Fusion protein for inhibiting wnt signaling pathway and application thereof

Abstract

The application provides a fusion protein for inhibiting a Wnt signal path and application thereof, and relates to the field of biological medicines, and comprises the following: a first peptide segment, wherein the first peptide segment comprises a FU1 fragment; a second peptide segment, wherein the second peptide segment comprises a DRPB fragment; and the first peptide segment is connected with the second peptide segment. The prepared fusion protein can effectively inhibit the Wnt signal path, can inhibit excessive proliferation of cells under a pathological condition of abnormal activation of Wnt, and can effectively regulate the Wnt signal path.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to fusion proteins that inhibit the Wnt signaling pathway and their applications, and more specifically, to fusion proteins, nucleic acid molecules, expression vectors, recombinant cells, compositions, the use of said fusion proteins, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of drugs or reagents, cultured tissues, cultured organoids, drugs, reagents, and methods for culturing organoids. Background Technology

[0002] Wnt (“wingless-associated integration site” or “wingless and Int-1” or “wingless-Int”) ligands and their signaling play a crucial role in controlling the development, homeostasis, and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, and many other tissues (e.g., reviewed by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of the Wnt signaling pathway has the potential to treat degenerative diseases and tissue damage.

[0003] Wnt signaling is transduced by a family of seven transmembrane-domain coiled proteins (Fzd). Wnt ligands bind to Fzd, activating the cytoplasmic protein Disheveled and phosphorylating Lrp5 / 6. This generates a signal that inhibits the phosphorylation and degradation of β-catenin, thereby stabilizing it. This stabilization is induced by Dvl's association with axonal proteins (scaffold proteins that hold various proteins together, including GSK3, APC, CK1, and β-catenin) to form a β-catenin degradation complex. The stabilized β-catenin then enters the nucleus, binds to TCF, and converts it into a transcriptional activator, which in turn transcribes and activates downstream target genes.

[0004] One of the challenges of modulating the Wnt signaling pathway for therapeutic purposes is the existence of multiple Wnt ligands and Wnt receptors, including coiled protein 1-10 (Fzd1-10), with many tissues expressing multiple and overlapping Fzds. Typical Wnt signaling also involves low-density lipoprotein (LDL) receptor-associated protein 5 (Lrp5) or low-density lipoprotein (LDL) receptor-associated protein 6 (Lrp6) as co-receptors, which are also widely expressed in various tissues in addition to Fzds.

[0005] Currently available inhibitors targeting the Wnt signaling pathway include β-Catenin / Tcf Inhibitor (FH535), XAV 939, IWP-2, IQ-1, and Dickkopf-1 (DKK1). DKK1 is a secreted protein that binds to Lrp5 / 6 and another transmembrane protein, Kremen1 / 2, forming a trimer that induces rapid endocytosis, reducing Lrp5 / 6 on the cell membrane and thus blocking the transmission of Wnt signals into the cell. However, current inhibitors that suppress the Wnt signaling pathway at the receptor level, especially DKK1, are not highly efficient and cannot completely inhibit the Wnt signaling pathway. Therefore, researching Wnt signaling pathway inhibitors at the receptor level could provide a method for efficiently inhibiting the Wnt signaling pathway, reducing excessive cell proliferation caused by excessive Wnt signaling, and providing a research model for disease treatment.

[0006] The novel Wnt inhibitor discovered in this invention can effectively inhibit the Wnt signaling pathway. Summary of the Invention

[0007] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0008] The Wnt signaling pathway is a key signal transduction pathway regulating various biological processes, including embryonic development, tissue homeostasis, and stem cell maintenance. In the classical Wnt pathway, R-spondin (Rspo), a secreted protein, mediates the endocytosis and degradation of the membrane-bound E3 ubiquitin ligases RNF43 / ZNRF3 by binding to LGR4 / 5 (Leucine-rich repeat-containing G protein-coupled receptor 4 / 5) on the cell membrane. Since RNF43 / ZNRF3 can promote the degradation of Wnt receptors such as FZD through ubiquitination, the regulation of RNF43 / ZNRF3 by Rspo reduces the degradation level of Wnt receptors and enhances the binding efficiency of Wnt ligands to FZD and LRP5 / 6, thereby amplifying the activation effect of the classical Wnt signaling pathway and promoting the transcription of β-catenin-dependent target genes (including genes involved in negative feedback regulation such as RNF43 / ZNRF3). Furthermore, inhibitors that suppress the Wnt signaling pathway at the receptor level, especially Dkk1, are not effective in inhibiting the Wnt signaling pathway. Therefore, the inventors designed a variety of fusion proteins targeting the classic Wnt signaling pathway. After extensive experimental screening, they unexpectedly obtained a superior fusion protein that can effectively inhibit the Wnt signaling pathway. This fusion protein can be used as an inhibitor of the Wnt signaling pathway, to treat or prevent diseases related to the Wnt signaling pathway, and to create research models that inhibit the Wnt signaling pathway.

[0009] Therefore, in a first aspect, the present invention proposes a fusion protein. According to embodiments of the present invention, the fusion protein comprises a first peptide segment containing a FU1 fragment; a second peptide segment containing a DRPB fragment; the first peptide segment and the second peptide segment are linked. The Wnt signaling pathway regulates gene transcription and plays an important role in the differentiation process of organisms, directly controlling cell fate such as proliferation and differentiation in early animal development. Simultaneously, activation of the Wnt signaling pathway promotes cell proliferation and survival without differentiation; therefore, the classical Wnt signaling pathway also participates in tumor formation. Over-activation of the Wnt signaling pathway causes abnormal cell proliferation, thereby promoting tumor development. Permanent inactivation of the APC complex, i.e., the loss of a "genetic regulatory gating gene," and abnormal activation of the Wnt signaling pathway can also promote tumor development, but without dependence on the binding of Wnt ligands to receptors. Therefore, the fusion protein proposed in this invention can effectively treat or prevent diseases related to the Wnt signaling pathway, such as colorectal cancer, breast cancer, early malignant precancerous lesions of the small intestine, and lung cancer, which are not dependent on APC mutations. Through extensive experiments and screening of various proteins, the inventors obtained a superior fusion protein. According to embodiments of the present invention, this fusion protein can antagonize the Wnt signaling pathway and inhibit the proliferation of small intestinal organoids under pathological conditions of abnormal Wnt activation, thereby potentially alleviating Wnt-driven hyperproliferative diseases (such as colorectal tumors). Specifically, the fusion protein obtained by fusing the FU1 and DRPB fragments possesses binding domains that bind to coiled protein (FZD) and E3 ubiquitin ligases (RNF43 / ZNRF3), effectively inhibiting the Wnt signaling pathway with significant inhibitory effects.

[0010] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the fusion protein described in the first aspect. The fusion protein encoded by the nucleic acid molecule according to embodiments of the invention can inhibit the excessive proliferation of small intestinal cells under pathological conditions of abnormal Wnt activation by antagonizing the Wnt signaling pathway, thereby potentially alleviating Wnt-driven hyperproliferative diseases (such as colorectal tumors).

[0011] In a third aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector may include optional control sequences operatively linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. These control sequences may be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The expression vector proposed in the embodiments of the present invention can efficiently express the fusion protein in suitable host cells. The fusion protein can effectively inhibit the Wnt signaling pathway and suppress excessive proliferation of small intestinal cells in a pathological model of abnormal Wnt activation, and can be used for the prevention or treatment of Wnt-driven diseases.

[0012] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect; or expressing the fusion protein described in the first aspect. Under certain conditions, the recombinant cell according to embodiments of the present invention can express and secrete a large amount of the fusion protein, and the obtained fusion protein can effectively inhibit the Wnt signaling pathway, thereby inhibiting the excessive proliferation of small intestinal cells.

[0013] In a fifth aspect, the present invention provides a composition. According to embodiments of the invention, the composition comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. As previously mentioned, the fusion protein according to embodiments of the present invention can effectively inhibit the Wnt signaling pathway, thereby inhibiting the excessive proliferation of small intestinal cells. Therefore, compositions containing the fusion protein, such as food compositions, pharmaceutical compositions, etc., can also effectively inhibit the Wnt signaling pathway and inhibit the excessive proliferation of small intestinal cells.

[0014] In a sixth aspect, the present invention provides the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the composition described in the fifth aspect in the preparation of a medicament. According to embodiments of the present invention, the use of the medicament includes at least one of the following: antagonism of the Wnt signaling pathway; treatment and / or prevention of Wnt signaling pathway-related diseases. The fusion protein of the present invention, or a fusion protein obtained using nucleic acid molecules, expression vectors, recombinant cells, or a portion thereof, can effectively inhibit the Wnt signaling pathway under certain conditions, thereby inhibiting the excessive proliferation of small intestinal cells and effectively treating or preventing Wnt signaling pathway-related diseases.

[0015] In a seventh aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the composition described in the fifth aspect. The Wnt signaling pathway regulates gene transcription and plays a crucial role in the differentiation process of organisms, directly controlling cell fate such as proliferation and differentiation in early animal development. Simultaneously, activation of the Wnt signaling pathway promotes cell proliferation and survival without differentiation; therefore, the classical Wnt signaling pathway also participates in tumor formation. Over-activation of the Wnt signaling pathway causes abnormal cell proliferation, thereby promoting tumor development. Permanent inactivation of the APC complex, i.e., the loss of a "genetic regulatory gating gene," and abnormal activation of the Wnt signaling pathway can also promote tumor development, but this does not depend on the binding of the Wnt ligand to its receptor. Therefore, the medicament proposed in this invention can effectively treat or prevent diseases related to the Wnt signaling pathway, such as colon cancer and breast cancer that are not dependent on APC mutations, early malignant precancerous lesions of the small intestine, and lung cancer. As mentioned above, the fusion protein of the present invention can effectively inhibit the Wnt signaling pathway, thereby regulating gene transcription and affecting cell proliferation and differentiation. According to specific embodiments of the present invention, nucleic acid molecules, expression vectors, recombinant cells, or drugs containing the above substances can also effectively inhibit the Wnt signaling pathway under suitable conditions, such as inhibiting the excessive proliferation of small intestinal cells, which can effectively treat or prevent diseases related to the Wnt signaling pathway.

[0016] In an eighth aspect of the invention, the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fourth aspect, or the composition described in the fifth aspect in the preparation of a reagent is provided. According to embodiments of the invention, the reagent is used to inhibit the proliferation of small intestinal cells. The fusion protein of the invention, or a portion of the fusion protein or composition obtained using nucleic acid molecules, expression vectors, or recombinant cells, can effectively inhibit the Wnt signaling pathway under certain conditions, thereby inhibiting the excessive proliferation of small intestinal cells.

[0017] In a ninth aspect, the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises: the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fourth aspect, or the composition described in the fifth aspect. As previously stated, the fusion protein of the embodiments of the present invention can effectively inhibit the Wnt signaling pathway, thereby inhibiting the excessive proliferation of small intestinal cells. Therefore, the reagent comprising the fusion protein and a series of related substances also has the above-mentioned effects.

[0018] In a tenth aspect of the invention, the invention provides the use of the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fourth aspect, the composition described in the fifth aspect, or the reagent described in the ninth aspect in cultured tissues and organoids, wherein the use is for non-therapeutic purposes. As previously stated, the fusion protein can effectively inhibit the Wnt signaling pathway. Therefore, the fusion protein, or the nucleic acid molecule, expression vector, or recombinant cell capable of expressing the fusion protein under suitable conditions, or the composition containing the fusion protein, can also effectively inhibit the Wnt signaling pathway. During tissue or organoid culture, contacting small intestinal cells with the fusion protein including the FU1 fragment and the DRPB fragment can effectively induce small intestinal cells to transform from a 3D structured small intestinal organoid into a state of structural disintegration and cell diffusion.

[0019] In an eleventh aspect, the present invention proposes a method for intervening in organoid growth. According to an embodiment of the present invention, the method includes the step of contacting small intestinal cells with the fusion protein described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fourth aspect, the composition described in the fifth aspect, or the reagent described in the ninth aspect. As previously mentioned, the fusion protein can effectively inhibit the Wnt signaling pathway. According to a specific embodiment of the present invention, after contacting small intestinal cells with the fusion protein including the FU1 fragment and the DRPB fragment, the cells will change from a 3D structured small intestinal organoid to a state of structural disintegration and cell diffusion.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0022] Figure 1 This is a graph showing the experimental results of dual-luciferase reporter gene detection of different types of proteins inhibiting the Wnt pathway according to an embodiment of the present invention. The data in the graph are expressed as Means±SEM (n=3), and the vertical axis (Relative luciferase activity (fold)) represents the relative fluorescence activity.

[0023] Figure 2 This is a diagram showing the results of normal culture of small intestinal organoids according to an embodiment of the present invention;

[0024] Figure 3 This is a diagram showing the results of treating small intestinal organoids with FU1-DRPB recombinant protein according to an embodiment of the present invention. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0027] In this article, "operable ligation" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0028] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0029] In this article, the term "amino acid" is represented by a single-letter or three-letter code with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Glycine; H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0030] For nucleotides, the terms "identity," "homology," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0031] For peptides, the terms "identity," "homology," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0032] The variant sequences described in this invention can have at least 95% identity (or homology) with the reference sequences, meaning at least 95%, and possibly 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0033] In this invention, the inventors constructed a plasmid corresponding to a Wnt signaling pathway inhibitor (FU1-DRPB) using molecular cloning methods. To verify its function, the effect of overexpression of this inhibitor on the Wnt signaling pathway was further examined in human 293T cells using a Top-flash dual-luciferase reporter gene assay system. The experimental results showed that overexpression of FU1-DRPB inhibited the Wnt signaling pathway by up to 99%, indicating that this Wnt inhibitor can effectively suppress the Wnt signaling pathway.

[0034] Furthermore, the inventors collected the supernatant of 293T cells overexpressing the inhibitor (FU1-DRPB) and cultured small intestinal organoids in a complete culture medium containing 50% FU1-DRPB supernatant for 48 hours. They found that the small intestinal organoids changed from a solid budding morphology to a state of structural disintegration and cell diffusion.

[0035] Therefore, this invention proposes a fusion protein containing FU1 fragment and DRPB fragment, a nucleic acid molecule, an expression vector, recombinant cells, a composition, and the use of the fusion protein, nucleic acid molecule, expression vector, recombinant cells, or composition in the preparation of drugs or reagents, the culture of tissues, and the culture of organoids, as well as methods for the drugs, reagents, and the culture of organoids, which will be described in detail below.

[0036] Fusion protein

[0037] In one aspect of the invention, a fusion protein is proposed. According to an embodiment of the invention, the fusion protein comprises a first peptide segment containing an FU1 fragment; a second peptide segment containing a DRPB fragment; and the first peptide segment is linked to the second peptide segment. Through extensive experiments, the inventors combined and screened various proteins to obtain a superior fusion protein. The fusion protein according to an embodiment of the invention can effectively inhibit the Wnt signaling pathway, specifically by inhibiting the excessive proliferation of small intestinal cells under pathological conditions of abnormal Wnt activation, thereby potentially alleviating Wnt-driven hyperproliferative diseases (such as colorectal tumors). The fusion protein obtained by fusing the FU1 and DRPB fragments includes a binding domain that binds to one or more coiled protein (FZD) receptors and E3 ubiquitin ligases (RNF43 / ZNRF3), which can effectively inhibit the Wnt signaling pathway.

[0038] It should be noted that in this invention, "fusion protein" and "recombinant protein" have the same meaning and can be used interchangeably.

[0039] According to some specific embodiments of the present invention, the above-mentioned fusion protein may further include at least one of the following additional technical features:

[0040] According to some specific embodiments of the present invention, the C-terminus of the first peptide segment is connected to the N-terminus of the second peptide segment; or the C-terminus of the second peptide segment is connected to the N-terminus of the first peptide segment.

[0041] According to some specific embodiments of the present invention, the FU1 fragment has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 95% identity with SEQ ID NO: 3.

[0042] According to some specific embodiments of the present invention, the DRPB fragment has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 95% identity with SEQ ID NO: 6.

[0043] According to some specific embodiments of the present invention, the fusion protein further includes a linker peptide. The linker peptide is not particularly limited; conventional linkers in the art can be used.

[0044] According to some specific embodiments of the present invention, the N-terminus of the linker peptide is connected to the C-terminus of the first peptide segment, and the C-terminus of the linker peptide is connected to the N-terminus of the second peptide segment; or the N-terminus of the linker peptide is connected to the C-terminus of the second peptide segment, and the C-terminus of the linker peptide is connected to the N-terminus of the first peptide segment.

[0045] According to some specific embodiments of the present invention, the linker peptide has an amino acid sequence as shown in SEQ ID NO: 10.

[0046] According to some specific embodiments of the present invention, the fusion protein further includes a signal peptide.

[0047] According to some specific embodiments of the present invention, the C-terminus of the signal peptide is connected to the N-terminus of the first peptide segment, or the C-terminus of the signal peptide is connected to the N-terminus of the second peptide segment.

[0048] According to some specific embodiments of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 4.

[0049] According to some specific embodiments of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 9.

[0050] Nucleic acid molecules, expression vectors

[0051] In another aspect of the invention, a nucleic acid molecule is proposed. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned fusion protein. The fusion protein encoded by the nucleic acid molecule according to an embodiment of the invention can effectively inhibit the Wnt signaling pathway and suppress excessive proliferation of small intestinal cells under pathological conditions of abnormal Wnt activation, thereby potentially alleviating Wnt-driven hyperproliferative diseases (such as colorectal tumors).

[0052] According to some specific embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:

[0053] According to some specific embodiments of the present invention, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 7.

[0054] It should be noted that those skilled in the art should understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. Furthermore, the nucleic acid sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0055] In another aspect, the present invention provides an expression vector. According to embodiments of the invention, the expression vector carries the aforementioned nucleic acid molecule. The expression vector may include optional control sequences operatively linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host; these control sequences may be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The expression vector proposed in the embodiments of the present invention can efficiently express the fusion protein in suitable host cells, and the fusion protein can effectively inhibit the Wnt signaling pathway, thereby inhibiting the excessive proliferation of small intestinal cells.

[0056] Recombinant cells, compositions

[0057] In another aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the aforementioned nucleic acid molecule or the aforementioned expression vector; or expresses the aforementioned fusion protein. Under certain conditions, the recombinant cell according to embodiments of the invention can express and secrete the fusion protein in large quantities. The obtained fusion protein can effectively inhibit the Wnt signaling pathway and, under pathological conditions of abnormal Wnt activation, can inhibit the excessive proliferation of small intestinal cells, thereby potentially alleviating Wnt-driven hyperproliferative diseases (such as colorectal tumors).

[0058] According to some specific embodiments of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into host cells.

[0059] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma fissicerum, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as small intestinal cells, BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including T cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0060] It should be noted that the "suitable conditions" mentioned in this specification refer to conditions suitable for the expression of the fusion protein described in this invention. Those skilled in the art will readily understand that suitable conditions for fusion protein expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion protein according to the specific environment of their laboratory.

[0061] In another aspect, the present invention provides a composition. According to embodiments of the present invention, the composition comprises: the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cells. As previously stated, the fusion protein can effectively inhibit the Wnt signaling pathway, thereby controlling cell proliferation and differentiation. According to some embodiments of the present invention, the fusion protein can inhibit the excessive proliferation of small intestinal cells. Therefore, compositions containing the fusion protein, such as food compositions and pharmaceutical compositions, can also effectively inhibit the Wnt signaling pathway and suppress the excessive proliferation of small intestinal cells. Furthermore, the classical Wnt signaling pathway is involved in tumor formation; therefore, the composition can effectively treat or prevent diseases related to the Wnt signaling pathway.

[0062] It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0063] Drugs and reagents

[0064] In another aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned composition. As previously stated, the fusion protein can effectively inhibit the Wnt signaling pathway, thereby regulating gene transcription and controlling cell proliferation and differentiation. Therefore, a medicament comprising the fusion protein and a series of related substances also possesses the aforementioned effects.

[0065] The medicament provided according to some specific embodiments of the present invention includes a pharmaceutically acceptable carrier and an effective amount of the active ingredient of the fusion protein.

[0066] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0067] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0068] The medicaments of this invention contain a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the medicaments of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The medicaments are preferably manufactured under aseptic conditions.

[0069] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0070] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0071] In another aspect, the present invention provides a reagent. According to embodiments of the present invention, the reagent comprises: the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned composition. As previously stated, the fusion protein of the embodiments of the present invention can effectively inhibit the Wnt signaling pathway, thereby regulating gene transcription and controlling cell proliferation and differentiation. According to some specific embodiments of the present invention, the fusion protein can effectively inhibit the excessive proliferation of small intestinal cells. Therefore, a reagent containing the fusion protein and a series of related substances also has the above-mentioned effects. The reagent can be in any form, such as solid or liquid.

[0072] use

[0073] In another aspect, the present invention provides for the use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned composition in the preparation of a medicament. According to embodiments of the present invention, the use of the medicament includes at least one of the following: antagonism of the Wnt signaling pathway; treatment and / or prevention of Wnt signaling pathway-related diseases. The fusion protein or fusion protein or composition obtained using the aforementioned nucleic acid molecule, expression vector, or recombinant cell can effectively inhibit the Wnt signaling pathway under certain conditions, such as inhibiting the excessive proliferation of small intestinal cells.

[0074] According to some specific embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0075] According to some specific embodiments of the present invention, the Wnt signaling pathway-related diseases include at least one of the following: colorectal cancer, liver cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, pancreatic cancer, lung cancer, pilomatoma, familial adenomatous polyposis, familial adenomatous polyposis (FAP), β-catenin-associated familial melanoma syndrome, and osteoarthritis.

[0076] In another aspect, the present invention proposes the use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned composition in the preparation of reagents. According to embodiments of the present invention, the reagent is used to inhibit cell proliferation. The fusion protein of the present invention, or a portion thereof obtained using nucleic acid molecules, expression vectors, recombinant cells, or other components, can effectively inhibit the Wnt signaling pathway under certain conditions, thereby regulating gene transcription and controlling cell proliferation and differentiation. According to some specific embodiments of the present invention, the fusion protein can effectively inhibit the excessive proliferation of small intestinal cells.

[0077] According to some specific embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0078] According to some specific embodiments of the present invention, the cells include small intestinal cells, colon cells, liver cells, gastric cells, or pancreatic cells.

[0079] In another aspect, the present invention proposes the use of the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned composition, or the aforementioned reagent in the culture of tissues and organoids. As previously stated, the fusion protein can effectively inhibit the Wnt signaling pathway. Therefore, the fusion protein, or a nucleic acid molecule, expression vector, or recombinant cell capable of expressing the fusion protein under suitable conditions, or a composition containing the fusion protein, can also effectively inhibit the Wnt signaling pathway. According to a specific embodiment of the present invention, during tissue or organoid culture, after contacting small intestinal cells with the fusion protein including the FU1 fragment and the DRPB fragment, the small intestinal cells can be transformed from a 3D structured small intestinal organoid into a state of structural disintegration and cell diffusion.

[0080] According to some specific embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0081] According to some specific embodiments of the present invention, the tissue includes: epithelial tissue, connective tissue, muscle tissue, or nerve tissue.

[0082] According to some specific embodiments of the present invention, the epithelial tissue includes: skin tissue, gastric lining, pancreatic lining, or liver.

[0083] According to some specific embodiments of the present invention, the connective tissue includes: the inner layer of skin, tendons, ligaments, cartilage, bone, fat, hair, or blood.

[0084] According to some specific embodiments of the present invention, the neural tissue includes glial cells and neurons.

[0085] According to some specific embodiments of the present invention, the organoids include: small intestinal organoids, colon organoids, liver organoids, stomach organoids, or pancreatic organoids.

[0086] method

[0087] In another aspect, the present invention provides a method for preparing the aforementioned fusion protein. According to an embodiment of the invention, the method includes: introducing the aforementioned expression vector into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the fusion protein. The method according to the embodiment of the invention can express the fusion protein in large quantities, and the fusion protein can effectively inhibit the Wnt signaling pathway, thereby inhibiting the excessive proliferation of small intestinal cells.

[0088] According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0089] According to some specific embodiments of the present invention, the cells are eukaryotic cells or prokaryotic cells.

[0090] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells.

[0091] According to some specific embodiments of the present invention, the mammalian cells include at least one selected from small intestinal cells, T cells, CHO-K1, CHOS, 293F, 293T, CAR-T, TCR-T, TIL, CAR-NK, CAR-M and CAR-DC.

[0092] According to some specific embodiments of the present invention, the mammalian cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0093] In another aspect, the present invention provides a method for culturing organoids or intervening in organoid growth. According to embodiments of the present invention, the method includes contacting cells with the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cells, or the aforementioned composition. As previously mentioned, the fusion protein can effectively inhibit the Wnt signaling pathway. According to some specific embodiments of the present invention, after contacting small intestinal cells with the fusion protein comprising FU1 and DRPB fragments, the small intestinal cells are transformed from a 3D structured small intestinal organoid into a state of structural disintegration and cell diffusion, which can efficiently inhibit small intestinal organoids.

[0094] According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0095] According to some specific embodiments of the present invention, the method further includes the following steps: 1) digesting the tissue to obtain the cells; 2) culturing the cells in 3D to obtain the organoids.

[0096] According to some specific embodiments of the present invention, the cells include: induced pluripotent stem cells, small intestinal cells, colon cells, liver cells, gastric cells, pancreatic cells, glial cells, neurons, muscle cells, or adipocytes. Those skilled in the art will understand that the cells are not particularly limited, and any cells whose cell state can be regulated (e.g., suppressed) by the Wnt signaling pathway can be cultured into 3D organoids using the above method.

[0097] In another aspect, the present invention provides a method for culturing tissues. According to embodiments of the present invention, the method includes the step of contacting small intestinal cells with the aforementioned fusion protein, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cells, the aforementioned composition, or the aforementioned reagent. As previously mentioned, the fusion protein can effectively inhibit the Wnt signaling pathway, regulate gene transcription, and control cell proliferation and differentiation. According to some specific embodiments of the present invention, after contacting small intestinal cells with the fusion protein comprising FU1 and DRPB fragments, the small intestinal cells are transformed from 3D intestinal organoids into a state of structural disintegration and cell diffusion, which can efficiently inhibit small intestinal organoids.

[0098] According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0099] According to some specific embodiments of the present invention, the cells include: induced pluripotent stem cells, small intestinal cells, colon cells, liver cells, gastric cells, pancreatic cells, glial cells, neuronal cells, muscle cells, or adipocytes.

[0100] According to some specific embodiments of the present invention, the cells include: small intestinal stem cells, colon stem cells, liver cells, gastric stem cells, or pancreatic stem / progenitor cells.

[0101] According to some specific embodiments of the present invention, the cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0102] It should be noted that the terms "plasmid" and "vector" used in the following implementation have the same meaning and can be used interchangeably.

[0103] The embodiments will be described in detail below. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0104] Example 1: Construction of expression vector

[0105] In this embodiment, an expression vector for FU1 was constructed. The specific experimental steps are as follows:

[0106] 1.1 Construction of FU1 plasmid

[0107] (1) A nucleotide sequence encoding a signal peptide (SEQ ID NO: 2) is added to the N-terminus of the nucleotide sequence encoding FU1 (SEQ ID NO: 1), and a nucleotide sequence encoding a linker is added to the C-terminus of the nucleotide sequence encoding FU1 (SEQ ID NO: 1). The amino acid sequence of FU1 is shown in SEQ ID NO: 3, and the signal peptide has the amino acid sequence shown in SEQ ID NO: 4.

[0108] The nucleotide sequence encoding FU1 is shown below:

[0109] GCCGAGGGGAGCCAGGCCTGTGCCAAAGGCTGTGAGCTCTGCTCTGAAGTCAACGGCTGCCTCAAGTGCTCACCCAAGCTGTTCATCCTGCTGGAGAGGAACGACATCCGCCAGGTGGGCGTCTGCTTGCCGTCCTGCCCACCTGGATACTTCGAC (SEQ ID NO: 1).

[0110] The nucleotide sequence encoding the signal peptide is shown below:

[0111] ATGGACGCCATGAAGAGAGGCCTGTGCTGCGTGCTGCTGCTGTGCGGCGCTGTGTTTGTGTCTGCC (SEQ ID NO: 2).

[0112] The amino acid sequence of FU1 is shown below:

[0113] AEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFD (SEQ ID NO: 3).

[0114] The amino acid sequence of the signal peptide is shown below:

[0115] MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO: 4).

[0116] (2) Insert the nucleotide sequence encoding FU1 with the signal peptide into the pcDNA3.1(+) overexpression vector and send it to the company to synthesize plasmids.

[0117] 1.2 Construction of DRPB plasmid

[0118] (1) A nucleotide sequence encoding a signal peptide (SEQ ID NO: 2) is added to the N-terminus of the nucleotide sequence encoding DRPB (SEQ ID NO: 5), wherein the amino acid sequence of DRPB is as shown in SEQ ID NO: 6, and the signal peptide has the amino acid sequence shown in SEQ ID NO: 4.

[0119] (2) Insert the DRPB nucleotide sequence with the signal peptide into the pcDNA3.1(+) overexpression vector and send it to the company to synthesize plasmid.

[0120] The nucleotide sequence encoding DRPB is shown below:

[0121] (SEQ ID NO: 5).

[0122] The amino acid sequence of DRPB is shown below:

[0123] SELGTRLIRAALDGNKDRVKDLIENGADVNASLMSGATPLHAAAMNGHKEVVKLLISKGADVNAQSVAGSTPLDAAAFSGHKEVVKLLISKGADVNAVNAAGLTPLHAAADNGHKEVVKLLISKGADVNAKADHGMTPLHFAAQRGHKEVVKLLISKGADLNTSAKDGATPLDMARESGNEEVVKLLEKQ (SEQ ID NO: 6).

[0124] 1.3 Construction of FU1-DRPB plasmid

[0125] (1) A nucleotide sequence encoding a signal peptide (SEQ ID NO: 2) is added to the N-terminus of the nucleotide sequence encoding FU1-DRPB (SEQ ID NO: 7), the signal peptide having the amino acid sequence shown in SEQ ID NO: 4; wherein, the FU1 and DRPB peptides are linked by a linker, the nucleotide sequence encoding the linker being shown in SEQ ID NO: 8. The amino acid sequence of FU1-DRPB is shown in SEQ ID NO: 9, and the amino acid sequence of the linker is shown in SEQ ID NO: 10.

[0126] (2) Insert the nucleotide sequence of FU1-DRPB with the nucleotide sequence encoding the signal peptide obtained in step (1) into the pcDNA3.1(+) overexpression vector and send it to the company to synthesize the plasmid. The synthesized precursor protein FU1-DRPB contains a signal peptide, which helps the above recombinant protein to be localized. After the recombinant protein matures, the signal peptide will be hydrolyzed and released. Therefore, the final recombinant protein does not contain the signal peptide sequence.

[0127] The nucleotide sequence encoding FU1-DRPB is as follows:

[0128] GCCGAGGGGAGCCAGGCCTGTGCCAAAGGCTGTGAGCTCTGCTCTGAAGTCAACGGCTGCCTCAAGTGCTCACCCAAGCTGTTCATCCTGCTGGAGAGGAACGACATCCGCCAGGTGGGCGTCTGCTTGCCGTCCTGCCCACCTGGATACTTCGACGCCCGCAACCCCGACAGCGAGCTGGGCACCAGACTGATCCGGGCCGCTCTGGATGGAAACAAGGACAGAGTGAAGGATCTGATTGAGAACGGTGCCGACGTGAATGCTAGCCTCATGAGCGGCGCTACCCCACTGCACGCCGCTGCAATGAATGGCCACAAGGAGGTGGTTAAACTCCTGATCAGCAAGGGCGCCGATGTGAATGCTCAATCTGTGGCCGGCAGCACACCTCTGGATGCCGCCGCCTTCAGCGGGCACAAGGAGGTGGTCAAGCTGCTGATCTCCAAGGGAGCTGACGTCAACGCCGTGAACGCCGCTGGCCTGACACCCCTGCATGCCGCCGCCGATAACGGCCACAAGGAAGTGGTGAAACTGCTGATCTCTAAGGGCGCTGATGTGAACGCCAAGGCTGACCACGGCATGACCCCACTGCACTTCGCCGCCCAGAGAGGCCACAAGGAAGTGGTGAAACTTCTGATCAGCAAGGGAGCCGACCTGAATACCTCCGCCAAGGACGGCGCCACCCCTCTGGATATGGCCCGCGAGAGCGGCAACGAGGAAGTCGTGAAGTTGCTGGAGAAGCAG (SEQ ID NO:7).

[0129] The nucleotide sequence encoding the linker peptide is shown below:

[0130] GCCCGCAACCCCGAC (SEQ ID NO: 8).

[0131] The amino acid sequence of FU1-DRPB is shown below:

[0132] AEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDSELGTRLIRAALDGNKDRVKDLIENGADVNASLMSGATPLHAAAMNGHKEVVKLLISKGADVNAQSVA GSTPLDAAAFSGHKEVVKLLISKGADVNAVNAAGLTPLHAAADNGHKEVVKLLISKGADVNAKADHGMTPLHFAAQRGHKEVVKLLISKGADLNTSAKDGATPLDMARESGNEEVVKLLEKQ (SEQ ID NO: 9).

[0133] The amino acid sequence of the linker is shown below:

[0134] ARNPD (SEQ ID NO: 10).

[0135] Example 2: Detection Inhibitors in a Dual-Luciferase Reporter Gene Detection System

[0136] 2.1 Experimental Procedure

[0137] (1) Prepare plasmids; plasmids expressing FU1, DRPB, and FU1-DRPB obtained in Example 1, TopFlash expressing firefly luciferase, Renilla expressing kidney luciferase, and empty vector plasmid pcDNA3.1(+);

[0138] (2) Resuscitate a healthy 293T cell and culture it in DMEM medium (containing 10% fetal bovine serum). After passage, seed it into 24-well plates with 500 μL of medium per well to achieve a cell density of 60%-80% at the time of transfection.

[0139] (3) 1-2 hours before transfection, replace the cells with antibiotic-free DMEM (catalog number: 12800017, company: Gibco) medium containing 10% fetal bovine serum (catalog number: F8318, company: Sigma);

[0140] (4) Preparation of transfection working solution: For each well of a 24-well plate, prepare an appropriate amount of plasmid and add it to the dilution buffer to a total volume of 50 μL. Mix gently and incubate at room temperature. The 24-well plate is divided into groups of 3 wells. The amount of DNA used in each group is prepared as follows:

[0141] Construction of FU1-DRPB inhibitor expression vector:

[0142] Experimental group: 200ng TopFlash + 50ng Renilla + 200ng Gene1 + 200ng pcDNA3.1(+), where Gene1 includes nucleotide sequences encoding pcDNA3.1(+), FU1, DRPB, and FU1-DRPB.

[0143] Control group: 200ng TopFlash + 50ng Renilla + 200ng pcDNA3.1(+) + 1025ng pcDNA3.1(+);

[0144] (5) For a 24-well plate, add 1 μl of transfection reagent (PEI) to each well. Add the transfection reagent to the transfection working solution prepared in step (4), mix gently, let stand at room temperature for 15 minutes, then gently and evenly drop it into each well of cells, and finally shake gently to mix. Place the cells in an incubator for culture.

[0145] (6) After co-transfecting the inhibitor plasmid prepared in step (4) for 12-16 h, change the medium: During the medium change, the co-transfection medium of the transfected plasmid was discarded in the experiment for detecting FU1-DRPB inhibitors, and the experimental group was replaced with DMEM medium containing 100 ng / mL Wnt3a growth factor (purchased from Bozhen, production batch number RWL003) and 10% FBS. The cells were then placed in an incubator and cultured for 24 h. All test samples were as follows: Figure 1 As shown, Figure 1 The "+Wnt3a" indicates that Wnt3a has been added to the detection system according to the method described in this step;

[0146] (7) After the culture is completed, the cell culture medium is discarded, the cells are washed twice with PBS, and an appropriate amount of Vazyme 1× cell lysis buffer (100 μL per well in a 24-well plate) is added to each well. The cells are shaken and lysed for 15 min. The cell lysis products are pipetted and aspirated into 1.5 mL centrifuge tubes and centrifuged at 12000 g at room temperature for 2 min.

[0147] (8) Add 100 μL of Luciferase Substrate reagent equilibrated to room temperature to a 96-well microplate. Carefully add 20 μL of cell lysis supernatant to the microplate, mix quickly, and immediately place it in a microplate reader to read the firefly luciferase activity. Then, immediately add 100 μL of Stop & Reaction Buffe (firefly luciferase stop solution and Renilla luciferase substrate) to the reaction solution, mix well, and immediately place it in a microplate reader to read the Renilla luciferase activity. The ratio of the firefly luciferase value to the Renilla luciferase value for each sample is the luciferase expression intensity.

[0148] 2.2 Analysis of Experimental Results

[0149] Specific results are as follows Figure 1 As shown, without the addition of exogenous Wnt 3a, the inhibition efficiency of the DRPB group was 23%, the FU1 group even activated 44%, while the inhibition efficiency of the FU1-DRPB group was 56%. After adding exogenous Wnt 3a, the control group with 100 ng / mL Wnt3a activated the Wnt signaling pathway by approximately 138 times. Compared with the Wnt3a control group, the inhibition efficiency of the Wnt3a DRPB group was 38%, the Wnt3a FU1 group had almost no inhibitory effect, while the inhibition efficiency of the Wnt3a FU1-DRPB group was as high as 98%. Therefore, the fusion protein of DRPB and FU1 has a highly efficient inhibitory effect on the Wnt signaling pathway.

[0150] Example 3: Effects of FU1-DRPB fusion protein on organoids

[0151] 3.1 Collect FU1-DRPB supernatant

[0152] (1) Plasmid preparation: Plasmid expressing FU1-DRPB obtained in Example 1, and secretible empty vector pcDNA3.1(+);

[0153] (2) After passage of 293T cells, seed them into 6-well plates with 2 mL of culture medium per well; so that the cell density reaches 60%-80% at the time of transfection;

[0154] (3) 1-2 hours before transfection, replace the cells with antibiotic-free DMEM (catalog number: 12800017, company: Gibco) medium containing 10% fetal bovine serum (catalog number: FSP500, company: Excell);

[0155] (4) Prepare transfection working solution (per well): Take 2 μg of FU1-DRPB plasmid or 2 μg of secretory empty vector pcDNA3.1(+) plasmid, add it to the dilution buffer to a total volume of 100 μL, mix gently and place at room temperature;

[0156] (5) Take 4 μL of transfection reagent (PEI) in each well of a 6-well plate, add the required amount of transfection reagent to the transfection working solution prepared in step (4), then mix gently, place at room temperature for 15 minutes, then gently and evenly drop into each well of cells, finally shake gently to mix, and put the cells into an incubator for culture.

[0157] (6) 12-16 h after plasmid transfection, replace with fresh DMEM medium containing 1% FBS;

[0158] (7) After changing the medium for 24 hours, replace it with fresh DMEM medium containing 1% FBS, and collect the supernatant of FU1-DRPB or empty pcDNA3.1(+) into a 15mL centrifuge tube. Sieve the obtained supernatant (0.22μm), and then store the sieved supernatant at 4℃ for later use.

[0159] (8) After changing the medium again for 24 hours, collect the supernatant of FU1-DRPB or empty pcDNA3.1(+) into a 15mL centrifuge tube, sieve the obtained supernatant (0.22μm), and then store the sieved supernatant at 4℃ for later use.

[0160] 3.2 Treatment of small intestinal organoids with FU1-DRPB inhibitors

[0161] Small intestinal organoids were isolated from wild-type C57 / B6 mice using the following specific method:

[0162] (1) Mice (6-8 weeks old) were euthanized by cervical dislocation. The abdominal cavity of the mice was cut open with sterile surgical instruments to fully expose the small intestine.

[0163] (2) Remove the small intestine and place it in pre-cooled DPBS buffer at 4 degrees Celsius for initial rinsing;

[0164] (3) Cut the small intestine longitudinally with surgical scissors and rinse it three times with DPBS buffer to remove the chyme;

[0165] (4) Gently and carefully scrape away the villi of the small intestine from the inner wall of the small intestine with a scalpel, and then rinse once with DPBS;

[0166] (5) Cut the remaining part into 5 mm long pieces and incubate on ice for 15 minutes with DPBS buffer containing 5 mM EDTA;

[0167] (6) Cut off the 1ml pipette tip and gently blow and beat the fragments repeatedly to make a large amount of small intestinal crypts detach from the fragments into the supernatant;

[0168] (7) The supernatant was filtered through a 70 μm pore size cell sieve (purchased from Jettech, catalog number CSS013070) to remove residual intestinal villi, and then centrifuged at 300g for 3 min to remove scattered single cells. After centrifugation, the supernatant was discarded, and the precipitate was a relatively pure intestinal pit;

[0169] (8) Add an appropriate amount of extracellular matrix to the separated pits and mix well on ice. After mixing, place on ice.

[0170] (9) Transfer the mixture of extracellular matrix and pits to the cell culture plate. Add 20-30 μL of the mixture to each well of the 24-well cell culture plate. The mixture must be added to the bottom of the culture well. After spreading the mixture, it should not touch the side wall of the culture well.

[0171] (10) Place the 24-well cell culture plate in a 37°C, 5% CO2 cell culture incubator and let it stand for 15 minutes. After confirming that the extracellular matrix has completely solidified, slowly add complete culture medium along the well wall (500 μL of complete culture medium for each well, the composition of which is shown in Table 1). Return the 24-well cell culture plate to the cell culture incubator. As the culture time increases, the pits will gradually grow into 3D organoids;

[0172] Table 1

[0173]

[0174] (11) After 48 hours of culture, the control group was replaced with a complete culture medium containing 50% empty pcDNA3.1(+) supernatant, and the experimental group was replaced with a complete culture medium containing 50% FU1-DRPB supernatant.

[0175] 3.2 Analysis of Experimental Results

[0176] Specific experimental results are as follows: Figure 2 and Figure 3 As shown, after culturing small intestinal organoids for 48 hours, the control group consisted of solid, budding organoids. Figure 2 ), while the organoids of the FU1-DRPB group changed from a solid budding morphology to a morphology of overall morphological disintegration ( Figure 3 The Wnt signaling pathway is known to play a crucial role in maintaining the intact three-dimensional structure and continuous proliferation capacity of small intestinal organoids. Therefore, the FU1-DRPB of this invention can inhibit this pathway, causing organoids to lose the support of the stem cell microenvironment, thereby leading to structural disintegration.

[0177] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0178] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

9.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

7.

4. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant cell, characterized in that, The recombinant cells include: Carrying the nucleic acid molecule of claim 2 or 3 or the expression vector of claim 4; or Express the fusion protein according to claim 1.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 4 into host cells; The recombinant cells include eukaryotic cells or prokaryotic cells.

7. A composition for inhibiting the Wnt signaling pathway, characterized in that, include: The fusion protein of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4, or the recombinant cell of any one of claims 5-6.

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