HSD17B13 gene modified non-human animal

Abstract

The invention belongs to the technical field of animal gene engineering, genetic modification and biological medicine, and particularly relates to an HSD17B13 gene modified non-human animal, a construction method thereof and application of the HSD17B13 gene modified non-human animal in the technical field of biological medicine. The construction method comprises the step of replacing a nucleotide sequence of endogenous HSD17B13 of a non-human animal with a nucleotide sequence containing human HSD17B13 at an endogenous HSD17B13 locus of the non-human animal, so that the obtained human or chimeric (e.g., humanized) HSD17B13 protein expressed by the non-human animal can be used as an animal model for researching the functions of the HSD17B13 gene and screening disease treatment drugs related to the HSD17B13, and the human or chimeric (e.g., humanized) HSD17B13 protein expressed by the non-human animal can be used as an animal model for researching the functions of the HSD17B13 gene and screening the disease treatment drugs related to the HSD17B13. The important application value is realized.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) HSD17B13 protein and methods of using the same. Background Art

[0002] Traditional drug development typically uses in vitro screening methods. However, these screening methods cannot provide the body's environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), resulting in a high rate of drug development failure. In addition, given the differences between humans and animals, the results of in vivo pharmacology trials using conventional experimental animals may not reflect the actual disease state and the interaction of target sites, resulting in significant differences between the results of many clinical trials and those of animal experiments.

[0003] Therefore, developing humanized animal models suitable for screening and evaluation of human drugs will significantly improve the efficiency of new drug development and reduce drug research and development costs. Summary of the Invention

[0004] The present application provides an animal model with a human or chimeric HSD17B13 protein. The animal model can express a human or chimeric HSD17B13 (e.g., humanized HSD17B13) protein. It can be used to study the function of the HSD17B13 gene, and can also be used to screen and evaluate HSD17B13 signaling pathway regulators (e.g., therapeutic agents targeting HSD17B13, including antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamic studies, and treatment studies of human HSD17B13 target-related diseases (e.g., immune diseases, tumors, inflammation (e.g., non-alcoholic steatohepatitis)); the animal model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of the HSD17B13 protein and provides a platform for screening drugs for the treatment of related diseases.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) protein. In some embodiments, the nucleotide sequence encoding the human or chimeric HSD17B13 protein can be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the chimeric HSD17B13 protein is a humanized HSD17B13 protein. In some embodiments, the amino acid sequence of the chimeric HSD17B13 protein comprises an amino acid sequence that is identical to a human HSD17B13 protein in at least 50 to 300, e.g., at least 50, 100, 200, 250, 270, 271, 274, 275, 280, 281, 290, 295, or 300, continuous or non-continuous amino acid sequences. In some embodiments, the nucleotide sequence encoding the human or chimeric HSD17B13 protein is operably linked to a human HSD17B13 regulatory element (e.g., a human HSD17B13 promoter, 5'UTR, and / or 3'UTR). In some embodiments, expression of the nucleotide sequence encoding the human or chimeric HSD17B13 protein is regulated by a human HSD17B13 regulatory element. In some embodiments, the amino acid sequence of the human or chimeric HSD17B13 protein comprises SEQ ID NO: 2 or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the human or chimeric HSD17B13 protein comprises SEQ ID NO: 23 or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 23. In some embodiments, the non-human animal is a mammal, such as a monkey or rodent. In some embodiments, the rodent comprises a mouse or rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal does not express endogenous HSD17B13 protein or expresses it at a reduced level compared to HSD17B13 in wild-type animals. In some embodiments, one or more cells of the non-human animal express a human or chimeric HSD17B13 protein.

[0006] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, comprising replacing a nucleotide sequence encoding endogenous HSD17B13 with a nucleotide sequence encoding human HSD17B13 at the endogenous HSD17B13 locus. In some embodiments, the nucleotide sequence encoding human HSD17B13 is operably linked to a human HSD17B13 regulatory element. In some embodiments, expression of the nucleotide sequence encoding human HSD17B13 is regulated by a human HSD17B13 regulatory element. In some embodiments, one or more cells of the non-human animal express a human or humanized HSD17B13 protein. In some embodiments, the endogenous HSD17B13 protein of the non-human animal is not expressed or is expressed at a reduced level compared to HSD17B13 in wild-type animals. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises all or part of exon 1 to exon 7 of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 50 bp, such as at least 50 bp, 100 bp, 500 bp, 1000 bp, 3000 bp, 5000 bp, 8000 bp, or 10000 bp, of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 50 bp, such as at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, or 500 bp, of consecutive nucleotides downstream of the 3'UTR of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 10 kb of continuous nucleotides upstream of the 5'UTR, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 500 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises any one of the following groups: 1) a nucleotide sequence identical or at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% to the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:26, SEQ ID NO:26 with a mutation at position 1269 from T to TT and / or at position 1522 from C to G, or SEQ ID NO:5 with a mutation at positions 22641-22642 from TT to T and / or at position 22895 from G to C;

[0007] 2) a nucleotide sequence comprising substitutions, deletions, and / or insertions of one or more nucleotides compared to the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 26, the nucleotide sequence of SEQ ID NO: 26 wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or the nucleotide sequence of SEQ ID NO: 5 wherein positions 22641-22642 are mutated from TT to T and / or position 22895 is mutated from G to C. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all of exon 1 to exon 6 of the endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50 bp, such as at least 50 bp, 100 bp, 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 5300 bp, 5301 bp, 5500 bp, or 6000 bp, of contiguous nucleotides upstream of the 5'UTR of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all or part of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50 bp, such as at least 50 bp, 100 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, 2311 bp, 2500 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, or 9488 bp of consecutive nucleotides of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50 bp of consecutive nucleotides upstream of the 5'UTR of an endogenous HSD17B13 gene of a non-human animal, all consecutive nucleotides from exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal, and at least 50 bp of consecutive nucleotides of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous HSD17B13 comprises at least 5301 bp of consecutive nucleotides upstream of the 5'UTR of the non-human animal's endogenous HSD17B13 gene, all consecutive nucleotides from exon 1 to exon 6 of the non-human animal's endogenous HSD17B13 gene, and at least 2311 bp of consecutive nucleotides in intron 6 of the non-human animal's endogenous HSD17B13 gene. In some embodiments, the modified HSD17B13 gene in the non-human animal's genome is homozygous or heterozygous for the endogenous replaced locus.

[0008] In one aspect, a method for constructing a genetically modified non-human animal is provided, wherein the nucleotide sequence of the endogenous HSD17B13 gene of the non-human animal is replaced with a nucleotide sequence comprising human HSD17B13 at the endogenous HSD17B13 locus of the non-human animal. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a nucleotide sequence encoding a human or chimeric HSD17B13 protein. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the nucleotide sequence of human HSD17B13 can be a genomic DNA sequence, a CDS sequence, or a cDNA sequence. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a nucleotide sequence identical to at least 50 to at least 29699 bp, for example, at least 50, 100, 1000, 5000, 10000, 15000, 20000, 29589, 29698, or 29699 bp of continuous or non-contiguous nucleotides of the human HSD17B13 gene. In some embodiments, the nucleotide sequence of human HSD17B13 comprises all of exon 1 to exon 7 of the human HSD17B13 gene. In some embodiments, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene, preferably further comprising a 5'UTR and / or a 3'UTR, and further preferably further comprising at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a nucleotide sequence upstream of the 5'UTR of the human HSD17B13 gene comprising at least 50 bp, for example, at least 50, 100, 500, 1000, 3000, 5000, 8000, or 10000 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the human HSD17B13 gene comprising at least 50 bp, for example, at least 50, 100, 200, 300, 400, or 500 bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a continuous nucleotide sequence from at least 50 bp upstream of the 5'UTR to at least 50 bp downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human HSD17B13 comprises a mutation.In some embodiments, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 26 in which position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 26 in which position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G. In some embodiments, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 5 wherein positions 22641-22642 are TT to T and / or position 22895 is G to C, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 5 wherein positions 22641-22642 are TT to T and / or position 22895 is G to C. In some embodiments, the construction method comprises a single targeting step, wherein the nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 5 wherein positions 22641-22642 are TT to T and / or position 22895 is G to C is replaced with the nucleotide sequence of endogenous HSD17B13 of a non-human animal. In some embodiments, the construction method comprises two or more targeting steps, such as a secondary targeting step, wherein the nucleotide sequence of SEQ ID NO: 5 is substituted for the nucleotide sequence of the endogenous HSD17B13 of the non-human animal, and then the nucleotide sequence of the human HSD17B13 mutation is substituted for the corresponding region of the nucleotide sequence of the human HSD17B13 at the endogenous HSD17B13 locus of the non-human animal. In some embodiments, the secondary targeting step targets exon 6 of the nucleotide sequence of the human HSD17B13. In some embodiments, the nucleotide sequence of the human HSD17B13 mutation comprises the nucleotide sequence of SEQ ID NO: 26; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the nucleotide sequence of the endogenous HSD17B13 of the non-human animal comprises a nucleotide sequence encoding an endogenous HSD17B13 protein of the non-human animal.In some embodiments, the nucleotide sequence of the endogenous HSD17B13 of the non-human animal comprises the nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the endogenous HSD17B13 of the non-human animal comprises all or part of exon 1 to exon 6 of the endogenous HSD17B13 gene of the non-human animal. In some embodiments, the nucleotide sequence of the endogenous HSD17B13 of the non-human animal further comprises all or part of intron 6. In some embodiments, the portion of intron 6 of the non-human animal endogenous HSD17B13 gene included in the nucleotide sequence of the non-human animal endogenous HSD17B13 comprises at least 50-9488 bp, such as at least 50 bp, 100 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, 2311 bp, 2500 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, or 9488 bp of consecutive nucleotides. In some embodiments, the nucleotide sequence of the non-human animal endogenous HSD17B13 comprises at least 50 bp, such as at least 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 5301, 5500, or 6000 bp of consecutive nucleotide sequence upstream of the 5'UTR of the non-human animal endogenous HSD17B13 gene. In some embodiments, the nucleotide sequence of endogenous HSD17B13 of a non-human animal comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR of the endogenous HSD17B13 gene of the non-human animal to all or part of intron 6 (e.g., at least 50 bp continuous nucleotide sequence of intron 6). In some embodiments, the nucleotide sequence of endogenous HSD17B13 of a non-human animal comprises a continuous nucleotide sequence of at least 5301 bp upstream of the 5'UTR of the endogenous HSD17B13 gene of the non-human animal to at least 2311 bp continuous nucleotide sequence of intron 6. In some embodiments, the nucleotide sequence encoding the human or chimeric HSD17B13 protein or the nucleotide sequence of human HSD17B13 is operably linked to an exogenous HSD17B13 regulatory element. In some embodiments, the expression of the nucleotide sequence encoding the human or chimeric HSD17B13 protein or the nucleotide sequence of human HSD17B13 is regulated by an exogenous HSD17B13 regulatory element. In some embodiments, the exogenous HSD17B13 regulatory element is preferably a human HSD17B13 regulatory element (eg, promoter, 5'UTR and / or 3'UTR).In some embodiments, the endogenous HSD17B13 protein in the non-human animal is not expressed or is expressed at a reduced level compared to HSD17B13 in wild-type animals. In some embodiments, the modified HSD17B13 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the mRNA transcribed from the modified HSD17B13 gene in the genome of the non-human animal comprises SEQ ID NO: 6 or SEQ ID NO: 24; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 6 or SEQ ID NO: 24. In some embodiments, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably include at least one of PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0009] In one aspect, the present invention provides a non-human animal or a method for constructing the same, comprising at least one cell comprising a nucleotide sequence encoding a human or humanized HSD17B13 protein. In some embodiments, the humanized HSD17B13 protein comprises an amino acid sequence that is identical to at least 50, 100, 200, 250, 270, 271, 274, 275, 280, 281, 290, 295, or 300 consecutive amino acids of a continuous amino acid sequence of a human HSD17B13 protein. In some embodiments, the non-human animal expresses the human or humanized HSD17B13 protein. In some embodiments, the nucleotide sequence encoding the human or humanized HSD17B13 protein is operably linked to a human HSD17B13 regulatory element (e.g., a promoter, 5'UTR, and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the human or humanized HSD17B13 protein can be integrated into the non-human animal's endogenous HSD17B13 locus. In some embodiments, the human or humanized HSD17B13 protein has at least one activity, eg, an endogenous HSD17B13 activity of a non-human animal and / or a human HSD17B13 activity.

[0010] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein, in at least one cell of the non-human animal, a nucleotide sequence encoding endogenous HSD17B13 is replaced at the non-human animal's endogenous HSD17B13 locus with a nucleotide sequence encoding human HSD17B13. In some embodiments, the endogenous HSD17B13 protein in the non-human animal is not expressed or is expressed at a reduced level compared to HSD17B13 in wild-type animals. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises all or part of exons 1 to 7 of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 50 bp (e.g., at least 50 bp, 100 bp, 500 bp, 1000 bp, 3000 bp, 5000 bp, 8000 bp, 10000 bp) of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 50 bp (e.g., at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp) of consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 10 kb of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 500 bp of consecutive nucleotides downstream of the 3'UTR of the human HSD17B13 gene. In some embodiments, the amino acid sequence of human HSD17B13 is SEQ ID NO: 2 or SEQ ID NO: 23, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises any one of the following groups: 1) a nucleotide sequence that is identical or at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 26, or SEQ ID NO: 26 with a mutation at position 1269 from T to TT and / or at position 1522 from C to G, or SEQ ID NO: 5 with a mutation at positions 22641-22642 from TT to T and / or from G to C;

[0011] 2) A nucleotide sequence comprising a substitution, deletion, and / or insertion of one or more nucleotides compared to the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 26, the nucleotide sequence shown in SEQ ID NO: 26 wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or the nucleotide sequence shown in SEQ ID NO: 5 wherein positions 22641-22642 are mutated from TT to T and / or position 22895 is mutated from G to C. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all or part of exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all or part of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50 bp of intron 6 of the endogenous HSD17B13 gene of a non-human animal, for example, at least 50 bp, 100 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, 2311 bp, 2500 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp or 9488 bp of consecutive nucleotides. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50, 500, 1000, 2000, 3000, 5000, 5301, 5500, or 6000 bp of consecutive nucleotides upstream of the 5'UTR of an endogenous HSD17B13 gene of a non-human animal, all of exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal, and at least 50 bp of consecutive nucleotides in intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 5301 bp of consecutive nucleotides upstream of the 5'UTR of an endogenous HSD17B13 gene of a non-human animal, all of contiguous nucleotides from exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal, and at least 2311 bp of consecutive nucleotides in intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding human HSD17B13 is operably linked to a human HSD17B13 regulatory element. In some embodiments, expression of the nucleotide sequence encoding human HSD17B13 is regulated by a human HSD17B13 regulatory element. In some embodiments, the human HSD17B13 regulatory element comprises a promoter, a 5'UTR, and / or a 3'UTR. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or rat).In some embodiments, the non-human animal further comprises a nucleotide sequence of a human or chimeric protein encoded by other genes, wherein the human or chimeric protein is selected from at least one of PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0012] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell that expresses human or chimeric HSD17B13. The method comprises replacing a nucleotide sequence encoding endogenous HSD17B13 at the non-human animal's endogenous HSD17B13 locus with a nucleotide sequence encoding human HSD17B13, thereby producing a genetically modified non-human animal cell that expresses human or chimeric HSD17B13 protein. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises all or part of exon 1 to exon 7 of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 50 bp (e.g., at least 50 bp, 100 bp, 500 bp, 1000 bp, 3000 bp, 5000 bp, 10000 bp) of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 50 bp of consecutive nucleotides downstream of the 3'UTR of the human HSD17B13 gene. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises at least 10 kb of consecutive nucleotides upstream of the 5'UTR of the human HSD17B13 gene, all of exon 1 to exon 7 of the human HSD17B13 gene, and at least 500 bp of consecutive nucleotides downstream of the 3'UTR of the human HSD17B13 gene. In some embodiments, the amino acid sequence of human HSD17B13 is SEQ ID NO: 2 or SEQ ID NO: 23; or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the nucleotide sequence encoding human HSD17B13 comprises any one of the following groups: 1) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 26, or SEQ ID NO: 26 with a mutation at position 1269 from T to TT and / or a mutation at position 1522 from C to G, or the nucleotide sequence of SEQ ID NO: 5 with a mutation at positions 22641-22642 from TT to T and / or a mutation at position 22895 from G to C; 2) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 26, or SEQ ID NO: 26 with a mutation at position 1269 from T to TT and / or a mutation at position 1522 from C to G, or the nucleotide sequence of SEQ ID NO: NO:5 The nucleotide sequence shown in which positions 22641-22642 are mutated from TT to T and / or position 22895 is mutated from G to C, comprising substitution, deletion and / or insertion of one or more nucleotides.In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all or part of exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises all or part of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50 bp, such as at least 50 bp, 100 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, 2311 bp, 2500 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, or 9488 bp of consecutive nucleotides of intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 50, 500, 1000, 2000, 3000, 5000, 5301, 5500, or 6000 bp of consecutive nucleotides upstream of the 5'UTR of an endogenous HSD17B13 gene of a non-human animal, all of exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal, and at least 50 bp of consecutive nucleotides in intron 6. In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 comprises at least 5301 bp of consecutive nucleotides upstream of the 5'UTR, all of exon 1 to exon 6 of an endogenous HSD17B13 gene of a non-human animal, and at least 2311 bp of consecutive nucleotides in intron 6 of an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding human HSD17B13 is operably linked to a regulatory element of human HSD17B13. In some embodiments, the expression of the nucleotide sequence encoding human HSD17B13 is regulated by a regulatory element of human HSD17B13. In some embodiments, the regulatory element of human HSD17B13 comprises a promoter, a 5'UTR, and / or a 3'UTR. In some embodiments, the non-human animal is a mouse.

[0013] In one aspect, the present invention provides a method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease associated with HSD17B13, the method comprising: 1) administering a therapeutic agent to a non-human animal or a non-human animal obtained by the construction method; and 2) determining the effect of the therapeutic agent on the non-human animal or the disease associated with HSD17B13. In some embodiments, the therapeutic agent comprises an antibody, nucleic acid drug, and / or polypeptide drug targeting HSD17B13. In some embodiments, the disease associated with HSD17B13 comprises non-alcoholic steatohepatitis (NASH).

[0014] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer (tumor), the method comprising:

[0015] 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a tumor; 2) determining the inhibitory effect of the therapeutic agent on the tumor. In some embodiments, the therapeutic agent comprises an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13, and / or a polypeptide drug. In some embodiments, the tumor comprises one or more tumor cells injected into the non-human animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the tumor involves measuring tumor volume in the non-human animal. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the cancer is hepatocellular carcinoma, clear cell renal carcinoma, breast cancer, head and neck squamous cell carcinoma, lymphocytic tumor, colorectal adenocarcinoma, head and neck cancer, liver cancer, or lung cancer. In some embodiments, the non-human animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1. In some embodiments, the tumor comprises one or more tumor cells injected into the non-human animal.

[0016] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating inflammation, the method comprising: 1) administering a therapeutic agent to a non-human animal or a non-human animal obtained by the construction method, wherein the non-human animal has inflammation; and 2) determining the effectiveness of the therapeutic agent in treating the inflammation. In some embodiments, the inflammation comprises one or more of non-alcoholic steatohepatitis or inflammatory bowel disease (IBD). In some embodiments, the therapeutic agent comprises an antibody, nucleic acid drug, and / or polypeptide drug targeting HSD17B13.

[0017] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a disease associated with HSD17B13, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a disease associated with HSD17B13; 2) determining the effectiveness of the therapeutic agent in treating a disease associated with HSD17B13. In some embodiments, the therapeutic agent comprises an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13, and / or a polypeptide drug. In some embodiments, the disease associated with HSD17B13 comprises one or more of the chronic fibrotic inflammatory liver diseases described above, including non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), liver fibrosis (including HCV-related cirrhosis), fatty liver disease (including non-alcoholic fatty liver disease (NAFLD) and / or alcoholic fatty liver disease (ALD)), cirrhosis, drug-induced liver damage, or hepatocellular necrosis.

[0018] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, comprising: 1) administering a therapeutic agent to a non-human animal described herein or a non-human animal obtained by the construction method described herein; and 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the therapeutic agent comprises an antibody, nucleic acid drug, and / or polypeptide drug targeting HSD17B13. In some embodiments, determining the effect of the therapeutic agent on the non-human animal involves measuring the non-human animal's weight or performing a blood test. In some embodiments, the blood test includes, but is not limited to, red blood cell count, hematocrit, and / or hemoglobin.

[0019] In one aspect, the present invention provides a mutant human HSD17B13 protein, wherein the amino acid sequence of the mutant human HSD17B13 protein comprises SEQ ID NO: 23; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 23.

[0020] In one aspect, the present invention provides a humanized HSD17B13 gene, wherein the humanized HSD17B13 gene expresses the mutated human HSD17B13 protein.

[0021] In one aspect, the present invention provides a humanized HSD17B13 gene, the humanized HSD17B13 gene comprising any one of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23; B) a nucleotide sequence as represented by SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 13, 24, 25, 26, 28, 29, 30, 31, or SEQ ID NO: 26, wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or a nucleotide sequence comprising positions 22641-22642 from TT to T and / or position 22895 from G to C compared to SEQ ID NO: 5; C) a nucleotide sequence as represented by SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 13, 24, 25, 26, 28, 29, 30, 31, or SEQ ID NO: 26, wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or a nucleotide sequence as represented by SEQ ID NO: 5, wherein position 12641-12642 is mutated from TT to T and / or position 22895 is mutated from G to C; NO: 26 wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 5 comprising a mutation from TT to T at position 22641-22642 and / or a mutation from G to C at position 22895; D) a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 13, 24, 25, 26, 28, 29, 30, 31, a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 26 wherein position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G, or a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: NO:5, wherein the nucleotide sequence identity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the nucleotide sequence comprising a mutation from TT to T at position 22641-22642 and / or a mutation from G to C at position 22895.

[0022] In one aspect, the present invention provides a cell, tissue or organ, which expresses human HSD17B13 protein and / or the mutated human HSD17B13 protein, and / or the genome of the cell, tissue or organ comprises the humanized HSD17B13 gene.

[0023] In one aspect, the present invention provides an animal model, wherein the animal model expresses human HSD17B13 protein and / or the mutated human HSD17B13 protein, and / or the genome of the animal model comprises the humanized HSD17B13 gene.

[0024] In one aspect, provided are uses of the above-mentioned non-human animals, the non-human animals obtained by the above-mentioned construction methods, the above-mentioned mutant human HSD17B13 proteins, the above-mentioned humanized HSD17B13 genes, the above-mentioned cells, tissues or organs, and the above-mentioned animal models, the uses comprising: A) use in the development of products involving immune processes related to HSD17B13 in human cells; B) use as a model system related to HSD17B13 in pharmacological, immunological, microbiological and medical research; C) use in the production and use of animal experimental disease models for etiological research related to HSD17B13 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) use in in vivo studies of the screening, efficacy testing, efficacy assessment, validation or evaluation of human HSD17B13 signaling pathway regulators; or, E) use in studying the function of the HSD17B13 gene, studying drugs and drug efficacy targeting human HSD17B13 target sites, and studying therapeutic drugs for diseases related to HSD17B13 (e.g., non-alcoholic steatohepatitis).

[0025] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that makes up the whole.

[0026] In the present invention, the term "locus" broadly refers to the location of a gene on a chromosome, and more narrowly refers to a DNA segment on a gene, which can be a gene, a portion of a gene, or a regulatory region of a gene. For example, the "HSD17B13 locus" includes a DNA segment selected from exons 1-7 of the HSD17B13 gene.

[0027] The term "exon XX to exon XXX" or "exon XX-XXX" or "all of exons XX-XXX" or "all of exon XX to exon XXX" in the present invention refers to the entire nucleotide sequence including exons and introns therebetween, for example, all of exons 1-7 include exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6 and exon 7.

[0028] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 6 refers to the intron between exon 6 and exon 7.

[0029] The terms "comprising" or "including" in the present invention are open-ended and encompass the specified components or steps being described, as well as other specified components or steps that do not substantially affect them. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid sequence, while still having the same or similar activity as the original sequence.

[0030] The term "and / or" in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in this application. For example, "A and / or B" includes "A", "B" and "A and B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This application describes methods and materials for use with the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of conflict, the present specification, including definitions, will control.

[0032] Those skilled in the art will readily appreciate other aspects and advantages of the present application from the following detailed description.

[0033] HSD17B13

[0034] In the human genome, the HSD17B13 gene (NCBI Gene ID: 345275, UniProt ID: Q7Z5P4, located at positions 87303794 to 87322882 on chromosome 4, NC_000004.12) contains seven exons, exon 1 to exon 7. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_178135.5 and its encoded protein NP_835236.2 (SEQ ID NO: 2) are shown in Table 1:

[0035] Table 1

[0036]

[0037] In the mouse genome, the HSD17B13 gene (NCBI Gene ID: 243168, UniProt ID: Q8VCR2, located at positions 104103308 to 104125254 on chromosome 5 NC_000071.7) contains seven exons, exon 1 to exon 7. The corresponding positions of each exon in the nucleotide and amino acid sequences of transcript NM_198030.2 and its encoded protein NP_932147.2 (SEQ ID NO: 1) are shown in Table 2:

[0038] Table 2

[0039]

[0040] HSD17B13 genes, proteins, and gene loci from other species are also known in the art. For example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), and Sus scrofa (pig). Relevant information (e.g., intron sequences, exon sequences, and amino acid sequences) for these genes can be found in NCBI, the entire contents of which are incorporated herein by reference.

[0041] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, for the purpose of best comparison, sequence is compared (for example, for best comparison, can introduce gap in one or both of the first and second amino acid or nucleotide sequences, and can ignore non-homologous sequences for the purpose of comparison).Then the amino acid residue or the nucleotide on the corresponding amino acid position or nucleotide position are compared.When a position in the first sequence is occupied by the amino acid residue or the nucleotide identical with the corresponding position in the second sequence, the molecule is identical at this position.The percent identity between the two sequences is a function of the quantity of the same positions shared by the sequences, and considering the number of gaps and the length of each gap, this needs to be introduced to realize the best comparison of the two sequences.For example, the determination of the percent identity between the comparison of the sequence and the two sequences can be completed using the Blossum 62 scoring matrix of gap penalty 12, gap extension penalty 4 and frameshift gap penalty 5.

[0042] The percentage ratio (homology percentage) of conserved residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine and isoleucine) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage ratio is higher than the identity percentage ratio.

[0043] carrier

[0044] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the switch region to be altered (5' arm or 5' homology arm), which is derived from the genomic DNA of the HSD17B13 gene and has a length of 100 to 10,000 nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the switch region to be altered (3' arm or 3' homology arm), which is derived from the genomic DNA of the HSD17B13 gene and has a length of 100 to 10,000 nucleotides.

[0045] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000071.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000071.7.

[0046] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000004.12; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% homology to NCBI Accession No. NC_000004.12.

[0047] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb.

[0048] In some embodiments, the switch region to be altered is located on exons 1-7 of the endogenous HSD17B13 gene of a non-human animal, preferably located on exon 1 to intron 6 of the endogenous HSD17B13 gene of a non-human animal, and further preferably located on the 5'UTR upstream nucleotide sequence to intron 6.

[0049] In some embodiments, the endogenous HSD17B13 locus of the non-human animal comprises a human HSD17B13 nucleotide sequence, and the switch region to be altered is located within the human HSD17B13 nucleotide sequence, preferably within exon 6 of the human HSD17B13 nucleotide sequence. In some embodiments, the 5' arm comprises SEQ ID NO: 3, 13, or 30. In some embodiments, the 3' arm comprises SEQ ID NO: 4, 25, or 31.

[0050] In some embodiments, the b) donor region comprises a human sequence. In some embodiments, the b) donor region comprises the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 26, SEQ ID NO: 26 with position 1269 mutated from T to TT and / or position 1522 mutated from C to G, or SEQ ID NO: 5 with positions 22641-22642 mutated from TT to T and / or position 22895 mutated from G to C.

[0051] In some embodiments, the targeting vector comprises one or more marker genes (or resistance genes). For example, a resistance gene for positive clone screening or a coding gene for a negative screening marker. In some embodiments, the resistance gene for positive clone screening comprises a neomycin phosphotransferase coding sequence Neo and / or a hygromycin B phosphotransferase coding sequence HygR. Preferably, the targeting vector further comprises two Frt recombination sites and / or Frt3 recombination sites arranged in the same direction on both sides of the marker gene. In some embodiments, the coding gene for the negative screening marker is a coding gene for the diphtheria toxin A subunit (DTA).

[0052] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets the HSD17B13 gene. In some embodiments, the target site of the sgRNA on the switch region to be altered is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'.

[0053] In some embodiments, the target site is shown as SEQ ID NO: 32 or 33. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 34 and 36. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 35 and 37. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 38 and 40. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 39 and 41.

[0054] In some embodiments, the present invention relates to a plasmid construct comprising an sgRNA (sgRNA vector, such as pT7-sgRNA) and / or a cell comprising the construct.

[0055] The present invention also relates to cells comprising the targeting vector and / or sgRNA vector as described above.

[0056] In some embodiments, the present invention provides a non-human mammalian cell comprising any of the above-described vectors, preferably further comprising one or more in vitro transcripts of the plasmid construct. In some embodiments, the non-human mammalian cell further comprises Cas9 mRNA or its in vitro transcript.

[0057] In some embodiments, the gene in the non-human mammalian cell is heterozygous. In some embodiments, the gene in the non-human mammalian cell is homozygous.

[0058] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the non-human mammalian cell is a fertilized egg cell. In some embodiments, the non-human mammalian cell is an embryonic stem cell. In some embodiments, the non-human mammalian cell is any cell capable of expressing HSD17B13.

[0059] Genetically modified non-human animals

[0060] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to at least one chromosome in the genome of the non-human animal having exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal or genetically modified non-human animal have exogenous DNA. In some embodiments, the cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (such as T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, which comprises a modified endogenous HSD17B13 locus, and the modified endogenous HSD17B13 locus comprises an exogenous sequence (such as a human sequence), for example, one or more non-human animal endogenous sequences are replaced with one or more human sequences, or one or more human and / or non-human sequences are inserted. Non-human animals are often able to pass genetic modifications to their offspring through germline transmission.

[0061] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more portions of the gene or nucleic acid are derived from different species, or at least one sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (x) gene or chimeric (x) nucleic acid has two or more different species origins, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized gene or humanized nucleic acid.

[0062] The "chimeric (X) protein" or "chimeric (X) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least one sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric (X) protein or chimeric (X) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a humanized protein or humanized polypeptide. In some embodiments, the chimeric (X) protein or chimeric (X) polypeptide refers to a human (X) protein or human (X) polypeptide.

[0063] As used herein, a "humanized (X) protein" or "humanized (X) polypeptide" refers to a protein or polypeptide, at least a portion of which is derived from a human (X) protein or polypeptide. In some embodiments, the humanized (X) protein or polypeptide refers to a human (X) protein or polypeptide.

[0064] As used herein, "humanized (x) nucleic acid" or "humanized (x) gene" refers to a nucleic acid or gene. In some embodiments, at least a portion of the nucleic acid or gene is derived from a human. In some embodiments, at least a portion of the nucleic acid or gene in the humanized (x) nucleic acid or humanized (x) gene is derived from a non-human animal. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, which can be a human exon or a chimeric exon.

[0065] In some embodiments, the chimeric HSD17B13 gene or chimeric HSD17B13 nucleic acid is a humanized HSD17B13 gene or humanized HSD17B13 nucleic acid. In some embodiments, at least a portion of the humanized HSD17B13 gene or humanized HSD17B13 nucleic acid is derived from a human HSD17B13 gene. In some embodiments, at least a portion of the humanized HSD17B13 gene or humanized HSD17B13 nucleic acid is derived from an endogenous HSD17B13 gene of a non-human animal. In some embodiments, the humanized HSD17B13 gene or humanized HSD17B13 nucleic acid comprises a sequence encoding an HSD17B13 protein. The encoded HSD17B13 protein has at least one activity, such as an activity of a human HSD17B13 protein and / or a non-human animal HSD17B13 protein.

[0066] In some embodiments, the chimeric HSD17B13 protein or polypeptide is a humanized HSD17B13 protein or polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the humanized HSD17B13 protein or polypeptide are derived from a human HSD17B13 protein. In some embodiments, at least one or more portions of the amino acid sequence of the humanized HSD17B13 protein or polypeptide are derived from a non-human animal HSD17B13 protein. The humanized HSD17B13 protein or polypeptide is functional, or has at least one activity, such as an activity of a human HSD17B13 protein and / or a non-human animal HSD17B13 protein.

[0067] The non-human animal of genetic modification can be various non-human animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) to form embryo in non-human animal under appropriate conditions.Above-mentioned construction method is known in the art, and is described in " A.Nagy, et al., " Manipulating the Mouse Embryo:A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", the entire contents of which are incorporated herein by reference.

[0068] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the rodent may be selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified non-human animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Malinomys (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), Spiny Mouse (e.g., spiny dormouse), and Murid (e.g., mole rats, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a mouse or rat (Muroidea), a gerbil, spiny mouse, and crested rat. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the non-human animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0069] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc. In some embodiments, the animal is a mouse of the C57BL strain, wherein the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, and 129 / Sv-ter / +. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as the BALB / c strain. In some embodiments, the mouse is a hybrid of the BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In certain embodiments, the non-human animal is a rodent. In certain embodiments, the non-human animal is a mouse with BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / 01a), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains. In certain embodiments, the non-human animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6 and Dark Agouti.In some embodiments, the rat strain is a hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6 and Dark Agouti. The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate or destroy all or part of the immune system of the non-human animal. Damage, inactivation or destruction of the immune system of the non-human animal can include, for example, by chemical means (e.g., administration of toxins), physical means (e.g., irradiation of animals), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γc. null Mice (Ito, M. et al., NOD / SCID / γc null mouse:an excellent recipient mouse model for engraftment of human cells, Blood 100(9):3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can optionally be irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of the endogenous HSD17B13 locus of a non-human animal, and also includes damage, inactivation or partial destruction of the immune system (or one or more cell types of the immune system) of the non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γc null Mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdc scid IL-2Rγ null Mouse, NOD Rag 1 - / - IL2rg - / - (NRG) mice, Rag2 - / - IL2rg - / - Modification of (RG) mice and combinations thereof. These transgenic animals are described, for example, in US Pat. No. 10,820,580 B2, which is incorporated herein by reference in its entirety. In some embodiments, the mice may include replacement of all or part of the mouse endogenous mature HSD17B13 coding sequence with all or part of the human mature HSD17B13 coding sequence.

[0070] Genetically modified non-human animals include modifications of an endogenous HSD17B13 gene site (locus) in a non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature HSD17B13 protein (e.g., comprising a nucleotide sequence that is identical to a nucleotide sequence encoding a mature HSD17B13 protein or that has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with a nucleotide sequence encoding a mature HSD17B13 protein). Although cells (e.g., ES cells, somatic cells) that may comprise the genetic modification are provided herein, in some embodiments, the genetically modified non-human animal includes modifications of an endogenous HSD17B13 gene site in a non-human animal.

[0071] The genetically modified non-human animal can express human HSD17B13 and / or chimeric (e.g., humanized) HSD17B13 at an endogenous locus of the non-human animal, wherein the endogenous HSD17B13 gene of the non-human animal has been replaced or inserted with a human HSD17B13 gene and / or a nucleotide sequence encoding a human HSD17B13 region, or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human HSD17B13 sequence. In some embodiments, the endogenous HSD17B13 locus of the non-human animal is modified by comprising all or part of a nucleic acid sequence encoding a mature human HSD17B13 protein.

[0072] In some embodiments, the genetically modified non-human animal can express human HSD17B13 and / or chimeric HSD17B13 (e.g., humanized HSD17B13) under the control of human HSD17B13 regulatory elements. Insertion or substitution at the endogenous locus of the non-human animal provides a non-human animal that expresses human HSD17B13 or chimeric HSD17B13 (e.g., humanized HSD17B13) in suitable cells and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. Human HSD17B13 or chimeric HSD17B13 (e.g., humanized HSD17B13) expressed in the non-human animal can maintain one or more functions of wild-type non-human animals or human HSD17B13 in the non-human animal. In addition, in some embodiments, the non-human animal does not express endogenous HSD17B13. In some embodiments, the expression level of endogenous HSD17B13 in the non-human animal is reduced compared to the expression level of HSD17B13 in wild-type animals. The term "endogenous HSD17B13" as used herein refers to the HSD17B13 protein expressed by the endogenous HSD17B13 nucleotide sequence of a non-human animal (eg, mouse) before any genetic modification.

[0073] In some embodiments, the humanized HSD17B13 gene comprises the 5'UTR of the human HSD17B13 gene. In some embodiments, the humanized HSD17B13 gene comprises the 3'UTR of the human HSD17B13 gene. In some embodiments, the humanized HSD17B13 gene comprises the 5'UTR of the endogenous (e.g., mouse endogenous) HSD17B13 gene of a non-human animal. In some embodiments, the humanized HSD17B13 gene comprises the 3'UTR of the endogenous (e.g., mouse endogenous) HSD17B13 gene of a non-human animal. Where appropriate, it is reasonable to assume that, based on the similarity of the non-human animal and human HSD17B13 gene sequences, they appear to be similarly regulated. As shown in the present application, humanized HSD17B13 mice comprising an insertion or replacement in the endogenous HSD17B13 locus of a non-human animal, which comprises humanization of the HSD17B13 coding sequence, do not exhibit pathological phenomena. Both genetically modified non-human animals that were heterozygous or homozygous for humanized HSD17B13 were normal.

[0074] The present invention further relates to the HSD17B13 genomic DNA sequence of humanized mice, a DNA sequence obtained by reverse transcription of mRNA that is identical to or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; and a tissue or organ comprising the cell thereof.

[0075] The present invention further relates to a non-human mammal produced by the above method. In some embodiments, its genome comprises a human HSD17B13 gene.

[0076] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0077] In some embodiments, the non-human mammal expresses a protein encoded by a humanized HSD17B13 gene.

[0078] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the construction method described in this application. In some embodiments, the non-human mammal is a rodent (eg, a mouse).

[0079] The present invention also provides a cell or cell line derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, or a primary cell culture derived from a non-human mammal or its progeny, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its progeny. In the case of a tumor, the cell or cell line is derived from a tumor tissue of a non-human mammal or its progeny, or a non-human mammal carrying a tumor.

[0080] The present invention provides a non-human mammal produced by any of the construction methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises human or humanized HSD17B13 DNA.

[0081] In some embodiments, a non-human mammal comprises a plasmid construct described herein. In some embodiments, a non-human mammal expressing a human or humanized HSD17B13 protein is provided. In some embodiments, a cell, tissue, or organ that specifically expresses a human or humanized HSD17B13 protein is provided.

[0082] In some embodiments, the expression of human or humanized HSD17B13 protein in non-human animals is controllable, such as by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0083] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferably, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0084] The non-human mammal described above is subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with a non-human mammal of the same genotype or another genotype.

[0085] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its descendants. For example, a cell culture-based model can be prepared by the following methods. The cell culture can be obtained by isolating cells from the non-human mammal, or by using the same construct and establishing a cell culture using standard cell transfection techniques. Integration of a genetic construct containing a DNA sequence encoding the human HSD17B13 protein can be detected by various methods.

[0086] There are many analytical methods available for detecting exogenous DNA, including nucleic acid level methods (including reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblotting analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well known to those skilled in the art. Many standard analytical methods can be used to perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcript levels, including RNase protection assays, Southern Blots, and RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized HSD17B13 protein.

[0087] In some embodiments, the genetically modified non-human animals described herein (eg, humanized homozygous HSD17B13 gene mice or humanized heterozygous HSD17B13 gene mice) can express human or humanized HSD17B13 in one or more cells.

[0088] Method for constructing genetically modified non-human animals

[0089] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology, CRISPR / Cas9 technology, homologous recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques using embryonic stem cells. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017) by Yin et al., which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0090] In some embodiments, the nucleotide sequence encoding endogenous HSD17B13 in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding human HSD17B13. In some embodiments, the expression level of the endogenous HSD17B13 protein in the non-human animal is reduced or absent compared to wild-type. In some embodiments, the replacement occurs in a cell, such as a germ cell, somatic cell, blastocyst, or fibroblast. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0091] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized HSD17B13 gene fragment, and a 3' homology arm. The process involves using homologous recombination to replace the endogenous HSD17B13 sequence with the human or humanized HSD17B13 gene fragment. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the endogenous HSD17B13 sequence of the non-human animal with the human or humanized HSD17B13 gene fragment.

[0092] In some embodiments, a method of making a genetically modified humanized animal comprises replacing a nucleotide sequence encoding endogenous HSD17B13 in the endogenous genome of a non-human animal with a nucleotide sequence encoding human HSD17B13 at the endogenous HSD17B13 locus (or site).

[0093] The present invention also provides a method for establishing an HSD17B13 gene humanized animal model, comprising the following steps:

[0094] (a) providing cells (e.g., fertilized egg cells) according to the method described in this application;

[0095] (b) culturing the cells (preferably culturing the cells in a liquid culture medium);

[0096] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, and allowing the cells to develop in the uterus of the female non-human mammal;

[0097] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0098] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0099] In some embodiments, the non-human mammal in step (c) is a pseudo-pregnant (or pregnant) female.

[0100] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0101] The fertilized egg can be from any non-human animal, such as any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The genetic construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which can then give birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0102] In some embodiments, the method for preparing a genetically modified non-human animal comprises modifying the coding frame of the HSD17B13 gene of the non-human animal, for example, by replacing the nucleic acid sequence encoding endogenous HSD17B13 with a nucleotide sequence encoding human HSD17B13 (e.g., a genomic DNA sequence, a CDS sequence, or a cDNA sequence) under the control of a human HSD17B13 gene regulatory element. For example, one or more functional region sequences of the HSD17B13 gene of the non-human animal can be knocked out or inserted into a sequence such that the endogenous HSD17B13 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the HSD17B13 gene of the genetically modified non-human animal can be all or part of the nucleotide sequence of exon 1 to exon 7 of the HSD17B13 gene of the non-human animal, preferably all of the nucleotide sequence of exon 1 to exon 6 of the HSD17B13 gene of the non-human animal.

[0103] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized HSD17B13 protein after the endogenous regulatory elements of the HSD17B13 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon, so that the HSD17B13 gene humanized animal model can express the human or humanized HSD17B13 protein in vivo, but does not express the HSD17B13 protein of the non-human animal. In some embodiments, the auxiliary sequence comprises WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA. In some embodiments, the sequence encoding the humanized HSD17B13 protein is operably linked to the human HSD17B13 regulatory elements.

[0104] In some embodiments, the genetically modified non-human animal does not express endogenous HSD17B13 protein.

[0105] In some embodiments, the nucleotide sequence encoding the HSD17B13 protein is deleted in the genome of the non-human animal. In some embodiments, all or part of exons 1-7 of the HSD17B13 gene are deleted in the genome of the non-human animal. In some embodiments, all of exons 1-6 of the endogenous HSD17B13 gene are deleted in the genome of the non-human animal. In some embodiments, the deleted portion of the non-human animal genome further comprises at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the endogenous HSD17B13 gene. In some embodiments, the deleted portion of the non-human animal genome further comprises at least 50 bp of consecutive nucleotides in intron 6 of the endogenous HSD17B13 gene. In some embodiments, the genome of the non-human animal is deleted from at least 50 bp of consecutive nucleotides upstream of the 5'UTR of the endogenous HSD17B13 gene to at least 50 bp of consecutive nucleotides in intron 6. In some embodiments, the genome of the non-human animal is deleted from at least 5301 bp of consecutive nucleotides upstream of the 5'UTR of the endogenous HSD17B13 gene to at least 2311 bp of consecutive nucleotides in intron 6.

[0106] In some embodiments, the construction method comprises replacing all or part of the nucleotide sequence encoding the endogenous HSD17B13 protein in the genome of a non-human animal with a nucleotide sequence encoding a human HSD17B13 protein or a mutant human HSD17B13 protein. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23 with the nucleotide sequence encoding SEQ ID NO: 1 in the genome of a non-human animal. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23 with the nucleotide sequence encoding SEQ ID NO: 1-271 in the genome of a non-human animal. In some embodiments, the construction method comprises replacing the entire nucleotide sequence of exon 1 to exon 6 of the endogenous HSD17B13 gene in the genome of a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the construction method comprises replacing the entire nucleotide sequence from the 5'UTR upstream nucleotide sequence to exon 6 of the endogenous HSD17B13 gene in the genome of a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the construction method comprises replacing the entire or partial nucleotide sequence from exon 1 to intron 6 of the endogenous HSD17B13 gene in the genome of a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23. In some embodiments, the construction method comprises replacing the entire or partial nucleotide sequence from the 5'UTR upstream nucleotide sequence to intron 6 of the endogenous HSD17B13 gene in the genome of a non-human animal with the nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23.

[0107] In some embodiments, the construction method comprises replacing the entire nucleotide sequence of exon 1 to exon 7 of the human HSD17B13 gene or the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene with the entire nucleotide sequence of exon 1 to exon 6 of the endogenous HSD17B13 gene in the genome of a non-human animal. In some embodiments, the construction method comprises replacing the entire nucleotide sequence of exon 1 to exon 7 of the human HSD17B13 gene or the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene with the entire nucleotide sequence from the 5'UTR upstream nucleotide sequence to exon 6 of the endogenous HSD17B13 gene in the genome of a non-human animal. In some embodiments, the construction method comprises replacing the entire nucleotide sequence of exon 1 to exon 7 of the human HSD17B13 gene or the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene with all or part of the nucleotide sequence of exon 1 to intron 6 of the endogenous HSD17B13 gene in the genome of a non-human animal. In some embodiments, the construction method comprises replacing the entire nucleotide sequence of exon 1 to exon 7 of the human HSD17B13 gene or the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene with the entire or partial nucleotide sequence from the 5'UTR upstream nucleotide sequence to intron 6 of the endogenous HSD17B13 gene in the non-human animal genome. In some embodiments, the construction method comprises replacing the entire or partial nucleotide sequence from the 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of at least 50 bp of the human HSD17B13 gene with the 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of at least 50 bp of the endogenous HSD17B13 gene in the non-human animal genome. In some embodiments, the construction method comprises replacing the at least 10,000 bp continuous nucleotide sequence upstream of the 5'UTR of the human HSD17B13 gene to the at least 500 bp continuous nucleotide sequence downstream of the 3'UTR with the at least 5,301 bp continuous nucleotide sequence upstream of the 5'UTR of the endogenous HSD17B13 gene in the non-human animal genome to the at least 2,311 bp continuous nucleotide sequence of intron 6. In some embodiments, the construction method comprises replacing the 5'UTR upstream nucleotide sequence of the human HSD17B13 gene to the 3'UTR downstream nucleotide sequence with all or part of the 5'UTR upstream nucleotide sequence of the endogenous HSD17B13 gene in the non-human animal genome to the intron 6. In some embodiments, the construction method comprises replacing the at least 50 bp continuous nucleotide sequence upstream of the 5'UTR of the human HSD17B13 gene to the at least 50 bp continuous nucleotide sequence downstream of the 3'UTR with the at least 50 bp continuous nucleotide sequence upstream of the 5'UTR of the endogenous HSD17B13 gene in the non-human animal genome to the at least 50 bp continuous nucleotide sequence of intron 6.In some embodiments, the construction method comprises replacing the at least 10,000 bp continuous nucleotide sequence upstream of the 5'UTR of the human HSD17B13 gene to the at least 500 bp continuous nucleotide sequence downstream of the 3'UTR of the human HSD17B13 gene with the at least 5,301 bp continuous nucleotide sequence upstream of the 5'UTR of the endogenous HSD17B13 gene in the non-human animal genome to the at least 2,311 bp continuous nucleotide sequence of intron 6. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or SEQ ID NO: 1 in the non-human animal genome with the nucleotide sequence represented by SEQ ID NO: 5, 6, 24, or SEQ ID NO: 5 at positions 22641-22642 from TT to T and / or at position 22895 from G to C. In some embodiments, the construction method comprises replacing the nucleotide sequence shown in SEQ ID NO: 5 or 6 with the nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1 in the genome of a non-human animal, and then performing a secondary targeting to replace the corresponding fragment of SEQ ID NO: 5 in the genome of the non-human animal (e.g., nucleotides 21373-23004 of SEQ ID NO: 5) with SEQ ID NO: 26 to introduce a mutation. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding a human or humanized HSD17B13 protein (e.g., a genomic DNA sequence, CDS sequence, or cDNA sequence) or a humanized HSD17B13 gene with the nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1 in the genome of a non-human animal.

[0108] Use of genetically modified non-human animals

[0109] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence at the non-human animal endogenous locus and under the control of human HSD17B13 regulatory elements (e.g., promoter, 5'UTR and / or 3'UTR), or inserting a homologous or orthologous human gene or human sequence into a non-human animal, can produce non-human animals with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the non-human animal and may integrate randomly into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and / or protein assays and / or functional assays.

[0110] Genetically modified non-human animals that express human or humanized HSD17B13 protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0111] The present invention also provides the use of the HSD17B13 gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0112] In some embodiments, the application comprises:

[0113] A) Applications in product development involving HSD17B13-related immune processes in human cells;

[0114] B) Application as a model system for HSD17B13-related research in pharmacology, immunology, microbiology, and medicine;

[0115] C) applications involving the production and use of animal experimental disease models for the study of the etiology of HSD17B13 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0116] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human HSD17B13 signaling pathway modulators; or

[0117] E) To study the function of the HSD17B13 gene, the efficacy of drugs targeting the human HSD17B13 target site, and the application of therapeutic drugs in the treatment of HSD17B13-related diseases (including tumors, inflammation (such as non-alcoholic steatohepatitis), and immune diseases).

[0118] The present invention provides a non-human animal expressing a human or humanized HSD17B13 protein, which can be used to screen for human HSD17B13-specific modulators (e.g., antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs). In some embodiments, the non-human animal is an animal model for a human disease. For example, the disease is genetically induced (knock-in or knock-out). In various embodiments, the genetically modified non-human animal further comprises a compromised immune system, such as a genetically modified human tissue xenograft, including a human solid tumor (e.g., breast cancer) or a hematologic tumor (e.g., a lymphocytic tumor (e.g., a B or T cell tumor)).

[0119] In some embodiments, therapeutic agents (e.g., antibodies, nucleic acid drugs, and / or polypeptide drugs targeting HSD17B13) block or inhibit HSD17B13-mediated signaling pathways. In some embodiments, the therapeutic agents described herein can block interactions between HSD17B13 complexes, thereby inhibiting the HSD17B13 signaling pathway.

[0120] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs) in treating various HSD17B13-related diseases. In some embodiments, the HSD17B13-related diseases include non-alcoholic steatohepatitis (NASH). In some embodiments, the HSD17B13-related diseases also include one or more of alcoholic steatohepatitis (ASH), liver fibrosis (including HCV-related cirrhosis), fatty liver disease (including non-alcoholic fatty liver disease (NAFLD) and / or alcoholic fatty liver disease (ALD)), cirrhosis, chronic fibrotic inflammatory liver disease, drug-induced liver damage, or hepatocellular necrosis.

[0121] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs) in treating various inflammatory conditions (e.g., infections). In some embodiments, the inflammation includes both acute and chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation includes non-alcoholic steatohepatitis or inflammatory bowel disease (IBD).

[0122] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs) in treating cancer. In some embodiments, a therapeutic agent is administered to a non-human animal, wherein the non-human animal has cancer or a tumor, and the inhibitory effect of the therapeutic agent on the cancer or tumor is detected. In some embodiments, the detection includes measuring the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry, and / or in vivo imaging detection of non-human animals. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, and the metabolic changes include changes in food consumption or water consumption. In some embodiments, the tumor cells include one or more cancer cells injected into an animal (e.g., cancer cells derived from humans or non-human animals). In some embodiments, the therapeutic agent inhibits or activates the HSD17B13 signaling pathway. In some embodiments, the therapeutic agent does not inhibit or activate the HSD17B13 signaling pathway.

[0123] In some embodiments, genetically modified non-human animals can be used to detect whether a therapeutic agent (e.g., an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13, and / or a polypeptide drug) is an agonist or antagonist. In some embodiments, the methods described herein can be used to detect the function of a therapeutic agent (e.g., an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13, and / or a polypeptide drug), for example, whether the therapeutic agent can upregulate or downregulate the immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can determine the effective dose of a therapeutic agent for treating a disease (e.g., a tumor, an immune disease, an inflammation (e.g., non-alcoholic steatohepatitis)). The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in a non-human animal, and / or determining the tumor (volume) growth inhibition rate (TGI). TV ). The tumor growth inhibition rate can be calculated using the formula TGI TV (%)=(1–T Vt / T Vc ) x 100, where T Vt and T Vc is the average tumor volume (or weight) of the treatment group and the control group.

[0124] In some embodiments, therapeutic agents (e.g., antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13, and / or polypeptide drugs) can be used to treat various cancers. As used herein, "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid cell growth and proliferation. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. As used herein, "tumors" include, but are not limited to, lymphomas, cervical cancer, leukemias, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, gliomas, lung cancer (e.g., non-small cell 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 myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer or meningioma. In some embodiments, the tumor comprises a solid tumor or a hematological tumor. In some embodiments, the tumor comprises hepatocellular carcinoma, breast cancer, lymphoma, renal clear cell carcinoma, head and neck squamous cell carcinoma, colorectal adenocarcinoma, head and neck cancer or lung cancer.

[0125] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13, and / or a polypeptide drug). The detection method comprises administering a therapeutic agent to the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, evaluating the weight change of the non-human animal or performing a blood test. In some embodiments, the blood test includes but is not limited to red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of a non-human animal not treated with a therapeutic agent).

[0126] The present invention also provides an animal model constructed by the method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or a model system for pharmacology, immunology, microbiology and medical research.

[0127] In some embodiments, an animal model generated by the methods described herein is provided for producing and utilizing animal experimental disease models of immune processes in human cells, studying pathogens, or developing new diagnostic and / or therapeutic strategies.

[0128] The present invention also provides an animal model generated by the method described herein to screen, verify, evaluate or study HSD17B13 gene function, human HSD17B13 antibodies, therapeutic drugs for human HSD17B13 target-related diseases (such as tumors, immune diseases or inflammation (such as non-alcoholic steatohepatitis)) or their effectiveness.

[0129] In some embodiments, the present disclosure provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method comprises transplanting human tumor cells into a non-human animal described herein, and applying human CAR-T to a non-human animal with human tumor cells. The effectiveness of CAR-T therapy can be determined and evaluated. In some embodiments, the non-human animal is selected from a HSD17B13 gene humanized non-human animal prepared by the method described herein, a double-gene or multi-gene humanized non-human animal (or its offspring) produced by the method described herein, a non-human animal expressing a human or humanized HSD17B13 protein, or the described tumor-bearing or inflammatory animal model. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat HSD17B13-related diseases described herein (e.g., tumors, immune diseases or inflammation (e.g., non-alcoholic steatohepatitis)). In some embodiments, TCR-T, CAR-T and / or other immunotherapies provide evaluation methods for treating HSD17B13-related diseases described herein, such as tumors, immune diseases, or inflammation (e.g., non-alcoholic steatohepatitis).

[0130] Non-human animal models with two or more human or chimeric genes

[0131] The present invention also provides an animal model or non-human animal having two or more human or chimeric genes. The non-human animal or animal model may comprise a human or chimeric HSD17B13 gene and a sequence encoding an additional human or chimeric protein.

[0132] In some embodiments, the additional human or chimeric protein comprises at least one of PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal further expresses at least one of human or humanized PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 protein.

[0133] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0134] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0135] (2) mating, in vitro fertilization, or direct gene editing of the non-human animal provided in step (1) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0136] In some embodiments, the other genetically modified non-human animals include non-human animals in which one or a combination of two or more of the genes PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 are humanized.

[0137] In some embodiments, HSD17B13 gene humanization is performed directly on non-human animals with modified human or chimeric PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 genes.

[0138] Since these proteins may be involved in different mechanisms, combination therapy targeting two or more of them may be a more effective treatment method. In fact, many related clinical trials are underway and have shown good results. Multi-gene modified non-human animal models can be used to determine the effectiveness of combination therapy targeting two or more proteins, for example, therapeutic agents (antibodies targeting HSD17B13, nucleic acid drugs targeting HSD17B13 and / or polypeptide drugs), and additional therapeutic agents for treating diseases (e.g., tumors, immune diseases or inflammation (e.g., non-alcoholic steatohepatitis)). The method comprises administering a therapeutic agent (an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13 and / or polypeptide drugs) and an additional therapeutic agent to a non-human animal, wherein the non-human animal has a disease (e.g., a tumor, an immune disease or inflammation (e.g., non-alcoholic steatohepatitis)), and determining the effect of the combination therapy on the disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4, or a nucleic acid drug and / or polypeptide drug targeting the above targets. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) or an anti-PD-L1 antibody. In some embodiments, the non-human animal further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0139] In some embodiments, the combination therapy is used to treat various cancers described herein. In some embodiments, the combination therapy is designed to treat immune diseases described herein, such as psoriasis. In some embodiments, the methods described herein can be used to evaluate combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include, for example, treating a subject with chemotherapy, for example, camphorine, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepate, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chloramphenicol, thiophene, nitrosoureas, daktarin, daunorubicin, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatin, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, in addition to this, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing part or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 : Schematic comparison of the mouse HSD17B13 locus and the human HSD17B13 locus (not to scale);

[0141] Figure 2 : Schematic diagram of HSD17B13 gene targeting strategy and targeting vector V1 design (not to scale);

[0142] Figure 3 : PCR identification results of the F2 generation of humanized mice expressing the HSD17B13 gene, where WT is the wild-type control and H2O is the water control;

[0143] Figure 4 : RT-PCR test results, where + / + represents wild-type C57BL / 6 mice, H / H represents HSD17B13 gene homozygous humanized mice, H2O represents water control, and GAPDH represents internal control;

[0144] Figure 5 : RT-qPCR test results, where + / + represents wild-type C57BL / 6 mice, and H / + represents humanized heterozygous mice harboring the HSD17B13 gene;

[0145] Figure 6 : Schematic diagram of HSD17B13 gene targeting strategy and targeting vector V2 design (not to scale);

[0146] Figure 7 : Schematic diagram of HSD17B13 gene targeting strategy and targeting vector V3 design (not to scale);

[0147] Figure 8: The inhibitory efficiency of nucleic acid drugs targeting human HSD17B13 on human HSD17B13 mRNA in HSD17B13 gene humanized mice;

[0148] Figure 9 : The inhibitory efficiency of nucleic acid drugs targeting human HSD17B13 on human HSD17B13 mRNA in HSD17B13 gene humanized mice;

[0149] Figure 10 : PCR identification results of the F1 generation of humanized mice with the HSD17B13 gene, wherein PC is a heterozygous positive control; WT is the humanized HSD17B13 mouse in Example 1, and H2O is a water control;

[0150] Figure 11 : RT-PCR test results, where + / + represents wild-type C57BL / 6 mice, H / H represents HSD17B13 gene homozygous humanized mice, H2O represents water control, and GAPDH represents internal control;

[0151] Figure 12 : Western blot detection results, where + / + represents wild-type C57BL / 6 mice, H / H represents HSD17B13 gene humanized homozygous mice, and GAPDH is used as the internal control. DETAILED DESCRIPTION

[0152] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0153] In each of the following examples, equipment and materials were obtained from the following companies:

[0154] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China Food and Drug Administration;

[0155] β-Tubulin mouse monoclonal antibody was purchased from Beyotime, catalog number AF1216;

[0156] Recombinant Anti-HSD17B13 antibody [EPR28344-54] was purchased from Abcam with the catalog number ab316153.

[0157] Example 1 HSD17B13 gene humanized mice

[0158] A schematic diagram of the comparison of the mouse HSD17B13 gene (NCBI Gene ID: 243168, located at positions 104103308 to 104125254 on chromosome 5 NC_000071.7, based on transcript NM_198030.2 and its encoded protein NP_932147.2 (SEQ ID NO: 1) and the human HSD17B13 gene (NCBI Gene ID: 345275, located at positions 87303794 to 87322882 on chromosome 4 NC_000004.12, based on transcript NM_178135.5 and its encoded protein NP_835236.2 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0159] To achieve the objectives of the present invention, a nucleotide sequence encoding a human HSD17B13 protein can be introduced into the endogenous HSD17B13 locus of a mouse, so that the mouse expresses a human or humanized HSD17B13 protein. Specifically, using gene editing technology, the entire sequence of mouse exons 1 to exon 6 and a partial sequence of intron 6, approximately 19.2 kb, are replaced with a nucleotide sequence comprising the regulatory elements of the human HSD17B13 gene and exon 1 to exon 7 (e.g., from upstream of the 5'UTR to downstream of the 3'UTR), approximately 29.6 kb, to obtain a humanized HSD17B13 locus, thereby achieving humanization of the mouse HSD17B13 gene.

[0160] In order to realize the targeting strategy of the present invention, a targeting vector V1 ( Figure 2 ), targeting vector V1 contains upstream and downstream homology arm sequences of the mouse HSD17B13 gene, and segment A containing the human HSD17B13 gene fragment. The upstream 5' homology arm sequence is SEQ ID NO: 3, the downstream 3' homology arm sequence is SEQ ID NO: 4, and the nucleotide sequence of the human HSD17B13 gene fragment is SEQ ID NO: 5. The connection design between the upstream of the human HSD17B13 gene fragment and the mouse sequence is: The sequence " AACCT The "T" in the sequence is the last nucleotide at the junction of the mouse sequence and the human HSD17B13 gene fragment. The first "C" in is the first nucleotide upstream of the human HSD17B13 gene fragment sequence. The connection between the downstream of the human HSD17B13 gene fragment and the mouse sequence is designed as follows: The sequence The "C" in the sequence is the last nucleotide downstream of the human HSD17B13 gene fragment sequence. TGGCA The "T" in " is the first nucleotide downstream of the mouse sequence connected to the human HSD17B13 gene fragment sequence.

[0161] The targeting vector also contains a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo. Two site-specific recombination system Frt recombination sites are arranged in the same direction on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human HSD17B13 gene is designed as follows: The sequence " AAACC The last "C" in the sequence is the last nucleotide of the human HSD17B13 gene connected to the 5' end of the Neo box. The "A" in the sequence is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human HSD17B13 gene is designed as follows:

[0162] The sequence " TCTGA "A" in the Neo box is the last nucleotide, the sequence The first "G" in the sequence is the first nucleotide connecting the human HSD17B13 gene to the 3' end of the Neo box. The mRNA sequence transcribed from the modified HSD17B13 gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 23.

[0163] Targeting vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary verification of the constructed targeting vector by enzyme digestion, it is sent to a sequencing company for sequencing verification. The sequencing-verified targeting vector is electroporated into embryonic stem cells of C57BL / 6 mice. The resulting cells are screened using a positive clone screening marker gene to identify the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into isolated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to culture medium for a short period of time and then transplanted into the oviducts of recipient female mice (white mice), producing F0 generation chimeric mice (black and white). F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice. F1 generation heterozygous mice are then mated with each other or with wild-type mice to obtain F2 generation mice. Alternatively, positive mice can be mated with Flp-expressing mice to remove the positive clone screening marker gene, and then mated with each other to obtain mice homozygous for the HSD17B13 gene.

[0164] The genotype of somatic cells of F2 generation mice can be identified by PCR, and the primers are shown in Table 3. The identification results of exemplary F2 generation mice are shown in Figure 3, among which mice numbered F2-1, F2-2 and F2-3 were positive.

[0165] Table 3 Primer sequences and recombinant fragment sizes for PCR detection of F2 generation genotypes

[0166]

[0167] The expression of mRNA in humanized HSD17B13 mice can be detected by RT-PCR. Specifically, 8-week-old wild-type C57BL / 6 mice (+ / +) and homozygous humanized HSD17B13 mice (H / H) of the same age were selected, and their livers were collected after euthanasia. The mRNA expression was detected by RT-PCR (primers are shown in Table 4). The test results are as follows: Figure 4 shown.

[0168] exist Figure 4 It can be seen that only mouse HSD17B13 mRNA was detected in wild-type C57BL / 6 mice, but no human HSD17B13 mRNA was detected; and only human HSD17B13 mRNA was detected in homozygous HSD17B13 gene humanized mice.

[0169] Table 4 RT-PCR primer sequences and target fragment sizes

[0170]

[0171]

[0172] In addition, RT-qPCR can be used to detect the expression of mRNA in HSD17B13 gene humanized mice. Specifically, 3 female and 3 male 5-week-old C57BL / 6 mice (+ / +) and 3 5-week-old HSD17B13 gene humanized mice heterozygous (H / +) prepared in this example were selected, and liver tissues were obtained after euthanasia. RT-qPCR detection was performed using the primer sequences shown in Table 5. The test results are shown in Figure 5. Figure 5 shown.

[0173] from Figure 5 As can be seen, human HSD17B13 mRNA can only be detected in HSD17B13 gene humanized mice heterozygous (H / +), while human HSD17B13 mRNA is not detected in wild-type C57BL / 6 mice, indicating that the modified HSD17B13 gene humanized mice can normally express human HSD17B13 protein.

[0174] Table 5 RT-qPCR primer sequences and target fragment sizes

[0175]

[0176] Example 2 HSD17B13 gene humanized mouse (V2)

[0177] To achieve the objectives of the present invention, similar to the above-described method, mutations can also be introduced into the targeting vector V1 to obtain HSD17B13 gene-humanized mice expressing the human HSD17B13 protein. For example, point mutations can be introduced into the human HSD17B13 fragment within the aforementioned targeting vector V1. In one embodiment, positions 22641-22642 in SEQ ID NO:5 are mutated from TT to T, and position 22895 is mutated from G to C. The modified HSD17B13 gene transcribed from the humanized mouse thus generated produces the mRNA sequence shown in SEQ ID NO:24, and the expressed protein sequence is shown in SEQ ID NO:2.

[0178] In another specific embodiment, secondary targeting was performed on the positive clone cells obtained in Example 1. Specifically, using gene editing technology, the human HSD17B13 gene fragment 2 (SEQ ID NO: 26) and the restriction site, totaling approximately 1659 bp, replaced the 1632 bp sequence at the corresponding position of the positive clone in Example 1. The mRNA sequence transcribed from the modified HSD17B13 gene in the modified humanized mouse is shown in SEQ ID NO: 24, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0179] In order to realize the targeting strategy of the present invention, a targeting vector V2 ( Figure 6 ), the targeting vector V2 contains upstream and downstream homology arm sequences of human HSD17B13 gene segment 2, and an A1 segment containing human HSD17B13 gene segment 2. The upstream 5' homology arm sequence is SEQ ID NO: 13, the downstream 3' homology arm sequence is SEQ ID NO: 25, and the nucleotide sequence of human HSD17B13 gene segment 2 is SEQ ID NO: 26.

[0180] The targeting vector also contains a resistance gene for positive clone screening, namely the hygromycin B phosphotransferase coding sequence HygR. Two Frt3 recombination sites, arranged in the same direction, are installed on either side of the resistance gene to form a HygR cassette. The 5' end of the HygR cassette is connected to the human HSD17B13 gene fragment 2 as follows: The sequence The "C" in the sequence is the last nucleotide of the human HSD17B13 gene segment 2 connected to the 5' end of the HygR box. GAGCTThe first "G" in " is the first nucleotide of the HygR box; the connection between the 3' end of the HygR box and the human HSD17B13 gene is designed as follows: The sequence The last "C" in the sequence is the last nucleotide of the HygR box. CCAA The first "C" in " is the first nucleotide connecting the human HSD17B13 gene to the 3' end of the HygR box.

[0181] The targeting vector can be constructed using conventional methods, such as enzyme digestion and ligation. The constructed targeting vector is initially verified by enzyme digestion and then sent to a sequencing company for sequencing verification. The correct targeting vector for sequencing verification is electroporated and transfected into the positive clone cells obtained in Example 1. The cells obtained are screened using a positive clone screening marker gene to screen the correct positive clone cells. The correct positive clone cells (black mice) screened out are imported into the separated blastocysts (white mice) according to techniques known in the art. The resulting chimeric blastocysts are transferred to the culture medium for a short period of time and then transplanted into the oviduct of the recipient female mouse (white mouse) to produce F0 generation chimeric mice (black and white). The F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then the F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. The positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then HSD17B13 gene humanized homozygous mice can be obtained by mating with each other.

[0182] In addition, CRISPR / Cas9 technology can be used for gene editing to design the targeting vector V3( Figure 7 ), wherein the targeting vector contains upstream and downstream homology arm sequences of human HSD17B13 gene segment 2, as well as segment A2 comprising human HSD17B13 gene segment 2. The upstream 5' homology arm sequence is SEQ ID NO: 30, the downstream 3' homology arm sequence is SEQ ID NO: 31, and the nucleotide sequence of human HSD17B13 gene segment 2 is SEQ ID NO: 26. The sequence of the modified HSD17B13 mRNA transcribed in the transformed humanized mouse is shown in SEQ ID NO: 24, and the sequence of the expressed protein is shown in SEQ ID NO: 2.

[0183] Targeting vector construction can be performed using conventional methods, such as enzyme digestion, ligation, and direct synthesis. After initial verification of the constructed targeting vector through enzyme digestion, it is sent to a sequencing company for sequencing verification. Targeting vectors that have been verified to be correct by sequencing are used in subsequent experiments.

[0184] The target site (target sequence) determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are prerequisites for constructing sgRNA expression vectors. Design and synthesize the sgRNA sequence that recognizes the target site. The target sequence of the exemplary sgRNA on the HSD17B13 gene is as follows:

[0185] sgRNA1 target site (SEQ ID NO: 32): 5′-CCTCCAGTTGTCATACTCCTTGG-3′;

[0186] sgRNA2 target site (SEQ ID NO: 33): 5′-AATAATAGCTCTATTGGGCCAGG-3′;

[0187] The activity of sgRNA was detected using a UCA kit. After confirming that it could mediate efficient cleavage efficiency, enzyme cleavage sites were added to its 5' end and complementary chain to obtain the forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 6. After annealing, the annealed products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) to obtain expression vectors pT7-HSD17B13-1 and pT7-HSD17B13-2.

[0188] Table 6 sgRNA1 and sgRNA2 sequences

[0189]

[0190] The pT7-sgRNA vector is synthesized by a plasmid synthesis company. The fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 42) is then digested with EcoRI and BamHI and ligated to a backbone vector (source: Takara, Cat. No. 3299). Sequencing verification by a professional sequencing company indicates that the target plasmid has been obtained. Pronuclear fertilized eggs of mice, such as C57BL / 6 mice, are taken. In vitro transcription products of the pT7-HSD17B13-1 and pT7-HSD17B13-2 plasmids (transcribed using the Ambion in vitro transcription kit according to the instructions), the targeting vector, and Cas9 mRNA are premixed and injected into the cytoplasm or nucleus of the mouse fertilized eggs using a microinjector. Microinjection of fertilized eggs was performed according to the method in the "Mouse Embryo Manipulation Experiment Manual (3rd Edition)" (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were expanded through hybridization and self-pollination to establish a stable HSD17B13 gene humanized mouse strain.

[0191] The genotype of F1 generation mouse somatic cells can be identified by PCR using the primers shown in Table 7. The exemplary results are shown in FIG. Figure 10 As shown, the PCR products of three mice numbered F1-1 to F1-3 were verified to be positive mice by sequencing.

[0192] Table 7 Primer sequences for PCR detection of F1 genotypes and sizes of recombinant fragments

[0193]

[0194] The expression of mRNA in HSD17B13 gene humanized mice can be detected by RT-PCR. Specifically, 8-week-old C57BL / 6 mice (+ / +) and 8-week-old male HSD17B13 gene humanized homozygotes (H / H) prepared in this example were selected, and liver tissues were obtained after euthanasia. RT-PCR detection was performed using the primer sequences shown in Table 8. The detection results are shown in FIG. Figure 11 As shown. Figure 11 As can be seen, only mouse HSD17B13 mRNA was detected in wild-type C57BL / 6 mice, but human HSD17B13 mRNA was not detected; human HSD17B13 mRNA was detected in HSD17B13 gene homozygous mice, but mouse HSD17B13 mRNA was not detected.

[0195] Table 8 RT-PCR primer sequences and target fragment sizes

[0196]

[0197]

[0198] In addition, the expression of human HSD17B13 protein in HSD17B13 gene humanized mice can be detected by conventional methods such as Western blot. Specifically, 8-week-old male C57BL / 6 mice (+ / +) and 8-week-old male HSD17B13 gene homozygous mice (H / H) prepared in this example were selected, and liver, lung, stomach, kidney and brain tissues were taken and detected using recombinant anti-HSD17B13 antibody [EPR28344-54] (Abcam, catalog number ab316153) that specifically recognizes human antibodies. The test results are as follows: Figure 12 shown.

[0199] The results showed that human HSD17B13 protein expression was detected only in the livers of mice homozygous for the humanized HSD17B13 gene, but not in the livers of wild-type C57BL / 6 mice. Furthermore, no human HSD17B13 protein was detected in the lungs, stomachs, kidneys, or brains of both wild-type C57BL / 6 mice and mice homozygous for the humanized HSD17B13 gene. Combined with the aforementioned RT-PCR results, this demonstrates that human HSD17B13 protein is successfully expressed in humanized HSD17B13 mice.

[0200] Example 3 Pharmacodynamic Model

[0201] The HSD17B13 humanized mice constructed in Example 1 or Example 2 of this application can be used to induce the preparation of various human disease models, including models of non-alcoholic steatohepatitis (NASH), and can be used to test the in vivo efficacy of small molecule drugs and small nucleic acid drugs. For example, HSD17B13 humanized mice can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of antagonists of the human-specific HSD17B13 signaling pathway in various disease models known in the art.

[0202] For example, the inhibitory efficiency of nucleic acid drugs on human HSD17B13 mRNA in the liver tissue of HSD17B13 gene humanized mice can be detected. Specifically, 8-week-old homozygous HSD17B13 gene humanized mice prepared in Example 1 were selected and randomly divided into two groups: a drug administration group and a control group. On the day of grouping (D0), the mice in the drug administration group were injected with nucleic acid drug 1 targeting human HSD17B13 (see Table 9), and the control group was injected with an equal volume of PBS. The nucleic acid drug was administered in the form of a PBS aqueous solution. The mice were killed on the 7th day (D7) after grouping, and the liver tissues were collected. The expression level of human HSD17B13 mRNA was detected by qPCR. The test results are as follows. Figure 9 As shown, the values are expressed as mean ± SEM.

[0203] from Figure 9 As can be seen, compared with the control group, the relative expression of human HSD17B13 mRNA in the drug-treated group was reduced, with an expression inhibition rate of approximately 75%. This indicates that the HSD17B13 gene humanized mice prepared by the present invention provide a powerful preclinical model for the in vivo evaluation of human HSD17B13-targeted nucleic acid drugs.

[0204] For another example, 15 10-week-old homozygous HSD17B13 humanized mice prepared in Example 2 were selected, including 6 females and 9 males. They were divided into drug administration groups (G2, G4 and G5) and control groups (G1 and G3). On the 0th day of grouping, the drug administration groups (G2, G4 and G5) were injected with human HSD17B13 nucleic acid drug 1 and human HSD17B13 nucleic acid drug 2 diluted to 5 μL / g by PBS (see Table 9), and the control groups (G1 and G3) were subcutaneously injected with an equal volume of PBS. The specific grouping and dosing regimen are shown in Table 10. On the 7th day of grouping, all mice were euthanized, and liver tissues were collected. The expression level of human HSD17B13 mRNA was detected by qPCR. The test results are as follows. Figure 8 shown.

[0205] Table 9 Sequences of nucleic acid drugs 1 and 2 targeting human HSD17B13

[0206]

[0207] wherein s represents a phosphorothioate linkage, f represents 2'-fluoro, (Agn) represents adenosine-diol nucleic acid (GNA), (Tgn) represents adenosine-diol nucleic acid (GNA) S-isomer, and L96 represents N-[tris(GalNac-alkyl)-amidodecanoyl]-4-hydroxyproline.

[0208] Table 10 Grouping and Dosage Scheme

[0209]

[0210] Depend on Figure 8 As shown, compared with the control group G1, the expression of human HSD17B13 mRNA in mice in the drug-treated group G2 was significantly reduced, with an expression inhibition rate of approximately 85%. Compared with the control group G3, the expression of human HSD17B13 mRNA in mice in the drug-treated groups G4 and G5 was significantly reduced, with expression inhibition rates of approximately 70% and 68%, respectively. This indicates that the HSD17B13 gene humanized mice prepared by the present invention provide a powerful preclinical model for the in vivo evaluation of human HSD17B13-targeted nucleic acid drugs.

[0211] For example, to prepare a non-alcoholic steatohepatitis (NASH) model, the HSD17B13 gene-humanized mice prepared in this invention can be fed a high-fat diet, D09100301 (shortening version), for at least 14 weeks. During the modeling process, the mice are weighed and observed daily. After developing the disease, the mice are grouped and administered via various routes, including oral gavage, intraperitoneal injection, or tail vein injection. The in vivo efficacy of different human drugs can be assessed using multiple assays, including behavioral scores, blood biochemistry, liver IHC pathology, HE pathology, and HSD17B13 mRNA expression.

[0212] Example 4 Preparation of double-gene or multi-gene humanized mice

[0213] The HSD17B13 gene humanized mice constructed and obtained in Example 1 or 2 of the present application can also be used to prepare a multi-gene humanized mouse model. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene containing PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. Alternatively, based on the humanized HSD17B13 mouse, a dual-gene humanized or multi-gene humanized mouse model can be obtained by isolating mouse ES embryonic stem cells and using gene recombination targeting technology. The homozygous or heterozygous humanized HSD17B13 gene mice obtained by this method can also be mated with other genetically modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice modified with the humanized HSD17B13 gene and other genes. Then, the heterozygotes can be mated with each other to obtain homozygous dual-gene or multi-gene modified mice.

[0214] The sequence involved in the present invention is as follows:

[0215] SEQ ID NO: 1

[0216] MNLILEFLLLVGVIIYSYLESLVKFFIPRRRKSVTGQTVLITGAGHGIGRLTAYEFAKQKSRLV

[0217] LWDINKRGVEETADKCRKLGAVVHVFVVDCSNRAEIYNSVDQVKREVGDVEIVVNNAGAIYPAD

[0218] LLSAKDEEITKTFEVNILGHFWIIKALLPSMLRRNSGHIVTVASVCGHGVIPYLIPYCSSKFAAVGFH

[0219] RALTAELDTLGKTGIQTSCLCPVFVNTGFTKNPSTRLWPVLEPEEVARSLINGILTNKKMIFVPSYIN

[0220] ISLILEKGPGFSSKHPHGGSQQPVTPIPGDLTPSSDFLKH

[0221] SEQ ID NO: 2

[0222] MNIILEILLLLITIIYSYLESLVKFFIPQRRKSVAGEIVLITGAGHGIGRQTTYEFAKRQSILVLW

[0223] DINKRGVEETAAECRKLGVTAHAYVVDCSNREEIYRSLNQVKKEVGDVTIVVNNAGTVYPADLL

[0224] STKDEEITKTFEVNILGHFWITKALLPSMMERNHGHIVTVASVCGHEGIPYLIPYCSSKFAAVGFHR

[0225] GLTSELQALGKTGIKTSCLCPVFVNTGFTKNPSTRLWPVLETDEVVRSLIDGILTNKKMIFVPSYINI

[0226] FLRLQKFLPERASAILNRMQNIQFEAVVGHKIKMK

[0227] SEQ ID NO:3

[0228] ctgtgccctctagttagtctggctaagggtttatctatcttgttgattttctcaaagaaccagctccaggtttggttgattctttgaatagttctttttgtttcca

[0229] cttggttgatttcacccctgagtttgattatttcctgccgtctactcctcttgggtgaatttgctttctttagttctagagcttctaggtgtgctgtcagtctgttagtgta

[0230] tgctctttctaatttctttttgggggcactcggggctctgagttttcctcttaggactgccttcattgtgtcccataagtttgggtatgttgtggcttcattttcattaacc

[0231] tctaaaaagtctttaatttctttatttcatccttgaccaaggaatcattgagtagagtgttgttcagtttccacatgaatgttggctttttattatttatgttgttattgaag

[0232] atcagccttagtccgtggtgatcagataggatacatgggataatttcaatatttttatatttgttgagtcctgttttgtgaccagttatatggtcagttttggaggagg

[0233] taccatgtggcactgagaagaaggtatatcctttcgttttaggataaaatgttctgtagatatctgttaagtccatttgtttcatcacttctgttagtttcactgtgtccc

[0234] tgtttagtttctgtttccatgatctgtccattgatgaaagtggtgtgttgaagtctcctactattattgtgtgaggtgcaatgtgtgctttgagctttactaaagtttcttt

[0235] aatgaatgtggctgcccttgtatttggagcatagatattcagaattgagagttcatcttggtagattttacctttgatgagtgtgaagtgcccttccttgtctttttttg

[0236] gtaactttgggttggaagtcaattttatcgatataaaatggctactccagcttttttctaaggaccatttgcttggaaaattgttttccagccttttactctgaggta

[0237] gcggctgtccttttccctgagatgggtttcctgcaagcaacaaaatgttgggtcctgtttgtgtagccagtctattagtctctctctttttattggggaattgagtcc

[0238] attggtgttaagagaaattaaagaaaagtaattgttgcttcctgttatttttgttgttaaagttgggattctgttcttagggctatcttcttttaggtttgttgaaggtttac

[0239] tttcttgctttttctagggcgtaatttccctccttgtattggtgttttccctttattatcctttgaagggttggattcgtggaaagatattgtgtgaatttggttttatcatgg

[0240] aatactttggtttctccatctatggtaattgagagttttgctgggtatagtagcctgggctggcatttgtgttctcttagggtctgtataacatctgtccaggatcttct

[0241] ggctttcatagtctctggtgagaagtctggtgtaattctaataggcctgcctttatatgttactggatctttttcccttactgcttttaatattctatctttagttagtgcat

[0242] ttgttgttctgattattatgtgttgggaggaatttcttttctggtcaagtctatttggagttctgtaagcttcttgtatgttcatgggcatctctttctttaggtttgggaag

[0243] ttttcttctataattttgttgaagatatttgctggccctttaagttgagaatcttcattctcatctactcctattatcagtaggtttggtcttcttattgtgtcctggatttcct

[0244] ggatgttttgagttaggatctttttgcattttgcatttttttttgattgttgtgcccatgttctctatggaatcttctgcacctgagattctctcttccatctcttatattctgtt

[0245] gctgatgctcatatctatggttccagatttctttcctagggtttctatctccagcgttgtctcattttgggttttctttattgtgtctcttccctttttagatcttgtatggtt

[0246] ttgttcaattctatcacctgtttggttgtgttttcctatatttctttaaggacttctacctttttagcagtgttctcctatatttctttaagtgagttattaaagtccttcttgat

[0247] gtcctctaccagcatcatgagatatgattttaaatccgaatcttgcttttcaggtgtgttggggtatccaggactggctgaggtgggagtgctgggttctgatgat

[0248] ggtgagtggtcttggtttctgttagtaagattcttacgtttgcctttagccatctggtagtctttggagttagttgttatagttgtctctggttggagcttgtttctcctgt

[0249] gattctgttagcctctgtcagcagacctgggtgtgtagctctatcctgagtttcagtgatcagagtactctctgcaggcaagctctcctcttgcaggaaaggtgc

[0250] acagatatctggcgtttggacctgcctcctagctgaagatgaaggcccgaaacagggcctatcccagaagctgtgtcgcttctgcagtctgcactctcacctg

[0251] cacagactagtctctgagggatctgagaaccaagatggctcccccaggtgctccacagcagagccctcctaagcgaggtgtgctcctctcctctggtgggg

[0252] aaggtgcccggatgtctggagcccgaaacggggtctgtcccagaagctgtgctgcttctgtggtccacatactcacttgtgcagactagtcacttccaagatg

[0253] gctcccctagttgctccacggcagaggccgctgccctttttggcacgatcgttgtttcttcttctttttttttatggccatcttggagccaagactcgaaagagcta

[0254] gtagattgttacatagctgctaaaggatgttctttagagataacttcagatagactgggcatgtagctcagttggtagtgtgctgacctaacaggctcaagctac

[0255] tgatcccagcactcccaaatcaggctgcagctggaggcaggggcagctcaaggtcacactcagctatgtagtagttggagcccagaatacctgggaccct

[0256] gcttttaaaaggaaatgaggctcggatgtgaagaacct

[0257] SEQ ID NO:4

[0258] tggcacgctgtagtgatttctctttcgatggagagattggtggtgttttaaatttgctctttgtggacaggatgaaaactctacctggtttccttgacatatg

[0259] aattaggaaggagtatttttgttccacattttttttctcagtgtgaggttttcttcacatcatgtgaatggacaatgaggtcattttatttacctaacttggggaagcaa

[0260] atattgacacagatttgcaactgtgaaatctcaacaccttgttctttgctttagaaagaccttggacacatttgtttttctttttattctattaggcaataactgaaaata

[0261] cccaaccgtatacatagtaacagtgacatgacctcccacacagatcccattatgaagcaggaaaccttcctgggttttcctttggcttctgtattttgctgcagg

[0262] ggactgatcccacagactaagacaggttacataggcagtctgtgttgacctcctgtctatcaacttgcagagttacttccacattttgctttgatacctttaaagtg

[0263] aagctcagagggatgcctcagtatgtgacaatgatctcttttgcccacgtctgcatattttgagtgtaaatatcttccctacaaggtccagctccatttctaaatatt

[0264] tttctatgataatcttcacagtcacagttctcactcctgctaaccattgcagccgcttaggctaccccaggaagcacagatccctaagtaagaccccatctacac

[0265] aaatcatttagttagagacacatcatagaattcctaggtccctgcgagcacggggtcatgatttattgatgagtatttcagaatggtggaagtatttgtttattgtct

[0266] actcagttcatggaaaacaacctttttggtaagcagacagtatctcctcttttttttaaatggtagaatatgatattgcatatacataagcacattttgaacatagctgaa

[0267] tataagtatcactgtttcatccttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttcttccggagcatataattttattgctcttctttttttaaagaatagaa

[0268] goesatcttttatactaggtccatgggctatctagtgtctggtttttgttcacccaagcagtgctagatacaagctccatcttgtgtagtaggctttaagtcaaaaca

[0269] gatttggttggttactctcacaacattgggctacctatttcctaccatatcttttaggcacaacaccaatgttgatccatgggtgtgtggctttgtttgttgttcttttcc

[0270] taggagcatgcagaatgacttctcacacctaagatactagtagatagggtgaagagtctatgtagtcaccagattgactttagatgttcagtgagttgagttg

[0271] cctttagcaataggaccttgttgacaggtggtgtagagaaaacatatggccatggtaacagcttgagttctttgcagattcccatgtaaacatttggacaacatct

[0272] footaaatgtaattgagtttgaatactggaagcttcttttggtgaggaaagataccctgttagaaacaagtctcctttgttatagggtgatttcatttatatgtgtaa

[0273] ttgcctttgtatgtgtatatttttctatcaatctgtctatgtatcatctatctaatctctctgtcatctaatatatgtatcatctgtcagttatccacctatcacacatatatag

[0274] ctattgtctatcaatcaccttttaatctaccttttcattctatcaaactattaatatatcaatcacttatgtatttgtgattgatctatcttttaaccatctaattatcacatatc

[0275] tttcttctattgatgatctattttcatctgtttctctgtctgcctctcatttccttttgaatagctctagctatcatatgcaaagggaagacattttcacaaaaaataagcag

[0276] tcttgagggtcctcatgatgttttcagtccctcagtgtctcattccaaaagcccaaagacaccatagaattcagacagaatgccatgtaacaaagccccacact

[0277] caatacccagttacaaccaccttagcagaacactaaagagattaaactatccttggctgccactaaaatcaggtagtcttcatggtaagtgtggatctagctttt

[0278] ggggaagtcctgtattctttgctaacccactttcatttcaagattgaacaaaacacaggaagatggtgccctgtgaggccttgacactcacatagcctcagtcc

[0279] agttagctccctgtccatgtgttcacaacagatgcttgtgataaagaaagccccacaggatgggtgaggtctgagtttctgaccacagatccttcctgttattttt

[0280] gatgttatttttatgtttgtgtggttatcttcttttgggtttgttgaaagaggatttctttttctttttctaaggtgtagtttcccttcttgtgttggcattttccatctattatcct

[0281] ttatagggctggatttgtggaaagatattgtgtaaatttggtttgtcatggaatatcttggtctctctgtctatgg

[0282] SEQ ID NO:5

[0283] cctatgaaaagacttaggtaaccacaaaataacagtgggagacatcaacacccaactgacaatattagacacatcattgaggcagaaaactaaca

[0284] gatttttcaggacctgactcacactgaccatatggcctagagatacctacagaactctccacccacacacaatatacattcttttttgcacatgg

[0285] cacatactctaaaatcaccacaatttggacataaaaacaatcctcagcaattaaaaaaaaaccgaatcataccaagcatattcttgaaccacacacacaatt

[0286] agtatagaaatcaatactagaaaattgttcaaaccataccattacatgaaaattgacaaactgctcctgaatgactatttggtaacaatcaattaagca

[0287] gaatcaaaatactttgaaactaatgagaacagagatacaaaaccagaatctctgggacagttaaagcaatgtcagagggagcttatggtgcaa

[0288] aatgcccaatcaaagtagaagacctcaattaaccacctacacacatgtagagaactagagaaatgagggcaaccaaccccaaagccagcag

[0289] aaaaagaaaaccaaaaacagagctgaaggaaggaatgaatgagatgcaaaaaaaaaagatcagcgaatccagaactggttcttttgaagaa

[0290] taatgagatagatagaccacatagctagactaatgaaaagagagagatccaaacaacaatcagcgacaaagggtaaccactaactacagaa

[0291] atacaaaaaaaccctcagagactactagaatactcctaggtagcagagttgtaaaacctagagaaatgaattctggaaatacaacctcccagatt

[0292] gaaccaggaagaaattgaatccctgaccagaccaataaggagttccaaaattgaatcagtaataaaagcctaacaacaagaataagcccaggaccagaca

[0293] gaatcacagccaaattctgccagatatattaagagctggtaccattcctactgaaactattccaaaaaaattgaggaggggggactcctccctaactcattcaa

[0294] tgaggccagcatcaccctgataccaaaacctggcagagacaccacaaaaatgaaaacttcaggccagtgcccttaatgaacatagatgtaaaaatcctcaa

[0295] caaaatactagcaaactgaactcagtagcacatcaaaaacctaatccaccacagtcaagtaggctttatacctgggatataaggttggttcaacatacgcaaa

[0296] tcaataaatatgattcatcacagaaacagaactaaaaacaaaaaccacatgatcatctcaataaatgcagaaaaggctttcaataaaattcaacatctcttcagg

[0297] ttaaaaactctcaacacactaggcgtcgaaggaacatacttcaaaataatagagccatctatgaaaaacccacagccaatgtaatactgaatgggcaaaagc

[0298] tggaagcattccccttgaaaactggcacaaaacaaggatgccctctctcacaactattcaacacagtactggaaatcctagccagagcaaccaggcaagag

[0299] aaagaaataaaaggcatccatataggaaaaaaggaagtcaaactctgcctgtttgtaggtgagatacaattctatatctaggaaaatcccataatctctgccca

[0300] aaagctccttgatctgtgatatggttaggctttgcgtccccacccaaatctcatgttgaattgtaatccccacgtgtcaagggagagaccaggtggaggtaatt

[0301] gaataacgggggcagtttctcccatgctgttatgatagtgagttctcacgagatctgatggttttataaggggtacttcccccttcactgggcacttctccttcctg

[0302] ctgccttgtgaagaaggtgccttgcttcccctttgccttctaccatgattgtaagtttccccaggcctccccagccatgatgaactgagtcaactaaaccttttttct

[0303] ttataaattgcccagtcttgggtggttctttatagcagtatgaacatggactaatacaatctaataaacaactttagcaatgcttcaggatacaaaatcaatgcaca

[0304] aaagtcagtagcattcatatacataaacaacatccaagctgagagccaaatcaagaagcaatcccattcacaatagctgtgaaaagaataaaatacctaaga

[0305] ataaaactaactaggaaagtgaaagatctctacaatgataattacaaacactgctcgaagaaatcagaaatgacacaaacaaacggaaaaatattccatgttc

[0306] atgagtggaaggaaatcaatattgttaaaatggccatactgcccaaagcaatttatagacttaatgctattcctatcgaactaccaatgacattcttcacaaaatta

[0307] gaaaaaaaaactattttaaaattcatatggaggacaggcacggtggctcacacctgtaatcccagcactttgggaggctgaggcaggcaaatcacaaggtc

[0308] aggagttcgagacgagcctggtcaacatggtgaaaccgcatctctactaaaagtacaaaaattatccaggcatggtggcgagcacctgtaatcccagctact

[0309] cgagaaactgaggcaggagaatcgcttgatcccaggaggcggagtgcagtgatccaagatcaagccactgcacttagacagcttgagcaacagaggga

[0310] gactccatctcaaaacaacaaaaaaaaacaaaaccatggatggagctggaggccattagcctaagcaaactaacacaggaacagaaaaccaaataccac

[0311] atgttctcacttatatgtaggagctaaacatcaagtacgtatagacataaagaagggaaaaacagacacaggggcctacttgagggtggagggtggtagga

[0312] gggtgaggatcgaaaaactacctattgggtattatgcttattacctgggtggcaaaataatctgtgtaccaaacccccaagacatgcaatttacctgtataacaa

[0313] acctgcacatgtactcctgaacctaaaaataaaagttaaaaaaagatatctgagattgtgtcatgcttcaggctcaaaatcctccattaatttctcatctaaaactt

[0314] aaagctaaagtacttaccatgactttacaaggcgatatgtaatccagccttttgtccctacaattaccccactaccttactacctgtataaatcagggttctccaga

[0315] gaaacagtaggagattgtgtatgcgtacatttatatatacacacagcacacacacacatacacggagagagagagatgtatttcaagaaattgaggaattgg

[0316] cttaagtgattgtgggaggctagcaggtctgagatctgtagggtagcctagaaagctgtaaactcttaggtgggaactgatggtgcaatgttgaagcagaac

[0317] ctcttttgactcagggaaacctcagttttgctttcacaaccttccaactaattggttaaggcccatccacattataaagaataatttcctttacttaaatactactgaat

[0318] atagatgttaaccgcatgtacaataccttcacaacaacacctagattagtgtttgattggataactggggactatagcctagtcatgttgacccatgaaactaact

[0319] gtcgcactatcctcaccctcattcaattctctccagctacataggtccccttgatgctccctcaacccaccaggcatgtttctacttcaaggccttagtatattact

[0320] gttcactctctccagatgctcttcctccagatatctacatagctagccccttctttcttttttttttttttttttttttttttttttgagacggaatctcgctctgtcgcccagg

[0321] ctggagtgcagtggcgcgatctcggctcactgcaagctccgcctcccgggttcacgccattctcctgcctcagcctcccaagtagctgggactacaggcgc

[0322] ccgccactacgcccgggtaattttttgtatttttagtagagacggggtttcaccgttttagccgggatggtctcgatcacctgacctcgtgatccgcccgcctcg

[0323] gcctcccaaagtgcccttctttcttttaagtctgtttgcttaggtctttcttttgaggtcttcttactctatacgtaaaactgcaaccctaccccacacagccataccct

[0324] acaagattccatttctccctctattctctatttttttccttggcatttatcactgtagagcattctatacattttgtttatttatcttctttattgtctacattgcccctagagtg

[0325] ttagtttcccaagggaacagatttgctttcatctattttgttcactggtattaccccaatacctggaatagtctctgaaacaaaccaagtgttttctatatatttaataa

[0326] ataaaggcctatatatcgtattattctgtttccttaatgtctcatttgccaccaccctcattcagggcactgtgacttctgggctacaggatgatgatgatgattttga

[0327] gacagggtctcactcctgggcccaggcctagagtgcagtggcgcaatctcagctgactgccacctccactttctaggctcaagctatcctcccacctcagcc

[0328] tcctaagtagctgggactataggcacatgccaccatgcccaaccactttttgtattttttgtaaagatgggggtctccctgtgtttcccaggctggtctcgagctc

[0329] ctgagctcaagagatccacccaccttggcctttcaaattgctgagattacaggcgtgagccactgtgcccagcctggtctagattaatgaaaagcttcctaact

[0330] ggtctccctccatctattcttgtcctcccttcaatgcattctccaaactgcagcatttctggggtgtgcatctgatggtatgggttccctgttcaacacctttcactag

[0331] cccttactgatcctatgctctgagtgattgagcctctgctaagttatctaacttctctctaccccacaccctgcttccaagacagaacttcttttttatttttgttgtttttt

[0332] gtttttgtttttttgagacagggtctcactctgtcaggctggagtgcagtgacacagtctcagctcactgcaacctccgactcccagattcaagtgattcttgtgct

[0333] tcggcctcctgagtagctgggactacaggcacataccaccaggcctggctaatttttgtattttagtagagacgggggttccaccgcgttgcccaggctggtc

[0334] tcgaactcctggcctcaagtgatccacctgcctcaggctcccaaagtgctaggattacaggcctgagccaccgtgcccagccagaacttcttattccttgacc

[0335] tcaccatgattcctcacctcctagtgttggcatacctggttttctgtatatggaacactcttatctccttcatcactggcttactcctgtcatcctcagggccccattt

[0336] atagctcaactcccacttcattctcagggctgggtaatttgcccttcctgtgaactcctgttgctccttatatgcagctgtcatagcactttgcaccaggtagaga

[0337] gtaaaagaggtaaaaggacaagagattttctgttttctttaaggaaaaggggaagccagaaaacagaggtaatcactgtagactaatttgacagtaaaatatgga

[0338] agacaggatgattttgttgaatatttataatgggagagatctgaaaatatttgaaagcaaaagacaacttttaggcaggagaaggtttggataaacagag

[0339] gaagagaattaggagaaataaagaaagattgagaaccagactatagataggggatgctctctttggtactacaaggaggattggtatggagtgat

[0340] agcaaaaggttttgtaatccaaaacatgccaaggctctgcatgaaatatgagtttggggtttgtgtatttggataggggtttttggaacctcaaggagaggtttat

[0341] atgtcttttaagaatacctaatactgtgttatcagtttgccagtatacatgaatagaacccttggcaatgaactaatgaaccccaccaggaaggaaaagaaaag

[0342] agtatgtatttgaagagattatattcccaaacctctacgtgtcagattactacagttagcctaaacaaaacagtagattaaatgggcataatttccataacgtcctcatt

[0343] ttatgatcttcagagaaatgatggcttagtgttgaccctttgtacaaagataattattcttactgaataaagataattacatttctaaaaccggtaggcagatggctc

[0344] tcaaaagggacattaatgaaaggaaaaaaaaagcagaccaaaatttagaaaatgtcttattttaataagattctaaatgctatggatgattttagctaagttgaga

[0345] aaattttactgaaagcatctcatcctaaaatgtgagtgaaagacaaattaatgataaatttaaggtaccttcattcatatcagtaggaaactgacctaaaatgtgat

[0346] caaagcaaattaaacatttttacttgttatgaaaatttgttgaaatgaagccagtgatgtcataaagacatacaaacaaaaagtctctaaaacttaaggaaaaaaa

[0347] tcaagggggggccaggtgcagtggctcacgcctgtaatcccaccattttgggaggccgagatgggtgaatcacttgaggtcaggagttcgagaccagcctg

[0348] gccaacatggtgaaaccccatctctattaaaaatacaaaaattagccaggcgtggcggcgggtgcctgtaatcccagctactcaggaggctgaggcacaa

[0349] gaatcacttgaacctgggaggcggaggttgcagcgagctgagattgtaccactgcactccagcctgggcgatagagcgagactccgtctcaaaaacaaac

[0350] aaaacaaaacaacaacaacaacaacaacaaaatcaagggacaaagtgtatgtttctagcattgttggggcaaatatgaaccctgtaattgaggaacatgaac

[0351] attccgtcccatagaaaacaagcaaaaagaaaaaaaaagagagagagctcactaggttgtctcatatgaaataaaatttctcagcaaccaaactatttgagaa

[0352] caatgttaagaagtgttgttcactgtctcaaagattcttaattcttgcataatacaaatatccagtgggtaaaataatctagattccactgtaaattttaaaactgcctt

[0353] tcaaaactgcatcagtactgtattgatttagatagcattggtctggccccctctgagcaagctggatattcaaatatagacattcaatctgaaaacagattcctgc

[0354] actgcagcccagcatgaggtctgatctaaagcacctatgtcaatatttaacagctccatatacatgtcacagcaaacctgcatgcagaaagatataaccaagt

[0355] actatttttttgccataaatttatccaaggttcaaactgaaagaaccaattacactgtctccaatataatataagtataggccaggtctcaaccttagttgagggtac

[0356] tcacgtttgggctaaataattcttggtttcaatgcacaaggctttcctatgaattgtaaagtgttcagcagcttccctggcctctacctattagatagatgacagtg

[0357] gcatgcctaaattgtggcaacaaagaatgtctccagacatgccaaaatgtaccctgtggaacaaaagtaatcctagttgagaaccactgatctaaggtaaaa

[0358] gttgaggctacagcatcaggcttcctgggtttaaatcctcactccacctcttgccagctgttactttatgcttacaaataagttacctaacttgcctatacatcagttt

[0359] ccacatctgtacaatgcagattttaatagcacctacttcaaatgtatgttgtgaagattaaacaagttgatatgtaaagcccttaaatcagtccatgatatatagtg

[0360] attaacacattcgcattcttactataattagtatttgatttatataaccattcactacttgcaaaactatttcataaacatattctcttatttgatccttatagccactttgtg

[0361] tgttggcatatgagggaaggttgaggttgttatgtgctctgaaggtctatgttgggtaatgtgtgcttttttaaaaaatatctattttctagttaaggaaactgtagca

[0362] tagagaaaataataagaggattgctagaaatcacacacacgtaacatacaatttaccattttaactattttgtaatgtacagatcagtagcattaagcacattcacatt

[0363] gttgtataaccatcaccaccattcacctgcggagctttcatcatcctaactgaaactccatactcattaaacaataattccccattccttttctcctcatcccctagta

[0364] accaccttactgctttctgtctctctctgtgtatttgactactctaggtacctcatataaatggagtcatacaatatttatacttttgcatctggcttatttcacttagcat

[0365] aatgtcattaaggttcatctatctagtagtatgtgtcagaatttccttcctttctaaggctgagtaatattccattgcatgtatatatcatattttgtttatctgttgatgaa

[0366] cactggggttgttcccattcttggctattggaagttgctagagtgctcatgtgttccttcaaaattcatattatgaaatcctatctcccagtgtgatggttttaggaa

[0367] gtgagacctttgggaggtgattaggtcatgagagctctgccatgggaataattgcccttatacaagagaacacggagggtttccttctccttctgccacttga

[0368] ggtgacagtgaaatgacagccatctatcaaccaggaagccagctcttaccagacaaagaatatgccagcgccttgatcttggacttcttgtgagaaataagtt

[0369] tctggaatttataaaccacctggtcaatggtaatttgttacagcagcagacaaagacagaagccaccccactttaaagttgagattcccgactcctggtctaat

[0370] gctctttccagtattatcacaaagggaactgatggttctgtatcctaaaatctcccccaaactcaagagattttcagggaaatggtcatcatgtgtaaaataatta

[0371] gccagttgaaatattgatgctgagcttttgtgaataaatgaatcaaatagctaagctgggttcattcagactttatctaagtttctttaagcttatctctacattctcctt

[0372] tttcatattgaaaaaaggtaacaatttccagaaagataccaacagatcattagctgatgcagtatctccagaattctttttcttttttttttagaccaggccttgttctgt

[0373] tgcttaggctggagcgcagtggcacaatcacagctcactgcaccacgaactcatggactgaagcaatcctcctgcctcagcctcctgggtagctgggacta

[0374] cagacacatgccaccatatccagctaatttttttctatagtttttttttttttttttgagacagggtcttactatgttgcccagactggtctcgaactcctgggctcaag

[0375] caatcctctgcctcagcctcccaaagtgctgggattacagatgtgagccactgcacctggcccctagaattgtttctagaggtgaaacttcaaggtgaaatata

[0376] gtacataactgcttttcagataaacaagtccagagagcacactctcttgtgctcttggcatcacttggcatcacttcatatttgaggtgtttcaaacccattagaac

[0377] acgtgaacaaggcctgcttccaaagctggcttccatctggtagtcccattaacaactgggcacaccccttccctagagctctgtgtagacagtacctcctccct

[0378] aggactacacaaggactgaaccagaaggaagaggacagagcaaagccatgaacatcatcctagaaatccttctgcttctgatcaccatcatctactcctact

[0379] tggagtcgttggtgaagtttttcattcctcagaggagaaaatctgtggctggggagattgttctcattactggagctgggcatggaataggcaggcagactact

[0380] tatgaatttgcaaaacgacagagcatattggttctgtgggatattaataaggtaatgtatacatcttccaactttttaaagtcacagagtaagatatgtattttaaga

[0381] attatttgacttaccatctacttatctttgtatttttgtttttcaaagtttgataaattccctggtcccttagtctgtatatgtgtcaggttagttagatgaagggaatgtaa

[0382] ttaagaactaagcagcgatttttatgacatggtgtgcaggttgatagaaagactcaggagccagtctccttccaagctgctaaatgaggcaagtcacatattat

[0383] ctctcagcctgttttcttggctctgaagtggggataataacttaggggatgggcaagaacgggatctgaaattacagctacaaacaaaagtcaaacgaaga

[0384] acttgcaacagaaacctttagtgcctcccctcatgcacaagcaacacagttctaaaatatttactgtctgaccctttacagaaaatgtttgccagtccgtagtcaa

[0385] aaggattaaataagtaatattttcagcacttagcatatgataaacgatacgtggcacatgataaacaataactgtgttaaataaaatatgtgcgcagtgagtcag

[0386] gcttttccttggacattagtatttttcctgtgttcttacttgtaaacactacattaacaaccccaaataaaactgaaggaactgaaatcttgtatcattttctctaaactt

[0387] gtaaattctggtaaggccatgaaaatatatgcagagaagtgtttacaggattttaggattggaaaaattgtgaagtactccttgagaatcacattttctgcaaatta

[0388] cagtggttttaattaccattatattattactttctcatgttctttgctgtcatgtttagttgaaacctaaaatgtctcttacacttagagaactaattcttttctgtttttttct

[0389] gaatagtgaagaatactatacaaaaaagctactacatttttatttaacagatatgagcatttatataatagaggagttgatgtatataaaaatgatttgccatcttttt

[0390] ggtctttgaagaaattcgaatgaactttctggaagatagcaagaatttacaaatagagaaaattgttgcctgctgttctcaggcatttgtccaaaaatataaataa

[0391] gtataaatctatgaaaagggcttgatgaaatctaaccttcaaatctctttccagatgtgtatttttggggaaagggctatatttattaagttttttttaaattttaaaattt

[0392] ccagagacaagagaaaagtaaattagaaggaagtcgtattaaaaatgacttaagggcgggtgcagtggctcacacctgtaatcccagcactttgggagac

[0393] ggaggtgggcagattgctggagcccaggagttcaagaccagcctgggcagcacagcaaaacccccaactctacaaaaaatacaaaaattagctgggtgc

[0394] gggggtgcacacccgtagtcccagctactcgggaggctgaggtgggaggatcgtttcagttcaggaagccaaggctgcaatgagctatgatggcatcatt

[0395] gcactccaagctgggcaatagagccaggctctgtctcaaaaaaaataaaaaaagacttaagaaaaataggtaacccaacctcaaaaattctctttgaatcatt

[0396] aaatttcatggttaaacatttaagctactgaatgattcactctaaggctgtaatgtaactcagatctcctttaggcgaggaagatgctggctgagttttcatcataa

[0397] ctggctccttttgccctgtgagatgagagacacagtagcagtttggctcttatgcaatctaaactgttgcgttgggaatacggttcaaaaaacacattggagttt

[0398] aagctaaagcaagtgttttgctaacaaaaagacaaggcatcacattttgcaattgtctagctcagttataaaacagaagaataggccggacgcggtggctcac

[0399] gcctgtaatcccagcactttgggaggccgagacgggcggatcacgaggtcaggagatcgagaccatcctggataacacagtgaaaccccgtctctactaa

[0400] aaatacaaaaaaattagccaggcgtagtggcgggcgcctgtagtcccagctactcgggaggctgaggcaggagaatggtgtgaacccgggaggcgga

[0401] gcttgcagtgagccgagatgacgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaactgaagaataat

[0402] taattcttcaatcaaaacatctgatgaatgctctggtaacttatgctctctactgacctagaaacaaatgagagagtatggtgtggtttgtgcaatctggcagtga

[0403] gcaagctaccaactaaatcagtgaaagactctcctattctttttttactcttctgcaatcccacaaaaggctatttgaggggatactgactttgagactgggtccta

[0404] acatccatgtttggggagttcaggctgctgctccagggtttagcctacagtagcgaaatacaaaggacccagagaccactcattcaaggtttgccctaaatag

[0405] cagcaacaccactgtcatctcaatacacgaagaatagggcttttcaggtatccttgcctctttgtcacagagaagagtttacagattgtgagacggaaaagtat

[0406] aatttttaaaaccttataaatattttctataaaagtcacctgaggtgaaaacttgaaaagaattataattttccagaatgtgagtcaagaaacattagagcaattttatc

[0407] ttaggaaagaggtctttgaatttaggctgaaagtaaattgctctgtctccatgtcctatggttatgggcaagtttggtacataaatgagaaatccatccagtggcc

[0408] ttgcccatctcactcccaaacacctgaaaatgtaatgttatatctcctagagtagcagcatggtctccctatgaaagtccttcttctttaaggagacttctttccct

[0409] tccctcctaggaggatgagtcagaatcatcaagaaaaatatgatgggcagaggcatacagtttaccattaccactagtttagaattactacttagcactttactg

[0410] cctattacatagttggtgctcaacaaatgtatgataaattaatggttgagtttttctttcttctccatattcatcttccatgacaccacgaagagcaatgtttttcaaga

[0411] atgttcttcaaggtttgaaagtagcctgctttagagaaactgcctactgtacagcctccaaccaagaggaaaagctgaaaaaagcatgaagggattttgttttgt

[0412] tttgtttgttttggttttaatatgagcattccctggcagaaaagccaggggtaatctcattgcaactaggcaatcactctcaagaaattttctaacaaataaggagg

[0413] ccaatttttattttattttgagacgaagtcccactctgtcacccaggttggagtgcaatggaatgatttcagctcactgcaacctccgcctcccgggttcaagtga

[0414] ttctcctgtctaaacttcccgagtagctgggattacaggctcccaccaccacgcccagctaattttttgtatttttagtagagatggggtttcaccattttggccag

[0415] actggtctcaaactcctgacctcaagtgatccaccctcctcggcctcctaaagtgctgggattacaggcgtgagccaccacacctgacccaggaggccaatt

[0416] tttaaaaggttaactaatcttcatgtccaaaatgaatgttaattgttcattttggacatgaatgttaattttttttttttttttttttgagacagagtctcactctgttgccca

[0417] ggctggagtccagtggcactatctccactcactgcaacttcctcctcccaggttcaagcaattatcctgcttcagcctcccaagtagctgggattacaggccca

[0418] caccatcaggcctggctaatttttgtatttttagtagagacggggtttcaccatgttggccaggctggtcttgaactcctgacctcgtgatccgccctcctcggc

[0419] caaccaaagtgctgggattacaggcgtgagccaccgcgcctagccgaatgttaattgtctaaaaatttttcttctccaatgtcttctcctccacttttttcggaaatt

[0420] gtttcttcctaattacagcgcggtgtggaggaaactgcagctgagtgccgaaaactaggcgtcactgcgcatgcgtatgtggtagactgcagcaacagaga

[0421] agagatctatcgctctctaaatcaggtgagactgcaggttcacaaatttcttcagattattttgtttcctaggacgctgacgtggaaaatgagaaaggtctttatga

[0422] ctgcctgatttaaattggattttagctgctaactgaagtagttatgtcaccaaggaaggatatatactttttttcttgtatgtaatccactcagctctgcccattattatt

[0423] gttcatattattaatcaatttcattctgatcagaagtgtgagcagtggcacagagtgactgacaaaagatttatcatcagggaatatggatcacttcctagttttgtt

[0424] ttagtcctattaactttgcagtaattccatcttctctttaattatttcccttgtgagattttattttggtgttaatgtagtcttctgtagaaaatgtaatattaataattattatc

[0425] acaattattttaaaagagtaaataccaaataatcacaatgaactaagcactctaacaaactttacattttttaattcaatccctacaataactctgtaaacttcatttta

[0426] cagataagcaaattatgactcagagaggttaagccagacccaggtcatgtagttattaggttatgaaaccaggatttctcaaccagcactttagaccaggtgc

[0427] ggtggttcacacatgtaatcccagcactttgtgaggccaaggtggaaggatcacatgagaccaagagttcaagaccagcccaggcaacatagtgagaccc

[0428] tatctctaaaaaaaaaaaaaaaaaaaaaaaaaagtttaaagaaaaacacatttttaaaaaatgaacactttaaaaatatttggtcagaatttatataggaattta

[0429] tcaacataaatgttaatttcactttactgataaacttgcaaaacatgatgtgctgggtactgaaatttagatgttaaaagaacagtttatcccacctttatgacagtgt

[0430] tcccttggcctccacgatttgagctcaacagtctgtcttgcctgaactctgagagacctcatacaatagaagaaagactctcatctctggattatattggtcccaa

[0431] aactttgagtttgaataatacacccagtgaaagtgttctttcaatttcaaaaggtgaagaaagaagtgggtgatgtaacaatcgtggtgaataatgctgggaca

[0432] gtatatccagccgatcttctcagcaccaaggatgaagagattaccaagacatttgaggtcaacatcctaggacatttttgggtgagtgtgagtcagaaacattt

[0433] ctgatttgtgcaccttctcttaagatacatgaaacttataacggagttcacatacttctggacaggaaactggccagatctttgccttaatcaagaatcattaaattt

[0434] gtttgagtagaagagccacagagtctctgacacaaaggacacagaattcaagtggacacaacacaccagaatgtaagctacttggtctgtcttgtccaccagt

[0435] atctgacacaaagcttggcatgtaccaggagctcaacaaatgtttgtggaggtttgttaagggttgtcagtgtacatcttttcaatgctgtcacttgtgacttcattt

[0436] ttttccctccacaccatgattttgtaatgtgtcctcattttgtggaattttagaatggaaaggacatcagaagtaattacttggatgtatataggatcgaggacacttt

[0437] tggacgagactctgaggcaagtgttctagatccatggggtgctggaactgagaaatgcagctatacagacctcatataattggttagttttgtgggagatgga

[0438] aatatcaacttcaactgcctttgtatagaaatttttatgattaatcttccagtgcctcaatattagtgtagaatctagggcagatctggattctagaagaaagaagaa

[0439] aaaaaagagatgtgtcccccttacctttaccagctcttcacatatgtgaattggctcccatgcccaccaaactacacggagacctcatacattagctacctatag

[0440] ctgcataacaaattatacaaaacttagtggtttaaagcaacaatgtatgttcactatcctctcacagtttctatgggttgggaatttggaggtagcttgggttggga

[0441] gttctagttctatgaatttgcataggatttattaaattcttataaaattttattgatgtttctcacaaaagaggtttttggaaaaaaagaaagacttgttttctgtaacatc

[0442] aacatataatatacaatattacaaatagggagatagtgaattcaatcatgattcattagtgtggtgtagaactctcagcttacactactcaactgtcttaatacagtt

[0443] acacaagatttcactcttttaattagaatgataaagccccaaaccaaaaaattatatgacaccaaattatcataaggaataattttagttctgaaaactctgaattttt

[0444] cccttaaatattgtttagatgacatatccaaaaaaggatctatttgattccttctgaagggaaggagggggagtactgagattagtgttggcatggggcttaccat

[0445] accaataaatttgtatctttatttctatcatttgtaaagaattaatcatggaatgcttggaagtattttatttcattgtataagttctctcaaatgcctttctgtcttaacaaa

[0446] aataaaactacctgatttggaaacctaacgtctatgtcattgtctttcttctttctgcaatgatccttaagatcacaaaagcacttcttccatcgatgatggagagaa

[0447] atcatggccacatcgtcacagtggcttcagtgtgcggccacgaagggattccttacctcatcccatattggtaagtatcacatgccagccatgttatatatttttat

[0448] actttgaagggagcattacacttcaaattgttaccactggagagtcctggttcttggcatcttgaacaaagaattggacaaaactcaccaacaaagccaggaa

[0449] agaatgaagcaacaaaagcagagatttattgaaaatgaaagtacgctttacagggtgggagtgggcccaagcacaggggctcaagagccaattacagaat

[0450] tttctggggtttaaataccccctagaggtttccactggttacttggtgtacgccctatgtaaatgaagaggatgaattaaagttacagagtcgtttactcagtgtac

[0451] accatatgtaaatggagaggatatttcctgtcatagctggagtgtttccatttgatttagttctaggaagtcagcatgaatcggccttatgttccctgcctccagac

[0452] cctgttctcctgcctcaagattacaatgctgagagcagagtgatttggatttacagaatttaaatttatagtagtttagaatgattttttaaatgactttttctaaaaca

[0453] atgaaaccaggttgtaattatatttaagatatttttagatttctgcaaactcctctgtagaacaatgagagaaaacagtaatgccaagcatgtttccattgtttcctg

[0454] gaaagaaacagaaaccccacagactgagaagcaaaacctacagaagctaaaatgaacacatgtctatgtcatggccttggtgcccaagataagacaat

[0455] cagagtggtccctggatcaaaacattttacagtgtgcttgtgccatgaaagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagagagagaaaacg

[0456] actctacctgactaaaagttgcagataccacactccatgcaccaccaaagacataaagggaaggaggtgagaggcgttaaggatgtactgctgtatttgcca

[0457] aatatcctttcctgtaaactcttctccagatcctcataataaaattaagaggccaaagtggcaaccattgtcaagagaaaaactatcaaccattgtcaagagaat

[0458] aactcagttattgagagagagaggagaaatgagcagagtcctacagaagtctgtcaacacagataccagttttgtagaatttctaaatgtatttttcctgattcat

[0459] atttttcaaaataaaagcagcaataaaaactgattagaaaacagtttgaagattcaatggaaaaaccttacatgtaggatggaaaactgaacattaagccaatc

[0460] aatagagttatttttgttcttttgttatcattgttgtttaagaaatgagatacgttcacaattctgcttaatcatgtaagaaaatgaaaatgaattgccatttatactctca

[0461] gaaaaatcacaagtggctgatttttggcttccacttgttcttaagccaaatgataccgccttctcacagaaagctgaggattggtttcactctcccttagctaaca

[0462] atgcttaataattctcttacagttccagcaaatttgccgctgttggctttcacagaggtctgacatcagaacttcaggccttgggaaaaactggtatcaaaacctc

[0463] atgtctctgcccagtttttgtgaatactgggttcaccaaaaatccaagcacaaaggtaaggtcaaaatcaagttagaatgggtatgtggtatgataaattgatatg

[0464] aaaactaatgagaaatgtttaggcaggccaactaatagaagaaaatgaaaaaggaaaaataatttttcttattattattatcttgaaattaaaggaataaagg

[0465] gggaaaacacattagggactagcaggaatgatcagccaccgatgaactgggatatttatttgtgtccgggagaaagcacatacatttgatcaccgttaccac

[0466] cctgtctttaaaatgcaaatgttccaaggaccagcaaataaattgagtatctagctccttagtcaaggtgaatttctgcaagaactcttgtctctggtgagacagg

[0467] atttgagaccacaagagaagaaaaattagtcctgaaaggagaagaaaaaagcaggaaggtgtggataagaacccgaaaattaagccatctgcttaacaaa

[0468] tttttctaatcctagtatatattctgctgcaggttaacaaaatatactaagcttaatgattcgaaaccaattttttactggaagggaattaatcctaaatatattcattca

[0469] aaagaactaaacaattctctgttgagtgccgcctcatttgaggatactgactcttacagcctgagttagctatgtggtctctgcagctggaatcactccctgcca

[0470] ctggagtccttcatggtgttagaccataggtactgttgactaaagaaaaaaaaaagtttttgtttttatttttgttttttttgagacagagtctcactctgtcacccagg

[0471] ctggagtacagtggcgcgatctcagctcaccgcaacctccgcctttctgggttcaagcaattctccttcctcagcctcctgagtatttggattacaggcgccca

[0472] ccaccacgcctggctaatttttgtatttttagtagagacggggtttcaccatgttggccaggctggtctcaaactcctgacctcaggtgtcctacctgccttggcc

[0473] tcctaaaatgctgggattacaggagtgagccaccatgcccggccaaaaaaataagtttttaaagaattaaaggtcatcctggctaacacagtgaaaccccgt

[0474] ctctactaaaaaacacaaaaaaattagccgggcgtggtggcgggcgcctgtagtcccagctgcgcgggaggctgaggcaggagaatggcgtgaacccg

[0475] ggaggcggagcttgcagtgagccgagatcgcgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaa

[0476] aaaaaagaattaaaggtgttaattttatttagaagccttactgaagactacagtcggaggcctatagcctgagagcagccctttagagaggttcagttgaactgt

[0477] tctgatagtgggggccatgtgctctatcctgtattgtcttcaaagcatctttccagagagctgcacattgtcacagagtcagggactttgtgaaattatgctgaca

[0478] accagaagtgagtaaacgtggcttcttacatttgctacgttgtctcacagtacttaataagtatgcaatatgtaagtaaatactatagtactattgcaactcctgatt

[0479] gttttcttagacaaggaattgggcccaataaaaaccctcttggtaggcattcaggcttcgtgtaccatgagctttcctaagggtatcctgccactcttggggaag

[0480] gcatgatagatgaggggagtaaggataatggaactctgggtacagggttcctgggggctaacttagaggtagacacaggcaatgctaaatatttgggattg

[0481] attttatagaggttgctagattgtgaatttccttagtaagggctaaggcattgatatgtaatgtcacacttggctccgaggctgggttgttggatccatgtagatga

[0482] aatcagggagagaaagggcagaacggagtaatttagaaatgtattgatttgtattactctctgttggcttgctattcaaggcagtggagaactcaatcacataat

[0483] aatctgcagcaaaccacagatcatcccagggaatgaagttttaacattcgctggctccctaactcctcacccagcctttacattcactggctgttcagtccatgc

[0484] ctggacatcttaatttgaatacaacatttaaatccatttttctgtcatcatcttgcactaacagacaattctacactaagcctatgtttatgaatatttctcaagagtac

[0485] atgtacacagccttcagtataaggaaaactggaagtatgacatacctccagttgtcatactccttgggcccctcttaaattctcattaaactgcaggataggcaa

[0486] gtcagaggtgaatctcaaatacgaaattcttaccggaaaggggttccaatccagaccccaagagagggttcttagatttctcgcaagaaataattcggggca

[0487] aggccatagtgcaaagcaaaagcaagtttattaggaaagtaaaggagtagagaacagctactccatggagaagaatggcttgagctgctccaccaagggt

[0488] atttagagttacttcttgattatatgctaaacaaggggtggattattcatgagttttccgggaaaagggtgagcaattcccagaactgagatttcctcccctttttag

[0489] gccatatagggtaacttcctgccattgccatggtatttgtaaactgtcatagtgctggtggaagtgtctcttagcttgctaatgtattatagttagcttataatgagc

[0490] agtgaggacaacagaggtcactttcatcaccatcttggttttggtgggttttggccggcttctttactgcaccctattttatcaacaaggtctttatgacctgaatct

[0491] tgtgccaacctcctatctcatcctgtgacaaagaatgccttaacttcctgggaatgcagcccagtaggtgtcagccttattttacccagaccctattcaagatgg

[0492] agttgctctgatttaaacgcctctgacaaaatgacgacctcaaaacaatccagctttatggaatacctccacaagaaagaaagtatacttagctatagaattttct

[0493] ccttgcatccaacagactttgagatgtcagatgtttccttcctgtcccatgattaatcctagccattcctctttcttgtctggctccactactccttaccatctaatgcc

[0494] tcgccaccattttgatattttgactaagtgagctatgaaacacacctactggatatgaaagtataagtttctgataacaaaacatcaacatgggatgtggaggaa

[0495] gtgggtagggtggcattaatgcagcaaatcctggaatattttaaatcttcattctaaatttagtaaaaatataggataattttcctgccatcatttacttataaaattaa

[0496] aattttagaaaataaaaataatattttcctctttttaatcacagattatggcctgtattggagacagatgaagtcgtaagaagtctgatagatggaatacttaccaat

[0497] aagaaaatgatttttgttccatcgtatatcaatatctttctgagactacagaagttaagtacagcacagaacacccaaatactaaaacaccaatagagcttttttttt

[0498] tgctttttttttttttagacagagtctcactctgtcaccctggctggattgcggtggttgcagtggcatgatcttggctcactgcaacctccgcctcctgggttcaag

[0499] caattctcatgcctcagacccccaagtaactgggattataggtgtgtgctgccacactacacccagctaatttttgtattttttgatagagagaggtttccccatgt

[0500] tggccaggctggactcgaactcctgacctcaagttatcctcctgtctcggcctcccaaagtgctgggattacagtcatgagccaccatgcctggcccaatag

[0501] agctattattatggagcatctttcagttgtgaaaattggcatggaaactctccatccctggggagaacagttatttcctctgttattttcctacccagtctataaaaa

[0502] gagagtgattcattttctctaccaaatctactgtctctgcccaaactttgctgaagactattctaactaaaggaaacacagtttaaaaagaatgcaatatagtgaa

[0503] gtagttaataataaagactccatttttaaaagtctgctggaagtttggttgggattgcactgaatctatagagcaattggggagtattgacatatcaacaatattga

[0504] gttttctaatccaagaacataatatctatttttaaaatcttcttcaaaatctttaaatctttaaattgtattttgtagtttttggtgtttaagtcttgcacatattttgtcagattt

[0505] attccaaagtatttcacgggttcttttttttttttttttttttttttttgagacagagtttcacccttgttgcccaggctggagtgcagtggcgtgatcttggctcactgca

[0506] gcttctgcctcctggcttcaagtgattctcctgcctcagcctcccaagtagctgggattacaggcacctgccccctcgcccaactaactttttgtgtttgtagtag

[0507] agacagggtttcaccatgttggccaggctggtctcgaactcctgacctcatgtgatccacctgcctcagcctcccaaagtgctgggattacaggcatgagcc

[0508] atcatgcccagccctatttgacggtttttgacgctaatgcaagtggcattttaaaaaattttatatttcccattgtttgttgtcagtatatattggatttttgtaatttgatc

[0509] tcatattttgcagtcttgctaaattgctaaacctctttttgctaaactcgataagcttttttttttttggtagattcctgggcctctaattttctttatgggaaagtttttaatt

[0510] acaaatttaatttctttaatagctacatggctattcaatttacttattaattcttggtaatgtgtgtctttcaaggaatttgtccatttcatctaagttgtagaatttctttgg

[0511] cataaatttgtacataacattcccttattatccttttaatgtctttagaatgtcttatttatttatttatttatttttattatatttttttgagacagagtctcgctctgttgccca

[0512] ggctggagtgcagtggcacaatcttggctcactgcaagctccgccttctgggttcatgccattctcctgcctcagcctccctagttgctgggactacaggcgc

[0513] ctgcaaccatgcccagcttattttttttttttttttttttttttttttttttttttttttttttttagtagagacggggtttcaccctgttagccaggatggtctcgatctcctgacc

[0514] tggtgatccgcccgcctcagcctcccaaagtgctgggattacaggcgtgagccaccaagcccagcctatttatttatttagtagagacagtctcactttgctgc

[0515] ccaggcaacaaaggttttgaatgcctggcctcaagcagtcctcctgccttggcctcccaaagtgctgggattacaggcatgagccactgcacctggccaaat

[0516] gaatatgctgataatatcttctttataaggatgacataagaataaaataatgtaatacaaacaaagcccctgtcactgaaaatgtatagacttcaaatgttaaagtc

[0517] ttagagaacagaatttatatgaaatagcaacagcaacaatttcccagaggaaatactctctcagctttcttctgaggagcagtttctaaattgaaattgtatcagt

[0518] gagaagataactatactaacttcataagccttgggcctttttgaaacaaatccatataaactatgaacaaacttgaaatagaacaatttgagaacagggtacaaa

[0519] ctgcattggtgtatcaatttcagtatttggttttagcttaaatagactgacttgagataacataaggagaaccttgacccccaagcaacatcatctcgcgagttga

[0520] ctaggccgggtgtggtgtctcacgcctgtaattccagcactttgggaggccacagcaggcagatcacttgaggtcaggcattcgagaccagcctggccaa

[0521] catggtgaaacctcagctctactaaagatacgaaaattagcaggcatagtggcctgcacctgtaataccaggcactcgcaggagaatcccttgaacccgga

[0522] aggcggagattgcagtaaaccatgattgtgccactgcactccagcctgggcaacaggagactctgtctcggaaaaataaattttttaaaaaaatgaaaaaaa

[0523] ataaaagttgactaaattagtgtcttggtactaagcactgtaggaagtgagtttcatggaaccccaactctcttggggcccaaagcaagtcatattaatattgaa

[0524] aattacatgcatatacatgcatatgaccaaggtgataaaaacaattattctgcctgagttggagaatagtatcccagtaaaataaacaagagtctcaaagtctttt

[0525] gtatcctttgaagctgtcatggtggtttgtaactaggcaacaggtatatattgttaatcttctttgcatttaattccttttatagagagacacaattttacgagcagatg

[0526] caattactagcatgaaggtttctttgtgagggtagttaaaaggcccacatgagctctcttcttatccttgtccttctttcagccagatcttccctgcccctttactcatt

[0527] ccatctttcacccacctacccccaaaacaaggaagtaaatcttgcattagtcaacaataccaaagtgattttcaatatgactttctctgcagaatgttattatttctg

[0528] cctctttacattcacatactgtcttcctttttttttttttttttttttttttttttagattgggtctcactctgttgcccaggctggagtgcagtggcttgatctcagctcactgt

[0529] aacctccacctcctgagttcaagcaattctcctgcctcagcctcctgagtagctgggattacaggcatgtgccaccacacctggctagtttttttgtatttttagta

[0530] gagacagggtttcaccatgttggtcaagctggtctcgaactcctgacctcatgatctgaccacctgtgcctctcaaagtgctgggattacaggcgtgagcacc

[0531] accgtgtgaatcggacccacaagagcactgaggtcggaagttcctattctctagaaagtataggaacttcatcagtccaggatacatagattaccacaactcc

[0532] caccgggccagccactctcttcctttcagttgcctactcatctcttatgcattcctggacatcagttgtccttttgaagctttcctccactatcccagcccatgtgaa

[0533] tcctccttccagttatagcccttaattctagatggctgatatttttcaataattgttttaagatgaccattttagcctatcagctaaacaatatcaaagacaatagctatt

[0534] tttcaagtactttagtttaccttattatagagtgcataatagatattcagtaaatagtaaaggagaggtgaaggcttgcatagaatggattctggtggtgtctcttgg

[0535] tgagcttttagcatcaagattaatcagcagtttcagcaatgagctcagaccttcagttttagatctttactcatatcagataagagagtgagaagagtggtatgtat

[0536] cagtgctttatttatatttgcatccaatttgaactatgaatattacaaaggtgcacacataggttcagacagattgatttaaaatgaccaaagatgacctgtcgtaa

[0537] gcaacctgggtatcttaagatgcactccttggagagggaatgttcctaaaaacattttcagagggacgaactgtatgaaattcagtaaaacataaatcatgagg

[0538] aaaactgattactctctttttgacatgaaatgagagttttaaatgcatggttacgattattaacgtactccgctgcaagacgttaataaagttactgttttgcaggcta

[0539] gaatgtcttgatgctgtaatcagaacacactttttcccctttcttccagcttcaaatgcagattcataattgggctgacttctaataactgcaatgttttctgccttgg

[0540] gcttgcagcagaagcctgacaaaatagtgtttgtttaggcaataatttatttatttatttattgagatggagtttcattcttgtcgcccaggctggagtgcaatggcg

[0541] tgatctcggctcactgcaacctctgtgttcaggcaataatttagactttaccttacttgtgattactatagcaattactatagccacaaggcataattttactgtctca

[0542] tttcaattttatgaatttgaatgtttttacacttttcctaatgaagtccactatgaagttatgtcaaaaaaaaaaagaaaaagaaagatgcacacgtaaaagagag

[0543] gtggttgcaagagaagaaaagaacggaggaaaattaaacgcaaaccagataactctcagcgtattctaaatgaccaaaaacagaactctgttgtcaaagatt

[0544] ttaaatggaaaatttttcaattgttttttcttttttgtacaggtttcttcctgaacgcgcctcagcgattttaaatcgtatgcagaatattcaatttgaagcagtggttgg

[0545] ccacaaaatcaaaatgaaatgaataaataagctccagccagagatgtatgcatgataatgatatgaatagtttcgaatcaatgctgcaaagctttatttcacattt

[0546] tttcagtcctgataatattaaaaacattggtttggcactagcagcagtcaaacggacaagattaattacctgtcttcctgtttctcaagaatatttacgtagtttttcat

[0547] aggtctgtttttcctttcatgcctctttaaaaacttctgtgcttacataaacatacttaaaaggttttctttaagatattttatttttccatttaaaggtggacaaaagctac

[0548] ctccctaaaagtaaatacaaagagaacttatttacacagggaaggtttaagactgttcaagtagcattccaatctgtagccatgccacagaatatcaacaagaa

[0549] cacagaatgagtgcacagctaagagatcaagtttcagcaggcagctttatctcaacctggacatattttaagattcagcatttgaaagatttccctagcctcttcc

[0550] tttttcattagcccaaaacggtgcaactctattctggactttattacttgattctgtcttctgtataactctgaagtccaccaaaagtggaccctctatatttcctccctt

[0551] tttatagtcttataagatcattatgaaaggtgaccgactctattttaaatctcagaattttaagttctagccccatgataacctttttctttgtaatttatgctttcatatat

[0552] ccttggtcccagagatgtttagacaattttaggctcaaaaattaaagctaacacaggaaaggaactgtactggctattacataagaaaaaatggacccaaga

[0553] gaaaaaggaagaaagaaaggttttttggtttttgt

[0554] gatctcagctcactgcaagctccacctccccgggttcacgccattctcctgcctcagcctcctgagtgctgggactacagggcgcccgccaccacgccaggc

[0555] taatttttgtattttttgtagagacggggtttcaccatgttagccaagatggtctcgatctcctgacctcgtgatccacccgctcggcctcccaaagtgctgggg

[0556] attacgggtgtgagccaccgtgcccagcctttttttttttaatagaaaaaataatccgactccactacatcaagactaatcttgttttgtgtgtttttcacatgtatta

[0557] tagaatgcttttgcatggactatcctcttgtttttattaaaaaacaaatgatttttttaaaagtcactaaaaaaacaattcactaaaaaataatatgtcattgtgctttaa

[0558] aaaaataacctcttgtagttataaaataaaacgtttgacttctaaactctgtctctatgagtgtccttcaagaaccaaggatgtggatggtaacagctacatctgg

[0559] cagttattaaatgaatccaggggttttctcaactctggtttctttgacagcaggagtgctggtttatcaactggaacttactgacccctttctatgtgtgagattaag

[0560] cacagtgctattgtttctaattttttttcttagtcaccggcaataatttttttcataacattcttttaatttcttgccttataatcaagaaatttatgagtaaaatgtattattttt

[0561] aaagtatggttacaaaaattcaaaagagaaacatggaaagtaaaaaatagaagcttacccccagcaatcccatttcccagaggtcacactgtcaatgcacaa

[0562] tcttccagtcttttactgatgcatgttcatgacaaatacattatattcttattgcatgggttgtatcagtagtttcataccatacattatgttctgtaacttgctctttttagc

[0563] SEQ ID NO: 6

[0564] acacaaggactgaaccagaaggaagaggacagagcaaagccatgaacatcatcctagaaatccttctgcttctgatcaccatcatctactcctactt

[0565] ggagtcgttggtgaagtttttcattcctcagaggagaaaatctgtggctggggagattgttctcattactggagctgggcatggaataggcaggcagactactt

[0566] atgaatttgcaaaacgacagagcatattggttctgtgggatattaataagcgcggtgtggaggaaactgcagctgagtgccgaaaactaggcgtcactgcgc

[0567] atgcgtatgtggtagactgcagcaacagagaagagatctatcgctctctaaatcaggtgaagaaagaagtgggtgatgtaacaatcgtggtgaataatgctg

[0568] ggacagtatatccagccgatcttctcagcaccaaggatgaagagattaccaagacatttgaggtcaacatcctaggacatttttggatcacaaaagcacttctt

[0569] ccatcgatgatggagagaaatcatggccacatcgtcacagtggcttcagtgtgcggccacgaagggattccttacctcatcccatattgttccagcaaatttgc

[0570] cgctgttggctttcacagaggtctgacatcagaacttcaggccttgggaaaaactggtatcaaaacctcatgtctctgcccagtttttgtgaatactgggttcac

[0571] caaaaatccaagcacaagattatggcctgtattggagacagatgaagtcgtaagaagtctgatagatggaatacttaccaataagaaaatgatttttgttccatc

[0572] gtatatcaatatctttctgagactacagaaggtttcttcctgaacgcgcctcagcgattttaaatcgtatgcagaatattcaatttgaagcagtggttggccacaaa

[0573] atcaaaatgaaatgaataaataagctccagccagagattatgcatgataatgatatgaatagtttcgaatcaatgctgcaaagctttattcacattttttcagtc

[0574] ctgataatattaaaaacattggtttggcactagcagcagtcaaacggacaagattaattacctgtcttcctgtttctcaagaatatttacgtagtttttcataggtctg

[0575] tttttcctttcatgcctcttaaaaactctctgtgcttacataaacatacttaaaaggtttctttaagatttttttttccatttaaaggtggacaaaagctacctccctaa

[0576] aagtaaaatacaaagagaacttatttacacagggaaggtttaagactgttcaagtagcattccaatctgtagccatgccacagaatatcaacaagaacacaga

[0577] tgagtgcacagctaagagatcaagtttcagcaggcagctttatctcaacctggacatattttaagattcagcatttgaaagatttccctagcctcttccttttcatt

[0578] agcccaaaacggtgcaactctattctggactttattacttgattctgtcttctgtataactctgaagtccaccaaaagtggaccctctatatttcctccctttttatagt

[0579] cttataagatcattatgaaaggtgaccgactctattttaaatctcagaattttaagttctagccccatgataaccttttctttgtaatttatgctttcatatatccttggt

[0580] cccagagatgtttagacaattttaggctcaaaaattaaagctaacacaggaaaaggaactgtactggctattacataagaaaacaatggacccaagagaagaa

[0581] aaagaagaaaggtttttttggtttttgtttttgtttttgttttttgttttttttttttgagatggagtctcactctgtcgcccaggctggagtgcagtggtatgatctca

[0582] gctcactgcaagctccacctccccgggttcacgccattctcctgcctcagcctcctgagtgctggactacaggcgcccgccaccacgccaggctaatttttt

[0583] gtatttttgtagagacggggtttcaccatgttagccaagatggtctcgatctcctgacctcgtgatccacccgctcggcctcccaaagtgctgggattacgg

[0584] gtgtgagccaccgtgcccagccttttttttttaatagaaaaaataatccgactccactacatcaagactaatcttgttttgtgtgtttttcacatgtattatagaatg

[0585] cttttgcatggactatcctcttgtttttattaaaaaacaaatgatttttttaaaagtcactaaaaaaacaattcactaaaaaaataatgtcattgtgctttaaaaaaaata

[0586] acctcttgtagttaaaaaaaacgtttgactttaaa

[0587] SEQ ID NO: 24

[0588] acaaaggactgaaccagaaggaagagcaagagcaaagccatgaaacatcatcctagaaaatccttctgcttctgatcaccatcatctactcctactt

[0589] ggagtcgttggtgaagtttttcattcctcagaggagaaaatctgtggctggggagattgttctcattactggagctgggcatggaataggcaggcagactactt

[0590] atgaatttgcaaaacgacagagcatattggttctgtgggatattaataagcgcggtgtggaggaaactgcagctgagtgccgaaaactaggcgtcactgcgc

[0591] atgcgtatgtggtagactgcagcaacagagaagagatctatcgctctctaaatcaggtgaagaaagaagtgggtgatgtaacaatcgtggtgaataatgctg

[0592] ggacagtatatccagccgatcttctcagcaccaaggatgaagagattaccaagacatttgaggtcaacatcctaggacatttttggatcacaaaagcacttctt

[0593] ccatcgatgatggagagaaatcatggccacatcgtcacagtggcttcagtgtgcggccacgaagggattccttacctcatcccatattgttccagcaaatttgc

[0594] cgctgttggctttcacagaggtctgacatcagaacttcaggccttgggaaaaactggtatcaaaacctcatgtctctgcccagtttttgtgaatactgggttcac

[0595] caaaaatccaagcacaagattatggcctgtattggagacagatgaagtcgtaagaagtctgatagatggaatacttaccaataagaaaatgatttttgttccatc

[0596] gtatatcaatatctttctgagactacagaagtttcttcctgaacgcgcctcagcgattttaaatcgtatgcagaatattcaatttgaagcagtggttggccacaaaa

[0597] tcaaaatgaaatgaataaataagctccagccagagatgtatgcatgataatgatatgaatagtttcgaatcaatgctgcaaagctttatttcacattttttcagtcct

[0598] gataatattaaaaacattggtttggcactagcagcagtcaaacggacaagattaattacctgtcttcctgtttctcaagaatatttacgtagtttttcataggtctgtt

[0599] tttcctttcatgcctcttaaaaacttctgtgcttacataaacatacttaaaaggttttctttaagatattttatttttccatttaaaggtggacaaaagctacctccctaaa

[0600] agtaaatacaaagagaacttatttacacagggaaggtttaagactgttcaagtagcattccaatctgtagccatgccacagaatatcaacaagaacacagaat

[0601] gagtgcacagctaagagatcaagtttcagcaggcagctttatctcaacctggacatattttaagattcagcatttgaaagatttccctagcctcttcctttttcatta

[0602] gcccaaaacggtgcaactctattctggactttattacttgattctgtcttctgtataactctgaagtccaccaaaagtggaccctctatatttcctccctttttatagtc

[0603] ttataagatacattatgaaaggtgaccgactctattttaaatctcagaattttaagttctagccccatgataacctttttctttgtaatttatgctttcatatatccttggtc

[0604] ccagagatgtttagacaattttaggctcaaaaattaaagctaacacaggaaaaggaactgtactggctattacataagaaacaatggacccaagagaagaaa

[0605] aggaagaaagaaaggttttttggtttttgttttgttttgttttgttttttgtttttttgagatggagtctcactctgtcgcccaggctggagtgcagtggtatgatctcag

[0606] ctcactgcaagctccacctcccgggttcacgccattctcctgcctcagcctcctgagtagctgggactacaggcgcccgccaccacgccaggctaattttttg

[0607] tattttttgtagagacggggtttcaccatgttagccaagatggtctcgatctcctgacctcgtgatccacccgcctcggcctcccaaagtgctgggattacgggt

[0608] gtgagccaccgtgcccagcctttttttttttaatagaaaaaataatccgactcccactacatcaagactaatcttgttttgtgtgtttttcacatgtattatagaatgctt

[0609] ttgcatggactatcctcttgtttttattaaaaacaaatgatttttttaaaagtcactaacaaaaacaattcactaaaaataaatatgtcattgtgctttaaaaaaataac

[0610] ctcttgtagttataaaataaaacgtttgacttctaaa

[0611] SEQ ID NO:23

[0612] MNIILEILLLLITIYSYLESLVKFFIPQRRKSVAGEIVLITGAGHGIGRQTTYEFAKRQSILVLW

[0613] DINKRGVEETAAECRKLGVTAHAYVVDCSNREEIYRSLNQVKKEVGDVTIVVNNAGTVYPADLL

[0614] STKDEEITKTFEVNILGHFWITKALLPSMMERNHGHIVTVASVCGHEGIPYLIPYCSSKFAAVGFHR

[0615] GLTSELQALGKTGICTSCLCPVFVNTGFTKNPSTRLWPVLETDEVVRSLIDGILTNKKMIFVPSYINI

[0616] FLRLQKVSS

[0617] SEQ ID NO:13

[0618] ctcttcacatatgtgaattggctcccat gcccaccaaactacacggagacctcatacatctacctatagctgcataaatacaaaacttagt

[0619] ggttaaagcaacaatgtatgttcactatcctctcacagtttctatgggttgggaatttggaggtagcttgggttgggagttctagttctatgaatttgcataggatt

[0620] tattaaattcttataaaattttattgatttctcacaaagaggttttggaaaaaagaaagactgttttctgtaacatcaacatataatatacaatattacaatatag

[0621] ggatagtgaattcaatcatcatcatcattagtgtggtgtagaactctcagcttacactactcactgtcttaatacagttacacaagatttcactcttttaattagaat

[0622] gataaagccccaaaccaaaaaattatatgacaccaaattatcataaggaataattttagttctgaaaactctgaatttttcccttaatattgtttagatgacatatcca

[0623] aaaaaggatctatttgattccttctgaagggaaggagggggagtactgagattagtgttggcatggggcttaccataccaataaatttgtatctttatttctatcat

[0624] ttgtaaagaattaatcatggaatgcttggaagtattttatttcattgtataagttctctcaaatgcctttctgtcttaacaaaaataaaactacctgatttggaaaccta

[0625] acgtctatgtcattgtctttcttctttctgcaatgatccttaagatcacaaaagcacttcttccatcgatgatggagagaaatcatggccacatcgtcacagtggctt

[0626] cagtgtgcggccacgaagggattccttacctcatcccatattggtaagtatcacatgccagccatgttatatatttttatactttgaagggagcattacacttcaaa

[0627] ttgttaccactggagagtcctggttcttggcatcttgaacaaagaattggacaaaactcaccaacaaagccaggaaagaatgaagcaacaaaagcagagatt

[0628] tattgaaaatgaaagtacgctttacagggtgggagtgggcccaagcacaggggctcaagagccaattacagaattttctggggtttaaataccccctagagg

[0629] tttccactggttacttggtgtacgccctatgtaaatgaagaggatgaattaaagttacagagtcgtttactcagtgtacaccatatgtaaatggagaggatatttcc

[0630] tgtcatagctggagtgtttccatttgatttagttctaggaagtcagcatgaatcggccttatgttccctgcctccagaccctgttctcctgcctcaagattacaatgc

[0631] tgagagcagagtgatttggatttacagaatttaaatttatagtagtttagaatgattttttaaatgactttttctaaaacaatgaaaccaggttgtaattatatttaagat

[0632] atttttagatttctgcaaactcctctgtagaacaatgagagaaaacagtaatgccaagcatgtttccattgtttcctggaataagaaacagaaaccccacagact

[0633] gagaagcaaaacctacagaagctaaaatgaacacatgtctatgtcatggccttggtgcccaagataagacaatcagagtggtccctggatcaaaacatttta

[0634] cagtgtgcttgtgccatgaaagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgagagagagagagagagaaaacgactctctacctgactaaaagttgcagatac

[0635] cacactccatgcaccaccaaagacataaagggaaggaggtgagaggcgttaaggatgtactgctgtatttgccaaatatcctttcctgtaaactcttctccag

[0636] atcctcataataaaattaagaggccaaagtggcaaccattgtcaagagaaaaactatcaaccattgtcaagagaataactcagttattgagagagagaggag

[0637] aaatgagcagagtcctacagaagtctgtcaacacagataccagttttgtagaatttctaaatgtatttttcctgattcatatttttcaaaataaaagcagcaataaaa

[0638] actgattagaaaacagtttgaagattcaatggaaaaaccttacatgtaggatggaaaactgaacattaagccaatcaatagagttatttttgttcttttgttatcatt

[0639] gttgtttaagaaatgagatacgttcacaattctgcttaatcatgtaagaaaatgaaaatgaattgccatttatactctcagaaaaatcacaagtggctgatttttggc

[0640] ttccacttgttcttaagccaaatgataccgccttctcacagaaagctgaggattggtttcactctcccttagctaacaatgcttaataattctcttacagttccagca

[0641] aatttgccgctgttggctttcacagaggtctgacatcagaacttcaggccttgggaaaaactggtatcaaaacctcatgtctctgcccagtttttgtgaatactgg

[0642] gttcaccaaaaatccaagcacaaggtaaggtcaaaatcaagttagaatgggtatgtggtatgataaattgatatgaaaactaatgagaaatgtttaggcaggc

[0643] caactaatagaagaaaatgaaaaaggaaaaataatttttcttattattattattatcttgaaattaaaggaataaagggggaaaacacattagggactagcagga

[0644] atgatcagccaccgatgaactgggatatttatttgtgtccgggagaaagcacatacatttgatcaccgttaccaccctgtctttaaaatgcaaatgttccaagga

[0645] ccagcaaataaattgagtatctagctccttagtcaaggtgaatttctgcaagaactcttgtctctggtgagacaggatttgagaccacaagagaagaaaaatta

[0646] gtcctgaaaggagaagaaaaaagcaggaaggtgtggataagaacccgaaaattaagccatctgcttaacaaatttttctaatcctagtatatattctgctgcag

[0647] gttaacaaaatatactaagcttaatgattcgaaaccaattttttactggaagggaattaatcctaaatatattcattcaaaagaactaaacaattctctgttgagtgc

[0648] cgcctcatttgaggatactgactcttcagcctgagttagctatgtggtctctgcagctggaatcactccctgccactggagtccttcatggtgttagaccatag

[0649] gtactgttgactaaagaaaaaaaaaagtttttgtttttattttgttttttttgagacagagtctcactctgtcacccaggctggagtacagtggcgcgatctcagct

[0650] caccgcaacctccgcctttctgggttcaagcaattctccttctccagcctcctgagtatttggattacaggcgcccaccaccacgcctggctaatttttgtattttt

[0651] agtagagacggggtttcaccatgttggccaggctggtctcaaactcctgacctcaggtgtcctacctgccttggcctcctaaaatgctgggattacaggagtg

[0652] agccaccatgcccggccaaaaaaaataagtttttaaagaattaaaggtcatcctggctaacacagtgaaaccccgtctctactaaaaaacacaaaaaaaattagc

[0653] cgggcgtggtggcgggcgcctgtagtcccagctgcgcgggaggctgaggcaggagaatggcgtgaacccgggaggcggagcttgcagtgagccga

[0654] gatcgcgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagaattaaaaggtgttaatttttt

[0655] agaagccttactgaagactacagtcggaggcctatagcctgagagcagccctttagagaggttcagttgaactgttctgatagtggggggccatgtgctctatc

[0656] ctgtattgtcttcaaagcatctttccagagagctgcacattgtcacagagtcagggactttgtgaaattatgctgacaaccagaagtgagtaaacgtggcttctt

[0657] acatttgctacgttgtctcacagtacttaataagtatgcaatatgtaagtaaatactatagtactattgcaactcctgattgttttcttagacaaggaattgggccca

[0658] ataaaaaccctcttggtaggcattcaggcttcgtgtaccatgagctttcctaagggtatcctgccactcttggggaaggcatgatagatgaggggagtaagga

[0659] taatggaactctgggtacagggttcctgggggctaacttagaggtagacacaggcaatgctaaatatttgggattgattttatagaggttgctagattgtgaattt

[0660] ccttagtaagggctaaggcattgatatgtaatgtcacacttggctccgaggctgggttgttggatccatgtagatgaaatcagggagaaagggcagaacg

[0661] gagtaatttagaaatgtattgatttgtattactctctgttggcttgctattcaaggcagtggagaactcaatcacataataatctgcagcaaaccacagatcatccc

[0662] agggaatgaagttttaacattcgctggctccctaactcctcacccagcctttacattcactggctgttcagtccatgcctggacatcttaatttgaatacaacatttt

[0663] aaatccatttttctgtcatcatcttgcactaacagacaattctacactaagcctatgtttatgaatatttctcaagagtacatgtacacagccttcagtataaggaaa

[0664] actggaagtatgacatacctccagttgtcatact

[0665] SEQ ID NO:25

[0666] ccaaagagctattattatggagcatctttcagttgtgaaaattggcatggaaactctccatccctggggagaacagttatttcctctgttattttcctacc

[0667] cagtctataaaaagagagtgattcattttctctaccaaatctactgtctctgcccaaactttgctgaagactattctaactaaaggaaacacagtttaaaaagaatg

[0668] caatatagtgaagtagttaataataaagactccatttttaaaagtctgctggaagtttggttgggattgcactgaatctatagatcaattggggagtattgacatat

[0669] caacaatattgagttttctaatccaagaacataatatctatttttaaaatcttcttcaaaatctttaaatctttaaattgtattttgtagtttttggtgtttaagtcttgcacat

[0670] attttgtcagatttattccaaagtatttcacgggttcttttttttttttttttttttttttttgagacagagtttcacccttgttgcccaggctggagtgcagtggcgtgatctt

[0671] ggctcactgcagcttctgcctcctggcttcaagtgattctcctgcctcagcctcccaagtagctgggattacaggcacctgccccctcgcccaactaattttttg

[0672] tgtttgtagtagagacagggtttcaccatgttggccaggctggtctcgaactcctgacctcatgtgatccacctgcctcagcctcccaaagtgctgggattaca

[0673] ggcatgagccatcatgcccagccctatttgacggtttttgacgctaatgcaagtggcattttaaaaaattttatatttcccattgtttgttgtcagtatatattggatttt

[0674] tgtaatttgatctcatattttgcagtcttgctaaattgctaaacctctttttgctaaactcgatcagcttttttttttttggtagattcctgggcctctaattttctttatggga

[0675] aagtttttaattacaaatttaatttctttaatagctacatggctattcaatttacttattaattcttggtaatgtgtgtctttcaaggaatttgtccatttcatctaagttgtag

[0676] aatttctttggcataaatttgtacataacattcccttattatccttttaatgtctttagaatgtcttatttatttatttatttatttttattatatttttttgagacagagtctcgctc

[0677] tgttgcccaggctggagtgcagtggcacaatcttggctcactgcaagctccgccttctgggttcatgccattctcctgcctcagcctccctagttgctgggact

[0678] acaggcgcctgcaaccatgcccagcttattttttttttttttttttttttttttttttttttttttttttttttagtagagacggggtttcaccctgttagccaggatggtctcgat

[0679] ctcctgacctggtgatccgcccgcctcagcctcccaaagtgctgggattacaggcgtgagccaccaagcccagcctatttatttatttagtagagacagtctc

[0680] actttgctgcccaggcaacaaaggttttgaatgcctggcctcaagcagtcctcctgccttggcctcccaaagtgctgggattacaggcatgagccactgcac

[0681] ctggccaaatgaatatgctgataatatcttctttataaggatgacataagaataaaataatgtaatacaaacaaagcccctgtcactgaaaatgtatagacttcaa

[0682] atgttaaagtcttagagaacagaatttatatgaaatagcaacagcaacaatttcccagaggaaatactctctcagctttcttctgaggagcagtttctaaattgaa

[0683] attgtatcagtgagaagataactatactaacttcataagccttgggcctttttgaaacaaatccatataaactatgaacaaacttgaaatagaacaatttgagaac

[0684] agggtacaaactgcattggtgtatcaatttcagtatttggttttagcttaaatagactgacttgagataacataaggagaaccttgacccccaagcaacatcatct

[0685] cgcgagttgactaggccgggtgtggtgtctcacgcctgtaattccagcactttgggaggccacagcaggcagatcacttgaggtcaggcattcgagacca

[0686] gcctggccaacatggtgaaacctcagctctactaaagatacgaaaattagcaggcatagtggcctgcacctgtaataccaggcactcgcaggagaatccct

[0687] tgaacccggaaggcggagattgcagtaaaccatgatgtgccactgcactccagcctgggcaacagagactctctcggaaaaataaatttttaaaaaa

[0688] atgaaaaaaataaaagttgactaaattagtgtcttggtactaagcactgtaggaagtgagtttcatggaaccccaacctcttggggcccaaagcaagtcata

[0689] ttaatattgaaaattacatgcatatacatgcatatgaccaaggtgataaaaacaattattctgcctgagttggaatagtatcccagtaaaataaacaagagtct

[0690] caaagtcttttgtatcctttgaagctgtcatggtggtttgtaactaggcaacggtatatattgttaatcttctttgcatttaattccttttatagagacacaatttac

[0691] gagcagatgcaattactagcatgaaggtttctttgtgagggtagttaaaggcccacatgagctctctcttatccttgtccttctttca

[0692] SEQ ID NO:26

[0693] ccttgggcccctcttaaattctcattaaactgcaggataggcaagtcagaggtgaatctcaaatacgaaattcttaccggaaaggggttccaatccag

[0694] accccaagagagggttcttagatttcgcaagaataattcggggcaaggccatagtgcaaagcaaaagcaagtttattaggaaagtaaaggagtagaga

[0695] acagctactccatggagaagaatggcttgagctgctccaccaagggtatttagagttacttcttgattatatgctaaacaaggggtggattattcatgagttttcc

[0696] gggaaaagggtgagcaattcccagaactgagatttcctcccctttttaggccatatagggtaacttcctgccattgccatggtatttgtaaactgtcatagtgctg

[0697] gtggaagtgtctcttagcttgctaatgtattatagttagcttataatgagcagtgaggacaacagaggtcactttcatcaccatcttggttttggtgggttttggcc

[0698] ggcttctttactgcaccctattttatcaacaaggtctttatgacctgaatcttgtgccaacctcctatctcatcctgtgacaaagaatgccttaacttcctgggaatg

[0699] cagcccagtaggtgtcagccttattttacccagaccctattcaagatggagttgctctgatttaaacgcctctgacaaaatgacgacctcaaaacaatccagctt

[0700] tatggaatacctccacaagaaagaaagtatacttagctatagaattttctccttgcatccaacagactttgagatgtcagatgtttccttcctgtcccatgattaatc

[0701] ctagccattcctctttcttgtctggctccactactccttaccatctaatgcctcgccaccattttgatattttgactaagtgagctatgaaacacacctactggatatg

[0702] aaagtataagtttctgataacaaaacatcaacatgggatgtggaggaagtgggtagggtggcattaatgcagcaaatcctggaatattttaaatcttcattctaa

[0703] atttagtaaaaatataggataattttcctgccatcatttacttataaaattaaaattttagaaaataaaaataatattttcctctttttaatcacagattatggcctgtattg

[0704] gagacagatgaagtcgtaagaagtctgatagatggaatacttaccaataagaaaatgatttttgttccatcgtatatcaatatctttctgagactacagaagtaag

[0705] tacagcacagaacacccaaatactaaaacaccaatagagcttttttttttgctttttttttttttagacagagtctcactctgtcaccctggctggattgcggtggttg

[0706] cagtggcatgatcttggctcactgcaacctccgcctcctgggttcaagcaattctcatgcctcagacccccaagtaactgggattataggtgtgtgctgccaca

[0707] ctacacccagctaatttttgtattttttgatagagacaggtttccccatgttggccaggctggactcgaactcctgacctcaagttatcctcctgtctcggcctccc

[0708] aaagtgctgggattacagtcatgagccaccatgcctggc

[0709] SEQ ID NO:30

[0710] gttctgatagtgggggccatgtgctctatcctgtattgtcttcaaagcatctttccagagagctgcacattgtcacagagtcagggactttgtgaaattat

[0711] gctgacaaccagaagtgagtaaacgtggcttcttacatttgctacgttgtctcacagtacttaataagtatgcaatatgtaagtaaatactatagtactattgcaac

[0712] tcctgattgttttcttagacaaggaattgggcccaataaaaaaccctcttggtaggcattcaggcttcgtgtaccatgagctttcctaagggtatcctgccactcttg

[0713] gggaaggcatgatagatgaggggagtaaggataatggaactctgggtacagggttcctgggggctaacttagaggtagacacaggcaatgctaaatattt

[0714] gggattgattttatagaggttgctagattgtgaatttccttagtaagggctaaggcattgatatgtaatgtcacacttggctccgaggctgggttgttggatccatg

[0715] tagatgaaatcagggagagaaagggcagaacggagtaatttagaaatgtattgatttgtattactctctgttggcttgctattcaaggcagtggagaactcaat

[0716] cacataataatctgcagcaaaccacagatcatcccagggaatgaagttttaacattcgctggctccctaactcctcaccacagcctttacattcactggctgttca

[0717] gtccatgcctggacatcttaatttgaatacaacatttaaatccatttttctgtcatcatcttgcactaacagacaattctacactaagcctatgtttatgaatatttctc

[0718] aagagtacatgtacacagccttcagtataaggaaaactggaagtatgacatacctccagttgtcatact

[0719] SEQ ID NO: 31

[0720] ccaaagagctattattatggagcatctttcagttgtgaaaattggcatggaaactctccatccctggggagaacagttatttcctctgttattttcctacc

[0721] cagtctataaaaagagagtgattcattttctctaccaaatctactgtctctgcccaaactttgctgaagactattctaactaaaggaaacacagtttaaaaagaatg

[0722] caatatagtgaagtagttaataataaagactccatttttaaaagtctgctggaagtttggttgggattgcactgaatctatagatcaattggggagtattgacatat

[0723] caacaatattgagttttctaatccaagaacataatatctatttttaaaatcttcttcaaaatctttaaatctttaaattgtattttgtagtttttggtgtttaagtcttgcacat

[0724] attttgtcagatttattccaaagtatttcacgggttcttttttttttttttttttttttttttgagacagagtttcacccttgttgcccaggctggagtgcagtggcgtgatctt

[0725] ggctcactgcagcttctgcctcctggcttcaagtgattctcctgcctcagcctcccaagtagctgggattacaggcacctgccccctcgcccaactaattttttg

[0726] tgtttgtagtagagacagggtttcaccatgttggccaggctggtctcgaactcctgacctcatgtgatccacctgcctcagcctcccaaagtgctgggattaca

[0727] ggcatgagccatcatgcccagccctatttgacggtttttgacgctaatgcaagtggcattttaaaaaattttatatttcccattgtttgttgtcagtatatattggatttt

[0728] tgtaatttgatctcatattttgcagtcttgctaaattgctaaacctctttttgctaaactcgatcagcttttttttttttggtagattcctgggcctctaattttctttatggga

[0729] aagtttttaattacaaatttaatttctttaatagctacatggctattcaatttacttattaattcttggtaatgtgtgtctttcaaggaatttgtccatttcatctaagttgtag

[0730] aatttctttggcataaatttgtacataacattcccttattatccttttaatgtctttagaatgtcttatttatttatttatttatttttattatatttttttgagacagagtctcgctc

[0731] tgttgcccaggctggagtgcagtggcacaatcttggctcactgcaagctccgccttctgggttcatgccattctc

[0732] SEQ ID NO:42

[0733] GAATTCTAATACGACTCACTATAGGGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAA

[0734] TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT

[0735] TAAAGGATCC

[0736] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0737] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0738] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) protein.

2. The construction method according to claim 1, characterized in that The amino acid sequence of the chimeric HSD17B13 protein comprises an amino acid sequence that is identical to at least 50 to 300 consecutive amino acids of the human HSD17B13 protein; Preferably, the amino acid sequence of the human or chimeric HSD17B13 protein comprises SEQ ID NO: 2 or SEQ ID NO: 23; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 2 or SEQ ID NO:

23.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous HSD17B13 locus, the nucleotide sequence of the non-human animal endogenous HSD17B13 is replaced with a nucleotide sequence comprising human HSD17B13.

4. The construction method according to claim 3, characterized in that The nucleotide sequence of human HSD17B13 comprises a nucleotide sequence encoding a human or chimeric HSD17B13 protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23; or comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 23; Preferably, the nucleotide sequence of human HSD17B13 comprises all of exon 1 to exon 7 of the human HSD17B13 gene; Preferably, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence from the start codon to the stop codon of the human HSD17B13 gene, further preferably further comprises a 5'UTR and / or a 3'UTR, and more preferably further comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR; Preferably, the nucleotide sequence of human HSD17B13 comprises a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR to a continuous nucleotide sequence of at least 50 bp downstream of the 3'UTR; Preferably, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 26 in which position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 26, or SEQ ID NO: 26 in which position 1269 is mutated from T to TT and / or position 1522 is mutated from C to G; Preferably, the nucleotide sequence of human HSD17B13 comprises the nucleotide sequence represented by SEQ ID NO: 5, or SEQ ID NO: 5 in which positions 22641-22642 are mutated from TT to T and / or position 22895 is mutated from G to C; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence represented by SEQ ID NO: 5, or SEQ ID NO: 5 in which positions 22641-22642 are mutated from TT to T and / or position 22895 is mutated from G to C.

5. The construction method according to any one of claims 3-4, characterized in that: The nucleotide sequence of the non-human animal endogenous HSD17B13 comprises a nucleotide sequence encoding an endogenous non-human animal HSD17B13 protein, preferably comprises a nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding SEQ ID NO: 1 or nucleotides 1-271 of SEQ ID NO: 1; Preferably, the nucleotide sequence of the non-human animal endogenous HSD17B13 comprises all or part of exon 1 to exon 6 of the non-human animal endogenous HSD17B13 gene, and further preferably further comprises all or part of intron 6; Preferably, the nucleotide sequence of the non-human animal endogenous HSD17B13 comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR of the non-human animal endogenous HSD17B13 gene; Further preferably, the nucleotide sequence of the non-human animal endogenous HSD17B13 comprises at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR of the non-human animal endogenous HSD17B13 gene to all or part of intron 6; The portion of intron 6 includes at least 50 bp of continuous nucleotide sequence.

6. The construction method according to any one of claims 1 to 5, characterized in that: The nucleotide sequence encoding the human or chimeric HSD17B13 protein or the nucleotide sequence of human HSD17B13 is operably linked to an exogenous HSD17B13 regulatory element, and the exogenous HSD17B13 regulatory element is preferably a human HSD17B13 regulatory element; Preferably, the endogenous HSD17B13 protein of the non-human animal is not expressed or is expressed at a reduced level compared to HSD17B13 in wild-type animals; Preferably, the modified HSD17B13 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

7. The construction method according to any one of claims 1 to 6, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified HSD17B13 gene in the genome of the non-human animal comprises SEQ ID NO: 6 or SEQ ID NO: 24; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 6 or SEQ ID NO: 24; Preferably, the non-human animal further comprises a nucleotide sequence encoding other human or chimeric proteins, and the other human or chimeric proteins preferably include at least one of PNPLA3, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

8. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease associated with HSD17B13, characterized in that The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 7; 2) Determining the effects of therapeutic agents on non-human animals or diseases associated with HSD17B13; Preferably, the therapeutic agent comprises an antibody targeting HSD17B13, a nucleic acid drug targeting HSD17B13 and / or a polypeptide drug; Preferably, the disease associated with HSD17B13 includes non-alcoholic steatohepatitis.

9. A cell, tissue or organ, characterized in that: The cells, tissues or organs are derived from non-human animals obtained by the construction method according to any one of claims 1 to 7.

10. Use of a non-human animal obtained by the construction method according to any one of claims 1 to 7 or a cell, tissue or organ according to claim 9, characterized in that: The applications include: A) Applications in product development involving HSD17B13-related immune processes in human cells; B) Application as a model system for HSD17B13-related research in pharmacology, immunology, microbiology, and medicine; C) applications involving the production and use of animal experimental disease models for the study of the etiology of HSD17B13 and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human HSD17B13 signaling pathway modulators; or E) Study the function of the HSD17B13 gene, study the drugs and their efficacy targeting the human HSD17B13 target site, and study the application of drugs in the treatment of diseases related to HSD17B13 (such as non-alcoholic steatohepatitis).

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