of the invention, a targeting vector is used to construct a humanized TMEM173 gene engineered animal model.

Preferably, the targeting vector comprises exon 5 to exon 8 of the human TMEM173 gene. Preferably, the nucleotide sequence comprises part of exon 5, intron 5-6, exon 6 through exon 7, intron 7-8 and part of exon 8 of the human TMEM173 gene, wherein said part of exon 5 comprises the nucleotide sequence of exon 5 excluding the first 1-5 (e.g., 1, 2, 3, 4, 5) amino acids, and said part of exon 8 comprises the entire coding region.

The targeting vector comprises a nucleotide sequence encoding SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 7.

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, selected from nucleotides having at least 90% homology with NCBI accession No. NC _ 000084.6; and a second DNA fragment, 3 'arm, homologous to the 3' end of the transition region to be altered, selected from the group consisting of nucleotides having at least 90% homology with NCBI accession number NC-000084.6.

Preferably, said transition region to be altered is located at the TMEM173 locus of the non-human animal. Further preferably, it is located on exon 5 to exon 8 of the TMEM173 gene of a non-human animal.

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

Preferably, to improve recombination efficiency, a non-human animal may be constructed using sgRNA targeting TMEM173 gene together with the above-described targeting vector. Wherein the sgRNA targets the non-human animal TMEM173 gene while the sequence of the sgRNA is on the target sequence on the TMEM173 gene to be altered.

Preferably, the target site of the sgRNA is located on exon 5 to exon 8 of the TMEM173 gene.

Preferably, the target site sequence at the 5' end targeted using the sgRNA is as set forth in SEQ ID NO: 29-35, and the 3' end target site sequence is shown in SEQ ID NO: 36-42. More preferably, the sgRNA target site sequence used is SEQ ID NO: 33 and/or SEQ ID NO: 38.

in a specific embodiment of the invention, the construction method comprises the steps of introducing the targeting vector, the sgRNA targeting the TMEM173 gene and the Cas9 into non-human animal cells, culturing the cells (preferably embryonic stem cells), transplanting the cultured cells into oviducts of female non-human animals, allowing the cells to develop, and identifying and screening the animal model obtained by humanized modification of the TMEM173 gene.

Preferably, the humanized TMEM173 protein comprises SEQ ID NO: 4 from 139 th to 379 th.

Further preferably, said humanized TMEM173 protein comprises SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the humanized TMEM173 protein comprises one of the following groups:

a) SEQ ID NO: 9 or SEQ ID NO: 4 part or all of the amino acid sequence shown in positions 139 to 379;

b) and SEQ ID NO: 9 or SEQ ID NO: 4 from 139 to 379, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

c) and SEQ ID NO: 9 or SEQ ID NO: 4 from 139 th to 379 th amino acid, with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference;

d) has the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 4, 139 to 379, comprising substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the humanized TMEM173 protein hybridizes with SEQ ID NO: 9, comprising one or more amino acid substitutions of H231R, R70H, G229A or R292Q.

In one embodiment of the invention, the humanized TMEM173 protein hybridizes to SEQ ID NO: 9, comprising one or more amino acid substitutions of H231R, R70H-G229A-R292Q, G229A-R292Q or R292Q.

Preferably, the genome of the animal model comprises a humanized TMEM173 gene, and the humanized TMEM173 gene encodes a humanized TMEM173 protein.

Preferably, the humanized TMEM173 gene comprises SEQ ID NO: 7. Further preferably, the mRNA sequence transcribed from the TMEM173 gene contained in said animal model comprises SEQ ID NO: 8.

In one embodiment of the present invention, the humanized TMEM173 gene comprises one of the following groups:

a) the mRNA sequence of the humanized TMEM173 gene is SEQ ID NO: 8, or a part or all of the sequence shown in fig. 8;

b) the mRNA sequence of the humanized TMEM173 gene is similar to that of SEQ ID NO: 8 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

c) the mRNA sequence of the humanized TMEM173 gene is similar to that of SEQ ID NO: 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide;

d) the mRNA sequence of the humanized TMEM173 gene has the sequence of SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

In one embodiment of the invention, the mRNA sequence of the humanized TMEM173 gene is SEQ ID NO: 8 or a variant of SEQ ID NO: 8 (CAT to CGG) at positions 1029-1031.

In one embodiment of the invention, the non-human animal is a mouse, and all or a partial fragment of the mRNA sequence of mouse TMEM173 is as set forth in SEQ ID NO: 1, and the whole or partial fragment of the protein sequence of mouse TMEM173 is shown as SEQ ID NO: 2 in whole or in part.

In another embodiment of the invention, all or a partial fragment of the mRNA sequence of human TMEM173 is as set forth in SEQ ID NO: 3, and the whole or partial fragment of the protein sequence of the human TMEM173 is shown as SEQ ID NO: 4 in whole or in part.

In one embodiment of the invention, the protein sequence of human TMEM173 is identical to SEQ ID NO: 4H 232R, R71H, G230A, or R293Q.

In another embodiment of the invention, the protein sequence of human TMEM173 is identical to SEQ ID NO: 4, H232R, R71H-G230A-R293Q, G230A-R293Q or R293Q.

The second aspect of the invention relates to an animal model obtained by adopting the construction method and through humanized modification of the TMEM173 gene.

In a third aspect, the invention relates to a construction method of an animal model with a deleted TMEM173 gene, which comprises the step of knocking out all or part of the sequence from exon 5 to exon 8 of an endogenous TMEM173 gene of a non-human animal so as to inactivate the endogenous TMEM173 protein.

In a specific embodiment of the invention, sgRNA sequences are used to target the endogenous TMEM173 gene of the animal, thereby knocking out all or part of the sequence of exons 5 to 8 of the endogenous TMEM173 gene. Preferably, the sgRNA sequence targets a target site sequence at the 5' end as set forth in SEQ ID NO: 29-35, and the 3' end target site sequence is shown in SEQ ID NO: 36-42; more preferably, the sgRNA target site sequence used is SEQ ID NO: 33 and/or SEQ ID NO: 38.

the fourth aspect of the invention relates to an animal model with TMEM173 gene deletion, which is obtained by the construction method.

In a fifth aspect, the present invention relates to a targeting vector of TMEM173 gene, wherein the targeting vector comprises exon 5 to exon 8 of human TMEM173 gene.

Preferably, the targeting vector comprises part of exon 5, intron 5-6, exon 6 through exon 7 all, intron 7-8 and part of exon 8 of the human TMEM173 gene, the part of exon 5 comprises a nucleotide sequence of exon 5 excluding the first 1-5 (e.g., 1, 2, 3, 4, 5) amino acids, and the part of exon 8 comprises the entire coding region.

Preferably, the transition region to be altered is located from exon 5 to exon 8 of the TMEM173 gene.

Preferably, the 5' arm sequence is as shown in SEQ ID NO: 5, respectively.

Preferably, the 3' arm sequence is as shown in SEQ ID NO: and 6.

Preferably, the targeting vector comprises SEQ ID NO: 7 or a sequence comprising a sequence identical to SEQ ID NO: 7 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 nucleotide.

In one embodiment of the invention, the targeting vector comprises SEQ ID NO: 7 or a sequence comprising a sequence identical to SEQ ID NO: 7 differ from position 2561 to position 2563, i.e. a sequence which changes from CAT to CGG.

The TMEM173 gene targeting vector also comprises a selectable gene marker.

Preferably, the marker gene is a gene encoding a negative selection marker. Further preferably, the gene encoding the negative selection marker is a gene encoding diphtheria toxin subunit a (DTA).

Preferably, the targeting vector further comprises a resistance gene for positive clone selection. Further preferably, the resistance gene selected by the positive clone is neomycin phosphotransferase coding sequence Neo.

Preferably, 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 number of the specific recombination systems is 2, and the specific recombination systems are respectively arranged at two sides of the resistance genes.

In a sixth aspect, the invention relates to a sgRNA of the TMEM173 gene, which targets the non-human animal TMEM173 gene, while the sequence of the sgRNA is on the target sequence on the TMEM173 gene to be altered.

Preferably, the sgRNA targets a target site sequence at the 5' end as set forth in SEQ ID NO: 29-35, and the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID NO: 36-42; .

In one embodiment of the invention, the sgRNA target site sequence used is preferably SEQ ID NO: 33 and/or SEQ ID NO: 38.

the seventh aspect of the invention relates to a DNA molecule encoding the sgRNA sequence described above.

The eighth aspect of the invention relates to a vector for constructing a humanized TMEM173 gene modified animal model, wherein the vector generates the sgRNA sequence and is used for genetically modifying part or all of exons 5 to 8 of the TMEM173 gene.

The ninth aspect of the present invention relates to a method for preparing the vector for constructing the humanized gene modified animal model, comprising the following steps:

1) the sequence is shown as SEQ ID NO: 29-35 and/or any sgRNA target sequence set forth in SEQ ID NO: 36-42, and preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence;

2) synthesizing fragment DNA containing a T7 promoter and sgRNA scafffold, carrying out enzyme digestion on the fragments by EcoRI and BamHI in turn, connecting the fragments to a skeleton vector pHSG299, and carrying out sequencing verification to obtain a pT7-sgRNAG2 vector;

3) respectively synthesizing the forward oligonucleotide and the reverse oligonucleotide in the step 1), and denaturing and annealing the synthesized sgRNA oligonucleotides to form a double chain which can be connected into the pT7-sgRNA 2 vector in the step 2);

4) respectively linking the double-stranded sgRNA oligonucleotides annealed in the step 3) with pT7-sgRNA 2 vectors, and screening to obtain sgRNA vectors.

In a tenth aspect, the present invention relates to a method for preparing a TMEM173 gene knock-out non-human animal, comprising the steps of:

a) obtaining the sgRNA vector according to steps 1) -4) of the method for preparing the sgRNA vector;

b) mixing an in-vitro transcription product of the sgRNA vector and Cas9mRNA to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of mouse fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, and then transplanting the fertilized eggs into an oviduct of a receptor mother mouse for development to obtain an F0 generation mouse;

c) testing the F0 mouse by using a PCR technology, and verifying that the TMEM173 gene in the cell is knocked out to obtain a TMEM173 gene knock-out positive mouse;

d) expanding the population quantity of the positive mice screened in the step c) in a hybridization and selfing mode, and establishing stable TMEM173 gene knockout mice.

The eleventh aspect of the present invention relates to a method for preparing a TMEM173 gene-humanized non-human animal, comprising the steps of:

the first step is as follows: obtaining an sgRNA vector according to steps 1) -4) of preparing the sgRNA vector as described above;

the second step is that: mixing an in-vitro transcription product of the sgRNA vector, the target vector of the TMEM173 gene and the Cas9mRNA, injecting the mixed solution into cytoplasm or nucleus of a fertilized egg of a female animal, transferring the fertilized egg after injection into a culture solution for culture, and then transplanting the fertilized egg into an oviduct of a recipient animal for development to obtain an F0 generation animal;

the third step: animals from the F0 generation were tested using PCR to verify that the TMEM173 gene was humanized in cells.

In a twelfth aspect, the invention relates to a cell comprising the targeting vector described above.

In a thirteenth aspect, the invention relates to the use of the above-mentioned targeting vector or cell in the gene editing of TMEM173 in animals.

In a fourteenth aspect, the invention relates to a humanized cell modified by TMEM173 gene, wherein the genome of the humanized TMEM173 gene modified cell comprises exon 5 to exon 8 of the human TMEM173 gene. Preferably, the human TMEM173 gene encodes SEQ ID NO: 4 or a nucleotide sequence comprising SEQ ID NO: 7, which is regulated by endogenous TMEM173 regulatory elements; the humanized TMEM173 gene engineered cells express human or humanized TMEM173 protein in vivo with reduced or absent expression of endogenous TMEM173 protein. Preferably, the human TMEM173 gene is regulated by endogenous TMEM173 regulatory elements.

In a fifteenth aspect, the invention relates to a TMEM173 gene-deleted cell that is deleted from exon 5 to exon 8 of an endogenous TMEM173 gene.

The sixteenth aspect of the present invention relates to a method for constructing a polygenic modified non-human animal, comprising the steps of:

(a) preparing an animal model by applying the construction method;

(b) mating the animal model obtained in the step (a) with a humanized animal except TMEM173, performing in vitro fertilization or directly performing gene editing, and screening to obtain the multi-gene humanized modified animal.

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

Preferably, the humanized animal except TMEM173 is selected from one or more than two humanized animals of genes PD-1, PD-L1, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, OX40, SIRPa, TIGIIT, TIM-3, CD40 or GITR.

Further preferably, said other humanized animal is selected from a human animal selected from the group consisting of PD-1 (201710505554.0, PCT/CN 2017/090320), PD-L1 (201710757022.6, PCT/CN 2017/099574), CTLA-4 (201710757917. X, PCT/CN 2017/099577), BTLA (201710948551.4, PCT/CN 2017/106024), CD27 (201711402264. X, PCT/CN 2017/117984), CD28 (28, PCT/CN 2018/28), CD137 (28, PCT/CN 2017/28), CD154 (28, 28), OX 28 (28, PCT/CN 2017/099575), sirpa (28, PCT/CN 2018/28), TIGIT (28, PCT/CN 2017/28), TIM-3 (28, PCT/CN 2017/28), CD 20172 (20172, 657172), or a combination of two or more of genes PD-1 (201710505554.0, PCT/CN 2017/099572, 28, 6572, GITR 28, or more.

The seventeenth aspect of the present invention relates to the polygenic-modified non-human animal or its offspring prepared by the above-described method for preparing a polygenic humanized engineered animal.

The eighteenth aspect of the present invention relates to a tumor-bearing animal model, wherein the method for preparing the animal model comprises the step of preparing an animal by the humanized TMEM173 gene modified animal model or the method for preparing a multi-gene humanized modified animal.

Preferably, the method for preparing the tumor-bearing animal model further comprises the step of implanting tumor cells into the humanized genetically modified animal prepared by the method or the progeny thereof.

The nineteenth aspect of the invention provides an application of the animal model obtained by the construction method or the polygene modified non-human animal obtained by the construction method in preparing a tumor-bearing animal model.

The twentieth aspect of the present invention relates to a cell or cell line or primary cell culture derived from the animal model obtained by humanizing the TMEM173 gene obtained by the above-mentioned construction method, the animal model obtained by humanizing the TMEM173 gene described above, the polygene-modified non-human animal obtained by the above-mentioned construction method, the polygene-modified non-human animal described above, or a progeny thereof or the tumor-bearing animal model described above.

In a twenty-first aspect, the present invention relates to a tissue or organ or a culture thereof derived from the animal model obtained by humanization of the TMEM173 gene obtained by the above-mentioned construction method, the animal model obtained by humanization of the TMEM173 gene, the multigene-modified non-human animal obtained by the above-mentioned construction method, the multigene-modified non-human animal or a progeny thereof, or the tumor-bearing animal model described above.

Preferably, the tissue or organ or culture thereof is spleen, tumor or culture thereof.

In a twenty-second aspect, the present invention relates to a humanized TMEM173 protein, said humanized TMEM173 protein comprising an amino acid sequence encoded by exon 5 to exon 8 of human TMEM173 gene, and an amino acid sequence of non-human animal TMEM173 protein.

Preferably, the humanized TMEM173 protein comprises SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the humanized TMEM173 protein is selected from one of the following groups:

a) the humanized TMEM173 protein sequence is SEQ ID NO: 9 or SEQ ID NO: 4 part or all of the amino acid sequence shown in positions 139 to 379;

b) the humanized TMEM173 protein sequence is identical to SEQ ID NO: 9 or SEQ ID NO: 4 from 139 to 379, is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

c) the humanized TMEM173 protein sequence is identical to SEQ ID NO: 9 or SEQ ID NO: 4 from 139 th to 379 th amino acid, with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid difference;

d) the humanized TMEM173 protein sequence has the sequence of SEQ ID NO: 9 or SEQ ID NO: 4, 139 to 379, comprising substitution, deletion and/or insertion of one or more amino acid residues.

In a twenty-third aspect, the present invention relates to a humanized TMEM173 gene encoding the above humanized TMEM173 protein, the humanized TMEM173 gene comprising exons 5 to 8 of a human TMEM173 gene, and a nucleotide sequence of a non-human animal TMEM173 gene.

Preferably, the humanized TMEM173 gene comprises SEQ ID NO: 7.

Preferably, the mRNA sequence transcribed from said humanized TMEM173 gene comprises SEQ ID NO: 8.

Preferably, the humanized TMEM173 gene comprises one of the following groups:

c) the mRNA sequence of the humanized TMEM173 gene hybridizes under stringent conditions to SEQ ID NO: 8;

d) the mRNA sequence of the humanized TMEM173 gene is similar to that of SEQ ID NO: 8 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 nucleotide;

e) the mRNA sequence of the humanized TMEM173 gene has the sequence of SEQ ID NO: 8, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted;

or

f) The part of the humanized TMEM173 gene derived from the human TMEM173 gene is SEQ ID NO: 3 or SEQ ID NO: 7, or a part or all of the sequence shown in seq id no;

g) the humanized TMEM173 gene has a part derived from the human TMEM173 gene which is similar to the part of the humanized TMEM173 gene shown in SEQ ID NO: 3 or SEQ ID NO: 7 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

h) the part of the humanized TMEM173 gene derived from the human TMEM173 gene is similar to the part of the humanized TMEM173 gene represented by SEQ ID NO: 3 or SEQ ID NO: 7;

i) the humanized TMEM173 gene has a part derived from the human TMEM173 gene which is similar to the part of the humanized TMEM173 gene shown in SEQ ID NO: 3 or SEQ ID NO: 7 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide;

j) the part of the humanized TMEM173 gene derived from the human TMEM173 gene has the sequence of SEQ ID NO: 3 or SEQ ID NO: 7, including substitution, deletion and/or insertion of one or more nucleotides.

In a twenty-fourth aspect, the present invention relates to a construct expressing the above humanized TMEM173 protein.

In a twenty-fifth aspect the invention relates to a cell comprising the above construct.

In a twenty-sixth aspect the present invention relates to a tissue comprising the above cells.

The twenty-seventh aspect of the present invention relates to an animal model obtained by the above construction method, a polygene-modified non-human animal obtained by the above construction method, the above cell or cell line or primary cell culture, the above tissue or organ or culture thereof, the above humanized TMEM173 protein or the above humanized TMEM173 gene, and their use in the preparation of a medicament for treating or preventing a tumor.

The twenty-eighth aspect of the present invention relates to an animal model obtained by the above construction method, a polygene-modified non-human animal obtained by the above construction method, the above cell or cell line or primary cell culture, the above tissue or organ or culture thereof, the above humanized TMEM173 protein or the above humanized TMEM173 gene, and applications thereof in studies related to TMEM173 gene or protein, wherein the applications comprise:

A) product development involving the immunological process of human cells, use in the manufacture or screening of human antibodies;

B) as model systems for pharmacological, immunological, microbiological and medical research;

C) the production of immune processes involving human cells and the use of animal experimental disease models for pathogenic research, for the development of diagnostic strategies or for the development of therapeutic strategies;

D) screening, drug effect detection, efficacy evaluation, validation or evaluation of human TMEM173 signal pathway modulators are studied in vivo; or,

E) the applications of the gene function of the TMEM173 gene, the human TMEM173 antibody, the medicine and the drug effect aiming at the target site of the human TMEM173, the medicine for immune-related diseases and the anti-tumor medicine are researched.

The "tumor" according to the present invention includes, but is not limited to, lymphoma, brain cancer, non-small cell lung cancer, cervical cancer, esophageal cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, 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 cervical cancer, esophageal cancer, renal cancer, brain cancer, breast cancer, ovarian cancer, prostate cancer, or gastric cancer.

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

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 TMEM173 protein comprises a part derived from human TMEM173 protein and a part of non-human STING protein. Wherein, the humanized TMEM173 protein comprises 5-379 amino acid sequences which are continuous or separated and are consistent with the amino acid sequence of the human TMEM173 protein.

The "humanized TMEM173 gene" according to the present invention includes a portion derived from the human TMEM173 gene and a portion of the non-human TMEM173 gene. Wherein, the 'humanized TMEM173 gene' comprises a continuous or alternate nucleotide sequence identical to the nucleotide sequence of the human TMEM173 gene.

The "exon x to exon xx" described herein includes a nucleotide sequence of an exon and an intron therebetween. For example, "exon 5 to exon 8" comprises the nucleotide sequence of exon 5, intron 5-6, exon 6, intron 6-7, exon 7, intron 7-8 and exon 8.

The "x-xx intron" described herein represents an intron between the x exon and the xx exon. For example, the "intron 5-6" refers to an intron between exon 5 and exon 6.

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 "TMEM 173 locus" refers to a DNA segment of an optional stretch of the TMEM173 gene. In one embodiment of the invention, the TMEM173 locus that is replaced may be a DNA fragment of an optional stretch from exon 5 to exon 8 of the TMEM173 gene.

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

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

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

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

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as a rhabdoid. In one embodiment, the non-human animal to which the gene is humanized is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from the family of cricotidae (e.g., mouse-like hamsters), cricotidae (e.g., hamsters, new world rats and mice, voles), muridae (true mice and rats, gerbils, spiny mice, crow rats), marmoraceae (mountaineers, rock mice, tailed rats, madagaska rats and mice), spiny muridae (e.g., spiny mice), and spale (e.g., mole rats, bamboo rats, and zokors). In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

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

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mullisetal 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., In-chief, Academic Press, Inc., New York), specific, volumes, 154 and 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: mouse and human TMEM173 gene comparison scheme (not to scale);

FIG. 2: humanized mouse TMEM173 gene schematic (not to scale);

FIG. 3: schematic targeting strategy (not to scale);

FIG. 4: mouse Southern blot results, where WT is wild type;

FIG. 5: FRT recombination process schematic (not to scale);

FIG. 6: f1 mouse tail PCR to identify somatic cell genotype, wherein WT is wild type, H₂O is water control, PC is positive control, and M is Marker;

FIG. 7: (ii) a phenotypic test result;

FIG. 8: schematic targeting strategy (not to scale);

FIG. 9: detecting the relative activity of the sgRNA, wherein Con is a negative control, and PC is a positive control; (A) the relative activity detection result of the sgRNA1-sgRNA7 is shown; (B) the relative activity detection result of the sgRNA8-sgRNA14 is shown.

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:

both the C57BL/6 mouse and the Flp tool mouse were purchased from the national rodent laboratory animal seed center of the Chinese food and drug assay institute;

PD-1 humanized mouse originated Beijing Baiosai map gene biotechnology limited company, product number is B-CM-001;

the AIO kit is from Beijing Baiosaixi map gene biotechnology limited company with the cargo number BCG-DX-004;

mouse colon cancer cells MC38 were purchased from Shanghai enzyme research Biotechnology, Inc.;

MEGASHORTscript ™ Kit (Ambion in vitro transcription Kit) was purchased from Thermo Fisher, cat # AM 1354;

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

TOP10 competent cells were purchased from Tiangen, Inc. under the accession number CB 104-02;

BglII, HindIII enzymes from NEB, cat # R0144M, R3104M, respectively;

VspI enzyme was purchased from Thermofoisher under the cat # ER 0911;

flow cytometer manufacturer BD, model Calibur.

Example 1TMEM173 Gene-humanized mouse

A comparison scheme of mouse TMEM173 Gene (NCBI Gene ID: 72512, Primary source: MGI: 1919762, UniProt ID: Q3TBT3, from position 35733678 to 35740554 on chromosome 18 NC-000084.6, based on transcript NM-028261.1 (SEQ ID NO: 1) and its encoded protein NP-082537.1 (SEQ ID NO: 2)) and human TMEM173 Gene (NCBI Gene ID: 340061, Primary source: HGNC:27962, UniProt ID: Q16552, from position 139475528 to 139482790 on chromosome 5 NC-000005.10, based on transcript NM-198282.3 (SEQ ID NO: 3) and its encoded protein NP-938023.1 (SEQ ID NO: 4)) is shown in FIG. 1.

For the purposes of the present invention, the gene sequence of human TMEM173 can be introduced at the endogenous mouse TMEM173 locus, such that the mouse expresses a human or humanized TMEM173 protein. Specifically, the mouse TMEM173 gene sequence can be replaced with the human TMEM173 gene sequence at the endogenous mouse TMEM173 locus by gene editing techniques, such as replacing the about 2.9kb (2898 bp) sequence containing at least exon 5 to exon 8 of the mouse TMEM173 gene with the corresponding human DNA sequence to obtain a humanized TMEM173 gene sequence (the schematic diagram is shown in fig. 2), thereby realizing the humanized modification of the mouse TMEM173 gene.

In the schematic of the targeting strategy shown in fig. 3, the targeting vector is shown to contain the homology arm sequences upstream and downstream of the mouse TMEM173 gene (mouse DNA of total 3444bp upstream of exon 5 of endogenous TMEM173 gene 5574bp and downstream of TGA) and 4635bp of human TMEM173 sequence (extending from exon 5 to the stop codon TAA in TGA). Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 5) is identical to the nucleotide sequence at positions 35744360 and 35738787 of NCBI accession No. NC-000084.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) is identical to the nucleotide sequence at positions 35733320 and 35729877 of NCBI accession No. NC-000084.6; the DNA fragment sequence of human TMEM173 (SEQ ID NO: 7) is identical to the nucleotide sequence at positions 139480895 and 139476261 of NCBI accession No. NC-000005.10. The mRNA sequence of the humanized mouse TMEM173 after being transformed and the protein sequence coded by the mRNA sequence are respectively shown as SEQ ID NO: 8 and SEQ ID NO: shown at 9.

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 linkage of the 5 'end of the Neo-box to the mouse locus is designed to be 5' -ACACAGGTGCCATACGTGGCCCATAGCTAAGCTTGATATCGAATTCCGAAGTTCCT-3' (SEQ ID NO: 10), wherein the sequence "TAGCT"the last" T "of a mouse is the last nucleotide, sequence"AAGCT"the first" A "of" is the first nucleotide of the Neo cassette. The junction of the 3 'end of the Neo cassette with the mouse locus was designed to be 5' -AACTTCATCAGTCAGGTACATAATGGTGGATCCACTAGTTCTAGAGCGGCCGCA

TTAATGTGTCCAGGGAAGGGACCACTGAACAAATGCTCCCA-3' (SEQ ID NO: 11), wherein the sequence "TTAAT"the last" T "is the last nucleotide, sequence of the Neo cassette"GTGTCThe first "G" of "is the first nucleotide of the mouse. In addition, a coding gene with a negative selection marker (diphtheria toxin a subunit coding gene (DTA)) was constructed downstream of the 3' homology arm of the targeting vector.

The vector construction can be carried out by conventional methods, such as enzyme digestion and ligation. 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 recombinant vector with correct sequencing verification is transfected into embryonic stem cells of a C57BL/6 mouse by electroporation, the obtained cells are screened by using a positive clone screening marker gene, the integration condition of an exogenous gene is checked by using PCR and Southern Blot technology, correct positive clone cells are screened, clones which are identified as positive by PCR are detected by Southern Blot (cell DNA is digested by BglII or VspI or HindIII respectively and hybridization is carried out by using 3 probes), the result is shown in figure 4, and the detection result shows that the 12 clones which are verified as positive by PCR are all positive heterozygous clones except 1-C7 and have no random insertion.

Wherein the PCR assay comprises the following primers:

F1：5’-GGTCACACAGCTAGAGAGACAGAAGTC-3’（SEQ ID NO：12），

R1：5’-CATAGCAACATCCTTCATGCCTTGGG -3’（SEQ ID NO：13）；

F2：5’-GCTCGACTAGAGCTTGCGGA-3’（SEQ ID NO：14），

R2：5’-CCCCTTTCTCTGGTACCTCAGATGC-3’（SEQ ID NO：15）；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

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

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

3 'Probe (3' Probe):

F：5’-CTCTGAGCGCTCAGAAAGGTATGG-3’（SEQ ID NO：18），

R：5’-GGACCTTAGCTTCAGGAAATGGG-3’（SEQ ID NO：19）；

neo Probe (Neo Probe):

F：5’-GGATCGGCCATTGAACAAGATGG -3’（SEQ ID NO：20），

R：5’- CAGAAGAACTCGTCAAGAAGGCG-3’（SEQ ID NO：21）。

the selected correct positive clone is introduced into the separated blastocyst (white mouse) according to the known technology in the field, the obtained chimeric blastocyst is transferred into the culture solution for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mouse (black and white alternate) can be produced. The F1 generation mice are obtained by backcrossing the F0 generation chimeric mice and the wild mice, and the F1 generation heterozygous mice are mutually mated to obtain the F2 generation homozygous son mice. The positive mice can also be mated with Flp tool mice to remove the positive clone selection marker gene (the process is schematically shown in figure 5), and then mated with each other to obtain TMEM173 gene humanized homozygote mice expressing humanized TMEM173 protein. The somatic cell genotype of the progeny mice can be identified by PCR, and the identification of an exemplary F1 mouse (with Neo removed) is shown in FIG. 6, in which the F1-1 mouse is a positive heterozygous mouse. The PCR assay included the following primers:

WT-F2：5’-CTCAGAGGCTGTGTGTTAGGTGG -3’（SEQ ID NO：22），

WT-R：5’-CTGGGCAGGGAACGCATTATGAC-3’（SEQ ID NO：23）；

Mut-F：5’-CTATCTCCCTGTTCCAGAACCTGC -3’（SEQ ID NO：24）

WT-R：5’-CTGGGCAGGGAACGCATTATGAC-3’（SEQ ID NO：23）；

Frt-F：5’-CAAGGCATAGGCAATGGGTGTCG -3’（SEQ ID NO：25）；

Frt-R：5’-CCTAAGGGTGTGTCCTCAGACG -3’（SEQ ID NO：26）；

Flp-F2：5’-GACAAGCGTTAGTAGGCACATATAC -3’（SEQ ID NO：27）；

Flp-R2：5’-GCTCCAATTTCCCACAACATTAGT-3’（SEQ ID NO：28）。

this shows that TMEM173 humanized gene engineering mice which can be stably passaged and have no random insertion can be constructed by using the method. The expression of the humanized TMEM173 protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry, etc., and the results are shown in fig. 7. The results show that the gene humanized mouse prepared in the embodiment can stably express the humanized TMEM173 protein and can be specifically combined with the human TMEM173 antibody.

In addition, a CRISPR/Cas system can be introduced for gene editing, so that a TMEM173 gene humanized mouse is obtained, and the designed targeting strategy is shown in FIG. 8. The target sequence in the system determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut a target gene, so that the selection and design of the high-efficiency specific target sequence are the premise for constructing an sgRNA expression vector. sgRNA sequences that recognize the 5 'target site (sgRNA 1-sgRNA 7), the 3' target site (sgRNA 8-sgRNA 14) were designed and synthesized. The 5 'end target site and the 3' end target site are respectively positioned on the No. 5 exon and the No. 8 exon of the TMEM173 gene, and the target site sequence of each sgRNA on the TMEM173 is as follows:

sgRNA1 target site sequence (SEQ ID NO: 29): 5'-GTACCCAATGTAGTATGACCAGG-3'

sgRNA2 target site sequence (SEQ ID NO: 30): 5'-TACTTGCGGTTGATCTTACCAGG-3'

sgRNA3 target site sequence (SEQ ID NO: 31): 5'-GTATGACCAGGCCAGCCCGTGGG-3'

sgRNA4 target site sequence (SEQ ID NO: 32): 5'-GCGGTTGATCTTACCAGGTAGGG-3'

sgRNA5 target site sequence (SEQ ID NO: 33): 5'-TGCGGTTGATCTTACCAGGTAGG-3'

sgRNA6 target site sequence (SEQ ID NO: 34): 5'-CAGACTGCAGAGACTTCCGCTGG-3'

sgRNA7 target site sequence (SEQ ID NO: 35): 5'-CATACTACATTGGGTACTTGCGG-3'

sgRNA8 target site sequence (SEQ ID NO: 36): 5'-GGTGCTCCGGCACATTCGTCAGG-3'

sgRNA9 target site sequence (SEQ ID NO: 37): 5'-TGGCTCTTGGGACAGTACGGAGG-3'

sgRNA10 target site sequence (SEQ ID NO: 38): 5'-AGATGAGGTCAGTGCGGAGT GGG-3'

sgRNA11 target site sequence (SEQ ID NO: 39): 5'-GCTGATCCATACCACTGATG AGG-3'

sgRNA12 target site sequence (SEQ ID NO: 40): 5'-CTCCGCACTGACCTCATCTG AGG-3'

sgRNA13 target site sequence (SEQ ID NO: 41): 5'-ACTGACCTCATCTGAGGCAT GGG-3'

sgRNA14 target site sequence (SEQ ID NO: 42): 5'-CACTGTCTCAGGAGGTGCTC CGG-3'

The activity of multiple sgrnas is detected by using a UCA kit, and the sgrnas have different activities as shown in the results, and the detection results are shown in fig. 9 and table 1. From these, 2 (sgRNA 5 and sgRNA10, respectively) were preferentially selected for subsequent experiments. A fertilized egg at a prokaryotic stage of a C57BL/6 mouse is taken, and a microinjector is used for injecting premixed in-vitro transcription products of the sgRNA5 and the sgRNA10 (the transcription is carried out by using an Ambion in-vitro transcription kit according to the method of the instruction) and Cas9mRNA and a targeting vector plasmid containing a DNA fragment sequence (SEQ ID NO: 7) of human TMEM173 into the cytoplasm or the nucleus of the fertilized egg of the mouse. Microinjection of embryos is performed according to the method in the manual for mouse embryo manipulation experiments (third edition), fertilized eggs after injection are transferred to a culture solution for short-term culture, and then are transplanted to the oviduct of a recipient mother mouse to produce a genetically modified humanized mouse, so that a founder mouse (i.e., a founder mouse, generation F0) is obtained. The obtained mice are crossed and selfed to enlarge the population quantity, and a stable mouse strain can be established.

Table 1 results of detection of sgRNA activity

Example 2 TMEM173 Gene humanized Point mutant mice

The TMEM173 gene in nature has great heterogeneity and population stratification (heterology and population stratification), and common human TMEM173 alleles include R232, HAQ (R71H-G230A-R293Q), AQ (G230A-R293Q), Q293 and H232 (see patent S, Jin L. TMEM173 variants and potential import to human biology and disease [ published connected line a head of print, 2018 May 1, Genes Immun.2018; 10.1038/S41435-018-0029-9. doi: 10.1038/S41435-018-. It has been shown that the human TMEM173 protein mutant can differentially recognize Cyclic Dinucleotides (CDN). To better mimic the gene types of different TMEM173 in human body, the inserted human protein-encoding nucleic acid sequence can be mutated, for example, when the targeting vector is constructed in the foregoing example 1, the inserted human TMEM173 sequence can be changed to directly obtain a point mutation mouse containing such allele, or the gene editing can be performed on the basis of the mouse obtained in example 1 to obtain a point mutation mouse.

Taking construction of R232 mutant as an example, in example 1, in constructing a targeting vector, the sequence of the synthesized human TMEM173 is similar to SEQ ID NO: 7, the sequence from position 2561 to position 2563 was different, i.e., from CAT to CGG, and the remaining preparation methods were identical, and the resulting humanized mouse expressed mRNA sequence was identical to that of SEQ ID NO: 8 differ at positions 1029-1031 (CAT to CGG) and encode a protein sequence identical to that of SEQ ID NO: the sequence shown in FIG. 9 differs only in one place, i.e., bit 231, H, becomes R. Tests prove that the humanized mouse containing the human R232 allele sequence is successfully obtained.

In addition, the mouse obtained by the method of example 1 can be genetically edited by using a conventional gene editing method, and taking the aforementioned R232 allele as an example, a variety of gene editing systems and preparation methods can be used, including but not limited to gene homologous recombination technology based on embryonic stem cells (ES), Zinc Finger Nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, homing endonuclease (megabase megaribozyme), common palindromic repeat cluster/common palindromic repeat cluster-associated protein system (clustered regularly modulated amplified sequences amplified polypeptides/CRISPR-assisted repeat polypeptides, CRISPR/Cas), or other molecular biology technologies, so as to obtain the humanized mouse of the R232 mutant.

Example 3 in vivo drug efficacy verification

The TMEM173 humanized mouse prepared by the method can be used for evaluating the drug effect of the regulator targeting the human TMEM 173. For example, TMEM173 humanized mouse homozygote is subcutaneously inoculated with mouse colon cancer cell MC38 to allow tumor growth to about 100mm³Then divided into control group or treatment group according to tumor volume, compound X, Y, Z targeting human TMEM173 and the like are randomly selected in the treatment group, and the control group is injected with equal volume of physiological saline or DMXAA. The tumor volume is measured regularly, the weight of the mouse is weighed, and the in vivo safety and the in vivo efficacy of the compound can be effectively evaluated by comparing the weight change of the mouse and the tumor size through results.

Example 4 preparation and use of Dual-or Multi-humanized mice

The TMEM173 mouse prepared by the method can also be used for preparing a double-humanized or multi-humanized mouse model. For example, on the basis of a mouse humanized by TMEM173, a two-gene or multi-gene modified mouse model modified by TMEM173 and other genes can be obtained by utilizing a mouse ES embryonic stem cell separation and gene homologous recombination targeting technology, and a two-gene or multi-gene modified mouse model modified by TMEM173 and other genes can be further obtained. The mouse TMEM173 obtained by the method can also be bred with other gene modified homozygous or heterozygous mice, the offspring of the mouse is screened, the mouse with double genes or multiple genes modified heterozygous with other gene modified TMEM173 and other gene can be obtained with a certain probability according to Mendelian genetic law, the heterozygotes are mutually bred to obtain the homozygotes with double genes or multiple genes modified, and the mouse with double genes or multiple genes modified can be used for in vivo efficacy verification of targeted human TMEM173 and other gene regulators and the like.

Taking a double humanized TMEM173/PD-1 mouse as an example, because mouse TMEM173 and PD-1 genes are respectively positioned on chromosome 18 and chromosome 1, selecting a TMEM173 humanized mouse to mate with a PD-1 humanized mouse, and finally obtaining the double humanized TMEM173/PD-1 mouse through screening positive progeny mice. The double humanized mice can be used to evaluate the potency, pharmacokinetics, and in vivo therapeutic efficacy of human specific modulators of the TMEM173 signaling pathway in combination with human PD-1 signaling pathway antagonists in a variety of disease models known in the art.

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> Beijing Baiosai map Gene Biotechnology Co., Ltd

Construction method and application of TMEM173 gene humanized modified animal model

<130> 1

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<170> SIPOSequenceListing 1.0

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tgaaactatt aaattccttg ctcagatttc aggaagtaaa gtgtgctgtt catctcaatc 60

tctcctgtct aacccctccc ctcccgattt ccgggggatc aatgatagta gagagctttg 120

gggcctctgg aaatcctgtg gggccctgtc acttttggtc cttgtatgga gtcctgctag 180

gtgtccactg gagtgtgtta catctcggga cctttagagg aattcggagt gcggggctgt 240

ggctgctgtc tccccattca gaagccactt gctagtagct actgaaaggc tcttcattgt 300

ctcttctgct ccaggaacac cggtctagga agcagaagat gccatactcc aacctgcatc 360

cagccatccc acggcccaga ggtcaccgct ccaaatatgt agccctcatc tttctggtgg 420

ccagcctgat gatcctttgg gtggcaaagg atccaccaaa tcacactctg aagtacctag 480

cacttcacct agcctcgcac gaacttggac tactgttgaa aaacctctgc tgtctggctg 540

aagagctgtg ccatgtccag tccaggtacc agggcagcta ctggaaggct gtgcgcgcct 600

gcctgggatg ccccatccac tgtatggcta tgattctact atcgtcttat ttctatttcc 660

tccaaaacac tgctgacata tacctcagtt ggatgtttgg ccttctggtc ctctataagt 720

ccctaagcat gctcctgggc cttcagagct tgactccagc ggaagtctct gcagtctgtg 780

aagaaaagaa gttaaatgtt gcccacgggc tggcctggtc atactacatt gggtacttgc 840

ggttgatctt accagggctc caggcccgga tccgaatgtt caatcagcta cataacaaca 900

tgctcagtgg tgcagggagc cgaagactgt acatcctctt tccattggac tgtggggtgc 960

ctgacaacct gagtgtagtt gaccccaaca ttcgattccg agatatgctg ccccagcaaa 1020

acatcgaccg tgctggcatc aagaatcggg tttattccaa cagcgtctac gagattctgg 1080

agaacggaca gccagcaggc gtctgtatcc tggagtacgc cacccccttg cagaccctgt 1140

ttgccatgtc acaggatgcc aaagctggct tcagtcggga ggatcggctt gagcaggcta 1200

aactcttctg ccggacactt gaggaaatcc tggaagatgt ccccgagtct cgaaataact 1260

gccgcctcat tgtctaccaa gaacccacag acggaaacag tttctcactg tctcaggagg 1320

tgctccggca cattcgtcag gaagaaaagg aggaggttac catgaatgcc cccatgacct 1380

cagtggcacc tcctccctcc gtactgtccc aagagccaag actcctcatc agtggtatgg 1440

atcagcctct cccactccgc actgacctca tctgaggcat gggacagcct tgtctgggct 1500

ctagtgatcc tttagcctcc tgactgagcc ttccttcaat ggttgggggc ctcagagact 1560

tcacatctcc agatgagtcc cacattcctg ggcaagccat ttatttcacc tctctgagcc 1620

tcaaccaacc ctactatgaa aggaggtcat aatgcgttcc ctgcccagcc aaaggatttt 1680

atatatgtag aagttggtgt caatgcctgg taaacttgag agaaaggcca agtacttccc 1740

gtggatgctg cagacattcc ctgctctctg ttgacctgtg tggatggtac cagcagactt 1800

ccaaccctcc agcttctggt cacgtgtgtt caatgggagc ttaagtagat ggcgagaggg 1860

agaaggaaca tttgttctgt tagctgtata caatcacagt gggctggcct gtcaactgcc 1920

ttcttaataa acatatctat tctcagattt ctagaatggc ctcttcccct tgtctctagc 1980

actggtattt gtgtgacact ggagtacttt ctgtctggtc tctttatatc atgtcccttg 2040

cacatggtgt tggcatcagg acgtcccaaa ctcatgacat cacataggcg acagcatgac 2100

ctgcaacctg cagaccggtt gccaagacaa caggcaccat attcccacct tccacttggc 2160

tcacctccca cctttacctg tgttacgtca tcttccatat cttccatacg tcttccatct 2220

tccatacgtc tctctcccct gcttctcttt ctgctgctac cttgtctctc ccttccaata 2280

aaacctcttc catgcggaac tg 2302

<210> 2

<211> 378

<212> PRT

<213> Mouse (Mouse)

<400> 2

Met Pro Tyr Ser Asn Leu His Pro Ala Ile Pro Arg Pro Arg Gly His

1 5 10 15

Arg Ser Lys Tyr Val Ala Leu Ile Phe Leu Val Ala Ser Leu Met Ile

20 25 30

Leu Trp Val Ala Lys Asp Pro Pro Asn His Thr Leu Lys Tyr Leu Ala

35 40 45

Leu His Leu Ala Ser His Glu Leu Gly Leu Leu Leu Lys Asn Leu Cys

50 55 60

Cys Leu Ala Glu Glu Leu Cys His Val Gln Ser Arg Tyr Gln Gly Ser

65 70 75 80

Tyr Trp Lys Ala Val Arg Ala Cys Leu Gly Cys Pro Ile His Cys Met

85 90 95

Ala Met Ile Leu Leu Ser Ser Tyr Phe Tyr Phe Leu Gln Asn Thr Ala

100 105 110

Asp Ile Tyr Leu Ser Trp Met Phe Gly Leu Leu Val Leu Tyr Lys Ser

115 120 125

Leu Ser Met Leu Leu Gly Leu Gln Ser Leu Thr Pro Ala Glu Val Ser

130 135 140

Ala Val Cys Glu Glu Lys Lys Leu Asn Val Ala His Gly Leu Ala Trp

145 150 155 160

Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Gly Leu Gln Ala

165 170 175

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

180 185 190

Gly Ser Arg Arg Leu Tyr Ile Leu Phe Pro Leu Asp Cys Gly Val Pro

195 200 205

Asp Asn Leu Ser Val Val Asp Pro Asn Ile Arg Phe Arg Asp Met Leu

210 215 220

Pro Gln Gln Asn Ile Asp Arg Ala Gly Ile Lys Asn Arg Val Tyr Ser

225 230 235 240

Asn Ser Val Tyr Glu Ile Leu Glu Asn Gly Gln Pro Ala Gly Val Cys

245 250 255

Ile Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln

260 265 270

Asp Ala Lys Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys

275 280 285

Leu Phe Cys Arg Thr Leu Glu Glu Ile Leu Glu Asp Val Pro Glu Ser

290 295 300

Arg Asn Asn Cys Arg Leu Ile Val Tyr Gln Glu Pro Thr Asp Gly Asn

305 310 315 320

Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Ile Arg Gln Glu Glu

325 330 335

Lys Glu Glu Val Thr Met Asn Ala Pro Met Thr Ser Val Ala Pro Pro

340 345 350

Pro Ser Val Leu Ser Gln Glu Pro Arg Leu Leu Ile Ser Gly Met Asp

355 360 365

Gln Pro Leu Pro Leu Arg Thr Asp Leu Ile

370 375

<210> 3

<211> 2223

<212> DNA/RNA

<213> human (human)

<400> 3

tataaaaata gctcttgtta ccggaaataa ctgttcattt ttcactcctc cctcctaggt 60

cacacttttc agaaaaagaa tctgcatcct ggaaaccaga agaaaaatat gagacgggga 120

atcatcgtgt gatgtgtgtg ctgcctttgg ctgagtgtgt ggagtcctgc tcaggtgtta 180

ggtacagtgt gtttgatcgt ggtggcttga ggggaacccg ctgttcagag ctgtgactgc 240

ggctgcactc agagaagctg cccttggctg ctcgtagcgc cgggccttct ctcctcgtca 300

tcatccagag cagccagtgt ccgggaggca gaagatgccc cactccagcc tgcatccatc 360

catcccgtgt cccaggggtc acggggccca gaaggcagcc ttggttctgc tgagtgcctg 420

cctggtgacc ctttgggggc taggagagcc accagagcac actctccggt acctggtgct 480

ccacctagcc tccctgcagc tgggactgct gttaaacggg gtctgcagcc tggctgagga 540

gctgcgccac atccactcca ggtaccgggg cagctactgg aggactgtgc gggcctgcct 600

gggctgcccc ctccgccgtg gggccctgtt gctgctgtcc atctatttct actactccct 660

cccaaatgcg gtcggcccgc ccttcacttg gatgcttgcc ctcctgggcc tctcgcaggc 720

actgaacatc ctcctgggcc tcaagggcct ggccccagct gagatctctg cagtgtgtga 780

aaaagggaat ttcaacgtgg cccatgggct ggcatggtca tattacatcg gatatctgcg 840

gctgatcctg ccagagctcc aggcccggat tcgaacttac aatcagcatt acaacaacct 900

gctacggggt gcagtgagcc agcggctgta tattctcctc ccattggact gtggggtgcc 960

tgataacctg agtatggctg accccaacat tcgcttcctg gataaactgc cccagcagac 1020

cggtgaccat gctggcatca aggatcgggt ttacagcaac agcatctatg agcttctgga 1080

gaacgggcag cgggcgggca cctgtgtcct ggagtacgcc acccccttgc agactttgtt 1140

tgccatgtca caatacagtc aagctggctt tagccgggag gataggcttg agcaggccaa 1200

actcttctgc cggacacttg aggacatcct ggcagatgcc cctgagtctc agaacaactg 1260

ccgcctcatt gcctaccagg aacctgcaga tgacagcagc ttctcgctgt cccaggaggt 1320

tctccggcac ctgcggcagg aggaaaagga agaggttact gtgggcagct tgaagacctc 1380

agcggtgccc agtacctcca cgatgtccca agagcctgag ctcctcatca gtggaatgga 1440

aaagcccctc cctctccgca cggatttctc ttgagaccca gggtcaccag gccagagcct 1500

ccagtggtct ccaagcctct ggactggggg ctctcttcag tggctgaatg tccagcagag 1560

ctatttcctt ccacaggggg ccttgcaggg aagggtccag gacttgacat cttaagatgc 1620

gtcttgtccc cttgggccag tcatttcccc tctctgagcc tcggtgtctt caacctgtga 1680

aatgggatca taatcactgc cttacctccc tcacggttgt tgtgaggact gagtgtgtgg 1740

aagtttttca taaactttgg atgctagtgt acttaggggg tgtgccaggt gtctttcatg 1800

gggccttcca gacccactcc ccacccttct ccccttcctt tgcccgggga cgccgaactc 1860

tctcaatggt atcaacaggc tccttcgccc tctggctcct ggtcatgttc cattattggg 1920

gagccccagc agaagaatgg agaggaggag gaggctgagt ttggggtatt gaatcccccg 1980

gctcccaccc tgcagcatca aggttgctat ggactctcct gccgggcaac tcttgcgtaa 2040

tcatgactat ctctaggatt ctggcaccac ttccttccct ggccccttaa gcctagctgt 2100

gtatcggcac ccccacccca ctagagtact ccctctcact tgcggtttcc ttatactcca 2160

cccctttctc aacggtcctt ttttaaagca catctcagat tacccaaaaa aaaaaaaaaa 2220

aaa 2223

<210> 4

<211> 379

<212> PRT

<213> human (human)

<400> 4

Met Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg Gly His

1 5 10 15

Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu Val Thr

20 25 30

Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr Leu Val

35 40 45

Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly Val Cys

50 55 60

Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg Gly Ser

65 70 75 80

Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg Arg Gly

85 90 95

Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro Asn Ala

100 105 110

Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu Ser Gln

115 120 125

Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala Glu Ile

130 135 140

Ser Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala

145 150 155 160

Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln

165 170 175

Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly

180 185 190

Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val

195 200 205

Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys

210 215 220

Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr

225 230 235 240

Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr

245 250 255

Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser

260 265 270

Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala

275 280 285

Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu

290 295 300

Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp

305 310 315 320

Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu

325 330 335

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

340 345 350

Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met

355 360 365

Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser

370 375

<210> 5

<211> 5574

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cttcgaggat taagtgatgc tgagcttgtg tgggccagag aggctctgag aggccagagc 60

ctctcacact ttgtcagggt gaaacttccc ctgaatggaa aaatgaggtg acatcttcca 120

agagcatcag tgatgagtgt tggagaagac agaagaactg gcctggaaat agaaataaga 180

acctgttcag atcagcattt cagggtatct tcacagaaga agcaactaca aaaaggcgtg 240

agagtaaagt ctgtatgtca gacagcaact tgaagctaac ttgcctccgt agcctctggc 300

cttctcttcg gctgatgaga gctgatcaca gacaaataca gactccttgc ctgtctgtaa 360

agctggtctg tctacatgtg gaagacagtc tcatgcaatc cctgtcaaat gattttttcc 420

aggcctggcc tcaaggtcct agtcaatttt tcaaacttct tgtgtcactg accaggtgag 480

agatttgatt gtatccccaa tgtgctgaag ccctgtcttc cagtattgtg tctatatgtc 540

tgtcttctgt tgctaaaagc acagtacaga ctggataagt tacagagaaa gcagttttat 600

ttcaatttgt aattctggtg caaggccaaa ggacttcatt cagtattaac tttgtttctg 660

gcaaaatacc aagggagggc attatgtggc cgaagacaag agacatttat tcattcattc 720

attcgttcgt tcgttcgttc atttattttg gatttctgag acaggttctt attcagtctg 780

ggctgtccta aactcactat ttatccaagg atgaccttgt actttgcgct tccactttcc 840

aagtgctgta actacaggag catgctgctg tgcctggctg aggcaagaga cattcatgtg 900

tatctccact gctcttttct tcttataaat ccaccagtat tagataacag ttgctccaca 960

ctaatgacct tctttaaacc caatcacttc ccaaacacct cgtctttgtg aaccatggtt 1020

ggactaagta cccaaccctg gaagggaagc cacatttaga ccatgcagtc tactgtggga 1080

catttttttg gacgtttttg aatggagtta aggcaaagta agctcactgg gtggagcact 1140

aatgccatct aactgggtcc atagaaaaag aaatgctagg taaagacaga tgctcagcag 1200

tgcagcaggt gaggtcggag gtagacattc agttcctgcc aaccaaggcc tgctagcagt 1260

gaagggagca aggagacttt ttcagagccc ttagacacct tggtccctgg ctaccaagct 1320

ttagagagaa taaaattgtg ttgtttgttt tgttttttgt ttgtttgttt tgttttgttt 1380

tgttttgttt ttttgagaga gggtttcttg gtgtagccct ggctctcctg gaactccttt 1440

tgtggaccag gctggcctcc atcttagaga tccacctgcc tctgcctcct gagtgctgcg 1500

atcaaaggaa taagccacca cccacctggt tgagataata catttatgtt gttttctgat 1560

gcttagttat agcaaccttg gagagtatat agatgcctct cctgcatgtt ctcataacat 1620

ctgcatcctc caccggccaa gatccagctg aggcagggtt tatacctctc ctttcagaat 1680

gttttatcga tgctgtcctc atctctctca taggccttcc aaattcggct ttaatggcac 1740

atctgcagta aagtccagac ttgggcattc cggttgccta ccagagcaac aagatgaacg 1800

catctttggt agccctcatc atgtgtgtag ctgaatactt ccttggatag ttatgttata 1860

tacatttgcc ccgcttggtt gttgatgcca gttcaaggcc tggcatcaca gattcatcag 1920

cagacctggt catgagtagg cacaatctct aggatgattc actgcatgag atatatggat 1980

aaaagatgta cagcctggca tagggctgca catctgcaat cccagctctt gggagggcag 2040

ggcaggaggc acaggcgctc aaagtcatcc ttggctacat attgagtttg aagacagcct 2100

gggctacatg agaccctgtc tcaaaatgaa acaaaagtaa ccctgcgcag gagctcctgg 2160

caggacgttt aaagagccag gcagtgggag cctgtccttc agcagcttta tcgctcttgc 2220

ccaatgcgcc ggaggttttg ctgtaatcag tccaagctaa cgtgcagacc acaatccatt 2280

caccaggccc tgctggctgc tctttttctt tggtctccac cattcccaac atctgacttc 2340

caggcacacc ttactattcc taagggacct gactcaatac tttttgtagc atgcatgcac 2400

tgcattgccc agaacagtgg atgagtgtac aggacagggc aagtagaaag cagtgcccag 2460

ataaaagtga tactaagagg ttttcttcta actagatatt aaaaacaaaa caaacaaata 2520

actaacatat taattaattg acttatttag tgtatatgat gctttgcctg tgtgtatccc 2580

tgtagcactt gtgtgcccgg tactggaaga gaccagaaga aacatcagag accctgaaga 2640

tggagttaga gatggttgtg aacagccatg taggtactga gaaactaact taggtcctct 2700

ggaagggcag ccaaggctct taacttcgga ttcatcttcc catcctctcc tttctgaata 2760

ctttgttatt gcttactgta tagatgaggc ttttctgaaa ctcagcccaa ccgtcctatc 2820

tcagcctccc cagtgctggg attacaggta tgtgccctcc tgcctgtcct tctgtataaa 2880

gtgtacatca tacaaataaa tttcatccca tgtagtgaag gaggggaaaa cggaatagaa 2940

atcactcaga ttcctacctc aaggaatctc atcctctctc atgctgtcat ccacccagtt 3000

agctctttat atgaggcaac tatgagggag agaaagtgta gggagttccc tgcagtttaa 3060

gataattgtt ttttaaaaat ccagaggttt tccaggaagc cagaatattc ctgggagttc 3120

aggggagcct ctgaatgccg gtgttctcct gtctctgctt gtgatacggg gaggattcag 3180

gttcttgcct ggaagggacg aactgctcct gtcccgggcc tcagcagcta gcttctcctg 3240

tgccagggaa actacatgaa ccatggcatc ttccatctgc tcctaaaaac aaggcacgga 3300

gccaactggc ttctgagctt ctgagcctca gggctcaggc tttacaacct caccgacttg 3360

ggtaaagtta ctgtacctct ctgggctcag atcaccctca tgtaaaaaaa aagaataaag 3420

agctggccag atggctagca gggaagagat gcttgtggct aagcctaagg atctgtgttg 3480

gatccctgat acccaccggg tgaaagggga gaatgactca taaattgtcc ggtgccctcc 3540

cctaacaaaa aaacaaaaca aaactataat tgtaaaaaca aacaaacaaa caaacaaaca 3600

aaaaacagga aagaatttga gccttcttcc taggggcaca gggagaactg aactgagcga 3660

aacaggatta gaagcctttg gctatctgga cctggacttc ccttcattta attctagtac 3720

cttcccactc aaccatcctg agactgggaa attagaggcg tggttatttc cccgaaaaga 3780

gtgctctccc ccctcccccg gaaggctgaa actattaaat tccttgctca gatttcagga 3840

agtaaagtgt gctgttcatc tcaatctctc ctgtctaacc cctcccctcc cgatttccgg 3900

gggatcaatg atagtagaga gctttggggc ctctggaaat cctgtggggc cctgtcactt 3960

ttggtccttg tatggagtcc tgctaggtgt ccactggagt gtgttacatc tcgggacctt 4020

tagaggaatt cggagtgcgg ggctgtggct gctggtgagt gactttttga acaccttcag 4080

tttgggggtt aggaaagagg aacagaggag ggcgatctga agcttcacct cccccaccct 4140

cccctgctat actcagacct ggtttatgga ggctggtagg aagggggggg ggtgctttgg 4200

caggaaacac caaaaagaat aaagagctgg ccagatggct agcttcctcc cagagcactg 4260

aaaactgtgc tctggcaagg gccagggctg tggttttact gaggaagcga gctgagttgc 4320

agttgttttt aacatttcgt tttcagtctc cccattcaga agccacttgc tagtagctac 4380

tgaaaggctc ttcattgtct cttctgctcc aggaacaccg gtctaggaag cagaaggtag 4440

gattagaaat ggggcagtat tctcctggta cttcgtggtg ggagaaaccc tgggctggca 4500

gaacactcta aggtctaggt cttggatcag tttcttcctt ctttctcaca ccagtgagct 4560

cagtggggct cacatgtaca cgctctgttt actatgaacc tctcctagac aggtgctgta 4620

ggatgctatg tgcccagggc gtctccttga ggtgtatcca agagtagccc atgggactag 4680

ctgggctggg agacccaagg agatggatga ctagtcagga ccttaggccc tgaaggacag 4740

gagaacccca gaagcatagc tgtggatttc ttgatgtcta cagatgccat actccaacct 4800

gcatccagcc atcccacggc ccagaggtca ccgctccaaa tatgtagccc tcatctttct 4860

ggtggccagc ctgatgatcc tttgggtggc aaaggatcca ccaaatcaca ctctgaagta 4920

cctagcactt cacctagcct cgcacgaact tggactactg ttgaaaaacc tctgctgtct 4980

ggctgaagag ctgtgccatg tccagtccag gtaaccctct gtggtctcca cgatgacttg 5040

atacccagca agtatcacac ccttgactcc taaccttggc cttccctgaa catcatcagc 5100

tgagttgggc tgggatctga ggctaggctg tcccctgcag gtaccagggc agctactgga 5160

aggctgtgcg cgcctgcctg ggatgcccca tccactgtat ggctatgatt ctactatcgt 5220

cttatttcta tttcctccaa aacactgctg acatatacct cagttggatg tttggccttc 5280

tggtcctcta taagtcccta agcatgctcc tgggccttca ggtatgaagc agaagaggtg 5340

ggtggtgtgg gaagagggga ctatggctag tgctggtgtg cctgagctca gtgctgagac 5400

tcagactaat ttaaaggttg gagacctggg tgtggagcta tgaaggcttg ggatgatggg 5460

tttaatagca gtgctgagag caagctggca gcaggttggg aaagttttct gcaagagaag 5520

ggctttggac atcccccttg aaagtccctc aggcccttct gctgtcttca gagc 5574

<210> 6

<211> 3444

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgtccaggg aagggaccac tgaacaaatg ctcccaaatg agagatctgt gaaaggatca 60

tggaggagag gaggtgcgtc tgaggacaca cccttaggaa tggttagtca ccaagcctac 120

ctgatgccgt tgtaacaata tcgtggtggc actgtcaccc ccagctagga ggtcaggcag 180

tccttcctat cagctttacc tagacttgac atgtgtcttc cctctacctc tgttttcatt 240

acaagggctc ggtattgcac tttgaacgtc cccttgtttt atgcgtgtgt tttctccagt 300

actgaggatc caattcatgg tcttgttcac ataggcaaac actctacccc tgagctgtgc 360

cctcagttct cattatgagt ttctgcatac agtgtcactg tagggggagg ttctgatgct 420

agttgctggg ctggaggtac agatgggact tctgggtttt tggatgagaa cgggctcgcc 480

atactttaga gacgaactaa gagccatgtg agatctcaaa atggagtagc cacacaggct 540

gctcctatag gcaggttgtc aggggagttt agcaactaaa gtttaggact ggtgggagag 600

gcagagataa gaaattggta aagggctggg tggtggtggt gcatgccttt aatctcagca 660

cttgggaggc agaggcaggt ggatttctga gttcaaagcc agcctggtct acaaagtgag 720

ttccaggaca gccagggcta tacagagaaa tcctgtttcg aaaaaaaaga aaaaagaaaa 780

aaaagaaaga aactggtaag ggcatgcttt tccaggcagg agataatagc acccagcaat 840

tgtgccaaga aggcaaattg aacacctgtg tgtctgtgtt tttatccttg gactcaaggg 900

aagccaggta agggctggta cctcaattga taccacaaaa ctacactcaa catgtcacta 960

tggagaaata tcacacacac acacacacac acacacacac acacacacac acacacacac 1020

acacacatca gaggaagcca gtcactctaa ggatagcaga accatgggga tcaagtgtgg 1080

ggcagtcttg ggactcgtga gatggctcag tgggtaagag cacccgacta ttcttccgaa 1140

ggtctgaagt tcaaatccca acaaccacat ggtggctcac aaccatccgt aatgagatct 1200

gactccctct tctggagtgt ctaaagacag ctacagtgta cttatatata ataaaaataa 1260

ataaatcttt aaaaaaaagt gtggggcagt ctcttctctc tctgtcacag ccatcttgtc 1320

ataacactga ggagcctcta tcacacccaa cacttccttc tctagcaacc aggcctagca 1380

taaacggcct cctcaggctg attgtgttca acccaccagt ccccgggatg gttcctaggc 1440

tgtgcggctg gtgaaactca gtctgaaatg ctcactttat tgcttggtac ctgtgtccac 1500

catggtgaaa tggggactgc atggcatctc tgccaaggtt gtgaggagtg aatgggactg 1560

acaggaccag caatctgcac atagatgaat agctgactgc taagcagcag catcaagcaa 1620

tgccagctgg cacctttttt tttttttttt tttttgagac agtgtttctc tgtgtagcct 1680

tggctgtcct ggaacacact ctgtagacca ggctggtctt gaactcacag agatctgcct 1740

gcttctgcct ccctagtggt aggattaaaa gcaagtacca ccattgcctg gctcttatgc 1800

agtcacaagg aggtgtgttc attctctgca cacatttcca cccattctgc ttctccccca 1860

cctccctggt tccctgcctc attctgtggg aggacagagc acgatgattt atccttagga 1920

ggtcccagtt gtgtttgttt gtttgtttgt ttgtttaaga tttcttttat ttatatgagt 1980

acactgtagc tgtcttcagc cacaccagaa ggggacattg atcccattac agatggctgt 2040

gagccaccat gtggttgcta ggatttgaac tcaggatctc tggaagagca gcactgctct 2100

taactgctga gccatccctc cagccttctt tttttttttt tttttttttt tttttaagaa 2160

agagcctcac tatgtagccc aggttagcat agaactcact atataaaatc aattcacctt 2220

gaactcagag atccatctgc ctctgcctct tgagtgctgg ggttaaaggt gtgcaccagc 2280

atatccaacc ctgctcttct cttctcttcc ttcctttttt tttttttttg taagttttta 2340

ctgattctct gtatcattca tatcgtgtac cccagcccac tcctctcctt caccttgtat 2400

ttgccttctg cccatgcaac ctcctcccca aataataaac aaacaagaaa taaaacaaag 2460

catggtgaac atctcgttgt ggaagctgta gtgtgtcaca gtgtgtccca caataaaccc 2520

ctctgtccac acaccttcac ttgggaatgt tcattgcact gagtcattgg tctgttctga 2580

gatctctggc ttccgtgacg ccgtcaatat tggatcctca ccaggacccc tcctggtttc 2640

ctgctgttgc cctgtgtcac agagatccta cagcttagga acagcaggac tgctttcatg 2700

tgtcccaaca gttcacagat gctgtagagg ttgtggtggt ggatctggat ctgggcgtgg 2760

atggaagctg agcgtcagcc tgccagctct tccttattca aaccgccagg gcgagctctc 2820

cagcactgct ctgactaggt cacccaatgc tgacctcggc aggaagcagg gtcagctctt 2880

ccgctcttat gccctccctc gcgcaggctc acccgcaccc acgctgccag agccctcggc 2940

tgtactgccc aatcaaggca gcctgctctc tcaagtgctg cacccggcaa ggggctggga 3000

ctgctctggc catgtcacac cttcggggct gcctcacctg agccatcatc ctcagggcca 3060

gctccactgt gttgcccaag cgaggtgcag agcctgcctt cagccagtga gggacaggga 3120

tagctctttc cctttcatga caggtggggg ggttgaccgg tgacaggtgg gggtggggag 3180

cagctgtccc gactactgca aggggagtgg ggtgggggag gggaaagggc ttcaccagtc 3240

gcccactcca cctcatttca cagcagatga gtggtggggc cagttctcct aaggactggc 3300

taagctgccc atgctctatc agggccaact ctactgtgag gttcagggct tgcttttatt 3360

tcacaacctc tagccagcca caggtggcta gtggcaaggc aggcctctcc tttctcttaa 3420

taaacaggtg ccatgtagtc tagg 3444

<210> 7

<211> 4635

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctggccccag ctgagatctc tgcagtgtgt gaaaaaggga atttcaacgt ggcccatggg 60

ctggcatggt catattacat cggatatctg cggctgatcc tgccaggtgg gccatcctct 120

ctgcccccat ttctcgatct acaaaacaga cactaagaca cacagcctct tgtgtttcta 180

aacacacaga actcactata tttttcaaag cattttcatg tctaccaaat cctttaatcc 240

caaggcatga aggatgttgc tatgatgcct agtttgcaga aaggaatatg gggcttagag 300

ccattaagtg acttgcccaa ggtcacttgt gaattctttt tttttctttt tgagaaggaa 360

tctccctctg tcgcccaggc tagagtgcag tggtaccatc tcggctactg caacctcctc 420

cttccaagtt caagtgatta tcctggctca ggttcccgag tagctgggat tataggcacc 480

caccacaacg cccggctaat ttttgtattt ttagtagaga cagggtttca ccgtattggc 540

caggccggta caaactcctg acctcaagtg atccgcctgc cttagcctcc caaagtgctg 600

ggattacagg cgtgagccca gctgtaaatt ctttcaataa atatttactg catgctgtgt 660

gctctcatgt gccttttcta attcacaccc tatgaggaag gaataatctt tattcctgtt 720

ttattttatt tatatatttt ttaaaaagaa gagggttttg ctatgttacc cagcctggtc 780

tcaactcctg ggctcaagca atcctcctgc cttggcttcc caaagtgccg agataacaag 840

caggagccac caagcccata cctattttag attttttttt tttttttttt ttgagacaaa 900

gtctcactct gttgcgcaga ctggagtgca gtagcacaat ctcagctcac tgcgacctct 960

gcctcctggg ttcagcctcc caagtagctg gaattacagg tccctgccac cggcatggct 1020

tttttttttt tttttttttt tttttttgta tttttagtag agacagggtt tcaccatgtt 1080

gcccaggctg gtcttgaatt cttgacctca agtaatccac ccaccttggc ctcccaaagt 1140

gctgggatta cagacatgag ccactgcacc agccttagat tttttttttt ttttaagatg 1200

gagtttcact ttttcacccg ggctgaagta gagtggcaca atctgggctc actgcaactt 1260

ctgcttccca ggttccagtg attctcctgc cttagcctcc caagtagctg ggattttagg 1320

tgcctgccac cacgcccggc taatttttgt attttcagta cagacggggt ttcaccatgt 1380

tgggtaggct agtctcaaac tcctgacctc aggtgatcca cccacctcgg cctcccaaag 1440

tgctagaact ataggcatga gccactgtgc ctggcctttt tttttaagtg cagttctgag 1500

aggtaaagtg atttatccga tatcacatag cttagagaga ggaagagaaa ggatttgaac 1560

ccaggtttgt ccagagcctg gaccctagac cactgcaccg tggagcctgt gtttgttgtt 1620

gttgttgttg ttgttgttgt tgttgttgtt gttgttgttg ttttgagatg aagtctcttt 1680

ctgttaccca gcctggagta cagtggtgac aagatctcag ctcactgcaa cctctgcctc 1740

ccaggttcaa gtgattctcc tgcctgagcc tcttgaatag ctaggattac aggcacgtgc 1800

caccatgcct ggctaatttt tgtattttta gtagagacaa tgtttcacca tgttgcccag 1860

gctagtctca aactcctgac ctcaaatgat ccacctgcct cagcctccca aagtgctgga 1920

ttacaggcgt gagccactgt gcctgcccta agcctgtgtg ttttattctt ctgacttgca 1980

ggctaaagcg gcagctcttc catatctcat tgctatctcc tagggcttcc gctaggagac 2040

tgatctgggg ctagaggcct ccctctgtgc acacgagaat gctggaaatg tcacctctca 2100

gggctctgcc tgcctctcag ccctgaaagc catggtggaa aggggtggcg ctgacataga 2160

catctgagga aagaagtgag ggagggtaaa gggtggtgca gtaagaggag gggtggggag 2220

ggcttttgga ggcgctgccc ctcctggcct cctgtacaat gagagtgcac tggactctcc 2280

attctctggc acccacacac tgcgggggcc aatgacctgg gtctcactcc tgaatcaggt 2340

gggagatagg gttagcagga ataacttctt gggcttccct gcctcagagc tccaggcccg 2400

gattcgaact tacaatcagc attacaacaa cctgctacgg ggtgcagtga gccagcggct 2460

gtatattctc ctcccattgg actgtggggt gcctgataac ctgagtatgg ctgaccccaa 2520

cattcgcttc ctggataaac tgccccagca gaccggtgac catgctggca tcaaggatcg 2580

ggtttacagc aacagcatct atgagcttct ggagaacggg cagcgggtaa gtgtgcaggg 2640

gagtgggggt ctctgaggag gggtcaggac cccagaaccc tgggccctag ccaagcactg 2700

atgaaaattc actgcccttc tctgagctgt agtgtcccta gctggtccca ggtctgggca 2760

ggattcagct ctgaatgata atgatgagta acatttatcg agcacttact acaagctgga 2820

tgctattctg ggtgtgtcat ctgatcacct cattgaattc tcaccaccac cctatggggt 2880

agggactgtt agcattccca ctttacagag gaggacactg aggctcagag acaccgtagg 2940

aaagtggacg atttaaactt gactgtacca tgctcttgac cataaagctg ccccagacga 3000

aggctgtgag aacatctgaa ggattcatgt gggtgcaggg gaacccagac cagagttgaa 3060

cccagagcct agccccagac ctgattccca gctggagcag ttgacagcct cgggtcttgc 3120

cctaggatct tggaagaagg atctgtgaag ggtaaattta gcccagccgt gtccctgatt 3180

tcagagttgg gtatcagagg caaaggcagg ccagacagca tagaccccat tagggtggcc 3240

acctcccagg actctatcgt tacaggctga gggagtgggg cctcagcccg tggcactggg 3300

ggccagagcc tggactggac cctccattct ccatccgcct ggcccctggc ttagtctggt 3360

cttcctctta cctcctctag gcgggcacct gtgtcctgga gtacgccacc cccttgcaga 3420

ctttgtttgc catgtcacaa tacagtcaag ctggctttag ccgggaggat aggcttgagc 3480

aggccaaact cttctgccgg acacttgagg acatcctggc agatgcccct gagtctcaga 3540

acaactgccg cctcattgcc taccagggtg aggggttggt agggtgaggg gttgataggc 3600

tggagggcag gaggagtcag gggtcccagg gtctcccaaa gagtcagaag gggctatgga 3660

gcaccatgcc ctgggcctgt ccttccttcc ctgggagaag ccccctcttc ccagcacacc 3720

agattgcttt ttaggtcttt gactgtaatc cctcagggga gcctgaagag gtgggttcta 3780

ggggcctctc acttcatcta ggattccaga ggaaacagaa gagtcccagg ccagtatgta 3840

ctgggcacag gagactaggg ctgcagcctc ggctctacta ctccctcact gtgtgacctc 3900

gggcaggttt ctgctcctct ctgagcatta gctttaaaag taagagggtg gaatttcatt 3960

ctctctgaaa tcactttttt tttttttttt tgagatggag tctcactctg ttgcccaggc 4020

tggagtgaag tggctcgatt tcgggtcact gcaacctcca cctcccgggt ccaagcgatt 4080

ctcctgcctc agcctcccaa gtagctggga ctacaggcgc ccgccaccat gcccagctaa 4140

tttttgtatt tttagtagag acggggtttc atcatattgg ccaggctggt cttgaattcc 4200

tgacctcatg atctacctgc ctcggcctcc caaagtgcta ggattacagg cgtgagccac 4260

tgtgccaggc ctgaaccact tctacctcta ggattctatg aaactctagt cccagtggct 4320

ggggcttggg taggaagggg ggtagggagg gttaggggat cttcccagca agttctctag 4380

gtatgtttga gtgggaatgg gtaagatcct cattactctc tcccctatct ccctgttcca 4440

gaacctgcag atgacagcag cttctcgctg tcccaggagg ttctccggca cctgcggcag 4500

gaggaaaagg aagaggttac tgtgggcagc ttgaagacct cagcggtgcc cagtacctcc 4560

acgatgtccc aagagcctga gctcctcatc agtggaatgg aaaagcccct ccctctccgc 4620

acggatttct cttga 4635

<210> 8

<211> 2302

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgaaactatt aaattccttg ctcagatttc aggaagtaaa gtgtgctgtt catctcaatc 60

tctcctgtct aacccctccc ctcccgattt ccgggggatc aatgatagta gagagctttg 120

gggcctctgg aaatcctgtg gggccctgtc acttttggtc cttgtatgga gtcctgctag 180

gtgtccactg gagtgtgtta catctcggga cctttagagg aattcggagt gcggggctgt 240

ggctgctgtc tccccattca gaagccactt gctagtagct actgaaaggc tcttcattgt 300

ctcttctgct ccaggaacac cggtctagga agcagaagat gccatactcc aacctgcatc 360

cagccatccc acggcccaga ggtcaccgct ccaaatatgt agccctcatc tttctggtgg 420

ccagcctgat gatcctttgg gtggcaaagg atccaccaaa tcacactctg aagtacctag 480

cacttcacct agcctcgcac gaacttggac tactgttgaa aaacctctgc tgtctggctg 540

aagagctgtg ccatgtccag tccaggtacc agggcagcta ctggaaggct gtgcgcgcct 600

gcctgggatg ccccatccac tgtatggcta tgattctact atcgtcttat ttctatttcc 660

tccaaaacac tgctgacata tacctcagtt ggatgtttgg ccttctggtc ctctataagt 720

ccctaagcat gctcctgggc cttcagagcc tggccccagc tgagatctct gcagtgtgtg 780

aaaaagggaa tttcaacgtg gcccatgggc tggcatggtc atattacatc ggatatctgc 840

ggctgatcct gccagagctc caggcccgga ttcgaactta caatcagcat tacaacaacc 900

tgctacgggg tgcagtgagc cagcggctgt atattctcct cccattggac tgtggggtgc 960

ctgataacct gagtatggct gaccccaaca ttcgcttcct ggataaactg ccccagcaga 1020

ccggtgacca tgctggcatc aaggatcggg tttacagcaa cagcatctat gagcttctgg 1080

agaacgggca gcgggcgggc acctgtgtcc tggagtacgc cacccccttg cagactttgt 1140

ttgccatgtc acaatacagt caagctggct ttagccggga ggataggctt gagcaggcca 1200

aactcttctg ccggacactt gaggacatcc tggcagatgc ccctgagtct cagaacaact 1260

gccgcctcat tgcctaccag gaacctgcag atgacagcag cttctcgctg tcccaggagg 1320

ttctccggca cctgcggcag gaggaaaagg aagaggttac tgtgggcagc ttgaagacct 1380

cagcggtgcc cagtacctcc acgatgtccc aagagcctga gctcctcatc agtggaatgg 1440

aaaagcccct ccctctccgc acggatttct cttgaggcat gggacagcct tgtctgggct 1500

ctagtgatcc tttagcctcc tgactgagcc ttccttcaat ggttgggggc ctcagagact 1560

tcacatctcc agatgagtcc cacattcctg ggcaagccat ttatttcacc tctctgagcc 1620

tcaaccaacc ctactatgaa aggaggtcat aatgcgttcc ctgcccagcc aaaggatttt 1680

atatatgtag aagttggtgt caatgcctgg taaacttgag agaaaggcca agtacttccc 1740

gtggatgctg cagacattcc ctgctctctg ttgacctgtg tggatggtac cagcagactt 1800

ccaaccctcc agcttctggt cacgtgtgtt caatgggagc ttaagtagat ggcgagaggg 1860

agaaggaaca tttgttctgt tagctgtata caatcacagt gggctggcct gtcaactgcc 1920

ttcttaataa acatatctat tctcagattt ctagaatggc ctcttcccct tgtctctagc 1980

actggtattt gtgtgacact ggagtacttt ctgtctggtc tctttatatc atgtcccttg 2040

cacatggtgt tggcatcagg acgtcccaaa ctcatgacat cacataggcg acagcatgac 2100

ctgcaacctg cagaccggtt gccaagacaa caggcaccat attcccacct tccacttggc 2160

tcacctccca cctttacctg tgttacgtca tcttccatat cttccatacg tcttccatct 2220

tccatacgtc tctctcccct gcttctcttt ctgctgctac cttgtctctc ccttccaata 2280

aaacctcttc catgcggaac tg 2302

<210> 9

<211> 378

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Pro Tyr Ser Asn Leu His Pro Ala Ile Pro Arg Pro Arg Gly His

1 5 10 15

Arg Ser Lys Tyr Val Ala Leu Ile Phe Leu Val Ala Ser Leu Met Ile

20 25 30

Leu Trp Val Ala Lys Asp Pro Pro Asn His Thr Leu Lys Tyr Leu Ala

35 40 45

Leu His Leu Ala Ser His Glu Leu Gly Leu Leu Leu Lys Asn Leu Cys

50 55 60

Cys Leu Ala Glu Glu Leu Cys His Val Gln Ser Arg Tyr Gln Gly Ser

65 70 75 80

Tyr Trp Lys Ala Val Arg Ala Cys Leu Gly Cys Pro Ile His Cys Met

85 90 95

Ala Met Ile Leu Leu Ser Ser Tyr Phe Tyr Phe Leu Gln Asn Thr Ala

100 105 110

Asp Ile Tyr Leu Ser Trp Met Phe Gly Leu Leu Val Leu Tyr Lys Ser

115 120 125

Leu Ser Met Leu Leu Gly Leu Gln Ser Leu Ala Pro Ala Glu Ile Ser

130 135 140

Ala Val Cys Glu Lys Gly Asn Phe Asn Val Ala His Gly Leu Ala Trp

145 150 155 160

Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu Leu Gln Ala

165 170 175

Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu Arg Gly Ala

180 185 190

Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys Gly Val Pro

195 200 205

Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu Asp Lys Leu

210 215 220

Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg Val Tyr Ser

225 230 235 240

Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala Gly Thr Cys

245 250 255

Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala Met Ser Gln

260 265 270

Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu Gln Ala Lys

275 280 285

Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala Pro Glu Ser

290 295 300

Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala Asp Asp Ser

305 310 315 320

Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg Gln Glu Glu

325 330 335

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

340 345 350

Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser Gly Met Glu

355 360 365

Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser

370 375

<210> 10

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acacaggtgc catacgtggc ccatagctaa gcttgatatc gaattccgaa gttcct 56

<210> 11

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aacttcatca gtcaggtaca taatggtgga tccactagtt ctagagcggc cgcattaatg 60

tgtccaggga agggaccact gaacaaatgc tccca 95

<210> 12

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggtcacacag ctagagagac agaagtc 27

<210> 13

<211> 26

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

catagcaaca tccttcatgc cttggg 26

<210> 14

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctcgactag agcttgcgga 20

<210> 15

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cccctttctc tggtacctca gatgc 25

<210> 16

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gacaaagatg gggtagagtc actaagg 27

<210> 17

<211> 28

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcttagaacc tgtccagtct aaacttag 28

<210> 18

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ctctgagcgc tcagaaaggt atgg 24

<210> 19

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggaccttagc ttcaggaaat ggg 23

<210> 20

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggatcggcca ttgaacaaga tgg 23

<210> 21

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cagaagaact cgtcaagaag gcg 23

<210> 22

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctcagaggct gtgtgttagg tgg 23

<210> 23

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctgggcaggg aacgcattat gac 23

<210> 24

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ctatctccct gttccagaac ctgc 24

<210> 25

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

caaggcatag gcaatgggtg tcg 23

<210> 26

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cctaagggtg tgtcctcaga cg 22

<210> 27

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gacaagcgtt agtaggcaca tatac 25

<210> 28

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gctccaattt cccacaacat tagt 24

<210> 29

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gtacccaatg tagtatgacc agg 23

<210> 30

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tacttgcggt tgatcttacc agg 23

<210> 31

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gtatgaccag gccagcccgt ggg 23

<210> 32

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcggttgatc ttaccaggta ggg 23

<210> 33

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tgcggttgat cttaccaggt agg 23

<210> 34

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cagactgcag agacttccgc tgg 23

<210> 35

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

catactacat tgggtacttg cgg 23

<210> 36

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ggtgctccgg cacattcgtc agg 23

<210> 37

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tggctcttgg gacagtacgg agg 23

<210> 38

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

agatgaggtc agtgcggagt ggg 23

<210> 39

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gctgatccat accactgatg agg 23

<210> 40

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ctccgcactg acctcatctg agg 23

<210> 41

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

actgacctca tctgaggcat ggg 23

<210> 42

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

cactgtctca ggaggtgctc cgg 23

Claims

1. A construction method of an animal model of TMEM173 gene humanized modification is characterized in that the genome of the animal model comprises a nucleotide sequence which codes SEQ ID NO: 4 from 139 to 379, said method comprising administering to a subject a nucleic acid sequence comprising a nucleotide sequence encoding SEQ ID NO: 4 from 139 th to 379 th nucleotide sequence to the non-human animal TMEM173 locus.

2. The method of claim 1, wherein the genome of said animal model comprises SEQ ID NO: 7; the construction method comprises the following steps of using a peptide containing SEQ ID NO: 7 to the non-human animal TMEM173 locus.

3. The method of claim 1 or 2, wherein said animal model expresses a human or humanized TMEM173 protein in vivo with reduced or absent expression of endogenous TMEM173 protein, said humanized TMEM173 protein comprising the amino acid sequence of SEQ ID NO: 4 from position 139 to 379 or SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1 or 2, wherein the animal model is constructed using a targeting vector and/or sgRNA, wherein the targeting vector comprises a nucleic acid sequence encoding SEQ ID NO: 4 or a nucleotide sequence comprising SEQ ID NO: 7, and the sequence of the target site at the 5' end of the sgRNA targeting is shown as SEQ ID NO: 29-35, the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID NO: 36-42.

5. The method of claim 1 or 2, wherein the non-human animal is a rodent, and the rodent is a rat or a mouse.

6. A targeting vector for the TMEM173 gene, said targeting vector comprising a nucleic acid sequence encoding SEQ ID NO: 4 or a nucleotide sequence comprising SEQ ID NO: 7.

7. The sgRNA of the TMEM173 gene is characterized in that the sequence of a target site at the 5' end of the sgRNA target is shown in SEQ ID NO: 29-35, and the sequence of the target site at the 3' end of the sgRNA target is shown in SEQ ID NO: 36-42.

8. A humanized TMEM173 protein, wherein said humanized TMEM173 protein comprises the amino acid sequence of SEQ ID NO: 4 from position 139 to 379 or comprises SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.

9. A humanized TMEM173 gene encoding the humanized TMEM173 protein of claim 8, wherein the humanized TMEM173 gene comprises the amino acid sequence of SEQ ID NO: 7.

10. The non-human animal constructed by the construction method of any one of claims 1 to 5, the humanized TMEM173 protein of claim 8, the use of the humanized TMEM173 gene of claim 9 in TMEM173 gene or protein-related studies, the use comprising: