Fusion protein of DR5 and MYDGF and application thereof

Abstract

The invention discloses a fusion protein of DR5 and MYDGF and application of the fusion protein. The fusion protein has DR5 and MYDGF activities, can block TRAIL-induced cell apoptosis, stimulate cell phosphorylation and promote cell tubulation, can target damaged heart, and plays a protective role in heart diseases or symptoms, especially myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to a fusion protein comprising DR5 and MYDGF and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) and its complications rank first in the cardiovascular disease mortality list due to their rapid progression and short golden rescue time, which seriously threatens the health of Chinese residents. Currently, the main treatments for AMI include thrombolysis, bypass surgery, and percutaneous coronary intervention (PCI). However, irreversible damage to myocardial tissue caused by ischemia and reperfusion is still a difficult problem in clinical practice, and there is no effective treatment drug in clinical practice.

[0003] The mechanism of reperfusion injury involves many cellular and molecular biological events such as ROS generation, intracellular calcium overload, mitochondrial dysfunction, cell inflammation, autophagy, apoptosis, etc., which intersect each other into a complex network and eventually lead to myocardial cell death. How to develop targeted drugs to effectively avoid possible reperfusion injury is the focus and difficulty of current scientific research and clinical work. Summary of the invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed. The purpose of the present invention is to provide a fusion protein comprising DR5 and MYDGF and its application. The present invention proves through experiments that the fusion protein has the activities of both DR5 and MYDGF, can block TRAIL-induced cell apoptosis, stimulate cell phosphorylation, promote cell tube formation, and can target the damaged heart, playing a protective role in heart diseases or conditions, especially in myocardial ischemia-reperfusion injury.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a fusion protein, which comprises a first polypeptide fragment including DR5 and a second polypeptide fragment including MYDGF.

[0007] Furthermore, the DR5 is a full-length DR5 or a fragment capable of binding to TRAIL, or a mutant of DR5, as long as it retains the activity of binding to TRIL ligand.

[0008] Furthermore, the DR5 is the entire amino acid sequence of the extracellular segment of DR5 or a partial amino acid sequence thereof, as long as it retains the activity of binding to the TRIL ligand.

[0009] In the present invention, mutation may refer to one or more conservative amino acid substitutions or one or more non-conservative amino acid substitutions, deletions or insertions. The mutated sequence is different from the wild-type sequence, wherein these mutations do not abolish the biological activity of the wild-type sequence. Conservative substitutions generally include substitutions of one amino acid by another amino acid with similar characteristics, such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art. Non-conservative substitutions, such as replacement of basic amino acids by hydrophobic amino acids, are also well known in the art.

[0010] Further, the DR5 comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 1. Alternatively, the DR5 has no more than 6, 5, 4, 3, 2, or 1 mutations relative to SEQ ID NO: 1 while retaining the basic biological activity of the polypeptide before mutation.

[0011] Furthermore, the amino acid sequence of DR5 is shown in SEQ ID NO:1.

[0012] Furthermore, the MYDGF is a full-length MYDGF or a fragment that can exert the function of MYDGF.

[0013] Further, the MYDGF comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 2. Alternatively, the MYDGF has no more than 6, 5, 4, 3, 2, or 1 mutations relative to SEQ ID NO: 2 while retaining the basic biological activity of the polypeptide before mutation.

[0014] Furthermore, the amino acid sequence of the MYDGF is shown in SEQ ID NO:2.

[0015] Furthermore, the fusion protein is also added with a protein tag for easy purification, and the protein tag includes but is not limited to His, GST, MBP, SUMO, and NusA.

[0016] Furthermore, the fusion protein also includes a third polypeptide fragment.

[0017] Furthermore, the third polypeptide fragment may have the function of connecting the first polypeptide fragment and / or the second polypeptide fragment, and in certain embodiments may also have the function of extending the half-life. Further, the third polypeptide fragment includes but is not limited to an immunoglobulin Fc region, a flexible linker, a rigid linker, a serum protein, a serum protein binding molecule, and an antibody.

[0018] The "immunoglobulin Fc region" in the present invention refers to an "immunoglobulin heavy chain constant region" and can be derived from antibodies belonging to the immunoglobulin classes called IgA, IgD, IgE, IgG, and IgM. In addition, it is expected that the immunoglobulin heavy chain constant region can be derived from any IgG antibody subclass called IgG1, IgG2, IgG3, and IgG4 in the art.

[0019] In the present invention, the immunoglobulin Fc region may be a natural immunoglobulin Fc region or an immunoglobulin Fc region variant. Further, the immunoglobulin Fc region variant comprises the following mutation: according to EU numbering, the amino acid at position 356 of IgG Fc is substituted with an amino acid other than D, E and C. For example, the amino acid at position 356 of IgG Fc is substituted with one of the following groups: G, S, A, T, V, N, L, I, Q, Y, F, H, P, M, K, R. Further, the immunoglobulin Fc region variant comprises the following mutation: according to EU numbering, the amino acid at position 439 of IgG Fc is substituted with an amino acid other than R, H, K and C. For example, the amino acid at position 439 of IgG Fc is substituted with one of the following groups: G, S, A, T, V, D, N, L, I, E, Q, Y, F, P, M. Further, the immunoglobulin Fc region variant comprises the following mutations in IgG Fc according to EU numbering: one or more of E356G, E356S, E356A, E356T, E356V, E356N, E356L, E356I, E356Q, E356Y, E356F, E356H, E356P, E356M, E356K, E356R, K439G, K439S, K439A, K439T, K439V, K439D, K439N, K439L, K439I, K439E, K439Q, K439Y, K439F, K439H, K439P, and K439M. Further, the immunoglobulin Fc region variant comprises the following mutations in IgG Fc according to EU numbering: one or more of E356K, E356R, E356Q, E356A, E356N, K439D, K439E, K439Q, K439A, and K439N. Further, the immunoglobulin Fc region variant also comprises the following mutations: according to EU numbering, the amino acids at positions 234 and 235 of IgG Fc are substituted with AA, and / or the amino acid at position 447 is deleted. For example, for IgG1 Fc, the following mutations are also included: L234A and L235A, and / or the amino acid at position 447 is deleted; for IgG4 Fc, the following mutations are also included: F234A and L235A, and / or the amino acid at position 447 is deleted.

[0020] In the present invention, the immunoglobulin Fc region may be human or non-human. In a specific embodiment of the present invention, the immunoglobulin Fc region is selected from IgG1 Fc. In a specific embodiment of the present invention, the IgG1 Fc is human immunoglobulin IgG1 Fc.

[0021] Further, the IgG1 Fc comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 3. Alternatively, the IgG1 Fc has no more than 6, 5, 4, 3, 2, or 1 mutations relative to SEQ ID NO: 3 while retaining the basic biological activity of the polypeptide before mutation.

[0022] Furthermore, the amino acid sequence of the IgG1 Fc is shown in SEQ ID NO:3.

[0023] In the present invention, the flexible linker includes glycine polymer (G)n, glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, LRQKD (GGGS) 2 ERP, DGGGS, LRQRDGERP, TGEKP, KESGSSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP, or (GGGGS)n.

[0024] Furthermore, the flexible linker is selected from (GGGGS)n.

[0025] Furthermore, the first polypeptide fragment and the second polypeptide fragment can be connected along the direction from the amino terminus to the carboxyl terminus, or the second polypeptide fragment and the first polypeptide fragment can be connected along the direction from the amino terminus to the carboxyl terminus. Regardless of the order in which they are connected, as long as the fusion protein described in the first aspect of the present invention can be expressed, it falls within the protection scope of the present invention.

[0026] Further, the first polypeptide fragment and the second polypeptide fragment may be directly connected to each other, or may be connected to one or more of the third polypeptide fragments as described in the fourth aspect of the present invention. The first polypeptide fragment may be connected to the second polypeptide fragment and then connected to the third polypeptide fragment together, or the first polypeptide fragment and the second polypeptide fragment may be connected to both ends of the third polypeptide fragment. In the present invention, the connection mode and connection order of the first polypeptide fragment and the second polypeptide fragment or the first polypeptide fragment and the second polypeptide fragment and the third polypeptide fragment are not limited. In some embodiments, the positional relationship of the first polypeptide fragment, the second polypeptide fragment, and the third polypeptide fragment may be any one of the following orders: the first polypeptide fragment-the second polypeptide fragment-the third polypeptide fragment, the first polypeptide fragment-the third polypeptide fragment-the second polypeptide fragment, the second polypeptide fragment-the first polypeptide fragment-the third polypeptide fragment, the second polypeptide fragment-the third polypeptide fragment-the first polypeptide fragment, the third polypeptide fragment-the second polypeptide fragment-the first polypeptide fragment, the third polypeptide fragment-the second polypeptide fragment-the first polypeptide fragment.

[0027] The second aspect of the present invention provides any one of the following biomaterials:

[0028] (1) A nucleic acid molecule encoding the fusion protein according to the first aspect of the present invention;

[0029] (2) A vector comprising the nucleic acid molecule described in (1);

[0030] (3) A recombinant host cell, comprising the nucleic acid molecule described in (1), or the expression vector described in (2).

[0031] Furthermore, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site.

[0032] In the present invention, nucleic acid molecules refer to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively connected". For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively connected to the coding sequence. In the present invention, nucleic acid molecules may also refer to codon-optimized nucleic acid molecules, or nucleic acid molecules obtained by substitution and / or deletion and / or addition of one or more nucleotides, as long as they are nucleic acid molecules capable of encoding the fusion protein described in the first aspect of the present invention, they all fall within the protection scope of the present invention.

[0033] Further, the type of vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus and cosmid, and can be changed according to the host cell to be introduced. Viral vector technology is well known in the art, and introduction can be carried out by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO 4 Precipitation, DEAE polydextrose, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. In some embodiments, viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In addition, in order to evaluate the expression of the target protein (e.g., monoclonal antibody), the vector introduced into the cell may also include any one or two of a selectable marker gene or reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector.

[0034] Further, the vector can be an expression vector, a cloning vector or an integration vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequence usually includes one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region and a selectable marker element for inserting a nucleic acid encoding a monoclonal antibody to be expressed.

[0035] Furthermore, the recombinant host cells include eukaryotic cells and prokaryotic cells.

[0036] Furthermore, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0037] Furthermore, the mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells.

[0038] Furthermore, the mammalian cells are 293F cells.

[0039] The third aspect of the present invention provides a multimeric fusion protein, which is composed of the fusion protein described in any one of the first aspects of the present invention.

[0040] Further, the multimeric fusion protein includes but is not limited to a dimeric fusion protein, a trimeric fusion protein, a tetrameric fusion protein, a pentameric fusion protein, and a hexameric fusion protein, and as long as the activity of the fusion protein described in the first aspect of the present invention is retained, it falls within the protection scope of the present invention. In a specific embodiment of the present invention, the multimeric fusion protein is a dimeric fusion protein.

[0041] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the fusion protein described in any one of the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, and the multimeric fusion protein described in the third aspect of the present invention as active ingredients, and the fusion protein and / or the biomaterial and / or the multimeric fusion protein are present in the pharmaceutical composition in a therapeutically effective amount.

[0042] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient.

[0043] The pharmaceutically acceptable carrier and / or adjuvant of the present invention includes any material suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, a material with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier and / or adjuvant are used to help the stability of the formulation or help improve its activity or its biological effectiveness or produce an acceptable taste or smell in the case of oral administration as needed. The pharmaceutically acceptable carrier and / or adjuvant include diluents, adhesives, surfactants, humectants, adsorption carriers, lubricants and / or disintegrants. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water. Adhesives include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and hexadecanol. Wetting agents include, but are not limited to, glycerol and starch. Adsorption carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and bentonite. Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talcum powder, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium dodecyl sulfate.

[0044] In some embodiments, the therapeutically effective amount of the fusion protein and / or the biomaterial and / or the multimeric fusion protein provided by the present invention can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, gender, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the desired therapeutically effective amount.

[0045] A fifth aspect of the present invention provides any of the following methods:

[0046] (1) A method for preparing the fusion protein of the first aspect of the present invention, the method comprising culturing the recombinant host cell in the biological material of the second aspect of the present invention to enable the recombinant host cell to express the fusion protein of the first aspect of the present invention;

[0047] (2) A method for preparing a recombinant host cell in the biological material according to the second aspect of the present invention, the preparation method comprising introducing the nucleic acid molecule or vector according to the second aspect of the present invention into a host cell;

[0048] (3) A method for blocking TRAIL-induced cell apoptosis in vitro, the method comprising the following steps: contacting the cells to be induced, TRAIL, the fusion protein described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, the multimeric fusion protein described in the third aspect of the present invention, and the pharmaceutical composition described in the fourth aspect of the present invention; and detecting cell apoptosis;

[0049] (4) A method for stimulating cell phosphorylation in vitro, the method comprising the following steps: contacting the cells to be stimulated with the fusion protein according to any one of the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, the multimeric fusion protein according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention; and detecting the cell phosphorylation status;

[0050] (5) A method for promoting cell tube formation in vitro, the method comprising the following steps: contacting the cells to be tube-formed with the fusion protein described in any one of the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, the multimeric fusion protein described in the third aspect of the present invention, or the pharmaceutical composition described in the fourth aspect of the present invention; and detecting the tube formation status.

[0051] Furthermore, the preparation method of the fusion protein also includes the purification method of the fusion protein described in the first aspect. The purification method includes utilizing the properties of the fusion protein itself or the tag it carries, and selecting a suitable purification method to enrich the fusion protein to be purified. In some embodiments, affinity purification, gel filtration, ion exchange purification, hydrophobic interaction, exclusion chromatography, and electrophoresis can be used for purification. In a specific embodiment of the present invention, the purification method is affinity purification.

[0052] Furthermore, the recombinant host cells include eukaryotic cells and prokaryotic cells.

[0053] Furthermore, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0054] Furthermore, the mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells.

[0055] Furthermore, the mammalian cells are 293F cells.

[0056] Further, the cells to be induced and the cells to be stimulated include, but are not limited to, adrenocortical carcinoma cells, bladder urothelial carcinoma cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma and paraganglioma cells, prostate cancer cells, rectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells, sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells.

[0057] Furthermore, the cells to be induced are selected from acute lymphoid leukemia cells, chronic lymphoid leukemia cells, and chronic myeloid leukemia cells. In a specific embodiment of the present invention, the cells to be induced or stimulated are Jurkat cells.

[0058] Furthermore, the cells to be stimulated and to form tubes include endothelial cells.

[0059] Furthermore, the endothelial cells include lymphatic endothelial cells and vascular endothelial cells. In a specific embodiment of the present invention, the vascular endothelial cells are HUVEC cells.

[0060] The second aspect of the present invention provides any of the following applications:

[0061] (1) Use of the fusion protein according to any one of the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, the multimeric fusion protein according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention in the preparation of a drug for treating a heart disease or condition;

[0062] (2) Use of the fusion protein according to any one of the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, the multimeric fusion protein according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention in blocking TRAIL-induced cell apoptosis in vitro;

[0063] (3) Use of the fusion protein according to any one of the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, the multimeric fusion protein according to the third aspect of the present invention, or the pharmaceutical composition according to the fourth aspect of the present invention to stimulate cell phosphorylation in vitro;

[0064] (4) Use of the fusion protein described in any one of the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, the multimeric fusion protein described in the third aspect of the present invention, or the pharmaceutical composition described in the fourth aspect of the present invention in promoting cell tube formation in vitro.

[0065] Preferably, the cardiac disease or disorder is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, myocarditis, myocardial infarction, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatic heart disease, arterial inflammation, or cardiovascular disease;

[0066] Preferably, the cardiac disease or disorder is selected from myocardial infarction, myocardial ischemia-reperfusion injury;

[0067] Furthermore, the cells in (2) include adrenocortical carcinoma cells, bladder urothelial carcinoma cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma and paraganglioma cells, prostate cancer cells, rectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells, sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells.

[0068] Furthermore, the cells in (2) are selected from acute lymphoid leukemia cells, chronic lymphoid leukemia cells, and chronic myeloid leukemia cells. In a specific embodiment of the present invention, the cells in (2) are Jurkat cells.

[0069] Furthermore, the cells in (3) and (4) include endothelial cells.

[0070] Furthermore, the endothelial cells include lymphatic endothelial cells and vascular endothelial cells. In a specific embodiment of the present invention, the vascular endothelial cells are HUVEC cells.

[0071] The present invention has the following advantages and beneficial effects:

[0072] The present invention provides a fusion protein comprising DR5 and MYDGF and an application thereof. The present invention proves through experiments that the fusion protein has the activities of both DR5 and MYDGF, can block TRAIL-induced cell apoptosis, stimulate cell phosphorylation, promote cell tube formation, and can target the damaged heart, thus playing a protective role in heart diseases or conditions, especially myocardial ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1Figures 1 and 2 are agarose gel electrophoresis diagrams of hDR5-Fc-MYDGF and hFc-MYDGF plasmids, wherein 1A is the PCR result diagram of the Fc part and the MYDGF part, and — indicates a negative control; 1B is the PCR result diagram of the Fc and MYDGF overlap extension, and — indicates a negative control; 1C is the PCR result diagram of the pcDNA3.1(+)-hDR5-Fc-MYDGF bacterial solution, and — indicates a negative control; 1D is the PCR product result diagram of Fc-MYDGF, and — indicates a negative control; 1E is the PCR result diagram of the pcDNA3.1(+)-hFc-MYDGF bacterial solution, and — indicates a negative control.

[0074] Figure 2 Figures 2A and 2B are graphs showing the UV280 absorption peak of purified hDR5-Fc-MYDGF; 2C is a graph showing the UV280 absorption peak of purified hFc-MYDGF; and 2D is a graph showing the SDS-PAGE identification of hFc-MYDGF protein expression and purification effect.

[0075] Figure 3 These are characterization and analysis diagrams of dual-target fusion proteins and two control proteins, among which, 3A is a diagram of the dimer pattern of the three fusion proteins; 3B is a diagram of the SDS-PAGE identification results of the three fusion proteins in reduced and non-reduced states, R represents the denatured reduced state, and NR represents the denatured non-reduced state; 3C is a diagram of the Western Blot identification of DR5 results of the three fusion proteins; 3D is a diagram of the Western Blot identification of Fc results of the three fusion proteins; 3E is a diagram of the Western Blot identification of MYDGF results of the three fusion proteins; 3F is a diagram of the affinity detection results of hTRAIL and hDR5-Fc-MYDGF; 3G is a diagram of the affinity detection results of hTRAIL and the three fusion proteins.

[0076] Figure 4 Figures 4A and 4B show the quantitative statistical results of Jurkat apoptosis detected by flow cytometry; and Figure 4C show the results of MTS detection of different concentrations of fusion protein blocking hTRAIL-induced Jurkat apoptosis.

[0077] Figure 5Figures 5A and 5B are the results of partial functional activity verification of the fusion protein MYDGF, where 5A is the phosphorylation result after the three fusion proteins stimulated HUVEC cells; 5B is the grayscale analysis of the P-Akt (S473) band and the Akt band in 5A using Image J, and the statistical analysis after the results were normalized; 5C is the grayscale analysis of the P-Akt (T308) band and the Akt band in 5A using Image J, and the statistical analysis after the results were normalized; 5D is the detection result of the effects of the three fusion proteins on the tube-forming ability of HUVEC cells.

[0078] Figure 6 The figures are the results of fusion protein targeting damaged heart after myocardial ischemia-reperfusion, among which 6A is the heart I40min / R6h model of mice (3 mice), the sham group is sham-operated mice given Cy7-labeled hDR5-Fc-MYDGF fusion protein, and the mice in each group were photographed using the small animal in vivo imaging system; 6B is the heart imaging result of the mouse in Figure A.

[0079] Figure 7 Figure 7 is the effect of fusion protein on myocardial infarction area after myocardial ischemia-reperfusion, where 7A is the TTC-Evensblue staining result, white represents the infarct area, red and white represent the danger zone, and blue represents the non-ischemic area; 7B is the statistical analysis of the results of 7A, AAR represents the danger zone area, LV represents the left ventricular area, and IS represents the infarct area. DETAILED DESCRIPTION

[0080] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0081] Example 1 Construction of hDR5-Fc-MYDGF and hFc-MYDGF Plasmids

[0082] 1. Experimental methods

[0083] 1.1 Design of recombinant protein

[0084] MYDGF was coupled to the N-terminus of hDR5-Fc. The hDR5-Fc nucleotide sequence was connected to the MYDGF nucleotide sequence using the bridge technology, and inserted into the pcDNA3.1(+) vector to form a complete plasmid pcDNA3.1(+)-hDR5-Fc-MYDGF, which was used as a template to construct the pcDNA3.1(+)-Fc-MYDGF plasmid. After obtaining the plasmid, it was transfected into 293F cells for expression. The specific sequence information of the recombinant protein is as follows:

[0085] The first polypeptide fragment, human DR5 ECD (SEQ ID NO: 1):

[0086] ITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKE

[0087] The second polypeptide fragment, human MYDGF (SEQ ID NO: 2):

[0088] VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL

[0089] The third polypeptide fragment, human IgG1 Fc (SEQ ID NO: 3):

[0090] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0091] 1.2 Primer synthesis

[0092] IGHGCH3-F: CCGGAATTCGAGCCCAAATCTTGTGACAAAAC;

[0093] IGHGCH3-R: CACGGTGGTCGGTTCGCTCACTTTACCCGGAGACAGGG AGAG;

[0094] IGHGCH3-MYDGF-F: CTCTCCCTGTCTCCGGGTAAAGTGAGCGAACCG ACCACCGTG;

[0095] MYDGF-R:CCGCTCGAGTCACAGTTCGGTGCGGCTCGCTTTC;

[0096] SP-F: CCCAAGCTTGCCACCATGGGATGGTCATGTATCATCCTTTTTCT GGTAGCAACTGCAACT;

[0097] SP-IGHGCH3-F:TCTGGTAGCAACTGCAACTGGAGTACATTCAGAGCC CAAATCTTGTGACAAAAC;

[0098] 1.3 Construction of pcDNA3.1(+)-hDR5-Fc-MYDGF plasmid

[0099] (1) Using the pcDNA3.1(+)-hDR5-Fc plasmid available in the inventor's laboratory as a template, PCR amplified the Fc fragment. The reaction system is shown in Table 1 below, and the reaction procedure is shown in Table 2.

[0100] Table 1 PCR system for amplifying Fc fragment

[0101]

[0102] Table 2 PCR program for amplifying Fc fragment

[0103]

[0104] (2) Prepare 1% agarose gel in advance. After the reaction is completed, add 10 μL 6× DNA Loading buffer and perform electrophoresis on all PCR products at a constant voltage of 125 V for 30 min.

[0105] (3) Glue recovery, the specific operations are as follows:

[0106] ① Add 500 μL of equilibrium solution BL to the adsorption column CA2 and centrifuge at 12000 rpm for 1 min. Discard the waste liquid.

[0107] ② Cut out the target band of gel using a gel cutter and place it in an EP tube. Add PN at a ratio of 100 μL PN per 0.1 g and heat in a 50°C metal bath. Flip the EP tube upside down and mix every 2 minutes until the gel block is completely melted.

[0108] ③ Transfer the solution from the previous step to the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge it at 12000rpm for 1 minute. Pour away the waste liquid and put the adsorption column back into the collection tube.

[0109] ④ Add 600 μL of rinse solution PW to the adsorption column and centrifuge at 12000 rpm for 1 min. Pour away the waste liquid and put the adsorption column back into the collection tube.

[0110] ⑤Repeat step ④.

[0111] ⑥Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2min, and remove as much rinsing liquid as possible. Open the cover of the adsorption column and let it stand for 10min to allow the liquid in the adsorption column to evaporate.

[0112] ⑦ Place the adsorption column in a new EP tube, add 40 μL of eluent EB to the middle of the adsorption column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 2 minutes to obtain the target fragment solution.

[0113] (4) PCR amplified the MYDGF fragment using the pcDNA3.1-myc-His(-)B-hMYDGF plasmid synthesized by Sangon Biotechnology Co., Ltd. as a template. The reaction system is shown in Table 3 below, and the reaction procedure is shown in Table 4.

[0114] Table 3 PCR system for amplifying MYDGF fragments

[0115]

[0116] Table 4 PCR program for amplifying MYDGF fragment

[0117]

[0118]

[0119] (5) After the PCR reaction is completed, gel electrophoresis is performed and gel recovery is performed after the band positions are correct.

[0120] (6) Using overlap extension PCR to connect the MYDGF fragment to the Fc fragment. The reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0121] Table 5 Overlap extension PCR system

[0122]

[0123] Table 6 Overlap extension PCR program

[0124]

[0125]

[0126] (7) After the PCR reaction is completed, gel electrophoresis is performed and the correct band is recovered.

[0127] (8) Double digestion: Both the vector and the PCR product were double digested with restriction endonucleases EcoRI and XhoI. The digestion system is shown in Table 7. Digestion was performed at 37°C for 4 h, and the band size was confirmed by gel electrophoresis and recovered.

[0128] Table 7 pcDNA3.1(+)-hDR5-Fc-MYDGF enzyme digestion system

[0129]

[0130] (9) The linear vector and target fragment were ligated using the enzyme ligation system shown in Table 8. The enzyme ligation was carried out at 16°C for 1 h.

[0131] Table 8 pcDNA3.1(+)-hDR5-Fc-MYDGF enzyme-linked system

[0132]

[0133] (10) Transformation: Take the DH5α competent cells out of the -80°C freezer and place them on ice. When the competent cells are half-thawed, add the enzyme-linked product, gently stir the EP tube to mix, place on ice for 30 minutes, heat shock at 42°C for 60 seconds, and place on ice for 5 minutes. Add 700 μL of LB medium and place on a shaker at 37°C and 220 rpm for 1 hour. Take 200 μL on an LB agar plate containing ampicillin resistance and spread it evenly using a spreader.

[0134] (11) Bacterial liquid PCR identification: Pick a single colony and place it in 1 mL of LB medium containing Amp, and culture it in a shaker at 37°C and 220 rpm for 4 hours. After bacterial liquid PCR, the bacterial liquid with the correct band is sent for sequencing.

[0135] 1.4 Construction of pcDNA3.1(+)-hFc-MYDGF plasmid

[0136] (1) Using the pcDNA3.1(+)-hDR5-Fc-MYDGF plasmid successfully constructed in 1.3 as a template, PCR amplified the Fc-MYDGF part. The reaction system is shown in Table 9, and the reaction procedure is the same as Table 4.

[0137] Table 9 PCR system of pcDNA3.1(+)-hFc-MYDGF

[0138]

[0139] (2) Both the vector and PCR product were double-digested with restriction endonucleases HindIII and XhoI. The band size was confirmed by gel electrophoresis and then recovered from the gel.

[0140] (3) Enzyme ligation: the steps are the same as those in Example 1.3.

[0141] (4) Transformation: The steps are the same as those in Example 1.3.

[0142] (5) Bacterial liquid PCR identification: the steps are the same as those in Example 1.3.

[0143] 2. Experimental results

[0144] In this example, the eukaryotic expression plasmids pcDNA3.1(+)-hDR5-Fc-MYDGF and pcDNA3.1(+)-hFc-MYDGF were first constructed. The human MYDGF gene sequence was synthesized at Sangon Biotech Co., Ltd. and spliced ​​with the Fc gene sequence already available in the laboratory by overlapping extension PCR. Since there is an EcoRI restriction site between DR5 and Fc in pcDNA3.1(+)-hDR5-Fc, the overlapping extended Fc-MYDGF fragment was inserted into the pcDNA3.1(+)-hDR5-Fc plasmid through the EcoRI and XhoI restriction sites to obtain the pcDNA3.1(+)-hDR5-Fc-MYDGF eukaryotic expression plasmid ( Figure 1 AC). After the sequencing was correct, the hFc-MYDGF gene sequence was amplified by PCR using it as a template, and inserted into the pcDNA3.1(+) vector through the HindⅢ and XhoI restriction sites to obtain the pcDNA3.1(+)-hFc-MYDGF eukaryotic expression plasmid ( Figure 1 DE). After sequencing was correct, the plasmid was extracted using an endotoxin-free extraction kit for subsequent protein expression.

[0145] Example 2 Expression and purification of hDR5-Fc-MYDGF and hFc-MYDGF

[0146] 1. Experimental methods

[0147] 1.1 Eukaryotic expression of hDR5-Fc-MYDGF and hFc-MYDGF

[0148] (1) Resuscitation of 293F cells: In order to implement the principle of slow freezing and quick dissolution of cells, open the water bath in advance and preheat it to 37°C. Take the cells out of liquid nitrogen and quickly put them into PE gloves and place them in a water bath. Gently shake them to melt the cells evenly. After the cells are completely thawed, transfer them to a 15mL centrifuge tube containing 5mL of complete culture medium in an ultra-clean workbench and centrifuge at 500rpm for 5min. Discard the supernatant, resuspend the cells with 3mL of culture medium, and then transfer them to a 125mL cell culture flask containing 25mL of complete culture medium. The cell density is 5×10 5 / mL. Place at 37°C, 5% CO 2 , cultured on a shaker at 125 rpm. Count them regularly and when the density reaches 2×10 6 Cells / mL were subcultured and the cell density was 5×10 5 Pieces / mL.

[0149] (2) Cell transfection and protein expression: After the cells are expanded to the required volume, transient transfection is performed and feeding is performed after 24 hours. The cell viability begins to decline around 6 days, at which time the protein yield is the highest. The cells are centrifuged at 4°C, 12,000 rpm, for 10 minutes and the supernatant is collected for purification.

[0150] 1.2 hDR5-Fc-MYDGF and hFc-MYDGF protein purification

[0151] (1) Preparation of reagents: All glassware used for purification of reagents were placed in an oven at 180°C for 3 h, and all plastic products were soaked in 0.2 M NaOH for more than 2 h to remove endotoxins.

[0152] (2) Cleaning the AKTA pipeline: Place the A1, A2 and B pipelines in 20% ethanol and pump them, and connect them to a 5mL HiTrap MabSelect SuRe column at a flow rate of 1mL / min. Use at least 5 column volumes of ultrapure water to clean the pipelines and the chromatography column. Then use 0.2M NaOH at a flow rate of 1mL / min to clean the chromatography column and pipelines to remove endotoxins (pay attention to cleaning the sample loading pipeline and the collection pipeline). After 30 minutes, clean the chromatography column in the order of 10 column volumes of PBS and 10 column volumes of ultrapure water.

[0153] (3) Equilibration: Equilibrate the column in the order of Elution, Wash2, and Wash1, with at least 5 column volumes of each buffer. Finally, return the UV value to zero.

[0154] (4) Sampling: Before loading, the sample was filtered using a 0.45 μm filter and then placed in an ice water bath for loading at a flow rate of 5 mL / min.

[0155] (5) Elution: After loading, the column was re-equilibrated with Wash1 and Wash2 to remove unbound proteins. Elution was then performed. When the UV value was greater than 10 mAu, the sample was collected and the pH of the collected solution was adjusted to about 7.4 with 1 M Tris.

[0156] (6) Cleaning the machine: Clean the machine with water and 20% ethanol, remove the chromatography column, and finally store the pipes and chromatography column in 20% ethanol.

[0157] (7) Ultrafiltration: Use a 10 kDa ultrafiltration tube to concentrate the protein to a small volume, add 10 times the volume of PBS and mix, continue to concentrate, and repeat this operation three times.

[0158] (8) BCA quantification and storage: After BCA quantification, the protein was packaged, first quick-frozen in liquid nitrogen, and then transferred to a -80°C refrigerator for storage.

[0159] 1.3SDS-PAGE identification

[0160] Add 5× Loading buffer to the sample, mix well, boil in boiling water for 10 minutes, and then add the sample after centrifugation. Perform electrophoresis at a constant voltage of 80V until the marker disperses, then adjust to a constant voltage of 120V and continue electrophoresis until bromophenol blue reaches the bottom of the glass plate. Take out the entire gel and stain it in Coomassie brilliant blue dye for 30 minutes, recover the dye, wash off the dye on the gel with single distilled water, and then decolorize it with a decolorizer. Decolorize until transparent and take pictures using a gel imaging system.

[0161] 2. Experimental results

[0162] In this case, the plasmid was transiently transfected into suspended 293F cells for the expression of fusion protein. The expression level of the target protein in the cell culture supernatant was monitored daily by HPLC. Generally, the protein expression level was the highest 6 days after transfection, and the cell supernatant was collected by centrifugation. The cell supernatant was purified using a HiTrap MabSelect SuRe 5mL affinity chromatography column. The theoretical molecular weight of the hDR5-Fc-MYDGF monomer is 64kDa, and the theoretical molecular weight of the hFc-MYDGF monomer is 41kDa. The SDS-PAGE test results showed that the sizes of the two proteins were consistent with the theoretical molecular weights. There was almost no target protein in the flow-through, and there were no obvious impurities in the eluate. The purity of hDR5-Fc-MYDGF was about 94%, and the purity of hFc-MYDGF was about 90%, which were relatively high ( Figure 2 AD). The above results indicate that the hDR5-Fc-MYDGF and hFc-MYDGF proteins were successfully purified with high purity and can be used for subsequent experiments.

[0163] Example 3 Characterization and Analysis of Fusion Protein

[0164] 1. Experimental methods

[0165] 1.1 SDS-PAGE identification

[0166] Add 5× Loading buffer to the sample that needs to be denatured and reduced, and boil it in boiling water for 10 minutes. Mix the sample that needs to be non-reduced with 5× non-reduced Loading buffer and then load it. Perform electrophoresis at a constant voltage of 80V until the marker disperses, then adjust to a constant voltage of 120V and continue electrophoresis until bromophenol blue reaches the bottom of the glass plate. Take out the entire gel and put it into Coomassie brilliant blue dye solution for staining for 30 minutes, recover the dye solution, wash off the dye solution on the gel with single distilled water, and then decolorize it with a decolorizing solution. Decolorize until transparent, and take pictures using a gel imaging system.

[0167] 1.2 Western Blot Identification of Fusion Protein

[0168] (1) Sample preparation: Add 5× Loading buffer to the sample, mix well, boil in boiling water for 10 min, and then centrifuge before loading.

[0169] (2) Sample loading: Carefully add the sample into the sample wells and fill the empty wells with the same volume of 2× Loading buffer.

[0170] (3) Electrophoresis: Run at a constant voltage of 80 V until the marker is dispersed, then adjust to a constant voltage of 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the glass plate.

[0171] (4) Transfer: Activate the PVDF membrane with methanol in advance, install the transfer sandwich in the transfer tank, and transfer the membrane at a constant current of 300 mA for 90 min.

[0172] (5) Blocking: Place the transferred membrane in 5% skim milk prepared in TBST and place on a shaker for 2 h.

[0173] (6) Incubation with primary antibody: dilute the antibody with 5% skim milk and incubate overnight at 4°C.

[0174] (7) Washing the membrane: Recover the primary antibody and store it at -20°C. Wash the membrane three times with TBST, each time for 10 min.

[0175] (8) Incubation with secondary antibody: Dilute the secondary antibody corresponding to the primary antibody with 5% skim milk and incubate the membrane for 2 h.

[0176] (9) Washing the membrane: Wash the membrane three times with TBST, each time for 10 min.

[0177] (10) Exposure: Place the membrane in a fully automatic chemiluminescence instrument and evenly add ECL luminescent liquid to perform chemiluminescence.

[0178] 1.3 BLI detection of affinity between fusion protein and hTRAIL

[0179] Since hTRAIL carries a His tag, a HIS1K sensor was used to immobilize hTRAIL, and the concentration of the immobilized substance was 5 μg / mL. The fusion protein was detected as an analyte, and the analyte concentration was first set to 1000 nM for a preliminary experiment. According to the results of the preliminary experiment, 5 concentration gradients were set for the formal experiment. The specific operation is as follows:

[0180] (1) Protein dilution: Dilute the analyte to 5 μL / mL with BLI buffer and add 200 μL to each well of a 96-well black plate. Dilute the analyte to 1000 nM (hDR5-Fc-MYDGF molecular weight is 128 kDa, hDR5-Fc molecular weight is 80 kDa, hFc-MYDGF molecular weight is 82 kDa), and add 200 μL to each well of the black plate. Set a reference well (200 μL BLI buffer).

[0181] (2) On-line operation: The sensor can be put on the machine after being pre-wetted in BLI buffer for 10 min. The on-line operation procedure is baseline1 (60 s), loading (120 s), baseline2 (120 s), association (180 s), dissociation (180 s), and regeneration.

[0182] (3) Data processing: Octet BLI Analysis software was used to process the data.

[0183] 2. Experimental results

[0184] In this case, SDS-PAGE analysis of the hDR5-Fc fusion protein already in the laboratory and the purified hDR5-Fc-MYDGF and hFc-MYDGF fusion proteins was performed on the reduced and non-reduced samples. According to the model diagram, the three proteins all exist in the form of dimers, with the theoretical molecular weight of hDR5-Fc-MYDGF being 128kDa, the theoretical molecular weight of hDR5-Fc being 80kDa, and the theoretical molecular weight of hFc-MYDGF being 82kDa. From the SDS-PAGE results, it can be seen that the band sizes of the reduced and non-reduced samples of the three proteins are consistent with the theoretical values ​​( Figure 3 B), it can be determined that it is a dimer protein.

[0185] Then, the three fusion proteins were identified by Western Blot using anti-DR5 antibody, anti-human IgG Fc antibody and anti-MYDGF antibody. The results showed that hDR5-Fc-MYDGF and hDR5-Fc contained DR5 part ( Figure 3 C), hDR5-Fc-MYDGF and hFc-MYDGF contain the MYDGF part ( Figure 3 D), hDR5-Fc-MYDGF, hDR5-Fc and hFc-MYDGF all contain the Fc part ( Figure 3 E), demonstrating that the three fusion proteins are structurally intact.

[0186] Next, the Octet molecular interaction instrument was used to analyze the affinity of the three fusion proteins hDR5-Fc-MYDGF, hDR5-Fc and hFc-MYDGF with hTRAIL (DR5 ligand). Because hTRAIL has a 6×His tag, the HIS1K sensor was selected for the experiment. The experiment was conducted with hTRAIL as the solid and the three fusion proteins hDR5-Fc-MYDGF, hDR5-Fc and hFc-MYDGF as the analytes. The results showed that hDR5-Fc-MYDGF and hDR5-Fc had a high affinity with hTRAIL ( Figure 3 FG), while hFc-MYDGF did not bind to hTRAIL.

[0187] SDS-PAGE, Western-Blot and molecular interaction analysis proved that the constructed fusion protein structure was complete and could be used for subsequent experiments.

[0188] Example 4 Fusion protein activity verification

[0189] 1. Experimental methods

[0190] 1.1 Flow cytometry analysis of the ability of the fusion protein to block TRAIL-induced Jurkat cell apoptosis to verify its DR5 activity

[0191] (1) Jurkat cell culture: Preheat the water bath to 37°C in advance, take the cells out of liquid nitrogen and quickly put them into PE gloves, place them in the water bath, and gently shake them to melt evenly. After the cells are completely thawed, transfer them to a 15mL centrifuge tube containing 5mL complete culture medium (5mL serum + 500μL double antibody + 500μL glutamine additive + 500μL sodium pyruvate additive, supplemented to 50mL with 1640 culture medium) in the clean bench, and centrifuge at 500rpm for 5min. Discard the supernatant, resuspend the cells with 3mL culture medium and transfer them to a T25 flask, and supplement the culture medium until the cell density is 5×10 5 / mL, shake the cells evenly according to the "cross" method, mark the recovery date, cell name and other information on the culture bottle, and place it in a 37℃, 5% carbon dioxide cell culture incubator. After 24 hours, observe the cell state and change the medium. The cell density reaches 2×10 6Cells were subcultured when the number of cells was about 10 / mL, usually 2 days. The cells in the T25 flask were transferred to a 15 mL centrifuge tube in a clean bench and centrifuged at 500 rpm for 5 min. The cells were resuspended in 2 mL of 1640 complete medium (with glutamine and sodium pyruvate additives), counted, and supplemented with medium to make the cell density 5×10 5 / mL, transfer to a T75 flask, shake the cells evenly using the "cross" method, and place in an incubator.

[0192] (2) Protein dilution: 100μL hTRAIL + 100μL fusion protein + 300μL cells need to be added to a 24-well plate. Therefore, both hTRAIL protein and dual-target fusion protein need to be diluted to 5 times their working concentration. That is, the working concentration of hTRAIL is 12.5ng / mL, and hTRAIL needs to be diluted to 62.5ng / mL using 1640 complete medium. The working concentrations of fusion protein are 10ng / mL, 100ng / mL, and 500ng / mL, that is, diluted to 50ng / mL, 500ng / mL, and 2500ng / mL respectively.

[0193] (3) Cell plating: Transfer cells from T75 flask to 15 mL centrifuge tube and centrifuge at 500 rpm for 5 min. Discard the supernatant, resuspend with 2 mL 1640 complete medium, count, and replenish medium until the cell density reaches 1.7 × 10 6 / mL (i.e. 5×10 5 After being evenly mixed, the cells were added to a 24-well plate.

[0194] (4) Drug stimulation: Add dual-target fusion protein and hTRAIL to the cells as needed, gently shake to mix, and then place in an incubator for 6 hours.

[0195] (5) Collect cells: Collect cells into flow cytometry tubes, add 750 μL PBS to each tube, centrifuge at 400 g for 5 min. Discard the supernatant, add 1 mL PBS to each tube, flick the flow cytometry tube with your fingers to disperse the cells (do not use a gun to blow), centrifuge at 400 g for 5 min, and discard the supernatant.

[0196] (6) Staining: Add 500 μL of mixed dye (100 μL Binding buffer + 400 μL ultrapure water + 5 μL Annexin V-APC + 10 μL 7-AAD) to each tube, use your fingers to flick the flow tube to disperse the cells, and incubate in the dark for 10 min.

[0197] (7) On-machine testing.

[0198] 1.2MTS detection of the ability of fusion protein to block TRAIL-induced Jurkat cell death and verify its DR5 activity

[0199] (1) Protein dilution: 50 μL hTRAIL + 50 μL fusion protein + 100 μL cells should be added to a 96-well plate. The concentration of hTRAIL is 200 ng / mL, and the concentrations of the dual-target fusion protein are 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL. Calculate and dilute in the same manner as in Example 1.1(2).

[0200] (2) Cell plating: As in Example 1.1, 5×10 4 cells.

[0201] (3) Drug stimulation: Same as Example 1.1, but incubation is required for 24 hours.

[0202] (4) Detection: Add 10 μL of MTS working solution to each well and incubate in an incubator away from light. Use an enzyme reader to detect OD at 2 h, 3 h, and 4 h. 490nm .

[0203] 1.3 Western Blot detection of fusion protein stimulating HUVEC cells to verify its MYDGF activity

[0204] (1) Protein dilution: The protein concentration used is 1 μg / mL.

[0205] (2) Cell plating: Plating cells one night in advance, 1×10 6 cells.

[0206] (3) Drug stimulation: Discard the culture medium in the dish, wash twice with PBS, quickly add the diluted protein, and place in the incubator.

[0207] (4) Cell collection: Take a small dish of cells at 5 min, 15 min and 30 min respectively, discard the culture medium, wash three times with PBS, add 200 μL of 2× Loading containing protease inhibitors and phosphatase inhibitors, scrape the cells with a cell scraper, lyse on ice for 30 min, then scrape all the cells with a cell scraper and transfer them to an EP tube, boil in boiling water for 10 min.

[0208] (5) Western Blot detection: the method is the same as step 1.2 in Example 3.

[0209] 1.4 Angiogenesis assay to detect the fusion protein promoting HUVEC cell tube formation and verify its MYDGF activity

[0210] (1) HUVEC cell culture: The recovery method is the same as step 1.1 of this example. After the cells are observed to be full under a microscope, they are passaged. The cells are placed in a clean bench, the culture medium is discarded, and they are washed twice with PBS. Trypsin is added for digestion for 40 seconds, and DMEM complete culture medium is used for neutralization. The cells are blown off and transferred to a 15 mL centrifuge tube, and centrifuged at 500 rpm for 5 minutes. The supernatant is discarded, the cells are resuspended with ECM culture medium, and evenly added to the dish containing ECM culture medium, shaken well, and placed in an incubator for culture.

[0211] (2) Melt Matrigel overnight at 4°C and pre-cool the 24-well plate and pipette tip at 4°C.

[0212] (3) Glue spreading: Take a 24-well plate and use the cut end of the gun to absorb Matrigel and slowly spread it from the edge of the well to the middle (be careful not to create bubbles). Observe under a microscope. If the glue surface is flat and there is no black hole in the middle of the well, it can be placed in a 37°C incubator and can be used after 4 hours.

[0213] (4) Cell preparation: The confluent HUVEC cells were digested, centrifuged, resuspended, and counted. The cell concentration was adjusted to 2×10 5 Take 200 μL of cell suspension and 200 μL of diluted protein, mix them evenly, and evenly drop them on the well plate covered with matrix gel to ensure that the number of cells in each well is 4×10 4 indivual.

[0214] (5) Photography: Observe and take photos under a microscope at 2h, 3h and 4h respectively.

[0215] 2. Experimental results

[0216] 2.1 Verification of partial functional activity of fusion protein DR5

[0217] TRAIL is a member of the tumor necrosis factor superfamily and can induce apoptosis by binding to DR4 / DR5. The fusion protein was used to block the apoptosis of Jurkat cells induced by hTRAIL, verifying the partial activity of the fusion protein DR5. The flow cytometry results show that after the Jurkat cells were stimulated with hTRAIL, the apoptosis rate increased from 3% to 38%, while the apoptosis rate dropped to 7% after the addition of hDR5-Fc fusion protein, and the apoptosis rate decreased with the increase of concentration. The addition of hDR5-Fc-MYDGF can achieve the same effect ( Figure 4 AB). The MTS experiment obtained the same results ( Figure 4C). The above results show that purified hDR5-Fc-MYDGF and hDR5-Fc can effectively block hTRAIL-induced Jurkat cell apoptosis and have DR5 activity. However, Fc-MYDGF has no effect of blocking hTRAIL-induced Jurkat cell apoptosis and has no DR5 activity.

[0218] 2.2 Verification of partial functional activity of fusion protein MYDGF

[0219] Studies have reported that MYDGF can phosphorylate Akt1 at S473 and T308 of endothelial cells through the PI3K / Akt signaling pathway. At the same time, MYDGF can promote endothelial cell angiogenesis. In order to verify the partial activity of the dual-target fusion protein MYDGF, three fusion proteins were used to stimulate HUVEC cells, and cells were collected for Western Blot experiments at 5min, 15min and 30min. The results showed that after adding hDR5-Fc-MYDGF or hFc-MYDGF stimulation, the expression level of P-Akt (S473) increased at 5min and 15min, and the increase in the expression level of phosphorylated Akt disappeared at 30min. When hDR5-Fc-MYDGF stimulated HUVEC cells for 5min and 15min, the expression level of P-Akt (T308) increased. When Fc-MYDGF stimulated HUVEC cells for 5min, the expression level of P-Akt (T308) increased. However, hDR5-Fc did not increase the phosphorylation level of HUVEC cells ( Figure 5 AC). Next, the effect of dual-target fusion proteins on the tube-forming ability of HUVEC cells was investigated. The results showed that hDR5-Fc-MYDGF or hFc-MYDGF could promote the tube-forming of HUVEC cells, while hDR5-Fc had no significant promoting effect ( Figure 5 D). This indicates that hDR5-Fc-MYDGF or hFc-MYDGF can stimulate the phosphorylation level of HUVEC cells and promote their tube formation, and has MYDGF activity, while hDR5-Fc does not have MYDGF activity.

[0220] Example 5 Targeting study of fusion protein in myocardial ischemia-reperfusion model

[0221] 1. Experimental methods

[0222] 1.1 Protein labeling with Cy7 dye

[0223] (1) Dye preparation: Dissolve Cy7 powder in DMSO to a final concentration of 10 mM, vortex to mix, and centrifuge. Store at -20°C in the dark after aliquoting and use within 2 weeks.

[0224] (2) Protein preparation: dilute the protein to 5 mg / mL with PBS (protein storage buffer is PBS), and add 100 μL of 1 M phosphate buffer with a pH of 9.0 to every 900 μL of protein to adjust the pH of the protein to improve labeling efficiency.

[0225] (3) Dye-labeled protein reaction: The dye and protein were mixed at a molar ratio of Cy7 dye / protein of 5:1, shaken to mix, and reacted on a rotating disk mixer at room temperature for 1 h, and then centrifuged.

[0226] (4) Ultrafiltration replacement: Transfer the reaction liquid to a 10 kDa concentrator tube, add 10 volumes of PBS, and centrifuge at 1200 g at 4°C to an appropriate volume. Repeat this step three times to remove unreacted free Cy7 dye.

[0227] (5) BCA quantification: Prepare the working solution in the BCA quantification kit with a ratio of 50:1 of solution A and solution B. Dilute the protein and BSA standard and add 25 μL to each well of a 96-well plate. Add 200 μL of the working solution and incubate at 37°C for 30 min. Detect the OD using an enzyme reader. 562nm , the protein concentration was calculated according to the standard curve.

[0228] 1.2 Establishment of mouse heart I40min / R6h model

[0229] (1) C57BL / 6J strain, 7 weeks old, 20-22 g male mice were purchased and randomly divided into groups after more than three days of adaptive feeding.

[0230] (2) Preparation before modeling: The mice were depilated the night before modeling. The abdominal hair of the mice was shaved with a razor, and depilatory cream was evenly applied. After 1 minute, they were wiped with a wet towel and wiped with absorbent cotton dipped in warm water. No food or water was allowed for 6 hours before modeling.

[0231] (3) After the mouse was anesthetized in an anesthesia induction box, it was fixed on the operating table in a supine position.

[0232] (4) Use alcohol cotton balls to wipe the mouse chest for disinfection. Use scissors to cut a small incision of about 1.5 cm between the third and fourth ribs of the mouse. Separate the extra-rib muscles and insert hemostatic forceps into the ribs to open a gap. Carefully squeeze out the mouse heart and quickly ligate the left anterior descending branch of the coronary artery with 6-0 sutures. Replace the heart and suture the skin.

[0233] (5) Drugs were injected into the tail vein at 35 minutes of ischemia. The chest cavity was opened again at 40 minutes of ischemia, the ligature on the heart was untied, the skin was sutured, and the mice were placed on a heating pad. After the mice woke up, they were returned to the cage and imaged 6 hours later.

[0234] 1.3 Small Animal In Vivo Imaging

[0235] Imaging was performed using an IVIS LuminaXRMS imaging system.

[0236] 1.4 Collection of materials

[0237] After in vivo imaging, the heart was removed in the dark, washed in PBS, and excess water was removed by blotting on filter paper before imaging.

[0238] 2. Experimental results

[0239] A mouse heart ischemia model of 40 min reperfusion was established, and Cy7-labeled hDR5-Fc-MYDGF fusion protein was injected into the tail vein. At the same time, sham-operated mice were given Cy7-labeled hDR5-Fc-MYDGF fusion protein as a control. After I40min / R6h, the small animal in vivo imaging system was used to image the mice in each group in the supine position. The results of supine imaging showed that there was an obvious fluorescent signal in the chest. The results of autopsy organ imaging showed that compared with the control group, the model mouse heart had an obvious fluorescent signal, which corresponded to the fluorescent signal of the chest in the supine position. The above results show that hDR5-Fc-MYDGF fusion protein can target the damaged heart in the heart I40min / R6h model ( Figure 6 AB).

[0240] Example 6 Study on the protective effect of fusion protein on myocardial ischemia-reperfusion model

[0241] 1. Experimental methods

[0242] 1.1 Establishment of the I1h / R24h cardiac injury model in Wistar rats

[0243] (1) Anesthesia: 2% isoflurane gas anesthesia was used, and the rats were fixed on the operating table in a supine position with their chest and abdomen exposed. Electrocardiograms were collected and preserved before surgery.

[0244] (2) Tracheal intubation: Prepare and disinfect the neck and chest of Wistar rats, cut the skin muscle layer of the front neck of the rat longitudinally, and gently use forceps to push aside the surrounding muscle tissue and the thyroid gland and surrounding muscle tissue to expose the trachea. Make a small cut in the trachea between the cricoid cartilages, gently insert the catheter along the direction of the trachea, and secure it with a knot. Turn on the small animal ventilator, connect the catheter, and observe whether the rat's chest rises and falls evenly.

[0245] (3) Heart ligation: Cut the chest skin and separate the surrounding muscle tissue with forceps, tear open the pericardium, and expose the heart. Find the left anterior descending coronary artery according to the position of the left atrial appendage and ligate the left anterior descending coronary artery. After ligation, observe whether there is bleeding, clamp the cut chest skin with hemostats to seal the heart. Measure and save the electrocardiogram, and confirm that the rat myocardial infarction model is successfully established by the increase of the ST segment on the electrocardiogram.

[0246] (4) Administration: 5 minutes before reperfusion (55 minutes after ligation), three fusion proteins (hDR5-Fc-MYDGF, hDR5-Fc, Fc-MYDGF) and PBS were administered via the tail vein in a double-blind manner at a dose of 15 mg / kg.

[0247] (5) Reperfusion: After 1 hour of ligation, remove the hemostatic forceps, loosen the ligature, clean the chest cavity with a cotton swab, and then suture the ribs, muscles, and skin in sequence. When the Wistar rat can breathe independently, press and clean the trachea with a cotton swab, and suture the trachea and neck skin respectively. After the Wistar rat wakes up, put it back in the cage and continue to raise it, and pay close attention to the rat's condition.

[0248] (6) Heart extraction: After 24 hours, the Wistar rats were anesthetized and fixed on the operating table. Blood was collected from the carotid artery into an EP tube. The heart was exposed by thoracotomy and flushed from the aortic arch with PBS. If used for TTC staining, after flushing with PBS, 4% Evans Blue was injected from the aorta. The whole heart was then completely cut out, excess Evans Blue was absorbed with filter paper, and then quickly frozen on dry ice and stored in a -40°C refrigerator for staining; 1.2 TTC staining

[0249] (1) Prepare 1% TTC dye solution with 1× PBS and dissolve it in a light-proof water bath at 37°C. Prepare it before use.

[0250] (2) The heart was taken out after being frozen at -40℃ for more than 30 minutes. The heart was cut into 5 slices starting from the apex of the heart. The thickness of each slice was 2-3 mm. The fourth slice spanned the ligature.

[0251] (3) The heart slices need to be rinsed in a 6-well plate with 1% TTC dye to wash away excess Evans Blue, then placed in a 5 mL EP tube with TTC dye and incubated in a 37°C water bath for 10 minutes to stain. Take out and invert once to mix.

[0252] (4) After staining, take out the heart slices and place them in a 24-well plate filled with PBS. Then, place the heart slices on a glass slide in the same order and fix them for photographing.

[0253] (5) After taking the photo, draw a picture to analyze the infarct area of ​​the heart, where the blue area is the safe area, the white area is the infarct area, and the red area is the non-infarct area.

[0254] 2. Experimental results

[0255] The heart I1h R24h model was established using 6-8 week old male Wistar rats. Three fusion proteins (hDR5-Fc-MYDGF, hDR5-Fc, Fc-MYDGF) were injected through the tail vein 5 minutes before reperfusion. The myocardial infarction area was observed and counted by TTC-Evans Blue staining. The statistical results showed that the three fusion proteins could reduce the myocardial infarction area after I1h / R24h in rats, among which hDR5-Fc-MYDGF had a more significant effect in reducing the infarction area. hDR5 and MYDGF played a synergistic role in reducing the infarction area ( Figure 7 AB).

[0256] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein includes a first polypeptide fragment including DR5 and a second polypeptide fragment including MYDGF.

2. The fusion protein according to claim 1, characterized in that The DR5 is a full-length DR5 or a fragment capable of binding to TRAIL; Preferably, the DR5 is the extracellular segment of DR5; Preferably, the DR5 comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 1; or, the DR5 has no more than 6, 5, 4, 3, 2, 1 mutations relative to SEQ ID NO: 1 while retaining the basic biological activity of the polypeptide before mutation; Preferably, the amino acid sequence of DR5 is as shown in SEQ ID NO:

1.

3. The fusion protein according to claim 1, characterized in that The MYDGF is a full-length MYDGF or a fragment that can exert the function of MYDGF; Preferably, the MYDGF comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with SEQ ID NO: 2; or, the MYDGF has no more than 6, 5, 4, 3, 2, 1 mutations relative to SEQ ID NO: 2 while retaining the basic biological activity of the polypeptide before mutation; Preferably, the amino acid sequence of MYDGF is as shown in SEQ ID NO:

2.

4. The fusion protein according to claim 1, characterized in that The fusion protein further comprises a third polypeptide fragment; Preferably, the third polypeptide fragment comprises an immunoglobulin Fc region, a flexible linker, a rigid linker, a serum protein, a serum protein binding molecule, and an antibody; Preferably, the third polypeptide fragment is an immunoglobulin Fc region; Preferably, the immunoglobulin Fc region is a natural immunoglobulin Fc region, or an immunoglobulin Fc region variant; Preferably, the immunoglobulin Fc region includes IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc; Preferably, the immunoglobulin Fc region is selected from IgG1 Fc; Preferably, the IgG1 Fc is human immunoglobulin IgG1 Fc; Preferably, the IgG1 Fc comprises an amino acid sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to SEQ ID NO: 3; or, the IgG1 Fc has no more than 6, 5, 4, 3, 2, 1 mutations relative to SEQ ID NO: 3 while retaining the basic biological activity of the polypeptide before mutation; Preferably, the amino acid sequence of the IgG1 Fc is as shown in SEQ ID NO: 3; Preferably, the flexible linker comprises a glycine polymer (G)n, a glycine-serine polymer, a glycine-alanine polymer, an alanine-serine polymer, LRQKD(GGGS)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKVD, GGRRGGGS, GGRR, LRQKDGGGSERP or (GGGGS)n; Preferably, the flexible linker is selected from (GGGGS)n.

5. The fusion protein according to claim 1, characterized in that The first polypeptide fragment and the second polypeptide fragment may be connected along the direction from the amino terminus to the carboxyl terminus; or the second polypeptide fragment and the first polypeptide fragment may be connected along the direction from the amino terminus to the carboxyl terminus; Preferably, the first polypeptide fragment and the second polypeptide fragment are directly connected to each other; Preferably, the first polypeptide fragment and the second polypeptide fragment can be linked to one or more of the third polypeptide fragments as described in claim 4; Preferably, the positional relationship of the first polypeptide fragment, the second polypeptide fragment, and the third polypeptide fragment can be any one of the following orders: first polypeptide fragment-second polypeptide fragment-third polypeptide fragment, first polypeptide fragment-third polypeptide fragment-second polypeptide fragment, second polypeptide fragment-first polypeptide fragment-third polypeptide fragment, second polypeptide fragment-third polypeptide fragment-first polypeptide fragment, third polypeptide fragment-second polypeptide fragment-first polypeptide fragment, third polypeptide fragment-first polypeptide fragment-second polypeptide fragment.

6. Any of the following biological materials: (1) A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to claim 1; (2) A vector, characterized in that the vector contains the nucleic acid molecule described in (1); (3) A recombinant host cell, characterized in that the host cell contains the nucleic acid molecule described in (1), or contains the expression vector described in (2); Preferably, the nucleic acid molecule further comprises a promoter, and / or a restriction enzyme cleavage site located after the promoter, and / or a kozak sequence located after the restriction enzyme cleavage site; Preferably, the recombinant host cell includes a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; Preferably, the mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells; Preferably, the mammalian cell is a 293F cell.

7. A multimeric fusion protein, characterized in that: The multimeric fusion protein is composed of the fusion protein according to any one of claims 1 to 5; Preferably, the multimeric fusion protein is a dimeric fusion protein.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, and the multimeric fusion protein according to the seventh aspect of the present invention as active ingredients, and the fusion protein and / or the biomaterial and / or the multimeric fusion protein are present in the pharmaceutical composition in a therapeutically effective amount; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

9. Any of the following methods: (1) A method for preparing the fusion protein according to any one of claims 1 to 5, characterized in that: The preparation method comprises culturing the recombinant host cell in the biomaterial according to the sixth aspect of the present invention so that the recombinant host cell can express the fusion protein according to any one of claims 1 to 5; (2) A method for preparing a recombinant host cell in the biomaterial according to the sixth aspect of the present invention, characterized in that the preparation method comprises introducing the nucleic acid molecule or vector according to the sixth aspect of the present invention into a host cell; (3) A method for blocking TRAIL-induced cell apoptosis in vitro, characterized in that the method comprises the following steps: contacting the cells to be induced, TRAIL, the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, and the pharmaceutical composition according to the eighth aspect of the present invention; detecting cell apoptosis; (4) A method for stimulating cell phosphorylation in vitro, characterized in that the method comprises the following steps: contacting the cells to be stimulated with the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention; and detecting the cell phosphorylation status; (5) A method for promoting cell tube formation in vitro, characterized in that the method comprises the following steps: contacting the cells to be tube-formed with the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention; and detecting the tube formation; Preferably, the cells to be induced or stimulated include adrenocortical carcinoma cells, bladder urothelial carcinoma cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma and paraganglioma cells, prostate cancer cells, rectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells, sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells; Preferably, the cells to be induced or stimulated are selected from acute lymphoid leukemia cells, chronic lymphoid leukemia cells, and chronic myeloid leukemia cells; Preferably, the cells to be induced are Jurkat cells; Preferably, the cells to be stimulated and to form tubes include endothelial cells; Preferably, the endothelial cells include lymphatic endothelial cells and vascular endothelial cells; Preferably, the vascular endothelial cells are HUVEC cells.

10. Any of the following applications: (1) Use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention in the preparation of a drug for treating a heart disease or condition; (2) Use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention in blocking TRAIL-induced cell apoptosis in vitro; (3) Use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention for stimulating cell phosphorylation in vitro; (4) Use of the fusion protein according to any one of claims 1 to 5, the biomaterial according to the sixth aspect of the present invention, the multimeric fusion protein according to the seventh aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention in promoting cell tube formation in vitro; Preferably, the cells in (2) and (3) include adrenocortical carcinoma cells, bladder urothelial carcinoma cells, cervical squamous cell carcinoma cells, endocervical adenocarcinoma cells, bile duct carcinoma cells, colon adenocarcinoma cells, lymphoid tumor cells, esophageal carcinoma cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe carcinoma cells, renal clear cell carcinoma cells, renal papillary cell carcinoma cells, acute myeloid leukemia cells, brain low-grade glioma cells, hepatocellular carcinoma cells, mesothelial carcinoma cells, ovarian cancer cells, pancreatic cancer cells, pheochromocytoma and paraganglioma cells, prostate cancer cells, rectal cancer cells, malignant sarcoma cells, melanoma cells, gastric cancer cells, testicular germ cell tumor cells, thyroid cancer cells, thymic cancer cells, endometrial cancer cells, uterine sarcoma cells, uveal melanoma cells, myeloma cells, acute lymphoid leukemia cells, chronic lymphoid leukemia cells, chronic myeloid leukemia cells, lymphoma cells, lung cancer cells, sarcoma cells, anal cancer cells, melanoma cells, retinoblastoma cells; Preferably, the cardiac disease or disorder is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, myocarditis, myocardial infarction, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatic heart disease, arterial inflammation, or cardiovascular disease; Preferably, the cardiac disease or disorder is selected from myocardial infarction, myocardial ischemia-reperfusion injury; Preferably, the cells in (2) are selected from acute lymphoid leukemia cells, chronic lymphoid leukemia cells, and chronic myeloid leukemia cells; Preferably, the cells in (2) are Jurkat cells; Preferably, the cells in (3) and (4) include endothelial cells; Preferably, the endothelial cells include lymphatic endothelial cells and vascular endothelial cells; Preferably, the vascular endothelial cells are HUVEC cells.

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