阅读说明：本技术 Sting基因人源化非人动物的构建方法及应用 (Construction method and application of humanized non-human animal of STING gene ) 是由 沈月雷 张美玲 黄蕤 白阳 郭朝设 周小飞 于 2021-09-02 设计创作，主要内容包括：本发明提供了一种人源化STING蛋白、一种人源化STING基因、一种STING基因的靶向载体、一种STING基因人源化的非人动物及其构建方法和其在生物医药领域的应用,利用同源重组的方式将编码人STING蛋白的核苷酸序列导入非人动物基因组中,该动物体内能正常表达人或人源化STING蛋白,可以作为人STING信号机理研究、肿瘤及自身免疫性疾病药物筛选的动物模型,对免疫靶点的新药研发具有重要的应用价值。(The invention provides a humanized STING protein, a humanized STING gene, a targeting vector of the STING gene, a humanized non-human animal of the STING gene, a construction method and application thereof in the field of biomedicine.)

1. A humanized STING protein comprising a portion of a human STING protein.

2. The humanized STING protein according to claim 1, comprising all or part of the amino acid sequence encoded by exon 3 to 8 of human STING gene, preferably all or part of the amino acid sequence encoded by exon 5 to 8 of human STING gene.

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

a) SEQ ID NO: 7 or 49, or a portion or all of the amino acid sequence shown in seq id no;

b) and SEQ ID NO: 7 or 49 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) and SEQ ID NO: 7 or 49 do not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by more than 1 amino acid; or the like, or, alternatively,

d) and SEQ ID NO: 7 or 49, comprising the substitution, deletion and/or insertion of one or more amino acid residues.

4. A humanized STING gene comprising a portion of a human STING gene.

5. The humanized STING gene according to claim 4, wherein the humanized STING gene comprises all or part of exon 3 to 8 of human STING gene, preferably all or part of exon 5, all of exon 6 to 7 and all or part of exon 8, wherein part of exon 5 comprises at least 50bp nucleotide sequence and part of exon 8 comprises at least 50bp nucleotide sequence.

6. The humanized STING gene according to claim 4 or 5, comprising a sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

7. The humanized STING gene according to any of claims 4-6, comprising one of the following group:

A) SEQ ID NO: 5 or SEQ ID NO: 47, or a portion thereof;

B) and SEQ ID NO: 5 or SEQ ID NO: 47 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 5 or SEQ ID NO: 47 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

D) and SEQ ID NO: 5 or SEQ ID NO: 47, comprising a nucleotide sequence in which one or more nucleotide residues are substituted, deleted and/or inserted.

8. The humanized STING gene according to any of claims 4-7, wherein the mRNA transcribed from the humanized STING gene comprises one of the group consisting of:

(i) comprises the amino acid sequence of SEQ ID NO: 6 or 48, or a portion thereof;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

(iv) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48, including substitution, deletion and/or insertion of one or more nucleotides.

9. A targeting vector, wherein said targeting vector comprises a human STING gene, preferably said targeting vector comprises all or part of exon 3 to 8 of a human STING gene, preferably comprises all or part of exon 5 to 8 of a human STING gene, more preferably comprises all or part of exon 5, all of exon 6 to 7, and all or part of exon 8, wherein part of exon 5 comprises at least a 50bp nucleotide sequence and part of exon 8 comprises at least a 50bp nucleotide sequence, further preferably said targeting vector comprises a nucleotide sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378, further preferably comprises the nucleotide sequence of the amino acid sequence shown in SEQ ID NO: 5 or 47.

10. The targeting vector according to claim 9, wherein said targeting vector further comprises a5 ' arm and/or a3 ' arm, said 5 ' arm being selected from the group consisting of nucleotides of 100-10000 in length of the genomic DNA of the STING gene of the non-human animal; preferably, said 5' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 5' arm sequence is identical to SEQ ID NO: 3 or 45 have at least 90% homology; the 3' arm is selected from 100-10000 nucleotides in length of the genomic DNA of the STING gene of the non-human animal; preferably, said 3' arm has at least 90% homology to NCBI accession No. NC _ 000084.6; further preferably, the 3' arm sequence is identical to SEQ ID NO: 4 or 46 have at least 90% homology.

11. A method for constructing a genetically humanized non-human animal, wherein the non-human animal expresses a human or humanized STING protein or comprises a human or humanized STING gene.

12. The method of claim 11, wherein the humanized STING protein is the humanized STING protein of any one of claims 1-3.

13. The method of claim 11 or 12, wherein the humanized STING gene is the humanized STING gene according to any one of claims 4-8.

14. The method of construction according to any one of claims 11-13, wherein the method of construction comprises introducing a nucleotide sequence comprising a human or humanized STING gene into a non-human animal STING locus, preferably a non-human animal STING locus comprising all or part of exons 5 to 8 of a human STING gene, more preferably all or part of exons 5, 6 to 7, and 8, wherein part of exons 5 comprises at least 50bp of nucleotide sequence and part of exons 8 comprises at least 50bp of nucleotide sequence, preferably the method of construction comprises introducing a nucleotide sequence comprising SEQ ID NO: 5 or SEQ ID NO: 47 into a non-human animal STING locus, optionally, the constructing method comprises introducing into the non-human animal STING locus a nucleotide sequence comprising a nucleotide sequence encoding SEQ ID NO: 7 at position 145-340 or SEQ ID NO: 49 at position 138-378, and more preferably, using the targeting vector of any of claims 9-10 for construction of a non-human animal.

15. The method of construction of any one of claims 11-14, wherein the human or humanized STING gene or nucleotide sequence encoding a human STING protein is regulated by endogenous regulatory elements.

16. The construct of any one of claims 11-15, wherein the non-human animal further expresses at least one of human or humanized PD-1, PD-L1, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, OX40, sirpa, TIGIT, TIM-3, CD40, and GITR proteins.

17. A cell, tissue or organ which is humanized by a gene, wherein the genome of said cell, tissue or organ comprises the humanized STING gene according to any one of claims 4 to 8, and said cell, tissue or organ expresses the humanized STING protein according to claims 1 to 3.

18. Use of the cell, tissue or organ derived from the humanized STING protein of any one of claims 1 to 3, the humanized STING gene of any one of claims 4 to 8, the non-human animal obtained by the construction method of any one of claims 11 to 16, or the cell, tissue or organ of claim 17, the use comprising:

use in product development involving immune processes in human cells, in the manufacture of antibodies, or as model systems for pharmacological, immunological, microbiological, medical research;

use in the production and use of animal experimental disease models for the development of new diagnostic and/or therapeutic strategies;

alternatively, the first and second electrodes may be,

the application in the aspects of screening, verifying, evaluating or researching the function of the STING pathway, the signal mechanism of the human STING pathway, the antibody of a target human, the medicine and the drug effect of the target human, the medicine for immune-related diseases and the anti-tumor medicine, screening and evaluating the medicine for the human and the drug effect research.

Technical Field

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

Background

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). The cytoplasmic DNA (whether the DNA is from a virus, a bacterium or an organism) can be combined with the enzyme of cyclic GMP-AMP synthase (cGAS) to form cGAMP (cyclic dinucleotide, CDN for short), the dimeric STING is combined with the enzyme to undergo conformational change, a downstream transcription factor TBK1(STAT-6, NF-kb) is activated, the transcription factor IRF3 is recruited and phosphorylated, interferon (interferon) (IFN mainly is type I) and other various cytokines (including TNF alpha, IL6 and the like) are caused to kill tumor cells and viruses, and thus, a cGAS-cGAMP-STING signal pathway is formed, and further research shows that small molecular compounds can also be combined with the STING to activate or inhibit the signal pathway. After recognizing the STING signaling pathway, small molecules with similar structures are designed, and the small molecules are expected to activate the STING signaling pathway to resist tumors and viruses and provide a basis for the activation and proliferation of T cells; or inhibiting STING signaling pathway to treat autoimmune diseases.

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. Similarity (poisitives) of human and murine STING proteins is as high as 81%, but due to species differences, it has been confirmed that STING targeting compound DMXAA (vadimezan) binds only murine STING, fails to activate human STING protein, and therefore, despite excellent efficacy in mouse anti-tumor models, DMXAA preclinical studies fail at the time of entry into phase III clinics. In recent years, international pharmaceutical companies such as BMS, nova and the like are increasingly searching for drugs capable of activating STING, and it is expected that more and more domestic and foreign pharmaceutical companies participate in drug development of targeted STING proteins in the future. Developing more animal models that can simulate human drug development will help to reduce the risk of development failure, 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 an "effective" humanized animal model for new drug development remains the greatest challenge.

Disclosure of Invention

In view of the fact that the STING gene has great application value in the fields of tumor and immunotherapy, the invention provides a novel method for establishing a STING gene humanized modified animal model and obtaining a STING gene humanized animal in order to further research the biological characteristics related to STING, improve the effectiveness of a drug effect test at an early stage in clinic and improve the success rate of research and development. Specifically, the invention aims to prepare a non-human animal model, the animal body can normally express the STING protein, the expressed STING protein can be identified and combined with a regulator targeting human STING, 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 STING 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:

in a first aspect of the present invention, there is provided a humanized STING protein comprising a portion of a human STING protein.

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

Preferably, the humanized STING protein comprises all or part of the amino acid sequence encoded by exon 3 to 8 of the human STING gene. Preferably, the amino acid sequence encoded by exons 5 to 8 of the human STING gene is contained in whole or in part.

Preferably, the humanized STING protein further comprises an amino acid sequence encoded by all or part of exons 1 to 4 of a non-human animal STING gene. Further preferably, the non-human animal STING gene further comprises an amino acid sequence encoded by a portion of exon 5 of the non-human animal STING gene.

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

A) SEQ ID NO: 2, all or part of the amino acid sequence at position 146-341 or 138-379;

B) and SEQ ID NO: 2, the amino acid sequence identity at position 146-341 or 138-379 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

C) and SEQ ID NO: 2 at position 146-341 or 138-379 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by no more than 1 amino acid; or

D) And SEQ ID NO: 2 at position 146-341 or 138-379, including substitution, deletion and/or insertion of one or more amino acid residues.

In one embodiment of the present invention, the humanized STING protein comprises a sequence identical to SEQ ID NO: 1, position 1-144, 341-378 or 1-136, or an amino acid sequence having at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, or at least 99% identity to SEQ ID NO: 1, 1-144, 341-378 or 1-136.

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

a) SEQ ID NO: 7 or 49, or a portion or all of the amino acid sequence shown in seq id no;

b) and SEQ ID NO: 7 or 49 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

c) and SEQ ID NO: 7 or 49 do not differ by more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or by more than 1 amino acid; or the like, or, alternatively,

d) and SEQ ID NO: 7 or 49, comprising the substitution, deletion and/or insertion of one or more amino acid residues.

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

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

Preferably, the humanized STING gene comprises all or part of exons 3 to 8 of a human STING gene. Further preferably, the combination comprises one or more than two exons from No. 3 to No. 8 exons of human STING gene, and more preferably, the part of No. 3 exon comprises from the start codon of No. 3 exon to the last nucleotide of No. 3 exon, and the part of No. 8 exon comprises from the first nucleotide of No. 8 exon to the stop codon.

In one embodiment of the present invention, exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8 of the human STING gene are included.

Preferably comprises all or part of exon 5 to 8, more preferably comprises all or part of exon 5, exon 6 to 7 and all or part of exon 8, even more preferably further comprises intron 5-6, intron 6-7 and/or intron 7-8, and part of exon 5 comprises at least 50bp of nucleotide sequence, such as at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 90, 100, 105, 106, 107, 108, 109bp of nucleotide sequence, even more preferably comprises 85 or 109bp of nucleotide sequence, and part of exon 8 comprises at least 50bp of nucleotide sequence, such as at least 50, 70, 75, 76, 77, 78, 79, 80, 100, 150, 170, 190, 191, 192, 193, 194, 195, 200, 500, 191, 192, 500, 194, and/or 195, 200, 500, and/or 5, 80, and/or 150, 170, 190, 191, 192, 193, 194, and/or 8 bp of nucleotide sequence, 700. 900, 922bp, further preferably 77 or 194 bp.

In one embodiment of the invention, all or part of the CDS sequence of the human STING gene or all or part of the full-length nucleotide sequence of human STING is included.

Preferably, the humanized STING gene comprises all or part of a nucleotide sequence encoding a human STING protein. Further preferred, comprises SEQ ID NO: 2 at position 146-341 or 138-379, further preferably comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

Preferably, the humanized STING gene further comprises a portion of a non-human animal STING gene. Further preferably, the non-human animal STING gene comprises all of exons 1 to 4and a part of exon 8. Still more preferably, the non-human animal STING gene further comprises a portion of exon 5.

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

A) SEQ ID NO: 5 or SEQ ID NO: 47, or a portion thereof;

B) and SEQ ID NO: 5 or SEQ ID NO: 47 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 5 or SEQ ID NO: 47 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

D) and SEQ ID NO: 5 or SEQ ID NO: 47, comprising a nucleotide sequence in which one or more nucleotide residues are substituted, deleted and/or inserted.

Preferably, the humanized STING gene further comprises a transcription termination sequence, preferably one or a combination of two or more of 3' UTR, polyA, WPRE or lox 2.

Preferably, the humanized STING gene further comprises a Neo cassette.

Preferably, the humanized STING gene further comprises endogenous regulatory sequences.

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

(i) comprises the amino acid sequence of SEQ ID NO: 6 or 48, or a portion thereof;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

(iv) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48, including substitution, deletion and/or insertion of one or more nucleotides.

In a third aspect of the present invention, there is provided a targeting vector comprising a portion of the human STING gene.

Preferably, the targeting vector comprises all or part of exons 3 to 8 of the human STING gene. It is further preferred that the combination comprises one or more than two exons from exon 3 to exon 8 of human STING gene, and more preferably, the part of exon 3 comprises from the start codon of exon 3 to the last nucleotide of exon 3, and the part of exon 8 comprises from the first nucleotide of exon 8 to the stop codon.

In one embodiment of the present invention, exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8 of the human STING gene are included.

Preferably comprises all or part of exon 5 to 8, more preferably comprises all or part of exon 5, even more preferably comprises all or part of exon 6 to 7 and all or part of exon 8, even more preferably further comprises intron 5-6, intron 6-7 and/or intron 7-8, part of exon 5 comprises at least a nucleotide sequence of 50bp, such as at least a nucleotide sequence of 50, 60, 70, 80, 81, 82, 83, 84, 85, 90, 100, 105, 106, 107, 108, 109bp, even more preferably comprises a nucleotide sequence of 85 or 109bp, and part of exon 8 comprises at least a nucleotide sequence of 50bp, such as at least a nucleotide sequence of 50, 70, 75, 76, 77, 78, 79, 80, 100, 150, 170, 190, 191, 192, 193, 194, 195, 200, 500, 700, 192, 200, 500, 700, and/or 8, 900. 922bp, further preferably 77 or 194 bp.

In one embodiment of the invention, all or part of the CDS sequence of the human STING gene or all or part of the full-length nucleotide sequence of human STING is included.

In one embodiment of the invention, the polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

In one embodiment of the invention, the polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 2, the nucleotide sequence of the amino acids shown in the positions 146-341 or 138-379.

In one embodiment of the invention, the polypeptide comprising SEQ ID NO: 5 or 47.

Preferably, the targeting vector further comprises a5 'arm and/or a 3' arm. The 5' arm is selected from 100-10000 nucleotides in length of the genomic DNA of the STING gene of the non-human animal. Preferably, the 5' arm has at least 90% homology with NCBI accession number NC _ 000075.6. Further preferably, the 5' arm sequence is identical to SEQ ID NO: 3 or 45 have at least 90% homology. The 3' arm is selected from 100-10000 nucleotides in length of the genomic DNA of the STING gene of the non-human animal. Preferably, the 3' arm has at least 90% homology with NCBI accession number NC _ 000084.6. Further preferably, the 3' arm sequence is identical to SEQ ID NO: 4 or 46 have at least 90% homology.

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

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

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

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

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

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

In a fourth aspect of the invention, there is provided a sgRNA that targets a STING gene in a non-human animal, with the sequence of the sgRNA on the target sequence on the STING gene to be altered.

Preferably, the target site of the sgRNA is located on exon 3 to exon 8 sequences of the STING gene. Further preferably, the target site of the sgRNA is located on exon 3, any intron between exon 3 and exon 8, and/or exon 8 sequence of the STING gene.

In a specific embodiment of the present invention, the target sites of the sgRNA are located on exon 5 and exon 8 of the STING gene.

Preferably, the sequence of the target site of the sgRNA is as shown in SEQ ID NO: 8-23,69-82, preferably, the sgRNA sequence targets a target site sequence at the 5' end as set forth in SEQ ID NO: 8-15 or 69-75, and the 3' end target site sequence is shown in SEQ ID NO: 16-23 or 76-82.

In a fifth aspect of the invention, a DNA molecule encoding the sgRNA described above is provided. Preferably, the double strands of the DNA molecules are the upstream and downstream sequences of the sgRNA, or the forward and reverse oligonucleotide sequences after the addition of the enzyme cleavage site.

In one embodiment of the invention, the DNA molecule is SEQ ID NO: 32.

in a sixth aspect of the present invention, a sgRNA vector is provided, which includes the sgRNA described above.

In a seventh aspect of the present invention, there is provided a cell comprising the targeting vector, the sgRNA, the DNA molecule, or the sgRNA vector.

In an eighth aspect of the present invention, there is provided a use of the targeting vector, the sgRNA, the DNA molecule, the sgRNA vector, or the cell for STING gene modification. Preferably comprising use in knocking out, inserting or replacing the STING gene.

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

Preferably, the non-human animal comprises a human or humanized STING gene comprising all or part of exon 3 to 8 of the human STING gene. Further preferably, the combination comprises one or more than two exons from No. 3 to No. 8 exons of human STING gene, and more preferably, the part of No. 3 exon comprises from the start codon of No. 3 exon to the last nucleotide of No. 3 exon, and the part of No. 8 exon comprises from the first nucleotide of No. 8 exon to the stop codon.

In one embodiment of the present invention, exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8 of the human STING gene are included.

Preferably, it comprises part or all of exon 5 to exon 8 of the human STING gene, and more preferably, it comprises all or part of exon 5, all of exon 6 to exon 7, and all or part of exon 8 of the human STING 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 STING gene, the part of exon 5 comprises at least 50bp of nucleotide sequence, for example, at least 50, 60, 70, 80, 81, 82, 83, 84, 85, 90, 100, 105, 106, 107, 108, 109bp nucleotide sequence, more preferably 85 or 109bp nucleotide sequence, at least 50bp nucleotide sequence in the 8 exon part, for example, a nucleotide sequence comprising at least 50, 70, 75, 76, 77, 78, 79, 80, 100, 150, 170, 190, 191, 192, 193, 194, 195, 200, 500, 700, 900, 922bp, and more preferably 77 or 194 bp.

Preferably, the part of exon 5 comprises the nucleotide sequence of exon 5 excluding the 1st to 10 th (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acids from the N-terminus, and the part of exon 8 comprises the nucleotide sequence of exon 8 excluding the 1st to 60 th (e.g. 1, 5, 10, 15, 20, 25, 30, 35, 38, 39, 40, 45, 50, 55, 60, etc.) amino acids from the C-terminus.

Preferably, the part of exon 5 comprises the last nucleotide from the start codon to exon 5, and the part of exon 8 comprises the first nucleotide from exon 8 to the stop codon.

Further preferably, the human STING gene of the non-human animal is derived from exon 5 to exon 8, and comprises at least the sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378 or a nucleotide sequence at least comprising the amino acid sequence as shown in SEQ ID NO: 2, the nucleotide sequence of the amino acids shown in the positions 146-341 or 138-379.

In one embodiment of the invention, all or part of the CDS sequence of the human STING gene or all or part of the full-length nucleotide sequence of human STING is included.

Preferably, the non-human animal comprises all or part of a nucleotide sequence encoding a human STING protein. Further preferred comprises a nucleic acid sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378. More preferably, the human STING protein encoded in said non-human animal is as set forth in SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

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

A) SEQ ID NO: 5 or SEQ ID NO: 47, or a portion thereof;

B) and SEQ ID NO: 5 or SEQ ID NO: 47 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

C) and SEQ ID NO: 5 or SEQ ID NO: 47 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

D) and SEQ ID NO: 5 or SEQ ID NO: 47, comprising a nucleotide sequence in which one or more nucleotide residues are substituted, deleted and/or inserted.

Preferably, the humanized STING gene further comprises a transcription termination sequence. Further preferred is one or a combination of two or more of 3' UTR, polyA, WPRE, and lox 2.

Preferably, the humanized STING gene further comprises a Neo cassette, and further preferably, the connection design sequence of the 5' end of the Neo cassette to the mouse locus is SEQ ID NO: 50, the connection design sequence of the 3' end of the Neo cassette and the mouse locus is SEQ ID NO: shown at 51.

Preferably, the humanized STING gene further comprises endogenous regulatory sequences.

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

(i) comprises the amino acid sequence of SEQ ID NO: 6 or 48;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%;

(iii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48 differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or no more than 1 nucleotide; or the like, or, alternatively,

(iv) comprises a nucleotide sequence substantially identical to SEQ ID NO: 6 or 48, including nucleotide sequences with one or more nucleotides substituted, deleted and/or inserted.

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

Preferably, the humanized STING protein comprises all or part of a human STING protein. Further preferably comprises all or part of the amino acid sequence encoded by exons 5 to 8 of the human STING gene. More preferably, the humanized STING protein further comprises at least SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

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

a) SEQ ID NO: 7 or SEQ ID NO: 49, or a portion or all of an amino acid sequence shown in seq id no;

b) and SEQ ID NO: 7 or SEQ ID NO: 49 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%;

c) and SEQ ID NO: 7 or SEQ ID NO: 49 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or by no more than 1 amino acid; or the like, or, alternatively,

d) and SEQ ID NO: 7 or SEQ ID NO: 49, including substitution, deletion and/or insertion of one or more amino acid residues.

Preferably, the genome of the non-human animal further comprises other gene modifications, and more preferably, the other genes comprise one or a combination of two or more of PD-1, PD-L1, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, OX40, sirpa, TIGIT, TIM-3, CD40, or GITR genes.

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

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

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

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

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

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

Preferably, the construction method comprises introducing a nucleotide sequence comprising a human or humanized STING gene into a non-human animal STING locus. It is further preferred that the STING locus is introduced into the non-human animal with all or part of exons 3 to 8 that comprise the human STING gene, and it is further preferred that the STING locus is introduced into the non-human animal with a combination that comprises one or more of exons 3 to 8 of the human STING gene, wherein part of exon 3 comprises the last nucleotide from exon 3 to exon 3 and part of exon 8 comprises the first nucleotide from exon 8 to stop codon.

In one embodiment of the present invention, exon 3, exon 4, exon 5, exon 6, exon 7 and exon 8 comprising the human STING gene are introduced into the STING locus of a non-human animal.

Preferably, the non-human animal STING locus is introduced with all or part of exon 5 to 8 comprising the human STING gene, further preferably with all or part of exon 5, exon 6 to 7 and all or part of exon 8, preferably further with intron 5-6, intron 6-7 and/or intron 7-8, wherein part of exon 5 comprises at least a 50bp nucleotide sequence, such as at least a 50, 60, 70, 80, 81, 82, 83, 84, 85, 90, 100, 105, 106, 107, 108, 109bp nucleotide sequence, further preferably 85 or 109bp nucleotide sequence, and part of exon 8 comprises at least a 50bp nucleotide sequence, such as at least a 50, 70, 75, 76, 77, 78, 79, 76, 77, 79, 80. 100, 150, 170, 190, 191, 192, 193, 194, 195, 200, 500, 700, 900, 922bp nucleotide sequence, and more preferably 77 or 194bp nucleotide sequence.

In one embodiment of the invention, the construction method comprises introducing the STING locus of the non-human animal with all or part of the CDS sequence comprising the human STING gene or all or part of the full-length nucleotide sequence of human STING.

Preferably, the construction method comprises introducing a cDNA sequence comprising a human STING gene into a non-human animal STING locus.

In one embodiment of the invention, the method of construction comprises the step of using a nucleic acid comprising SEQ ID NO: 5 or 47 into the STING locus of a non-human animal.

Preferably, the construction method comprises introducing the STING locus into a non-human animal with a nucleic acid sequence comprising all or part of a nucleotide sequence encoding a human STING protein. Further preferred is a polypeptide comprising a sequence encoding SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378, and more preferably, the human STING gene from exon 5 to exon 8 in said non-human animal, and comprising at least the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7 at positions 145-340, most preferably, the human STING protein encoded in said non-human animal is as set forth in SEQ ID NO: 7, position 145-340 or SEQ ID NO: 49 at position 138-378.

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

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

Preferably, the position of said insertion or substitution is located after the endogenous regulatory element of the STING gene.

Preferably, the position of the insertion or substitution is located on exons 3 to 8 of the STING gene in the non-human animal. Further preferably located from the start codon to the stop codon of the STING gene.

Preferably, the position of the insertion or substitution is located on exons 5 to 8 of the STING gene in the non-human animal. Further preferably located in exon 5.

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

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

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

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

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

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

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

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

Preferably, the target site of the sgRNA is located on exon 3 to exon 8 sequences of the STING gene. Further preferably, the target site of the sgRNA is located on exon 5, intron 4-5, and/or exon 8 sequences of the STING gene.

In a specific embodiment of the present invention, the target sites of the sgRNA are located on exon 5 and exon 8 of the STING gene.

Preferably, the sequence of the target site of the sgRNA is as shown in SEQ ID NO: 8-23,69-82, preferably, the sgRNA sequence targets a target site sequence at the 5' end as set forth in SEQ ID NO: 8-15 or 69-75, and the 3' end target site sequence is shown in SEQ ID NO: 16-23 or 76-82.

Preferably, the above replacement is an in situ replacement.

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

In an eleventh aspect of the present invention, there is provided a polygene-modified non-human animal, wherein the non-human animal is the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, and the genome of the non-human animal comprises a modification of one or a combination of two or more of the genes PD-1, PD-L1, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, OX40, sirpa, TIGIT, TIM-3, CD40, or GITR.

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

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

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

Preferably, the other genetically modified non-human animal comprises a non-human animal humanized with a combination of one or more of the genes PD-1, PD-L1, CTLA-4, LAG-3, BTLA, CD27, CD28, CD47, CD137, CD154, OX40, SIRPa, TIGIIT, TIM-3, CD40, or GITR.

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

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

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

In a fifteenth aspect of the present invention, there is provided a method for constructing a tumor-bearing or inflammation model of an animal, the method comprising the step of obtaining a humanized non-human animal of STING gene or a multi-gene-modified non-human animal by using the above-mentioned construction method.

In a sixteenth aspect of the present invention, an application of the above non-human animal or the non-human animal obtained by the above construction method in preparing an animal model with tumor or inflammation is provided.

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

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

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

In a twentieth aspect of the invention, there is provided a cell which is humanized with a STING gene, said cell comprising a human or humanized STING gene. Preferably, the humanized STING gene described above.

Preferably, the cell expresses a human or humanized STING protein. Further preferred is the above-mentioned humanized STING protein of the present invention. Preferably, the cells are not capable of developing into an individual animal.

In a twenty-first aspect of the present invention, there is provided a cell deficient in STING gene, said cell being deficient in all or part of exons 1 to 8 of STING gene. Preferably, all or part of exons 3 to 8 are deleted, more preferably all or part of exons 5 to 8 are deleted.

In a specific embodiment of the invention, the deletion encodes SEQ ID NO: 2 position 145-340 or SEQ ID NO: 2, 137-378 position.

In another embodiment of the present invention, the nucleotide sequence of STING gene from start codon to stop codon is deleted. Preferably, the cells are not capable of developing into an individual animal.

In a twenty-second aspect of the present invention, there is provided a non-human animal in which the STING gene is deleted, the non-human animal being deleted for all or part of exons 1 to 8 of the STING gene. Preferably, all or part of exons 3 to 8 are deleted, more preferably all or part of exons 5 to 8 are deleted.

In a specific embodiment of the invention, the deletion encodes SEQ ID NO: 2 position 145-340 or SEQ ID NO: 2, 137-378 position.

In a twenty-third aspect of the present invention, there is provided a method for constructing a non-human animal or cell in which STING gene is deleted, the method comprising deleting a gene using the sgRNA.

In a twenty-fourth aspect of the present invention, there is provided a construct comprising the humanized STING gene described above.

Preferably, the construct expresses a human or humanized STING protein, such as the humanized STING protein described above.

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

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

A twenty-seventh aspect of the present invention provides a non-human animal derived from the above non-human animal, the non-human animal obtained by the above construction method, the above humanized STING gene, the above humanized STING protein, the above tumor-bearing or inflammatory model, the above cell or cell line or primary cell culture, the above tissue or organ or culture thereof, the above tumor-bearing tissue, the above cell, the above construct, the above cell or the above tissue, and uses thereof, wherein the uses comprise: use in product development involving immune processes in human cells, in the manufacture of antibodies, or as model systems for pharmacological, immunological, microbiological, medical research; or in the production and use of animal experimental disease models for the development of new diagnostic and/or therapeutic strategies; or screening, verifying, evaluating or researching the function of the STING pathway, the signal mechanism of the human STING pathway, the antibody of the target human, the medicine of the target human, the drug effect, the medicine of immune related diseases and the anti-tumor medicine, screening and evaluating the medicine of the human and the research of the drug effect.

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

In a twenty-eighth aspect of the invention, there is provided a method of screening for a human STING-specific modulator, the method comprising administering the modulator to an individual; wherein the individual is selected from the non-human animal, the non-human animal obtained by the construction method or the tumor-bearing or inflammation model, and the regulator is evaluated and screened.

Preferably, the screening method further comprises implanting tumor cells into the individual.

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

Preferably, the screening method further comprises detecting tumor suppression.

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

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

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

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

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

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

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

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

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

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

The humanized non-human animal of the STING gene can normally express human or humanized STING protein in vivo, can be used for drug screening, drug effect evaluation, autoimmune diseases and tumor treatment aiming at a target site of a human STING pathway, can accelerate the development process of a new drug, and can save time and cost.

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 "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.

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

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

The "exon nos. 3 to 8" or "all of the exon nos. 3 to 8" described in the present invention includes nucleotide sequences of exons and introns therebetween, i.e., exon No. 3, intron No. 3-4, exon No.4, intron No. 4-5, exon No. 5, intron No. 5-6, exon No. 6, intron No. 6-7, exon No. 7, intron No. 7-8, and exon No. 8.

The "intron 3-4" described in the present invention means an intron between the exon 3 and the exon 4.

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 "STING locus" refers to a DNA fragment of any one of exons 1 to 8 of the STING gene. In one embodiment of the present invention, the STING locus to be inserted or replaced may be a DNA fragment of an optional stretch of exon 5 to 8 of the STING 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); DNACloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mulliserial.u.s.pat.no. 4, 683, 195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds. inchief, Academic Press, Inc., New York), specific, Vols.154and 155(Wuetal. eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Caloseds, 1987, Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

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

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

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

Drawings

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

FIG. 1: schematic representation of the comparison of mouse STING gene and human STING gene loci (not to scale).

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

FIG. 3: STING gene targeting strategies and targeting vector design schemes (not to scale).

FIG. 4: and (5) detecting the activity of the sgRNA, wherein Con is a negative control, and PC is a positive control.

FIG. 5: f0 mouse genotype identification results, wherein M is Marker, WT is wild type control, H₂O is water control.

FIG. 6: f1 mouse PCR genotype identification result, wherein PC is positive control, WT is wild control, H₂O is water control.

FIG. 7: southern Blot assay results.

FIG. 8: ELISA detection results, wherein the +/+ wild type C57BL/6 mouse and H/+ are hybrid of humanized mice with the STING gene.

FIG. 9: PCR assay of STING Gene knock-out mice, where WT is wild-type control, H₂O is water control.

FIG. 10: the STING gene targeting strategy in example 2.

FIG. 11: mouse Southern blot results, where WT is wild type;

FIG. 12: FRT recombination process schematic (not to scale);

FIG. 13: 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. 14: schematic targeting strategy (not to scale);

FIG. 15: 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 sgRNA17-sgRNA23 is shown; (B) the relative activity detection result of the sgRNA24-sgRNA30 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:

NcoI, HindIII, BbsI, EcoRI and BamHI enzymes were purchased from NEB under the respective accession numbers R0193M, R3104M, R0539L, R0101M, R0136M;

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

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

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

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

erythrocyte lysates were purchased from Biyuntian, Inc., cat # C3702;

LEGEND MAX^TMmouse TNF-. alpha.ELISA Kit was purchased from Biolegend, cat. 430907;

r & D Mouse IFN-beta DuoSet ELISA was purchased from R & D, Inc., cat # DY 8234-05;

LEGEND MAX^TMmouse IL-6ELISA Kit was purchased from Biolegend, cat # 431307.

Example 1 humanized mouse of STING Gene

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

To achieve the object of the present invention, a human STING gene sequence can be introduced at the endogenous mouse STING locus such that the mouse expresses a human or humanized STING protein. For example, a method of directly inserting a nucleotide sequence containing a human STING gene or a coding sequence of a human STING protein into a mouse endogenous STING locus may be adopted, and an auxiliary sequence (e.g., a stop codon, etc.) or other methods (e.g., inversion or knock-out) may be added after the insertion of the sequence such that the mouse endogenous STING sequence after the insertion site cannot be normally expressed; in situ replacement strategies, i.e., direct replacement of the nucleotide sequence of the human STING gene at the mouse endogenous STING locus, can also be used. This example will illustrate how to humanize the STING gene in an in situ replacement strategy.

Specifically, the mouse STING gene sequence can be replaced by a human STING gene sequence at an endogenous mouse STING gene locus by using a gene editing technology, for example, a humanized STING gene sequence (a schematic diagram is shown in fig. 2) is obtained by replacing about 4.1kb (4144bp) sequence from exon 5 to exon 8 of the mouse STING gene with a corresponding human DNA sequence, so that the humanized STING gene sequence is realized.

The CRISPR/Cas system was used for gene editing and further designing the targeting strategy as shown in fig. 3, which shows the homology arm sequences containing the upstream and downstream of the mouse STING gene on the targeting vector, and the fragment containing the DNA sequence encoding the human STING gene. Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 3) is identical to the nucleotide sequence at positions 35740014 and 35738766 of NCBI accession No. NC-000084.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence at positions 35734621 and 35733137 of NCBI accession No. NC-000084.6; the DNA fragment sequence of human STING (SEQ ID NO: 5) was identical to the nucleotide sequence AT positions 139480874 and 139476378 of NCBI accession No. NC-000005.10, except that mutations were made AT positions 139483414 and 139483415, which were mutated from AT to GG, thereby mutating the encoded amino acid from histidine to arginine. The mRNA sequence of the humanized mouse STING after modification and the protein sequence coded by the mRNA sequence are respectively shown as SEQ ID NO: 6 and SEQ ID NO: 7, wherein, SEQ ID NO: 6 position 771-1358 human sequence, SEQ ID NO: 7, 145-340 bit is human sequence.

The construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection, direct synthesis and the like. And carrying out preliminary verification on the constructed recombinant vector by enzyme digestion, and then sending the recombinant vector to a sequencing company for sequencing verification. The targeting vector, which was sequence verified to be correct, was used for subsequent experiments.

sgRNA sequences that recognize the 5 'target site (sgRNA1-sgRNA8), the 3' target site (sgRNA9-sgRNA16) were designed and synthesized. The 5 'end target site and the 3' end target site are respectively positioned on the No. 5 exon and the No. 8 exon of the STING gene, and the target site sequence of each sgRNA on the STING gene is as follows:

sgRNA1 target site sequence (SEQ ID NO: 8): 5'-GTACCCAATGTAGTATGACCAGG-3'

sgRNA2 target site sequence (SEQ ID NO: 9): 5'-GCGGTTGATCTTACCAGGTAGGG-3'

sgRNA3 target site sequence (SEQ ID NO: 10): 5'-ATTTAACTGTATGGCAGACATGG-3'

sgRNA4 target site sequence (SEQ ID NO: 11): 5'-AAGAAGTTAAATGTTGCCCACGG-3'

sgRNA5 target site sequence (SEQ ID NO: 12): 5'-TACTTGCGGTTGATCTTACCAGG-3'

sgRNA6 target site sequence (SEQ ID NO: 13): 5'-TGGCCTGGTCATACTACATTGGG-3'

sgRNA7 target site sequence (SEQ ID NO: 14): 5'-GCTTTAGGAATTTAACTGTATGG-3'

sgRNA8 target site sequence (SEQ ID NO: 15): 5'-CATACTACATTGGGTACTTGCGG-3'

sgRNA9 target site sequence (SEQ ID NO: 16): 5'-GGTGCTCCGGCACATTCGTCAGG-3'

sgRNA10 target site sequence (SEQ ID NO: 17): 5'-GCTGTGTGTTAGGTGGCAAGAGG-3'

sgRNA11 target site sequence (SEQ ID NO: 18): 5'-ACCAAGGCACATTAGAGTCAAGG-3'

sgRNA12 target site sequence (SEQ ID NO: 19): 5'-CAGCAAGATGCAGGCACCCTTGG-3'

sgRNA13 target site sequence (SEQ ID NO: 20): 5'-CATCTCTAAAGCAGGAGGGTGGG-3'

sgRNA14 target site sequence (SEQ ID NO: 21): 5'-TGAGAAACTGTTTCCGTCTGTGG-3'

sgRNA15 target site sequence (SEQ ID NO: 22): 5'-GGTTCTGGAATAGAGATGGGGGG-3'

sgRNA16 target site sequence (SEQ ID NO: 23): 5'-TTTCTTCCTGACGAATGTGCCGG-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. 4and table 1. From which sgRNA1 and sgRNA10 were preferentially selected for subsequent experiments. The 5' end and the complementary strand are respectively added with enzyme cutting sites to obtain a forward oligonucleotide and a reverse oligonucleotide (the sequences are shown in a table 2), and after annealing, the annealing products are respectively connected to pT7-sgRNA plasmids (the plasmids are firstly linearized by BbsI), so as to obtain expression vectors pT7-STING-1 and pT 7-STING-10.

Table 1 results of detection of sgRNA activity

TABLE 2 sgRNA1 and sgRNA10 sequence Listing

pT7-sgRNA vector was synthesized by plasmid synthesis company as a fragment DNA (SEQ ID NO: 32) containing the T7 promoter and sgRNA scaffold, and ligated to a backbone vector (Takara, cat. No. 3299) by enzyme digestion (EcoRI and BamHI) in sequence, and sequencing by the professional sequencing company was verified, and the result indicated that the objective plasmid was obtained.

Taking a C57BL/6 mouse prokaryotic stage fertilized egg, and injecting premixed in-vitro transcription products of pT7-STING-1 and pT7-STING-10 (transcribed by using an Ambion in-vitro transcription kit according to the method of the specification) and Cas9mRNA and a targeting vector plasmid containing a DNA fragment sequence of human STING into the cytoplasm or nucleus of the mouse fertilized egg by using a microinjection instrument. Microinjection of embryos is carried out according to the method in the experimental manual for mouse embryo manipulation (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 female mouse for development, and F0 generation first-built mice can be produced.

The somatic cell genotype of F0 mouse can be identified by conventional detection methods (such as PCR detection, etc.), and the genotype identification results of some F0 mice are shown in FIG. 5. As seen by combining the results of 5 'primer detection and 3' primer detection, 2 mice numbered F0-01 and F0-02 in FIG. 5 were positive mice. The PCR analysis included the following primers:

5' end primer:

L-GT-F(SEQ ID NO：33)：5’-GCTTTGGCAGGAAACACCAAAAAG-3’

L-GT-R(SEQ ID NO：34)：5’-GGTTGTTGTAATGCTGATTGTAAGTT-3’

3' end primer:

R-GT-F(SEQ ID NO：35)：5’-CATTCTCTGGCACCCACACACTG-3’

R-GT-R(SEQ ID NO：36)：5’-TCAGGGGTAGAGTGTTTGCCTAT-3’

wherein, the position of the primer L-GT-F is positioned at the left side of the 5 'homology arm, the position of the primer R-GT-R is positioned at the right side of the 3' homology arm, and the L-GT-R and the R-GT-F are both positioned on the human sequence.

The obtained F0 generation mice were backcrossed with wild type mice to obtain F1 generation mice, and the genotype of the F1 generation mice was identified by the same PCR method, and the results are shown in FIG. 6, which shows that 7F 1 generation mice numbered F1-01 to F1-07 were positive mice.

The PCR primer sequence comprises:

L-GT-F(SEQ ID NO：33)：5’-GCTTTGGCAGGAAACACCAAAAAG-3’

L-GT-R(SEQ ID NO：34)：5’-GGTTGTTGTAATGCTGATTGTAAGTT-3’

R-GT-F(SEQ ID NO：35)：5’-CATTCTCTGGCACCCACACACTG-3’

R-GT-R(SEQ ID NO：36)：5’-TCAGGGGTAGAGTGTTTGCCTAT-3’

WT-F(SEQ ID NO：37)：5’-GGAGACCTGGGTGTGGAGCTATG-3’

WT-R(SEQ ID NO：38)：5’-CATGAAGACAGCTACAGTGTATC-3’

Mut-R(SEQ ID NO：39)：5’-TTAGAAACACAAGAGGCTGTGTGT-3’

southern blot assays were performed on 7 mice identified as positive for F1 PCR to confirm the presence of random insertions. Cutting rat tail to extract genome DNA, digesting genome with BglII enzyme or NcoI enzyme, transferring membrane and hybridizing. The 5 'probe and the 3' probe are located outside the 5 'homology arm and on the 3' homology arm, respectively, and the lengths of the specific probes and the target fragment are shown in Table 3. The Southern blot assay results are shown in FIG. 7, which shows that the mice numbered F1-01 to F1-07 were all positive heterozygous and had no random insertions.

TABLE 3 lengths of the particular probes and target fragments

Restriction enzyme	Probe needle	Wild type fragment size	Recombinant sequence fragment size
				BglII	5 'Probe (5' Probe)	13.2kb	9.4kb
NcoI	3 'Probe (3' Probe)	3.1kb	4.8kb

The Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

F：5’-CCTCCCCTCCCGATTTCCGGGGGA-3’(SEQ ID NO：40)

R：5’-TGGCCAGCTCTTTATTCTTTTTGG-3’(SEQ ID NO：41)

3 'Probe (3' Probe):

F：5’-ACCATGAATGCCCCCATGACCTCAG-3’(SEQ ID NO：42)

R：5’-CTCTCAAGTTTACCAGGCATTGACAC-3’(SEQ ID NO：43)

this indicates that the method can be used for constructing transgenic mice which can be stably passaged and are humanized by the STING gene without random insertion.

The humanized mice with STING gene can be tested for the normality of STING signaling pathway by STING modulators. Such as an ELISA assay. Specifically, 3 mice of wild type C57BL/6 and humanized heterozygote mice of STING gene prepared by the method are respectively selected, bone marrow cells of tibia and femur are taken after neck-removing euthanasia, the cells are resuspended and cultured by RPMI-1640 culture solution containing 10ng/mL M-CSF after red blood cells are lysed, when the cells are cultured to the 8 th day, trypsase is used for digestion and collection of F4/80+ CD11b + cells (namely macrophages), the differentiated macrophages are inoculated to a 96-well plate for culture for 1-2 hours, 100 μ L of three compounds ADU-S100 analogs (human-mouse cross-recognition STING agonist), DMXAA analogs (mouse-specific STING agonist), and (human-specific STING agonist Z) were added to the culture medium at final concentrations of 0, 2.5 μ M, 10 μ M, 40 μ M, and 80 μ M, respectively, and the culture was continued for 5 hours, and then the supernatants were collected for ELISA. As shown in FIG. 8, it can be seen that the expression levels of TNFA (FIG. 8B), IFNB (FIG. 8D) and IL6 (FIG. 8F) in the hybrid of the humanized mouse with the STING gene significantly increased with the increase in the concentration of the human-specific STING agonist Z, as compared with the wild-type C57BL/6 mouse (+/+). The signal path in the humanized mouse of the STING gene prepared by the method is normal.

In addition, since the cleavage of Cas9 causes double strand break of genomic DNA, insertion/deletion mutations are randomly generated by the repair mode of chromosome homologous recombination, and a knockout mouse with loss of STING protein function may be obtained. For this purpose, a pair of primers was designed for detecting knockout mice, wild type mice should have no PCR band, knockout mice should have 1 PCR band, and the product length should be about 341bp, as shown in FIG. 9, wherein numbers KO-01 to KO-06 are STING knockout mice. The primers are respectively positioned on the left side of a5 'end target site and the right side of a 3' end target site, and have the following sequences:

WT-F(SEQ ID NO：37)：5’-GGAGACCTGGGTGTGGAGCTATG-3’

WT-R1(SEQ ID NO：44)：5’-TCAGGAGGCTAAAGGATCACTAG-3’。

example 2 humanized mouse with STING Gene

In order to prepare humanized mice with STING gene, in addition to the method described in example 1, the present invention also designed and validated another in situ replacement strategy as follows: in the schematic diagram of the targeting strategy shown in fig. 10, the targeting vector is shown to contain the homologous arm sequences upstream and downstream of the mouse STING gene (a total of 3444bp of mouse DNA upstream of 5574bp of exon 5 of the endogenous STING gene and downstream of TGA), and 4635bp of the human STING sequence (extending from exon 5 to the stop codon TGA). Wherein the upstream homology arm sequence (5 'homology arm, SEQ ID NO: 45) 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: 46) is identical to the nucleotide sequence at positions 35733320 and 35729877 of NCBI accession No. NC-000084.6; the DNA fragment sequence of human STING (SEQ ID NO: 47) 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 STING after modification and the protein sequence coded by the mRNA sequence are respectively shown as SEQ ID NO: 48 and SEQ ID NO: 49, wherein SEQ ID NO: position 750-1475 of 48 is a human sequence, SEQ ID NO: 49 amino acid 138-378 is human sequence.

The targeting vector also comprises a resistance gene used for positive clone screening, namely neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites which are arranged in the same direction are arranged on two sides of the resistance gene to form a Neo cassette (Neo cassette). Wherein the linkage of the 5 'end of the Neo-box to the mouse locus is designed to be 5' -ACACAGGTGCCATACGTGGCCCATAGCTAAGCTTGATATCGAATTCCGAAGTTCCT-3' (SEQ ID NO: 50), 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' -AACTTCATCAGTCAGGTACATAATGGTGGATCCACTAGTTCTAGAGCGGCCGCATTAATGTGTCCA GGGAAGGGACCACTGAACAAATGCTCCCA-3' (SEQ ID NO: 51), 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 construction of the targeting vector can be carried out by adopting a conventional method, such as enzyme digestion connection and the like. And carrying out preliminary verification on the constructed targeting vector by enzyme digestion, and then sending the targeting vector to a sequencing company for sequencing verification. The 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 11, 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：52)，

R1：5’-CATAGCAACATCCTTCATGCCTTGGG-3’(SEQ ID NO：53)；

F2：5’-GCTCGACTAGAGCTTGCGGA-3’(SEQ ID NO：54)，

R2：5’-CCCCTTTCTCTGGTACCTCAGATGC-3’(SEQ ID NO：55)；

the Southern Blot detection comprises the following probe primers:

5 'Probe (5' Probe):

F：5’-GACAAAGATGGGGTAGAGTCACTAAGG-3’(SEQ ID NO：56)，

R：5’-GCTTAGAACCTGTCCAGTCTAAACTTAG-3’(SEQ ID NO：57)；

3 'Probe (3' Probe):

F：5’-CTCTGAGCGCTCAGAAAGGTATGG-3’(SEQ ID NO：58)，

R：5’-GGACCTTAGCTTCAGGAAATGGG-3’(SEQ ID NO：59)；

neo Probe (Neo Probe):

F：5’-GGATCGGCCATTGAACAAGATGG-3’(SEQ ID NO：60)，

R：5’-CAGAAGAACTCGTCAAGAAGGCG-3’(SEQ ID NO：61)。

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. Alternatively, positive mice and Flp tool mice may be mated to remove the positive clone selection marker gene (see FIG. 12 for a schematic diagram of the process), and then mated with each other to obtain humanized homozygous mice expressing humanized STING gene for humanized STING 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. 13, 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：62)，

WT-R2：5’-CTGGGCAGGGAACGCATTATGAC-3’(SEQ ID NO：63)；

Mut-F：5’-CTATCTCCCTGTTCCAGAACCTGC-3’(SEQ ID NO：64)

WT-R：5’-CTGGGCAGGGAACGCATTATGAC-3’(SEQ ID NO：63)；

Frt-F：5’-CAAGGCATAGGCAATGGGTGTCG-3’(SEQ ID NO：65)；

Frt-R：5’-CCTAAGGGTGTGTCCTCAGACG-3’(SEQ ID NO：66)；

Flp-F2：5’-GACAAGCGTTAGTAGGCACATATAC-3’(SEQ ID NO：67)；

Flp-R2：5’-GCTCCAATTTCCCACAACATTAGT-3’(SEQ ID NO：68)。

this shows that stable passage and no random insertion can be constructed by using the method. The expression of the humanized STING protein in positive mice can be confirmed by conventional detection methods, such as flow cytometry. The results show that the humanized mouse prepared by the present example can stably express the humanized STING protein and can specifically bind to the human STING antibody.

In addition, a CRISPR/Cas system can be introduced for gene editing, so that a STING gene humanized mouse is obtained, and the designed targeting strategy is shown in figure 14. 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 (sgRNA17-sgRNA23), the 3' target site (sgRNA24-sgRNA30) 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 STING gene, and the target site sequence of each sgRNA on the STING is as follows:

sgRNA17 target site sequence (SEQ ID NO: 69): 5'-GTACCCAATGTAGTATGACCAGG-3'

sgRNA18 target site sequence (SEQ ID NO: 70): 5'-TACTTGCGGTTGATCTTACCAGG-3'

sgRNA19 target site sequence (SEQ ID NO: 71): 5'-GTATGACCAGGCCAGCCCGTGGG-3'

sgRNA20 target site sequence (SEQ ID NO: 72): 5'-GCGGTTGATCTTACCAGGTAGGG-3'

sgRNA21 target site sequence (SEQ ID NO: 73): 5'-TGCGGTTGATCTTACCAGGTAGG-3'

sgRNA22 target site sequence (SEQ ID NO: 74): 5'-CAGACTGCAGAGACTTCCGCTGG-3'

sgRNA23 target site sequence (SEQ ID NO: 75): 5'-CATACTACATTGGGTACTTGCGG-3'

sgRNA24 target site sequence (SEQ ID NO: 76): 5'-GGTGCTCCGGCACATTCGTCAGG-3'

sgRNA25 target site sequence (SEQ ID NO: 77): 5'-TGGCTCTTGGGACAGTACGGAGG-3'

sgRNA26 target site sequence (SEQ ID NO: 78): 5'-AGATGAGGTCAGTGCGGAGT GGG-3'

sgRNA27 target site sequence (SEQ ID NO: 79): 5'-GCTGATCCATACCACTGATG AGG-3'

sgRNA28 target site sequence (SEQ ID NO: 80): 5'-CTCCGCACTGACCTCATCTG AGG-3'

sgRNA29 target site sequence (SEQ ID NO: 81): 5'-ACTGACCTCATCTGAGGCAT GGG-3'

sgRNA30 target site sequence (SEQ ID NO: 82): 5'-CACTGTCTCAGGAGGTGCTC CGG-3'

The activity of multiple sgrnas was detected by using UCA kit, and the sgrnas showed different activities as shown in fig. 15 and table 4. From these, 2 (sgRNA 21 and sgRNA26, respectively) were preferentially selected for subsequent experiments. A fertilized egg of a C57BL/6 mouse in a prokaryotic stage is taken, and a microinjector is used for injecting premixed in-vitro transcription products of the sgRNA21 and the sgRNA26 (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: 47) of human STING 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 4 detection results of sgRNA activity

Example 3 preparation of double humanized or multiple double humanized mice

The humanized mouse with the STING gene prepared by the method can also be used for preparing a double humanized or multi-humanized mouse model. For example, in example 1 or 2, the embryonic stem cells used for blastocyst microinjection may be selected from mice containing other gene modifications such as PD-1 and PD-L1, or alternatively, on the basis of mice humanized with STING gene, a two-gene or multi-gene modified mouse model of STING and other gene modifications may be obtained by using isolated mouse ES embryonic stem cells and gene recombination targeting techniques. The homozygous or heterozygous strain of STING mouse obtained by the method can also be mated with homozygous or heterozygous mouse modified by other genes, the offspring of the mouse is screened, the humanized STING mouse and the heterozygous mouse modified by other genes and double genes or multiple genes can be obtained with a certain probability according to the mendelian genetic law, the heterozygous strain is mated with each other to obtain the homozygous or multiple genes modified, and the mouse modified by the double genes or multiple genes can be used for in vivo efficacy verification of targeted human STING and other gene regulators and the like.

Taking a double humanized STING/PD-1 mouse as an example, because the STING and PD-1 genes of a mouse are located on chromosome 18 and chromosome 1, the STING humanized mouse is selected to mate with the PD-1 humanized mouse, and the double humanized STING/PD-1 mouse is finally obtained by 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 STING 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> Jiangsu Gene Biotechnology Co., Ltd, Baiosai map

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

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tctccccatt cagaagccac ttgctagtag ctactgaaag gctcttcatt gtctcttctg 60

ctccaggaac accggtctag gaagcagaag gtaggattag aaatggggca gtattctcct 120

ggtacttcgt ggtgggagaa accctgggct ggcagaacac tctaaggtct aggtcttgga 180

tcagtttctt ccttctttct cacaccagtg agctcagtgg ggctcacatg tacacgctct 240

gtttactatg aacctctcct agacaggtgc tgtaggatgc tatgtgccca gggcgtctcc 300

ttgaggtgta tccaagagta gcccatggga ctagctgggc tgggagaccc aaggagatgg 360

atgactagtc aggaccttag gccctgaagg acaggagaac cccagaagca tagctgtgga 420

tttcttgatg tctacagatg ccatactcca acctgcatcc agccatccca cggcccagag 480

gtcaccgctc caaatatgta gccctcatct ttctggtggc cagcctgatg atcctttggg 540

tggcaaagga tccaccaaat cacactctga agtacctagc acttcaccta gcctcgcacg 600

aacttggact actgttgaaa aacctctgct gtctggctga agagctgtgc catgtccagt 660

ccaggtaacc ctctgtggtc tccacgatga cttgataccc agcaagtatc acacccttga 720

ctcctaacct tggccttccc tgaacatcat cagctgagtt gggctgggat ctgaggctag 780

gctgtcccct gcaggtacca gggcagctac tggaaggctg tgcgcgcctg cctgggatgc 840

cccatccact gtatggctat gattctacta tcgtcttatt tctatttcct ccaaaacact 900

gctgacatat acctcagttg gatgtttggc cttctggtcc tctataagtc cctaagcatg 960

ctcctgggcc ttcaggtatg aagcagaaga ggtgggtggt gtgggaagag gggactatgg 1020

ctagtgctgg tgtgcctgag ctcagtgctg agactcagac taatttaaag gttggagacc 1080

tgggtgtgga gctatgaagg cttgggatga tgggtttaat agcagtgctg agagcaagct 1140

ggcagcaggt tgggaaagtt ttctgcaaga gaagggcttt ggacatcccc cttgaaagtc 1200

cctcaggccc ttctgctgtc ttcagagctt gactccagcg gaagtctct 1249

<210> 4

<211> 1485

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

accatgaatg cccccatgac ctcagtggca cctcctccct ccgtactgtc ccaagagcca 60

agactcctca tcagtggtat ggatcagcct ctcccactcc gcactgacct catctgaggc 120

atgggacagc cttgtctggg ctctagtgat cctttagcct cctgactgag ccttccttca 180

atggttgggg gcctcagaga cttcacatct ccagatgagt cccacattcc tgggcaagcc 240

atttatttca cctctctgag cctcaaccaa ccctactatg aaaggaggtc ataatgcgtt 300

ccctgcccag ccaaaggatt ttatatatgt agaagttggt gtcaatgcct ggtaaacttg 360

agagaaaggc caagtacttc ccgtggatgc tgcagacatt ccctgctctc tgttgacctg 420

tgtggatggt accagcagac ttccaaccct ccagcttctg gtcacgtgtg ttcaatggga 480

gcttaagtag atggcgagag ggagaaggaa catttgttct gttagctgta tacaatcaca 540

gtgggctggc ctgtcaactg ccttcttaat aaacatatct attctcagat ttctagaatg 600

gcctcttccc cttgtctcta gcactggtat ttgtgtgaca ctggagtact ttctgtctgg 660

tctctttata tcatgtccct tgcacatggt gttggcatca ggacgtccca aactcatgac 720

atcacatagg cgacagcatg acctgcaacc tgcagaccgg ttgccaagac aacaggcacc 780

atattcccac cttccacttg gctcacctcc cacctttacc tgtgttacgt catcttccat 840

atcttccata cgtcttccat cttccatacg tctctctccc ctgcttctct ttctgctgct 900

accttgtctc tcccttccaa taaaacctct tccatgcgga actgccttgg cctagtgtgt 960

tgttgagatg cgacccgcta ttctatcaca tcacatggcc cttcaatagg cactccttga 1020

attacccggt tctaggacac atctttttct gctaggacat gatagctatg aggcattgcc 1080

cctgacatct gagtctgagg gcatatacag cacttcctat actcttcatc ctgccacaag 1140

gcataggcaa tgggtgtcgt ttcaacccaa acagagccca ccattcacac tgagggcaca 1200

ccccacctta ctttcagtgt ccctgcctcc agtggcaggc tgaaggatca ggagcggggc 1260

tgagtggagc agcacacagg tgccatacgt ggcccatagc tgtgtccagg gaagggacca 1320

ctgaacaaat gctcccaaat gagagatctg tgaaaggatc atggaggaga ggaggtgcgt 1380

ctgaggacac acccttagga atggttagtc accaagccta cctgatgccg ttgtaacaat 1440

atcgtggtgg cactgtcacc cccagctagg aggtcaggca gtcct 1485

<210> 5

<211> 4497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcagtgtgtg aaaaagggaa tttcaacgtg gcccatgggc tggcatggtc atattacatc 60

ggatatctgc ggctgatcct gccaggtggg ccatcctctc tgcccccatt tctcgatcta 120

caaaacagac actaagacac acagcctctt gtgtttctaa acacacagaa ctcactatat 180

ttttcaaagc attttcatgt ctaccaaatc ctttaatccc aaggcatgaa ggatgttgct 240

atgatgccta gtttgcagaa aggaatatgg ggcttagagc cattaagtga cttgcccaag 300

gtcacttgtg aattcttttt ttttcttttt gagaaggaat ctccctctgt cgcccaggct 360

agagtgcagt ggtaccatct cggctactgc aacctcctcc ttccaagttc aagtgattat 420

cctggctcag gttcccgagt agctgggatt ataggcaccc accacaacgc ccggctaatt 480

tttgtatttt tagtagagac agggtttcac cgtattggcc aggccggtac aaactcctga 540

cctcaagtga tccgcctgcc ttagcctccc aaagtgctgg gattacaggc gtgagcccag 600

ctgtaaattc tttcaataaa tatttactgc atgctgtgtg ctctcatgtg ccttttctaa 660

ttcacaccct atgaggaagg aataatcttt attcctgttt tattttattt atatattttt 720

taaaaagaag agggttttgc tatgttaccc agcctggtct caactcctgg gctcaagcaa 780

tcctcctgcc ttggcttccc aaagtgccga gataacaagc aggagccacc aagcccatac 840

ctattttaga tttttttttt tttttttttt tgagacaaag tctcactctg ttgcgcagac 900

tggagtgcag tagcacaatc tcagctcact gcgacctctg cctcctgggt tcagcctccc 960

aagtagctgg aattacaggt ccctgccacc ggcatggctt tttttttttt tttttttttt 1020

ttttttgtat ttttagtaga gacagggttt caccatgttg cccaggctgg tcttgaattc 1080

ttgacctcaa gtaatccacc caccttggcc tcccaaagtg ctgggattac agacatgagc 1140

cactgcacca gccttagatt tttttttttt tttaagatgg agtttcactt tttcacccgg 1200

gctgaagtag agtggcacaa tctgggctca ctgcaacttc tgcttcccag gttccagtga 1260

ttctcctgcc ttagcctccc aagtagctgg gattttaggt gcctgccacc acgcccggct 1320

aatttttgta ttttcagtac agacggggtt tcaccatgtt gggtaggcta gtctcaaact 1380

cctgacctca ggtgatccac ccacctcggc ctcccaaagt gctagaacta taggcatgag 1440

ccactgtgcc tggccttttt ttttaagtgc agttctgaga ggtaaagtga tttatccgat 1500

atcacatagc ttagagagag gaagagaaag gatttgaacc caggtttgtc cagagcctgg 1560

accctagacc actgcaccgt ggagcctgtg tttgttgttg ttgttgttgt tgttgttgtt 1620

gttgttgttg ttgttgttgt tttgagatga agtctctttc tgttacccag cctggagtac 1680

agtggtgaca agatctcagc tcactgcaac ctctgcctcc caggttcaag tgattctcct 1740

gcctgagcct cttgaatagc taggattaca ggcacgtgcc accatgcctg gctaattttt 1800

gtatttttag tagagacaat gtttcaccat gttgcccagg ctagtctcaa actcctgacc 1860

tcaaatgatc cacctgcctc agcctcccaa agtgctggat tacaggcgtg agccactgtg 1920

cctgccctaa gcctgtgtgt tttattcttc tgacttgcag gctaaagcgg cagctcttcc 1980

atatctcatt gctatctcct agggcttccg ctaggagact gatctggggc tagaggcctc 2040

cctctgtgca cacgagaatg ctggaaatgt cacctctcag ggctctgcct gcctctcagc 2100

cctgaaagcc atggtggaaa ggggtggcgc tgacatagac atctgaggaa agaagtgagg 2160

gagggtaaag ggtggtgcag taagaggagg ggtggggagg gcttttggag gcgctgcccc 2220

tcctggcctc ctgtacaatg agagtgcact ggactctcca ttctctggca cccacacact 2280

gcgggggcca atgacctggg tctcactcct gaatcaggtg ggagataggg ttagcaggaa 2340

taacttcttg ggcttccctg cctcagagct ccaggcccgg attcgaactt acaatcagca 2400

ttacaacaac ctgctacggg gtgcagtgag ccagcggctg tatattctcc tcccattgga 2460

ctgtggggtg cctgataacc tgagtatggc tgaccccaac attcgcttcc tggataaact 2520

gccccagcag accggtgacc gggctggcat caaggatcgg gtttacagca acagcatcta 2580

tgagcttctg gagaacgggc agcgggtaag tgtgcagggg agtgggggtc tctgaggagg 2640

ggtcaggacc ccagaaccct gggccctagc caagcactga tgaaaattca ctgcccttct 2700

ctgagctgta gtgtccctag ctggtcccag gtctgggcag gattcagctc tgaatgataa 2760

tgatgagtaa catttatcga gcacttacta caagctggat gctattctgg gtgtgtcatc 2820

tgatcacctc attgaattct caccaccacc ctatggggta gggactgtta gcattcccac 2880

tttacagagg aggacactga ggctcagaga caccgtagga aagtggacga tttaaacttg 2940

actgtaccat gctcttgacc ataaagctgc cccagacgaa ggctgtgaga acatctgaag 3000

gattcatgtg ggtgcagggg aacccagacc agagttgaac ccagagccta gccccagacc 3060

tgattcccag ctggagcagt tgacagcctc gggtcttgcc ctaggatctt ggaagaagga 3120

tctgtgaagg gtaaatttag cccagccgtg tccctgattt cagagttggg tatcagaggc 3180

aaaggcaggc cagacagcat agaccccatt agggtggcca cctcccagga ctctatcgtt 3240

acaggctgag ggagtggggc ctcagcccgt ggcactgggg gccagagcct ggactggacc 3300

ctccattctc catccgcctg gcccctggct tagtctggtc ttcctcttac ctcctctagg 3360

cgggcacctg tgtcctggag tacgccaccc ccttgcagac tttgtttgcc atgtcacaat 3420

acagtcaagc tggctttagc cgggaggata ggcttgagca ggccaaactc ttctgccgga 3480

cacttgagga catcctggca gatgcccctg agtctcagaa caactgccgc ctcattgcct 3540

accagggtga ggggttggta gggtgagggg ttgataggct ggagggcagg aggagtcagg 3600

ggtcccaggg tctcccaaag agtcagaagg ggctatggag caccatgccc tgggcctgtc 3660

cttccttccc tgggagaagc cccctcttcc cagcacacca gattgctttt taggtctttg 3720

actgtaatcc ctcaggggag cctgaagagg tgggttctag gggcctctca cttcatctag 3780

gattccagag gaaacagaag agtcccaggc cagtatgtac tgggcacagg agactagggc 3840

tgcagcctcg gctctactac tccctcactg tgtgacctcg ggcaggtttc tgctcctctc 3900

tgagcattag ctttaaaagt aagagggtgg aatttcattc tctctgaaat cacttttttt 3960

tttttttttt gagatggagt ctcactctgt tgcccaggct ggagtgaagt ggctcgattt 4020

cgggtcactg caacctccac ctcccgggtc caagcgattc tcctgcctca gcctcccaag 4080

tagctgggac tacaggcgcc cgccaccatg cccagctaat ttttgtattt ttagtagaga 4140

cggggtttca tcatattggc caggctggtc ttgaattcct gacctcatga tctacctgcc 4200

tcggcctccc aaagtgctag gattacaggc gtgagccact gtgccaggcc tgaaccactt 4260

ctacctctag gattctatga aactctagtc ccagtggctg gggcttgggt aggaaggggg 4320

gtagggaggg ttaggggatc ttcccagcaa gttctctagg tatgtttgag tgggaatggg 4380

taagatcctc attactctct cccctatctc cctgttccag aacctgcaga tgacagcagc 4440

ttctcgctgt cccaggaggt tctccggcac ctgcggcagg aggaaaagga agaggtt 4497

<210> 6

<211> 2302

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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 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 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> 7

<211> 378

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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 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 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> 8

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtacccaatg tagtatgacc agg 23

<210> 9

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcggttgatc ttaccaggta ggg 23

<210> 10

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atttaactgt atggcagaca tgg 23

<210> 11

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aagaagttaa atgttgccca cgg 23

<210> 12

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tacttgcggt tgatcttacc agg 23

<210> 13

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tggcctggtc atactacatt ggg 23

<210> 14

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctttaggaa tttaactgta tgg 23

<210> 15

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

catactacat tgggtacttg cgg 23

<210> 16

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggtgctccgg cacattcgtc agg 23

<210> 17

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gctgtgtgtt aggtggcaag agg 23

<210> 18

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

accaaggcac attagagtca agg 23

<210> 19

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cagcaagatg caggcaccct tgg 23

<210> 20

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

catctctaaa gcaggagggt ggg 23

<210> 21

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgagaaactg tttccgtctg tgg 23

<210> 22

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggttctggaa tagagatggg ggg 23

<210> 23

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tttcttcctg acgaatgtgc cgg 23

<210> 24

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gtacccaatg tagtatgacc 20

<210> 25

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

taggtaccca atgtagtatg acc 23

<210> 26

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ggtcatacta cattgggtac 20

<210> 27

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aaacggtcat actacattgg gtac 24

<210> 28

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gctgtgtgtt aggtggcaag 20

<210> 29

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

taggctgtgt gttaggtggc aag 23

<210> 30

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cttgccacct aacacacagc 20

<210> 31

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aaaccttgcc acctaacaca cagc 24

<210> 32

<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

tttaaaggat cc 132

<210> 33

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gctttggcag gaaacaccaa aaag 24

<210> 34

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggttgttgta atgctgattg taagtt 26

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cattctctgg cacccacaca ctg 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

tcaggggtag agtgtttgcc tat 23

<210> 37

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ggagacctgg gtgtggagct atg 23

<210> 38

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

catgaagaca gctacagtgt atc 23

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ttagaaacac aagaggctgt gtgt 24

<210> 40

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

cctcccctcc cgatttccgg ggga 24

<210> 41

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

tggccagctc tttattcttt ttgg 24

<210> 42

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

accatgaatg cccccatgac ctcag 25

<210> 43

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ctctcaagtt taccaggcat tgacac 26

<210> 44

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tcaggaggct aaaggatcac tag 23

<210> 45

<211> 5574

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

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> 46

<211> 3444

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

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> 47

<211> 4635

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

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> 48

<211> 2302

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

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> 49

<211> 378

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

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> 50

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

acacaggtgc catacgtggc ccatagctaa gcttgatatc gaattccgaa gttcct 56

<210> 51

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aacttcatca gtcaggtaca taatggtgga tccactagtt ctagagcggc cgcattaatg 60

tgtccaggga agggaccact gaacaaatgc tccca 95

<210> 52

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

ggtcacacag ctagagagac agaagtc 27

<210> 53

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

catagcaaca tccttcatgc cttggg 26

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

gctcgactag agcttgcgga 20

<210> 55

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

cccctttctc tggtacctca gatgc 25

<210> 56

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

gacaaagatg gggtagagtc actaagg 27

<210> 57

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gcttagaacc tgtccagtct aaacttag 28

<210> 58

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ctctgagcgc tcagaaaggt atgg 24

<210> 59

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

ggaccttagc ttcaggaaat ggg 23

<210> 60

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

ggatcggcca ttgaacaaga tgg 23

<210> 61

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

cagaagaact cgtcaagaag gcg 23

<210> 62

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

ctcagaggct gtgtgttagg tgg 23

<210> 63

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

ctgggcaggg aacgcattat gac 23

<210> 64

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

ctatctccct gttccagaac ctgc 24

<210> 65

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

caaggcatag gcaatgggtg tcg 23

<210> 66

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

cctaagggtg tgtcctcaga cg 22

<210> 67

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

gacaagcgtt agtaggcaca tatac 25

<210> 68

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

gctccaattt cccacaacat tagt 24

<210> 69

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

gtacccaatg tagtatgacc agg 23

<210> 70

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

tacttgcggt tgatcttacc agg 23

<210> 71

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

gtatgaccag gccagcccgt ggg 23

<210> 72

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

gcggttgatc ttaccaggta ggg 23

<210> 73

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

tgcggttgat cttaccaggt agg 23

<210> 74

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

cagactgcag agacttccgc tgg 23

<210> 75

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

catactacat tgggtacttg cgg 23

<210> 76

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ggtgctccgg cacattcgtc agg 23

<210> 77

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

tggctcttgg gacagtacgg agg 23

<210> 78

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

agatgaggtc agtgcggagt ggg 23

<210> 79

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

gctgatccat accactgatg agg 23

<210> 80

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

ctccgcactg acctcatctg agg 23

<210> 81

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

actgacctca tctgaggcat ggg 23

<210> 82

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

cactgtctca ggaggtgctc cgg 23