Construction method and application of S100A8 / A9 humanized mouse model

Abstract

The invention relates to a construction method and application of an S100A8 / A9 humanized mouse model. Specifically, the invention relates to an expression vector which comprises a first gene expression cassette for expressing human S100A8 and a second gene expression cassette for expressing human S100A9, and the expression directions of the first gene expression cassette and the second gene expression cassette are opposite. The invention further particularly relates to a method for expressing the S100A8 and S100A9 heterodimers, a construction method of the animal model and application of the animal model constructed according to the method in drug screening or disease mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the construction method and application of the S100A8 / A9 humanized mouse model, and also specifically relating to the expression vector used in the construction process. Background Technology

[0002] In inflammatory responses, neutrophils and monocytes are among the first innate immune cells to arrive at the site of inflammation. The recruitment of immune cells during inflammation is a highly regulated process. It requires not only classic adhesion molecules for leukocyte recruitment, such as selectins, integrins, and their ligands, but also cell-activating factors, such as chemokines and chemokine receptors, as well as alarm molecules and their respective pattern recognition receptors. Among alarm molecules, the S100A8 / A9 heterodimer has been shown to play a crucial role in inflammatory responses.

[0003] S100A8 / A9 is present in the cytoplasm of neutrophils and monocytes and belongs to the Ca2+ family. 2+ S100A8 / A9 belongs to the large family of binding proteins. After being passively or actively released from neutrophils or monocytes, S100A8 / A9 primarily exerts its effector function by binding to two pattern recognition receptors: Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE). Since inflammation-induced release of S100A8 / A9 from neutrophils and monocytes begins during the intravascular phase of leukocyte recruitment, plasma S100A8 / A9 levels have been used as an inflammatory biomarker for various inflammatory diseases. Furthermore, the abundant extravasation of neutrophils (e.g., in the intestines of patients with active inflammatory bowel disease) is also a rich source of S100A8 / A9 release, thus fecal S100A8 / A9 levels can serve as an indicator of disease activity in these patients.

[0004] In summary, S100A8 / A9 is a key regulator in the inflammatory response, and this alarm factor is an interesting drug target that can block the activation of innate immune cells in autoimmune diseases. Furthermore, increasing basic and clinical research has found that S100A8 / A9 can serve not only as a diagnostic or prognostic biomarker for various diseases (such as acute myocardial infarction and malignant solid tumors), but also participate in disease progression. However, there is currently a lack of fully humanized S100A8 / A9 animal models available for research on pathological processes, pathogenesis, and drug screening.

[0005] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0006] In view of this, this application provides a novel expression vector, based on which a mouse model expressing humanized S100A8 / A9 heterodimers can be constructed with high efficiency and stability, laying a solid foundation for studying the pathological process, pathogenesis, and drug screening of S100A8 / A9-related diseases. Furthermore, the sequence introduced into the mouse model contains all the genes of the coding regions and non-coding regions between the coding regions of human S100A8 and S100A9, not only satisfying the expression of humanized S100A8 / A9 heterodimer proteins but also allowing for research on the non-coding regions of the S100A8 and S100A9 genes.

[0007] According to one embodiment of this application, an expression vector is provided, the expression vector comprising: a first gene expression cassette for expressing human S100A8; and a second gene expression cassette for expressing human S100A9; wherein the expression directions of the first gene expression cassette and the second gene expression cassette are opposite.

[0008] According to one embodiment of this application, a method for expressing S100A8 and S100A9 heterodimers is also provided, the method comprising introducing the expression vector described in this application into cells.

[0009] According to one embodiment of this application, a method for constructing an animal model is also provided, the method comprising introducing the expression vector described in this application into a specific site of the animal genome.

[0010] According to one embodiment of this application, the use of the expression vector described in this application in expressing S100A8 and S100A9 heterodimers is also provided.

[0011] According to one embodiment of this application, the use of the expression vector described in this application in constructing animal models is also provided.

[0012] According to one embodiment of this application, the use of an animal model obtained by the method described in this application in drug screening or disease mechanism research is also provided.

[0013] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0014] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0015] Figure 1 This is a schematic diagram of the elements of the expression carrier in Example 1.

[0016] Figure 2 The image shows the electrophoresis results of the targeting vector constructed in Example 1 after restriction endonuclease digestion.

[0017] Figure 3 This is a diagram showing the expected results of mouse genotype identification in Example 2.

[0018] Figure 4 This is an electrophoresis result of genotyping of S100A8 / A9 humanized mice in Example 2.

[0019] Figure 5 The relative expression levels of mouse S100A9 mRNA in peripheral blood mononuclear cells of S100A8 / A9 humanized mice and wild-type mice in Example 2.

[0020] Figure 6 The relative expression levels of human S100A9 mRNA in peripheral blood mononuclear cells of S100A8 / A9 humanized mice and wild-type mice in Example 2.

[0021] Figure 7 The expression level of human S100A8 / A9 heterodimer in the peripheral blood of S100A8 / A9 humanized mice and wild-type mice in Example 2. Detailed Implementation

[0022] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0023] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0024] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0026] When used in this document, the terms “include,” “contain,” and “comprising” mean that other elements are not excluded in addition to the listed elements.

[0027] The terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” used in this application are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues.

[0028] In this application, the terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acids of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included in the definition of polypeptide.

[0029] It should be noted that, in the context of this application, upstream refers to the 5' end of the nucleic acid or the N-terminus of the polypeptide, and downstream refers to the 3' end of the nucleic acid or the C-terminus of the polypeptide. The direction from upstream to downstream is from the 5' end to the 3' end or from the N-terminus to the C-terminus.

[0030] The terms “S100A8 / S100A9”, “S100A8 / A9”, “S100A8 / S100A9 dimer”, “S100A8 / S100A9 heterodimer”, “S100A8 / S100A9 polypeptide”, “S100A8 / S100A9 protein” or “heterodimer of S100A8 and S100A9” used in this application have the same meaning and can be used interchangeably. They refer to a protein complex in the form of a heterodimer formed by calcium-binding protein A8 (S100A8) and calcium-binding protein A9 (S100A9) through hydrophobic interactions in the presence of calcium and zinc ions.

[0031] The term "vector" as used in this application refers to a self-replicating DNA molecule that transfers a foreign target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0032] In this application, the terms "expression vector" and "recombinant vector" are used interchangeably, referring to a vector containing a foreign gene and also including regulatory elements for expression in a specified host organism. Introducing the expression vector into a suitable host organism enables it to express the inserted target gene.

[0033] The terms "exogenous" and "heterogeneous" used in this application are used interchangeably and refer to sources different from the native (original) organism, such as organisms derived from another species. The terms "heterogeneous gene" or "exogenous gene" used in this application refer to genes that do not naturally exist in the host organism and are introduced into the host organism through gene transfer.

[0034] The term "operably linked" or "operable ligation" as used in this application refers to the connection of multiple nucleic acid fragments in a functional relationship. It is an "operably linked" sequence when one nucleic acid is functionally associated with another. For example, if a promoter or other transcriptional regulatory sequence affects the transcription of a coding sequence (or gene), then the ligation sequence is operably linked in a manner that allows the promoter to induce transcription of the gene. Operable ligation means that the linked nucleotide sequences may be continuous or discontinuous.

[0035] In this application, "transformation," "introduction," and "importation" refer to the transfer of a foreign gene into a host organism, such as a host cell, resulting in stable genetic inheritance. The transformed gene can be in plasmid form retained in the host organism or integrated into the host organism's genome. A host organism containing the transformed gene is referred to as a "transgenic," "recombinant," "transformed," or "engineered" organism. Transformation of the host organism using an expression vector can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method; the steps used are well known in the art. If necessary, methods such as microinjection, electroporation, or liposome packaging can also be used. These are well-known techniques in the art and will not be described in detail here.

[0036] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0037] S100A8 / A9 heterodimers have been shown to play a key role in inflammatory responses. However, both S100A8 and S100A9 readily form homodimers or even high-molecular-weight (e.g., tetramers) protein products, making it difficult for heterodimers to form or significantly reducing their formation during in vitro and in vivo expression. This has become a challenge in constructing humanized mouse models of S100A8 / A9.

[0038] Therefore, there is an urgent need to construct mouse models with high-purity expression of S100A8 / S100A9 heterodimers using genetic engineering techniques. This would greatly facilitate the screening of drugs targeting S100A8 / S100A9 heterodimers, the research of antibody drugs targeting human S100A8 / S100A9 heterodimers, and would also help to conduct in-depth research on the structure and biological function of this heterodimer.

[0039] expression carrier According to one embodiment of this application, an expression vector is provided, the expression vector comprising: a first gene expression cassette for expressing human S100A8; and a second gene expression cassette for expressing human S100A9; wherein the expression directions of the first gene expression cassette and the second gene expression cassette are opposite.

[0040] In some implementations, the first gene expression cassette is read in order from the 5' end to the 3' end, and the second gene expression cassette is read in the opposite direction to the first gene expression cassette, that is, in order from the 3' end to the 5' end.

[0041] In some implementations, the second gene expression cassette is read in order from the 5' end to the 3' end, and the first gene expression cassette is read in the opposite direction to the second gene expression cassette, that is, in order from the 3' end to the 5' end.

[0042] When the expression directions of the first and second gene expression cassettes are opposite, it can effectively improve the transcriptional silencing mechanism (TGS) caused by the co-expression of two exogenous genes, thereby promoting the binding of transcription factors to RNA polymerase and avoiding the silencing or significant downregulation of expression levels caused by the simultaneous expression of the first and second gene expression cassettes. Therefore, in the expression vector proposed in this application, the two oppositely arranged gene expression cassettes can effectively reduce the risk of transcriptional interference and can efficiently express the S100A8 / S100A9 heterodimer at both the cellular level and in vivo.

[0043] In some embodiments, the first gene expression cassette sequentially includes: a first promoter, a nucleic acid sequence expressing human S100A8, and a first polyA tail.

[0044] In some embodiments, the second gene expression cassette sequentially includes: a second promoter, a nucleic acid sequence expressing human S100A9, and a second polyA tail.

[0045] In some embodiments, "nucleic acid sequence expressing human S100A8" or "nucleic acid sequence expressing human S100A9" refers to the genomic nucleic acid sequence between the start codon and the stop codon of human S100A8 or S100A9. In some embodiments, "nucleic acid sequence expressing human S100A8" or "nucleic acid sequence expressing human S100A9" may contain both exons and introns. In some embodiments, "nucleic acid sequence expressing human S100A8" or "nucleic acid sequence expressing human S100A9" contains exons but does not contain introns. Those skilled in the art will understand that regardless of whether a nucleic acid sequence contains introns, it can express the corresponding protein; therefore, such nucleic acid sequences should be covered within the scope of protection of this application.

[0046] To achieve opposite expression directions, the first and second gene expression cassettes can be linked head-to-head via their respective promoters. In some embodiments, the first and second gene expression cassettes are operatively linked via a first and a second promoter. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes: a reverse sequence of a first polyA tail, a reverse sequence of the expressed human S100A8 nucleic acid sequence, a reverse sequence of the first promoter, a second promoter, the expressed human S100A9 nucleic acid sequence, and a second polyA tail. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes: a reverse sequence of a second polyA tail, a reverse sequence of the expressed human S100A9 nucleic acid sequence, a reverse sequence of the second promoter, a first promoter, the expressed human S100A8 nucleic acid sequence, and a first polyA tail.

[0047] In some preferred embodiments, to achieve opposite expression directions, the first gene expression cassette and the second gene expression cassette can also be linked "tail-to-tail" via their respective polyA tails. In some embodiments, the first gene expression cassette and the second gene expression cassette are operatively linked via a first polyA tail and a second polyA tail. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes operatively linked: a first promoter, a nucleic acid sequence expressing human S100A8, a first polyA tail, a reverse sequence of the second polyA tail, a reverse sequence of the nucleic acid sequence expressing human S100A9, and a reverse sequence of the second promoter. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes operatively linked: a second promoter, a nucleic acid sequence expressing human S100A9, a second polyA tail, a reverse sequence of the first polyA tail, a reverse sequence of the nucleic acid sequence expressing human S100A8, and a reverse sequence of the first promoter.

[0048] Those skilled in the art will understand that the "reverse sequence" described in this application refers to a sequence in which the nucleotides are arranged in a completely opposite order to the original sequence, but which can still function normally or express its product when read in the reverse direction. For example, if the nucleotide sequence from the 5' end to the 3' end of the original sequence is ATCGATCG, its reverse sequence from the 5' end to the 3' end should be GCTAGCTA, but the reading order is from the 3' end to the 5' end.

[0049] First, when the first gene expression cassette and the second gene expression cassette express in opposite directions and are connected "tail-to-tail" through their respective polyA tails, spatial interference to the transcription complex driven by the other promoter can be effectively avoided during the extension of the transcription complex driven by one promoter, thereby effectively improving the transcription efficiency of the two expression cassettes. Secondly, when the expression directions of the first gene expression cassette and the second gene expression cassette are opposite and they are connected "tail-to-tail" through their respective polyA tails, this arrangement can make the two exogenous proteins (human S100A8 and human S100A9) closer in space after translation termination, further improving their efficiency in forming heterodimers. Therefore, this "tail-to-tail" connection method can further improve the expression of S100A8 / S100A9 heterodimers at the cellular level and in vivo, and is a more preferred embodiment of this application.

[0050] Due to the degeneracy of codons, those skilled in the art will understand that the nucleic acid sequences capable of encoding human S100A8 (amino acid sequence as shown in SEQ ID NO: 24; NCBI accession number: NM_001319201.1) or human S100A9 (amino acid sequence as shown in SEQ ID NO: 25; NCBI accession number: NM_002965.3) are not limited to those shown in SEQ ID NO: 5, 26, or SEQ ID NO: 8, 27, and are not limited herein. Those skilled in the art can select appropriate encoding nucleic acid sequences for use in the expression vector provided in this application, based on the amino acid sequences of human S100A8 or human S100A9, depending on the expression purpose or the host. In some specific embodiments, the nucleic acid sequence expressing human S100A8 contains exons and introns, and the nucleic acid sequence expressing human S100A8 contains the sequence shown in SEQ ID NO: 5. In some specific embodiments, the nucleic acid sequence of the expressed human S100A8 contains exons but no introns, and the nucleic acid sequence of the expressed human S100A8 contains the sequence shown in SEQ ID NO: 26. In some specific embodiments, the nucleic acid sequence of the expressed human S100A9 contains both exons and introns, and the nucleic acid sequence of the expressed human S100A9 contains the sequence shown in SEQ ID NO: 8. In some specific embodiments, the nucleic acid sequence of the expressed human S100A9 contains exons but no introns, and the nucleic acid sequence of the expressed human S100A9 contains the sequence shown in SEQ ID NO: 27.

[0051] The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates polypeptide expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to those of the host cell. In some embodiments, the first promoter includes CAG, EF1A, CMV, SV40, PGK, UbC, human beta-actin, TRE, UAS, Ac5, GFAP, Polyhedrin promoter, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL. In some embodiments, the second promoter includes CAG, EF1A, CMV, SV40, PGK, UbC, human beta-actin, TRE, UAS, Ac5, GFAP, Polyhedrin promoter, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL. In some preferred embodiments, the first promoter is CAG, with the sequence shown in SEQ ID NO: 3. In some preferred embodiments, the second promoter is EF1A, with the sequence shown in SEQ ID NO: 9.

[0052] Similarly, those skilled in the art can select any suitable polyA tail sequence as needed. In some embodiments, the first polyA tail includes BGH polyA, rBG polyA, or SV40 polyA. In some embodiments, the second polyA tail includes BGH polyA, rBG polyA, or SV40 polyA. In some preferred embodiments, the first polyA tail is BGH polyA, and its sequence is shown in SEQ ID NO: 6. In some preferred embodiments, the second polyA tail is rBG polyA, and its sequence is shown in SEQ ID NO: 7.

[0053] In some embodiments, the expression vector further includes a Kozak sequence. In some embodiments, the Kozak sequence comprises the nucleotide sequence shown in GCCACC (SEQ ID NO: 4). In the expression vector provided in this application, a Kozak sequence can also be added to further optimize the expression of the encoding nucleic acid sequence; for example, the Kozak sequence can bind to translation initiation factors to mediate the translation initiation of mRNA containing a 5' cap structure.

[0054] In some embodiments, the first gene expression cassette sequentially includes: a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, and a first polyA tail. In some embodiments, the second gene expression cassette sequentially includes: a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, and a second polyA tail.

[0055] For example, the expression vector used for tail-to-tail ligation can further incorporate a Kozak sequence. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes: a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, a first polyA tail, a reverse sequence of a second polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A9, a reverse sequence of the Kozak sequence, and a reverse sequence of the second promoter. In some embodiments, the expression vector, from its 5' end to its 3' end, sequentially includes: a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, a second polyA tail, a reverse sequence of the first polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A8, a reverse sequence of the Kozak sequence, and a reverse sequence of the first promoter.

[0056] For example, the expression vector used for head-to-head ligation can further incorporate a Kozak sequence. In some embodiments, the expression vector comprises, from the 5' end to the 3' end, the following operably linked sequences: a reverse sequence of a first polyA tail, a reverse sequence of the nucleic acid sequence expressing human S100A8, a reverse sequence of the Kozak sequence, a reverse sequence of a first promoter, a second promoter, a Kozak sequence, the nucleic acid sequence expressing human S100A9, and a second polyA tail. In some embodiments, the expression vector comprises, from the 5' end to the 3' end, the following operably linked sequences: a reverse sequence of a second polyA tail, a reverse sequence of the nucleic acid sequence expressing human S100A9, a reverse sequence of the Kozak sequence, a reverse sequence of the second promoter, a first promoter, a Kozak sequence, the nucleic acid sequence expressing human S100A8, and a first polyA tail.

[0057] In some embodiments, the expression vector further includes homology arms sequences that are at least partially complementary to a sequence at a specific genomic site, to enable the insertion of the first and second gene expression cassettes in the expression vector at the specific genomic site. If insertion at a specific genomic site is not desired, and instead, insertion at a random site is chosen, homology arms sequences may not be included in the expression vector; this can be chosen by those skilled in the art as needed.

[0058] The insertion site can be any suitable site in the genome, and those skilled in the art can select it as needed and design suitable homologous arm sequences for that specific site. In some embodiments, the specific genomic site includes the Rosa26 gene, the AAVS1 gene, the CMAH gene, or the β4GalNT2 gene. In some embodiments, the specific genomic site is the Rosa26 gene, the 5' homologous arm sequence has the sequence shown in SEQ ID NO: 2, and the 3' homologous arm sequence has the sequence shown in SEQ ID NO: 10.

[0059] The length of homologous arms is typically tens, hundreds, or even thousands of base pairs to ensure efficient recombination. In some embodiments, the length of the homologous arm sequence is 1000-3000 bp.

[0060] Homologous arms can be simultaneously positioned on both sides of the 5' and 3' ends to further increase the accuracy of recombination. In some embodiments, the homologous arm sequences include 3' homologous arm sequences and / or 5' homologous arm sequences. In some embodiments, the 5' homologous arm sequence is complementary to at least a portion of the 5' end sequence at a specific genomic site. In some embodiments, the 3' homologous arm sequence is complementary to at least a portion of the 3' end sequence at a specific genomic site.

[0061] For example, expression vectors used for tail-to-tail ligation can further incorporate homologous arm sequences. In some embodiments, the expression vector comprises, from 5' to 3', the following operably linked sequences: a 5' homologous arm sequence, a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, a first polyA tail, a reverse sequence of a second polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A9, a reverse sequence of the Kozak sequence, a reverse sequence of the second promoter, and a 3' homologous arm sequence. In some embodiments, the expression vector comprises, from 5' to 3', the following operably linked sequences: a 5' homologous arm sequence, a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, a second polyA tail, a reverse sequence of the first polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A8, a reverse sequence of the Kozak sequence, a reverse sequence of the first promoter, and a 3' homologous arm sequence.

[0062] For example, expression vectors used for head-to-head ligation can further incorporate homologous arm sequences. In some embodiments, the expression vector comprises, from 5' to 3', the following operably linked sequences: a 5' homologous arm sequence, a reverse sequence of a first polyA tail, a reverse sequence of the expressed human S100A8 nucleic acid sequence, a reverse sequence of the Kozak sequence, a reverse sequence of a first promoter, a second promoter, a Kozak sequence, the expressed human S100A9 nucleic acid sequence, a second polyA tail, and a 3' homologous arm sequence. In some embodiments, the expression vector comprises, from 5' to 3', the following operably linked sequences: a 5' homologous arm sequence, a reverse sequence of a second polyA tail, a reverse sequence of the expressed human S100A9 nucleic acid sequence, a reverse sequence of the Kozak sequence, a reverse sequence of the second promoter, a first promoter, a Kozak sequence, the expressed human S100A8 nucleic acid sequence, a first polyA tail, and a 3' homologous arm sequence.

[0063] The expression vector can be any suitable vector, such as plasmids, viruses, bacteria, bacteriophages, and insertable DNA fragments, etc., and is not limited thereto. In some preferred embodiments, the expression vector includes, but is not limited to, plasmid vectors or viral vectors. Those skilled in the art can select appropriate types of expression vectors for expressing this application according to the expression purpose or the different hosts.

[0064] The expression vector of this application can be constructed using methods known in the art. For example, appropriate restriction enzyme sites can be added to both ends of the expression vector of this application according to the restriction enzyme sites contained in the backbone vector used, and then the vector can be loaded into the backbone vector.

[0065] In some specific embodiments, the expression vector comprises the sequence shown in SEQ ID NO: 1.

[0066] Construction methods and uses According to one embodiment of this application, a method for expressing S100A8 and S100A9 heterodimers is also provided, characterized in that the method includes introducing the expression vector described in this application into cells.

[0067] According to one embodiment of this application, the use of the expression vector described in this application in expressing S100A8 and S100A9 heterodimers is also provided.

[0068] In some embodiments, the cells comprise mouse cells. In some embodiments, the cells comprise mouse zygotes. The expression vector provided in this application can be introduced into mouse zygotes to construct a mouse model that efficiently expresses the S100A8 / A9 heterodimer.

[0069] In another embodiment, the cell can be any cell type of interest. Not limited to constructing animal vectors, the expression vectors provided in this application are also suitable for efficiently expressing S100A8 and S100A9 heterodimers in a variety of cell systems.

[0070] Those skilled in the art can introduce the constructed expression vector into cells using commonly used transfection methods, such as electroporation and liposome transfection. These commonly used transfection methods are also well known in the art.

[0071] According to one embodiment of this application, a method for constructing an animal model is also provided, characterized in that the method includes introducing the expression vector described in this application into a specific site of the animal genome.

[0072] According to one embodiment of this application, the use of the expression vector described in this application in constructing animal models is also provided.

[0073] In some embodiments, the animal includes mice. In some embodiments, the mice include C57BL / 6 mice, FVB / N mice, 129 mice, BALB / c mice, DBA / 1 mice, or DBA / 2 mice. In some embodiments, the method of introducing the expression vector provided in this application into a specific site of the mouse genome includes the following steps: introducing the expression vector, the sgRNA targeting a specific site of the genome, and the Cas9 protein described in this application into the fertilized egg of the recipient mouse via microinjection; transplanting the fertilized egg into the oviduct of a surrogate mouse to obtain F0 generation mice; mating the F0 generation mice with wild-type mice to obtain F1 generation heterozygous mice; mating the F1 generation heterozygous mice with each other to obtain homozygous mice; and mating the homozygous mice with S100A9 gene knockout mice to obtain an S100A8 / A9 humanized mouse model.

[0074] To increase the occurrence of double-strand breaks, enzymes capable of cleaving specific genomic sites are typically introduced simultaneously when the expression vector is introduced into a specific site on the animal genome. In some embodiments, components of a CRISPR (clustered regularly spaced short palindromic repeats) / Cas system or such systems may be introduced simultaneously, allowing for site-specific cleavage of the genomic site via the CRISPR complex. In some embodiments, the CRISPR complex comprises a single-guide RNA (sgRNA) complexed with a Cas protein. Furthermore, any suitable Cas protein known in the art may be used in this application and is not limited thereto. In some preferred embodiments, the Cas protein is the Cas9 protein.

[0075] The genomic specific site can be any site suitable for introducing a first gene expression cassette and / or a second gene expression cassette. Those skilled in the art can select such sites as needed and design suitable homologous arm sequences and sgRNA sequences for the specific site. In some embodiments, the genomic specific site includes the Rosa26 gene, AAVS1 gene, ROSA26 gene, CMAH gene, or β4GalNT2 gene. In some embodiments, the genomic specific site is the ROSA26 gene, the introduction is achieved via CRISPR / Cas, and the sgRNA sequence is shown in SEQ ID NO: 11 and SEQ ID NO: 23.

[0076] According to one embodiment of this application, the use of an animal model obtained by the method described in this application in drug screening or disease mechanism research is also provided.

[0077] Within the scope of knowledge in this field, a variety of diseases are associated with elevated S100A8 / S100A9 dimer expression levels, including but not limited to: sepsis (Am J Respir Crit Care Med, 2017, 196(3): 315-327), autoimmune diseases (Autoimmun Rev, 2023, 22(5): 103295), acute lung injury (Blood, 2022, 140(24):2626-2643), acute pancreatitis (Dis Markers, 2018, 2018: 6457347), acute kidney injury (Adv Sci(Weinh), 2022, 9(12): e2103675), tumors (Sci Transl Med, 2020, 12(572):eabb5817), and organ ischemia / reperfusion injury (Circulation, 2019, 140(9): 751-764; Kidney Int, 2015, 87(1): 85-94), and atherosclerosis-related cardiovascular and cerebrovascular diseases (Pharmacol Res, 2020, 161:105212), the entire contents of which are incorporated herein by reference. Therefore, the animal models obtained according to the methods described in this application can be used for mechanistic studies of various diseases associated with elevated heterodimer expression levels of S100A8 and S100A9, or for screening drugs to alleviate or treat these diseases. In some embodiments, the diseases are associated with elevated heterodimer expression levels of S100A8 and S100A9. In some preferred embodiments, the diseases include cardiovascular diseases or tumors.

[0078] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0079] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0080] Example Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0081] Example 1: Construction of S100A8 / A9 humanized mouse model 1.1 Determine the source sequence to be inserted The human S100A8 gene sequence (NCBI Reference Sequence: NM_002964.5) and the human S100A9 gene sequence (NCBI Reference Sequence: NM_002965.4) were selected as exogenous genes to be inserted into the mouse genome.

[0082] 1.2 Identification of sgRNAs targeting the Rosa26 gene The mouse Rosa26 gene (NCBI Reference Sequence: NR_027008.1) was selected as the insertion site for the foreign gene. Using CRISPR / Cas9 technology, sgRNAs targeting intron 1 of the Rosa26 gene were designed and synthesized (crRNA sequences are shown in SEQ ID NO: 11, and tracrRNA sequences are shown in SEQ ID NO: 23).

[0083] 1.3 Design and Construction of Expression Carrier A mouse genome fragment containing a homologous arm was amplified from a BAC clone (Cyagen Biosciences) using high-fidelity Taq DNA polymerase (Nanjing Novizan), and then constructed into an expression vector. The specific sequence of the expression vector is SEQ ID NO: 1.

[0084] Schematic diagram of the expression carrier as follows Figure 1 As shown, from the 5' end to the 3' end, it includes, in sequence: 5' homologous arm (SEQ ID NO: 2) — CAG promoter (SEQ ID NO: 3) — Kozak sequence (nucleotide sequence GCCACC) — genomic nucleic acid sequence between the start codon and stop codon of human S100A8 (SEQ ID NO: 5) — BGHpoly (SEQ ID NO: 6) — reverse sequence of rBGpoly (SEQ ID NO: 7) — reverse sequence of genomic nucleic acid sequence between the start codon and stop codon of human S100A9 (SEQ ID NO: 8) — reverse sequence of Kozak (nucleotide sequence GCCACC) — reverse sequence of EF1A promoter (SEQ ID NO: 9) — 3' homologous arm (SEQ ID NO: 10).

[0085] The constructed expression vector was digested with restriction endonucleases (FspI, DrdI, NcoI, and BamHI) to confirm its size and linearize it. Figure 2 As shown, the appearance of bands of the corresponding size after digestion with restriction endonucleases proves that the expression vector was successfully constructed.

[0086] 1.4 Construction of S100A8 / A9 humanized mouse model The mixture of the linearized expression vector, sgRNA, and Cas9 protein (M0646M, NEB) was injected into mouse zygotes via microinjection and then transplanted into the oviducts of surrogate mice. Genotypes of the mice were identified by PCR and DNA sequencing after birth, thus obtaining positive F0 generation mice.

[0087] F0 generation mice were bred with wild mice to obtain F1 generation heterozygous mice, and F1 generation heterozygous mice were bred with each other to obtain homozygous Rosa26-S100A8-S100A9[KI / KI] mice; Rosa26-S100A8-S100A9 [KI / KI] mice were bred with S100a9 gene knockout mice (Cyagen Biosciences, S-KO-04180) to obtain S100A8 / A9 humanized mice, namely Rosa26-S100A8-S100A9. [KI / KI];S100a9[- / -].

[0088] Example 2: Identification of the S100A8 / A9 humanized mouse model 2.1 DNA Extraction 2.1.1 Reagent Kit Extraction Method DNA was extracted from mouse tails using a kit (TaKaRa MiniBEST Universal Genomic DNA Extractionkit (Ver.5.0_Code No. 9765)).

[0089] (1) Preparation: Prepare anhydrous ethanol; Before using Western blotting, 65 mL of anhydrous ethanol needs to be added; (Note: The kit recommends 56 mL, but long-term data show that adding 65 mL of anhydrous ethanol results in higher genomic purity).

[0090] If Buffer GL precipitates, dissolve it by heating at 56°C and use it after it has returned to room temperature. When eluting DNA from the membrane, DNA eluted with 0.25 x TE buffer has a longer shelf life; TE buffer is preheated to 56°C before use for higher DNA elution efficiency.

[0091] (2) Experimental procedure: Add 180 μL Buffer GL, 20 μL Proteinase K (use 20 μL of the Proteinase K from the kit, which is 20 mg / mL), and 10 μL RNase to each EP tube containing the sample (2-5 mm rat tail). (Note: The rat tail should not be too long, generally 2-5 mm; if the rat tail is too thick, the lysis volume can be increased to 300 μL.) Incubate at 56℃ overnight (generally 12-16 hours); Centrifuge at 12000 rpm for 2 min to remove hair and other impurities to the bottom of the tube; Transfer the supernatant to another clean EP tube (Note: Try not to aspirate hair and impurities from the bottom of the tube), then add 200 μL of Buffer GB and 200 μL of anhydrous ethanol, cap the tube and mix by inverting the tube. After mixing, transfer the liquid to the prepared adsorption column, centrifuge at 12000 rpm for 2 min, and discard the filtrate; Add 500 μL of Buffer WA to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate; Add 700 μL of buffer WB to the adsorption column, let stand for 1 min, then centrifuge at 12000 rpm for 1 min; discard the filtrate (Note: Ensure that the specified volume of anhydrous ethanol has been added to the WB; adding WB around the perimeter of the adsorption column helps to completely wash away any salts adhering to the column wall). Repeat the above steps and discard the filtrate; Empty centrifugation: Centrifuge at 12000 rpm for 2 min; Place the adsorption column onto a new 1.5 mL centrifuge tube with the cap open and incubate at room temperature for 10 min to remove residual ethanol (Note: residual ethanol will inhibit polymerase amplification). Add 50 μL of preheated 0.25 x TE buffer to the center of the adsorption column membrane, cover it, and let it stand at room temperature for 10 min (Note: Preheated sterile water can improve elution efficiency). Elute DNA by centrifuging at 12000 rpm for 2 min; the eluted liquid can be added back to the center of the membrane, let stand for 5 min, and then centrifuge at 12000 rpm for 2 min to obtain a higher concentration of DNA; The concentration of the obtained DNA can be detected, and its purity can also be observed through electrophoresis.

[0092] 2.1.2 Coarse Extraction Method The coarse-cutting method is used to coarsely extract genomic DNA from mouse tails or paws, which is suitable for amplifying bands <1 kb.

[0093] (1) Preparation of Triton lysis buffer (1 L): Weigh the following items into beakers using an electronic balance: KCl: 3.7 g; Tris: 1.2 g; Dissolve in 300-400 mL of sterile water; Add 1 mL of Triton X-100 (Sigma, T8787-50 mL) and dissolve completely. If it cannot be completely dissolved, it can be dissolved in a 56°C oven. (Note: Triton X-100 is an oily liquid. Rinse the pipette before aspirating. After emptying the Triton X-100, you can repeatedly pipette tip into the solution to avoid a large amount of Triton X-100 remaining on the inner wall of the tip.) Add 80 μL to 1 L and store at room temperature until used. (Note: Do not autoclave Triton X-100, as autoclaving will produce a large amount of precipitate.)

[0094] (2) Sample pyrolysis: Take a mouse tail or paw (~2 mm) into an EP tube, add 98 μL of lysis buffer and 2 μL of 20 mg / mL proteinase K (Merck, Cat. No. MK539480) (Note: The mouse tail should not exceed 3 mm, otherwise lysis will be incomplete; if the mouse tail is too thick, the amount of lysis buffer can be increased appropriately; proteinase K should not be repeatedly frozen and thawed, otherwise its activity will be affected). After adding lysis buffer and proteinase K, place the EP tube in a 56°C constant temperature device (oven or water bath) and run overnight. The next day, the sample was removed from the constant temperature device and placed in a metal bath or PCR instrument, and reacted at 98°C for 15 min to inactivate proteinase K. Centrifuge the sample for 15 min, and the supernatant can be used as a PCR template (Note: Generally, 1.5 μL of supernatant is added to a 25 μL PCR system).

[0095] 2.2 PCR reaction (1) Design forward and reverse primers for human S100A8 / A9 insertion.

[0096] Primer 1: (annealing temperature 60.0℃) F1:5'-CTTCTTTATGCATTCATTTTCCACCG-3' (SEQ ID NO: 12); R1: 5'-AAAATGAGATAGAGTGCGCTGTG-3' (SEQ ID NO: 13); Primer 2: (annealing temperature 60.0℃) F2: 5'-CACTTGCTCTCCCAAAGTCGCTC-3' (SEQ ID NO: 14); R2: 5'-ATACTCCGAGGCGGATCACAA-3' (SEQ ID NO: 15); (2) Primers were designed for the knockout of the mouse S100A9 gene as follows: Primer 3: (annealing temperature 60.0℃) F3: 5'-GTATATGTGGAGGGAAGCTGTCTC-3' (SEQ ID NO: 16); R3: 5'-GTGAAAGGAGGCAGAAAGGACATG-3' (SEQ ID NO: 17); Primer 4: (annealing temperature 60.0℃) F3: 5'-GTATATGTGGAGGGAAGCTGTCTC-3' (SEQ ID NO: 16); R4: 5'-CTATCCACAGAACTGTCTCACCATC-3' (SEQ ID NO: 18); The extracted DNA was subjected to PCR. The PCR reaction system is shown in Table 1, where all primers were at a concentration of 10 μM. The PCR reaction conditions are shown in Table 2. Under standard conditions, 35 cycles of PCR were performed using 25 µL of primers, with the following two primers added for each reaction.

[0097] Table 1 PCR reaction system

[0098] Table 2 PCR reaction conditions

[0099] PCR products were identified by agarose gel electrophoresis. Agarose gels were prepared using agarose (BIOWEST AGAROSE, REGULAR) and TBE (Tris Bio Basic Inc, TBO194-500g; EDTA Shanghai Sangon, 0105-500g; BoricAcid, Shanghai Sangon, 0588-500). Control groups included a water control (without DNA template) and a wild-type control (mouse genomic DNA). DNA markers (Thermo Scientific GeneRuler 2000bp DNA Ladder #MK001) were used for identification.

[0100] The expected result should be as follows Figure 3 As shown: When identifying the insertion of the human S100A8 / A9 gene, homozygotes were represented by a single band of 252 bp; heterozygotes were represented by two bands of 252 bp and 453 bp, respectively; and wild-type individuals were represented by a single band of 453 bp.

[0101] When identifying the S100A9 gene knockout in mice, homozygotes showed a single band of 339 bp; heterozygotes showed two bands of 339 bp and 614 bp, respectively; and wild-type mice showed a single band of 614 bp. This validated the genotype of S100A8 / A9 humanized mice.

[0102] The actual test results are as follows Figure 4 As shown, the boxes indicate the mouse numbers that conform to the humanized S100A8 / A9 mouse genotype, namely No. 4, No. 6, No. 8, and No. 14-15.

[0103] 2.3 Verification of human and mouse S100A9 mRNA levels in S100A8 / A9 humanized mice The levels of human and mouse S100A9 mRNA in S100A8 / A9 humanized mice were detected by real-time PCR.

[0104] The steps are as follows: 2.3.1 Isolation of mouse peripheral blood mononuclear cells Peripheral blood mononuclear cells were isolated using a mouse peripheral blood mononuclear cell isolation kit (Solopro, P6340).

[0105] Take fresh EDTA-anticoagulated whole blood from mice and dilute it with an equal volume of whole blood and tissue diluent.

[0106] Add 3 ml of separation solution to the centrifuge tube, and spread the diluted blood evenly on the surface of the separation solution, making sure to keep the interface between the two liquids clear.

[0107] Centrifuge at room temperature, horizontal rotor, 500 xg, for 20 minutes.

[0108] After centrifugation, distinct layers will appear: the top layer is a diluted plasma layer, the middle layer is a clear separation medium layer, and the white membrane layer between the plasma and the separation medium is the mononuclear cell layer. Carefully aspirate the white membrane layer cells into a clean 15 ml centrifuge tube and wash the white membrane layer cells with 10 ml of PBS. Centrifuge at 250 xg for 10 minutes.

[0109] Discard the supernatant, resuspend the cells in 5 mL of PBS, centrifuge at 250 xg for 10 minutes. Repeat once.

[0110] Discard the supernatant and resuspend the cells in 1 ml of PBS for later use.

[0111] 2.3.2 Extraction of RNA from peripheral blood mononuclear cells The peripheral blood mononuclear cells prepared above were added to 1 mL of TRIzol reagent (Invitrogen) for total RNA extraction.

[0112] After standing at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 12,000 rpm and 4°C for 15 minutes.

[0113] Carefully aspirate the upper aqueous phase into a 1.5 mL nuclease-free EP tube, add an equal volume of isopropanol, mix well, incubate at room temperature for 20 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and a white RNA precipitate will be visible at the bottom of the tube.

[0114] Carefully aspirate the supernatant, leaving the RNA precipitate; add 1 mL of 75% ethanol, wash the precipitate twice, and centrifuge at 5000 rpm and 4°C for 5 minutes.

[0115] Discard the 75% ethanol, air dry the precipitate at room temperature until it becomes translucent, add 20 μL of nuclease-free water, mix well by pipetting, and fully dissolve the RNA. Measure the RNA concentration using a NanoDrop2000 (Thermo Fisher).

[0116] 2.3.3 RNA reverse transcription into cDNA The extracted RNA was reverse transcribed into cDNA using the RevertAid first-strand cDNA synthesis kit (ThermoFisher, K16225). The reverse transcription reaction system (step 1) was prepared according to Table 3, mixed thoroughly, and incubated at 65°C for 5 minutes, then placed on ice. The reverse transcription reaction system (step 2) was prepared according to Table 4, mixed thoroughly, and incubated at 42°C for 60 minutes, followed by incubation at 70°C for 5 minutes to inactivate the reverse transcriptase.

[0117] Table 3 Reverse transcription reaction system (first step)

[0118] Table 4. Reverse transcription reaction system (step 2)

[0119] 2.3.4 RT-PCR The cDNA obtained from the reverse transcription was diluted 10-fold with nuclease-free water and used as a template for real-time PCR. The RT-PCR reaction system was prepared according to Table 5 using Nanjing Novizan SYBR qPCR Master Mix (Q412-02). The reaction program was as follows: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 40 cycles. This was followed by a melting curve program: 95℃ for 15 seconds; 60℃ for 1 minute; 95℃ for 1 second. After the experiment, GADPH was used as an internal control, and the relative gene expression level was analyzed using the ΔCt method.

[0120] Table 5 RT-PCR reaction system

[0121] The primer sequences are as follows: Human S100A9 qPCR forward primer: 5'-GGTCATAGAACACATCATGGAGG-3' (SEQ ID NO: 19) Human S100A9 qPCR reverse primer: 5'-GGCCTGGCTTATGGTGGTG-3' (SEQ ID NO: 20) Mouse GAPDH qPCR forward primer: 5'-AATGCATCCTGCACCACC-3' (SEQ ID NO: 21) Reverse primer for mouse GAPDH qPCR: 5'-ATGCCAGTGAGCTTCCCG-3' (SEQ ID NO: 22) The results are as follows Figure 5 As shown, in mouse peripheral blood mononuclear cells, compared to the wild-type group, there was almost no expression of mouse S100A9 mRNA in S100A8 / A9 humanized mice. Figure 6 As shown, in mouse peripheral blood mononuclear cells, S100A8 / A9 humanized mice showed significant mRNA expression of human S100A9 compared to the wild-type group.

[0122] 2.4 Verification of human S100A8 / A9 protein expression levels in S100A8 / A9 humanized mice The expression levels of human S100A8 / A9 protein in wild-type and S100A8 / A9 humanized mice were detected using a human S100A8 / A9 heterodimer ELISA kit (R&D, catalog number DS8900).

[0123] The steps are as follows: Blood was collected from mice and anticoagulated with EDTA. The mice were centrifuged at 1000 xg for 15 minutes at 4°C. The supernatant was collected and diluted 50 times (10 μL sample + 490 μL calibration diluent RD5-10).

[0124] Using a monoclonal antibody coated with a specific human S100A8 / A9 heterodimer, add 50 μL of detection diluent RD1-34 to each well, followed by 50 μL of standard, control mouse serum, and S100A8 / A9 humanized mouse serum. Seal the wells with rubber bands and incubate at room temperature for 2 hours. Discard the liquid in the wells, add 400 μL of washing buffer to each well, wash thoroughly, and remove all liquid. Pat dry on clean paper and repeat the washing process 4 times.

[0125] Add 200 μL of HRP-labeled human S100A8 / A9 heterodimer antibody to each well and incubate at room temperature for 2 hours. Discard the liquid in the wells, add 400 μL of washing buffer to each well, wash thoroughly, remove all liquid, pat dry on clean paper, and repeat the washing process 4 times.

[0126] Add 200 μL of substrate solution to each well. Incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.

[0127] The absorbance of each well was measured at 450 nm using an ELISA reader within 30 minutes.

[0128] Calculate the concentration of the sample to be tested based on the standard curve (note the need to multiply by the dilution factor).

[0129] The results are as follows Figure 7 As shown, in the peripheral blood of mice, the expression level of human S100A8 / A9 protein was higher in S100A8 / A9 humanized mice compared with the wild-type group.

[0130] In summary, the S100A8 / A9 humanized mouse model was successfully constructed.

[0131] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. An expression vector, characterized by, The expression vector comprises: a first gene expression cassette for expressing human S100A8; and a second gene expression cassette for expressing human S100A9; wherein the expression direction of the first gene expression cassette is opposite to that of the second gene expression cassette.

2. The expression vector of claim 1, wherein: the first gene expression cassette comprises, in sequence, a first promoter, a nucleic acid sequence for expressing human S100A8, and a first polyA tail; and / or the second gene expression cassette comprises, in sequence, a second promoter, a nucleic acid sequence for expressing human S100A9, and a second polyA tail.

3. The expression vector of claim 2, wherein: the first gene expression cassette and the second gene expression cassette are operably linked by the first polyA tail and the second polyA tail; or the first gene expression cassette and the second gene expression cassette are operably linked by the first promoter and the second promoter.

4. The expression vector of claim 3, wherein the expression vector comprises, in sequence from 5' end to 3' end, operably linked: a first promoter, a nucleic acid sequence for expressing human S100A8, a first polyA tail, a reverse sequence of a second polyA tail, a reverse sequence of a nucleic acid sequence for expressing human S100A9, and a reverse sequence of a second promoter; a second promoter, a nucleic acid sequence for expressing human S100A9, a second polyA tail, a reverse sequence of a first polyA tail, a reverse sequence of a nucleic acid sequence for expressing human S100A8, and a reverse sequence of a first promoter; a reverse sequence of a first polyA tail, a reverse sequence of a nucleic acid sequence for expressing human S100A8, a reverse sequence of a first promoter, a second promoter, a nucleic acid sequence for expressing human S100A9, and a second polyA tail; or a reverse sequence of a second polyA tail, a reverse sequence of a nucleic acid sequence for expressing human S100A9, a reverse sequence of a second promoter, a first promoter, a nucleic acid sequence for expressing human S100A8, and a first polyA tail.

5. The expression vector of claim 4, wherein: the amino acid sequence of the human S100A8 is SEQ ID NO: 24; the amino acid sequence of the human S100A9 is SEQ ID NO: 25; the nucleic acid sequence for expressing human S100A8 comprises a sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 26; and / or the nucleic acid sequence for expressing human S100A9 comprises a sequence set forth in SEQ ID NO: 8 or SEQ ID NO:

27.

6. The expression vector of claim 4, wherein the first promoter and / or second promoter independently comprises CAG, EF1A, CMV, SV40, PGK, UbC, human beta actin, TRE, UAS, Ac5, GFAP, Polyhedrin promotor, TBG, ALB, ApoE HCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL.

7. The expression vector of claim 4, wherein the first polyA tail and / or second polyA tail independently comprises BGH polyA, rBG polyA, or SV40 polyA.

8. The expression vector of claim 4, wherein: the first promoter is CAG, and has a sequence as set forth in SEQ ID NO: 3; the second promoter is EF1A, and has a sequence as set forth in SEQ ID NO: 9; the first polyA tail is BGH polyA, and has a sequence as set forth in SEQ ID NO: 6; and / or the second polyA tail is rBG polyA, and has a sequence as set forth in SEQ ID NO:

7.

9. The expression vector of claim 2, wherein the expression vector further comprises a Kozak sequence.

10. The expression vector of claim 9, wherein the Kozak sequence has a nucleotide sequence of GCCACC.

11. The expression vector of claim 9, wherein: the first gene expression cassette comprises, in order: a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, and a first polyA tail; and / or the second gene expression cassette comprises, in order: a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, and a second polyA tail.

12. The expression vector of claim 11, wherein the expression vector comprises, in order from 5’ end to 3’ end, operably linked: a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, a first polyA tail, a reverse sequence of a second polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A9, a reverse sequence of a Kozak sequence, and a reverse sequence of a second promoter; a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, a second polyA tail, a reverse sequence of a first polyA tail, a reverse sequence of a nucleic acid sequence expressing human S100A8, a reverse sequence of a Kozak sequence, and a reverse sequence of a first promoter; the reverse sequence of the second polyA tail, the reverse sequence of the nucleic acid sequence expressing human S100A9, the reverse sequence of the Kozak sequence, the reverse sequence of the second promoter, the first promoter, the Kozak sequence, the nucleic acid sequence expressing human S100A8, and the first polyA tail. the reverse sequence of the second polyA tail, the reverse sequence of the nucleic acid sequence expressing human S100A9, the reverse sequence of the Kozak sequence, the reverse sequence of the second promoter, the first promoter, the Kozak sequence, the nucleic acid sequence expressing human S100A8, and the first polyA tail.

13. The expression vector of any one of claims 1-12, wherein the expression vector further comprises a homology arm sequence complementary to at least a portion of a sequence of a specific genomic site.

14. The expression vector of claim 13, wherein the homology arm sequence is 1000-3000 bp in length.

15. The expression vector of claim 13, wherein the homology arm sequence comprises: a 3’ end homology arm sequence complementary to at least a portion of a sequence of the 3’ end of the specific genomic site; and / or a 5’ end homology arm sequence complementary to at least a portion of a sequence of the 5’ end of the specific genomic site.

16. The expression vector of claim 15, wherein the expression vector comprises, in order from the 5’ end to the 3’ end, the operably linked: a 5’ end homology arm sequence, a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, a first polyA tail, the reverse sequence of a second polyA tail, the reverse sequence of a nucleic acid sequence expressing human S100A9, the reverse sequence of a Kozak sequence, the reverse sequence of a second promoter, and a 3’ end homology arm sequence; a 5’ end homology arm sequence, a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, a second polyA tail, the reverse sequence of a first polyA tail, the reverse sequence of a nucleic acid sequence expressing human S100A8, the reverse sequence of a Kozak sequence, the reverse sequence of a first promoter, and a 3’ end homology arm sequence; a 5’ end homology arm sequence, the reverse sequence of a first polyA tail, the reverse sequence of a nucleic acid sequence expressing human S100A8, the reverse sequence of a Kozak sequence, the reverse sequence of a first promoter, a second promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A9, a second polyA tail, and a 3’ end homology arm sequence; or a 5’ end homology arm sequence, the reverse sequence of a second polyA tail, the reverse sequence of a nucleic acid sequence expressing human S100A9, the reverse sequence of a Kozak sequence, the reverse sequence of a second promoter, a first promoter, a Kozak sequence, a nucleic acid sequence expressing human S100A8, a first polyA tail, and a 3’ end homology arm sequence.

17. The expression vector of claim 13, wherein the specific genomic site comprises a Rosa26 gene, an AAVS1 gene, a CMAH gene, or a b4GalNT2 gene. ​ 18. The expression vector of claim 17, wherein the specific genomic locus is Rosa26 gene, the 5’ homology arm sequence has the sequence of SEQ ID NO: 2, and the 3’ homology arm sequence has the sequence of SEQ ID NO:

10.

19. The expression vector of claim 18, wherein the expression vector comprises the sequence of SEQ ID NO:

1.

20. A method of expressing a S100A8 and S100A9 heterodimer, characterized in that, The method comprises introducing the expression vector of any one of claims 1-19 into a cell.

21. The method of claim 20, wherein the cell comprises a mouse zygote.

22. A method of constructing an animal model, comprising: The method comprises introducing the expression vector of any one of claims 1-19 into a specific genomic locus of an animal.

23. The method of claim 22, wherein the animal comprises a mouse, and the mouse comprises a C57BL / 6 mouse, a FVB / N mouse, a 129 mouse, a BALB / c mouse, a DBA / 1 mouse, or a DBA / 2 mouse.

24. The method of claim 22, wherein the animal is a mouse, and the introducing a specific genomic locus of an animal comprises the following steps: introducing the expression vector of any one of claims 1-19, sgRNA targeting the specific genomic locus, and Cas9 protein into the recipient mouse zygote by microinjection; implanting the zygote into the oviduct of a surrogate mouse to obtain F0 generation mice; mating the F0 generation mice with wild type mice to obtain F1 generation heterozygous mice; mating the F1 generation heterozygous mice to obtain homozygous mice; and mating the homozygous mice with S100A9 knockout mice to obtain S100A8 / A9 humanized mouse model.

25. The method of claim 24, wherein the specific genomic locus comprises Rosa26 gene, AAVS1 gene, ROSA26 gene, CMAH gene, or β4GalNT2 gene.

26. The method of claim 25, wherein the specific genomic locus is ROSA26 gene.

27. The method of claim 26, wherein the introducing is achieved by CRISPR / Cas, and the sgRNA has the sequence of SEQ ID NO: 11 and SEQ ID NO:

23.

28. Use of the expression vector of any one of claims 1-19 in expressing S100A8 and S100A9 heterodimer.

29. Use of the expression vector of any one of claims 1-19 in constructing an animal model.

30. Use of the animal model obtained by the method of any one of claims 22-27 in drug screening or disease mechanism study.

31. The use of claim 30, wherein the disease comprises cardiovascular disease or tumor.

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