A fusion protein of DR5 and MYDGF and its application

Abstract

The present invention discloses a fusion protein of DR5 and MYDGF and its application. The fusion protein possesses the activities of DR5 and MYDGF, can block TRAIL-induced cell apoptosis, stimulate cell phosphorylation, promote cell tube formation, and can target damaged hearts, exerting a protective effect in heart diseases or conditions, especially myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular 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 cardiovascular mortality rates due to their rapid progression and limited critical rescue window, posing a serious health threat to the Chinese population. Current treatments for AMI primarily include thrombolysis, coronary bypass surgery, and percutaneous coronary intervention (PCI). However, irreversible damage to myocardial tissue caused by ischemia and reperfusion remains a challenging clinical problem, with no effective treatment available.

[0003] The mechanism of reperfusion injury involves numerous cellular and molecular biological events, including ROS generation, intracellular calcium overload, mitochondrial dysfunction, cellular inflammation, autophagy, and apoptosis. These events intersect in a complex network, ultimately leading to cardiomyocyte death. Developing targeted drugs to effectively prevent potential reperfusion injury remains a key and challenging area of ​​research and clinical practice. Summary of the Invention

[0004] In view of this, and to overcome the deficiencies of the prior art, the present invention is proposed. The present invention aims to provide a fusion protein comprising DR5 and MYDGF and its use. Experiments have shown that the fusion protein possesses the activities of both DR5 and MYDGF, can block TRAIL-induced apoptosis, stimulate cell phosphorylation, promote cell tube formation, and can target damaged hearts, exerting a protective effect in heart diseases or conditions, particularly myocardial ischemia-reperfusion injury.

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

[0006] In a first aspect, the present invention provides a fusion protein, comprising a first polypeptide fragment comprising DR5 and a second polypeptide fragment comprising 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, a 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 differs from the wild-type sequence, wherein the mutations do not abolish the biological activity of the wild-type sequence. Conservative substitutions generally include substitutions of one amino acid with another amino acid having 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 a basic amino acid with a hydrophobic amino acid, are also well known in the art.

[0010] Furthermore, 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 to 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 essential 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] Furthermore, 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 to 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 essential biological activity of the polypeptide prior to the mutation.

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

[0015] Furthermore, the fusion protein is further 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 function to connect the first polypeptide fragment and / or the second polypeptide fragment, and in certain embodiments, may also function to extend the half-life. Furthermore, 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 referred to as IgA, IgD, IgE, IgG, and IgM. Furthermore, it is contemplated that the immunoglobulin heavy chain constant region can be derived from any of the IgG antibody subclasses referred to in the art as IgG1, IgG2, IgG3, and IgG4.

[0019] In the present invention, the immunoglobulin Fc region may be a native immunoglobulin Fc region or an immunoglobulin Fc region variant. Furthermore, the immunoglobulin Fc region variant comprises the following mutation: substitution of the amino acid at position 356 of IgG Fc, according to EU numbering, 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 group: G, S, A, T, V, N, L, I, Q, Y, F, H, P, M, K, or R. Furthermore, the immunoglobulin Fc region variant comprises the following mutation: substitution of the amino acid at position 439 of IgG Fc, according to EU numbering, 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 group: G, S, A, T, V, D, N, L, I, E, Q, Y, F, P, or M. Furthermore, the immunoglobulin Fc region variant comprises one or more of the following mutations in IgG Fc according to EU numbering: 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. Furthermore, 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. Furthermore, the immunoglobulin Fc region variant further comprises the following mutations: substitution of amino acids 234 and 235 of IgG Fc with AA, and / or deletion of amino acid position 447, according to EU numbering. For example, for IgG1 Fc, the variant further comprises the following mutations: L234A and L235A, and / or deletion of amino acid position 447; for IgG4 Fc, the variant further comprises the following mutations: F234A and L235A, and / or deletion of amino acid position 447.

[0020] In the present invention, the immunoglobulin Fc region can 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] Furthermore, 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. 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 essential biological activity of the polypeptide prior to the 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)2ERP, DGGGS, LRQRDGERP, TGEKP, KESGSVSSEQLAQFRSLD, 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 in the direction from the amino terminus to the carboxyl terminus, or the second polypeptide fragment and the first polypeptide fragment can be connected in the direction from the amino terminus to the carboxyl terminus. Regardless of the order of connection, as long as the fusion protein described in the first aspect of the present invention can be expressed, it falls within the scope of protection of the present invention.

[0026] Furthermore, 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 and the second polypeptide fragment may be connected together with the third polypeptide fragment, 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 method and connection order of the first polypeptide fragment and the second polypeptide fragment or the first polypeptide fragment, 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 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-second polypeptide fragment-first polypeptide fragment, and third polypeptide fragment-first polypeptide fragment-second 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 an enzyme cleavage site located after the promoter, and / or a Kozak sequence located after the 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, then the promoter or enhancer is effectively connected to the coding sequence. In the present invention, nucleic acid molecules can also refer to codon-optimized nucleic acid molecules, and can also refer to nucleic acid molecules obtained by replacing and / or deleting and / or adding one or more nucleotides. As long as it is a nucleic acid molecule that can encode the fusion protein described in the first aspect of the present invention, it falls within the protection scope of the present invention.

[0033] In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.In some embodiments, the vector of the present invention can be used to express the host cell of the present invention.

[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 for integrating the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences usually include 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 for inserting a nucleic acid encoding the monoclonal antibody to be expressed, and a selectable marker element.

[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] Furthermore, 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 scope of protection 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 carriers and / or excipients described herein include any substance suitable for use in humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit / risk ratio. The pharmaceutically acceptable carriers and / or excipients are used, as needed, to enhance the stability of the formulation, improve its activity or bioavailability, or produce an acceptable taste or odor in the case of oral administration. The pharmaceutically acceptable carriers and / or excipients include diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants. Diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water. Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, 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, stearyl monoglyceride, and cetyl alcohol. Humectants include, but are not limited to, glycerin and starch. Adsorbent 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, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl 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, comprising culturing the recombinant host cell in the biomaterial of the second aspect of the present invention to enable the 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 biomaterial according to the second aspect of the present invention, the method comprising introducing the nucleic acid molecule or vector according to the second aspect of the present invention into the host cell;

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

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

[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 method for preparing the fusion protein also includes the method for purifying the fusion protein described in the first aspect. The purification method comprises utilizing the properties of the fusion protein itself or the tag it carries to select an appropriate purification method to enrich the fusion protein to be purified. In some embodiments, purification can be performed using affinity purification, gel filtration, ion exchange purification, hydrophobic interaction, size exclusion chromatography, or electrophoresis. 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] Furthermore, 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 cancer cells, glioblastoma multiforme cells, head and neck squamous cell carcinoma cells, renal chromophobe cell 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 cell 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, and 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 heart diseases or conditions;

[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, and 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 pectoris, 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 adrenal cortical 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 cell 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 its application. The present invention has demonstrated 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, thereby 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 — represents the negative control; 1B is the PCR result diagram of the Fc and MYDGF overlap extension, and — represents the negative control; 1C is the PCR result diagram of the pcDNA3.1(+)-hDR5-Fc-MYDGF bacterial solution, and — represents the negative control; 1D is the PCR product result diagram of Fc-MYDGF, and — represents the negative control; 1E is the PCR result diagram of the pcDNA3.1(+)-hFc-MYDGF bacterial solution, and — represents the 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 Figures 3A and 3B show the characterization and analysis of dual-target fusion proteins and two control proteins, among which 3A is a diagram showing the dimer pattern of the three fusion proteins; 3B is a diagram showing the SDS-PAGE identification results of the three fusion proteins in the reduced and non-reduced states, where R represents the denatured reduced state and NR represents the denatured non-reduced state; 3C is a diagram showing the Western Blot identification of DR5 of the three fusion proteins; 3D is a diagram showing the Western Blot identification of Fc of the three fusion proteins; 3E is a diagram showing the Western Blot identification of MYDGF of the three fusion proteins; 3F is a diagram showing the affinity test results of hTRAIL and hDR5-Fc-MYDGF; and 3G is a diagram showing the affinity test results of hTRAIL and the three fusion proteins.

[0076] Figure 4 Figures 4A and 4B show the results of partial functional activity verification of fusion protein DR5, where 4A shows the results of fusion protein blocking hTRAIL-induced Jurkat apoptosis; 4B shows the quantitative statistical results of flow cytometry detection of Jurkat apoptosis; and 4C shows the results of MTS detection of different concentrations of fusion protein blocking hTRAIL-induced Jurkat apoptosis.

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

[0078] Figure 6 Figures 6A and 6B show the results of fusion protein targeting damaged hearts after myocardial ischemia-reperfusion. Figure 6A shows the cardiac I40min / R6h model of mice (3 mice). The sham group consists of sham-operated mice given Cy7-labeled hDR5-Fc-MYDGF fusion protein. The mice in each group were photographed using a small animal in vivo imaging system. Figure 6B shows the cardiac imaging results of the mice in Figure A.

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

[0080] The present invention will be further described below in conjunction with specific examples. The specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods in the following examples where specific conditions are not specified are generally tested under conventional conditions or according to the 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 linked to the MYDGF nucleotide sequence using a bridge technique and inserted into the pcDNA3.1(+) vector to form the complete plasmid pcDNA3.1(+)-hDR5-Fc-MYDGF, which was used as a template to construct the pcDNA3.1(+)-Fc-MYDGF plasmid. The resulting plasmid was then transfected into 293F cells for expression. The specific sequence information of the recombinant protein is shown below:

[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 inventors' laboratory as a template, PCR amplified the Fc fragment. The reaction system is shown in Table 1, 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 of 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 equilibration solution BL to adsorption column CA2 and centrifuge at 12,000 rpm for 1 min. Discard the waste solution.

[0107] ② Using a gel cutter, cut out the gel at the target band and place it in an EP tube. Add PN at a ratio of 100 μL PN per 0.1 g of gel and heat in a 50°C metal bath. Invert the EP tube every 2 minutes to mix thoroughly until the gel 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 then centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid and return the adsorption column to the collection tube.

[0109] ④ Add 600 μL of rinse solution PW to the adsorption column and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid and return the adsorption column to the collection tube.

[0110] ⑤Repeat step ④.

[0111] ⑥ Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much rinse solution as possible. Open the cap of the adsorption column and let it sit for 10 minutes 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 amplification of the MYDGF fragment was performed 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 fragments

[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) Overlap extension PCR was used 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 enzyme digestion: Both the vector and PCR product were double digested with restriction endonucleases EcoRI and XhoI. The enzyme digestion system is shown in Table 7. Digestion was carried out at 37°C for 4 h. Band size was confirmed by gel electrophoresis and the gel was recovered.

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

[0129]

[0130] (9) Ligate the linearized vector and target fragment using the enzyme ligation system shown in Table 8. Ligate at 16°C for 1 h.

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

[0132]

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

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

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

[0136] (1) Using the pcDNA3.1(+)-hDR5-Fc-MYDGF plasmid constructed in 1.3 as a template, PCR amplify the Fc-MYDGF portion. The reaction system is shown in Table 9, and the reaction procedure is the same as in 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 sizes were 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) PCR identification of bacterial liquid: the steps are the same as those in Example 1.3.

[0143] 2. Experimental results

[0144] In this example, the pcDNA3.1(+)-hDR5-Fc-MYDGF and pcDNA3.1(+)-hFc-MYDGF eukaryotic expression plasmids 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 overlap extension PCR. Since there is an EcoRI restriction site between DR5 and Fc in pcDNA3.1(+)-hDR5-Fc, the overlap extension 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 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 and preheat it to 37°C in advance. Take the cells out of liquid nitrogen and quickly put them into PE gloves and place them in the water bath. Gently shake them to melt the cells evenly. After the cells are completely melted, transfer them to a 15mL centrifuge tube containing 5mL complete culture medium in the clean bench and centrifuge at 500rpm for 5min. Discard the supernatant, resuspend the cells with 3mL culture medium, and then transfer them to a 125mL cell culture flask containing 25mL complete culture medium. The cell density is 5×10 5 / mL. Place at 37℃, 5% CO2, 125rpm shaker to culture. Count them regularly and when the density reaches 2×10 6 Cells / mL were passaged and the cell density was 5×10 5 pieces / mL.

[0149] (2) Cell transfection and protein expression: Transient transfection was performed after the cells were expanded to the desired volume and fed 24 hours later. Cell viability began to decline around day 6, at which time protein yield was highest. The cells were centrifuged at 4°C, 12,000 rpm, for 10 minutes, and the supernatant was 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 tubing: Place tubing A1, A2, and B in 20% ethanol and pump wash, then connect them to a 5 mL HiTrap MabSelect SuRe column at a flow rate of 1 mL / min. Use at least 5 column volumes of ultrapure water to clean the tubing and column. Then, use 0.2 M NaOH at a flow rate of 1 mL / min to clean the column and tubing to remove endotoxins (pay attention to cleaning the sample loading and collection tubing). After 30 minutes, clean the 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, using at least 5 column volumes of each buffer. Finally, return the UV value to zero.

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

[0155] (5) Elution: After loading, the column was equilibrated again with Wash1 and Wash2 to remove unbound proteins. Elution was then performed. When the UV value was greater than 10 mAU, the collected solution was collected and the pH 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 aliquoted, 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 thoroughly, boil in boiling water for 10 minutes, and centrifuge immediately before loading. Run electrophoresis at 80V until the marker disperses. Then, adjust the voltage to 120V and continue electrophoresis until the bromophenol blue reaches the bottom of the glass plate. Remove the entire gel and stain it in Coomassie Brilliant Blue for 30 minutes. Recover the stain, wash the gel with distilled water, and destain with a destaining solution. Destain until transparent and photograph using a gel imaging system.

[0161] 2. Experimental results

[0162] In this case, the plasmid was transiently transfected into suspended 293F cells for expression of the 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 weight. There was almost no target protein in the flow-through, and there was no obvious miscellaneous band 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 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 samples requiring denaturation and reduction, then boil in boiling water for 10 minutes. For samples requiring non-reduction, mix thoroughly with 5× Non-Reducing Loading Buffer before loading. Run electrophoresis at 80V until the marker disperses. Adjust the voltage to 120V and continue electrophoresis until bromophenol blue reaches the bottom of the glass plate. Remove the entire gel and stain it in Coomassie Brilliant Blue for 30 minutes. Recover the stain, wash the gel with distilled water, and destain with a destaining solution. Destain until transparent and photograph 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 immediately before loading.

[0169] (2) Sample loading: Carefully add the sample to the sample wells and fill the remaining 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 minutes.

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

[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 at -20°C, then wash 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) Membrane washing: 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 solution to perform chemiluminescence.

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

[0179] Because hTRAIL carries a His tag, a HIS1K sensor was used to immobilize hTRAIL at a concentration of 5 μg / mL. The fusion protein was detected as the analyte. A preliminary experiment was performed, with the analyte concentration set at 1000 nM. Based on the preliminary results, five concentration gradients were set for the final experiment. The specific steps are 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, and hFc-MYDGF molecular weight is 82 kDa) and add 200 μL to each well of the black plate. Set up a reference well (200 μL BLI buffer).

[0181] (2) On-line operation: The sensor can be put into the machine after being pre-wetted in BLI buffer for 10 minutes. The on-line procedure is baseline1 (60 seconds), loading (120 seconds), baseline2 (120 seconds), association (180 seconds), dissociation (180 seconds), 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 reduced and non-reduced samples of the hDR5-Fc fusion protein already available in the laboratory, as well as purified hDR5-Fc-MYDGF and hFc-MYDGF fusion proteins, was performed. According to the schematic diagram, all three proteins exist as dimers, with the theoretical molecular weights of hDR5-Fc-MYDGF being 128 kDa, hDR5-Fc being 80 kDa, and hFc-MYDGF being 82 kDa. The SDS-PAGE results show that the band sizes of the three proteins in the reduced and non-reduced samples are consistent with the theoretical values ​​( Figure 3 B), it was confirmed to be 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 carries a 6×His tag, the HIS1K sensor was selected for the experiment. The experiment was conducted using 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 higher affinity with hTRAIL ( Figure 3 FG), while hFc-MYDGF did not bind to hTRAIL.

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

[0188] Example 4 Verification of fusion protein activity

[0189] 1. Experimental methods

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

[0191] (1) Jurkat cell culture: preheat the water bath to 37°C in advance, remove the cells from 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 melted, transfer them to a 15 mL centrifuge tube containing 5 mL of complete culture medium (5 mL of serum + 500 μL of double antibody + 500 μL of glutamine additive + 500 μL of sodium pyruvate additive, supplemented to 50 mL with 1640 culture medium) in a clean bench, and centrifuge at 500 rpm for 5 minutes. Discard the supernatant, resuspend the cells with 3 mL of culture medium and transfer them to a T25 flask, and supplement the culture medium until the cell density is 5 × 10 5 Cells were shaken evenly using the "cross" method. The culture flask was clearly marked with the recovery date, cell name and other information. The flask was then placed in a 37°C, 5% CO2 cell culture incubator. After 24 hours, the cell status was observed and the medium was changed. The cell density reached 2×10 6Cells were passaged when the cell density was about 5 × 10 cells / mL, usually after 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 supplements), counted, and supplemented with medium to a cell density of 5 × 10 cells / mL. 5 Cells were transferred to a T75 flask, shaken evenly using the "cross" method, and placed in an incubator.

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

[0193] (3) Cell plating: Transfer cells from T75 flasks to 15 mL centrifuge tubes and centrifuge at 500 rpm for 5 min. Discard the supernatant and resuspend in 2 mL of 1640 complete medium. Count the cells and replenish the medium until the cell density reaches 1.7 × 10 6 / mL (i.e. 5×10 5 After being evenly distributed, 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) Cell collection: Collect cells into flow cytometry tubes, add 750 μL PBS to each tube, and centrifuge at 400 g for 5 min. Discard the supernatant, add 1 mL PBS to each tube, and use your fingers to move the tube 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 move the flow tube to disperse the cells, and incubate in the dark for 10 min.

[0197] (7) Computer testing.

[0198] 1.2 MTS assay to verify the DR5 activity of the fusion protein in blocking TRAIL-induced Jurkat cell death

[0199] (1) Protein dilution: Add 50 μL hTRAIL + 50 μL fusion protein + 100 μL cells 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 using the same method 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 a dark incubator. Detect OD at 2 h, 3 h, and 4 h using a microplate reader. 490nm .

[0203] 1.3 Western Blot detection of fusion protein-stimulated HUVEC cell phosphorylation 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 harvesting: Take a small dish of cells at 5 min, 15 min, and 30 min, discard the culture medium, and wash three times with PBS. Add 200 μL of 2× Loading containing protease inhibitors and phosphatase inhibitors, scrape the cells with a cell scraper, and lyse on ice for 30 min. Then scrape all the cells with a cell scraper and transfer them to an EP tube and 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 that in step 1.1 of this example. After the cells are observed to be fully grown 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 40 seconds, and the cells are neutralized with DMEM complete culture medium. 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, and the cells are resuspended in ECM culture medium and evenly added dropwise to a dish containing ECM culture medium. After shaking, the cells are 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 pipette to slowly spread the Matrigel from the edge of the well to the center (be careful not to create bubbles). Observe under a microscope. If the surface of the gel is flat and there are no black holes in the middle of the well, place it in a 37°C incubator and use it 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 matrigel 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 cell 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 Jurkat cells were stimulated with hTRAIL, the apoptosis rate increased from 3% to 38%, while the apoptosis rate was reduced to 7% after the addition of hDR5-Fc fusion protein. Moreover, the apoptosis rate decreased with increasing 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 indicate that both purified hDR5-Fc-MYDGF and hDR5-Fc can effectively block hTRAIL-induced apoptosis in Jurkat cells and possess DR5 activity. However, Fc-MYDGF does not block hTRAIL-induced apoptosis in Jurkat cells and lacks 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 in endothelial cells through the PI3K / Akt signaling pathway. Furthermore, MYDGF can promote endothelial cell angiogenesis. To verify the partial activity of the dual-target fusion protein MYDGF, HUVEC cells were stimulated with the three fusion proteins and harvested for Western blotting at 5, 15, and 30 minutes. The results showed that after stimulation with hDR5-Fc-MYDGF or hFc-MYDGF, P-Akt (S473) expression levels increased at 5 and 15 minutes, while the increase in phosphorylated Akt expression disappeared at 30 minutes. P-Akt (T308) expression levels were increased at 5 and 15 minutes after hDR5-Fc-MYDGF stimulation of HUVEC cells. P-Akt (T308) expression levels were also increased after 5 minutes of Fc-MYDGF stimulation of HUVEC cells. 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 formation ability of HUVEC cells was investigated. The results showed that both hDR5-Fc-MYDGF and hFc-MYDGF could promote the tube formation of HUVEC cells, while hDR5-Fc had no significant promoting effect ( Figure 5 D) The results showed that hDR5-Fc-MYDGF or hFc-MYDGF could stimulate the increase of phosphorylation level in HUVEC cells and promote their tube formation, indicating that hDR5-Fc 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 Cy7 dye-labeled protein

[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 (the 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 the 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 hour, and then centrifuged instantly.

[0226] (4) Ultrafiltration: 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 of the BCA quantification kit with a ratio of A solution to B solution = 50:1. 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 value using a microplate reader. 562nm , and 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-week-old male mice weighing 20-22 g were purchased and randomly divided into groups after adaptive feeding for more than three days.

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

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

[0232] (4) Use an alcohol cotton ball 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 muscles outside the ribs and insert hemostatic forceps into the ribs to open a gap. Carefully squeeze out the mouse heart and quickly ligate the left anterior descending coronary artery with 6-0 suture. Replace the heart and suture the skin.

[0233] (5) The drug was injected into the tail vein at 35 minutes of ischemia. At 40 minutes of ischemia, the chest cavity was opened again, the ligature on the heart was untied, the skin was sutured, and the mouse was placed on a heating pad. After the mouse woke up, it was returned to the cage for breeding and imaging was performed 6 hours later.

[0234] 1.3 Small Animal In Vivo Imaging

[0235] Imaging was performed using the 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 and reperfusion of 6 h 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 supine chest. The above results show that the 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. The rats were fixed on the operating table in a supine position with their chest and abdomen exposed. Electrocardiograms were collected and stored before surgery.

[0244] (2) Intubation: Prepare and disinfect the neck and chest of Wistar rats. Cut the skin and muscle layer of the rat's neck longitudinally. 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 open the chest skin and separate the surrounding muscle tissue with forceps, tear the pericardium, and expose the heart. Locate 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 for bleeding and clamp the cut chest skin with hemostats to seal the heart. Measure and save the electrocardiogram (ECG). The successful establishment of the rat myocardial infarction model is confirmed by the elevation of the ST segment on the ECG.

[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. The administration followed the double-blind principle and the dosage was 15 mg / kg.

[0247] (5) Reperfusion: After 1 hour of ligation, remove the hemostat, 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 separately. After the Wistar rat wakes up, return it to the cage and continue to raise it, closely monitoring the rat's condition.

[0248] (6) Heart removal: After 24 hours, anesthetize the Wistar rats and fix them on the operating table. Blood is collected from the carotid artery into an EP tube. The heart is opened to expose the heart and flushed with PBS from the aortic arch. If used for TTC staining, after flushing with PBS, inject 4% Evans Blue from the aorta. Then, completely remove the heart, absorb the excess Evans Blue with filter paper, and quickly freeze it on dry ice. Store it in a -40℃ refrigerator and wait for staining. 1.2 TTC staining

[0249] (1) Prepare 1% TTC staining solution with 1× PBS, dissolve in a dark water bath at 37°C, and prepare immediately 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, each slice was 2-3 mm thick, and the fourth slice was across the ligature line.

[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 in the middle to mix.

[0252] (4) After staining, remove 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 graph to analyze the infarcted area of ​​the heart. The blue area represents the safe zone, the white area represents the infarcted area, and the red area represents the non-infarcted area.

[0254] 2. Experimental results

[0255] A cardiac I1h R24h model was established using 6-8 week old male Wistar rats. Three fusion proteins (hDR5-Fc-MYDGF, hDR5-Fc, and Fc-MYDGF) were injected via the tail vein 5 minutes before reperfusion. The myocardial infarction area was observed and statistically analyzed using TTC-Evans Blue staining. The statistical results showed that all three fusion proteins could reduce the myocardial infarction area after I1h / R24h in rats, with hDR5-Fc-MYDGF showing 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 above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall 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 DR5; a second polypeptide fragment MYDGF; and a third polypeptide fragment IgG1 Fc. The amino acid sequence of the DR5 is shown in SEQ ID NO: 1; the amino acid sequence of the MYDGF is shown in SEQ ID NO: 2; the amino acid sequence of the IgG1 Fc is shown in SEQ ID NO: 3; The first polypeptide fragment, the third polypeptide fragment, and the second polypeptide fragment are connected along the direction from the amino end to the carboxyl end.

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

3. The nucleic acid molecule according to claim 2, characterized in that The nucleic acid molecule further comprises a promoter, and / or an enzyme cleavage site located after the promoter, and / or a Kozak sequence located after the enzyme cleavage site.

4. A carrier, characterized in that The vector contains the nucleic acid molecule according to any one of claims 2-3.

5. A recombinant host cell, characterized in that The host cell comprises the nucleic acid molecule according to any one of claims 2 to 3, or comprises the vector according to claim 4.

6. The recombinant host cell according to claim 5, characterized in that The recombinant host cells include eukaryotic cells and prokaryotic cells.

7. The recombinant host cell according to claim 6, characterized in that The eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

8. The recombinant host cell according to claim 7, characterized in that The mammalian cells include immune cells, CHO cells, 293T cells, and 293F cells.

9. The recombinant host cell according to claim 8, characterized in that The mammalian cells are 293F cells.

10. A multimeric fusion protein, characterized in that The multimeric fusion protein is composed of the fusion protein according to claim 1.

11. The multimeric fusion protein according to claim 10, characterized in that The multimeric fusion protein is a dimeric fusion protein.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein of claim 1, the nucleic acid molecule of any one of claims 2-3, the vector of claim 4, the recombinant host cell of any one of claims 5-9, and the multimeric fusion protein of claim 10 as active ingredients, and the fusion protein and / or the nucleic acid molecule and / or the vector and / or the recombinant host cell and / or the multimeric fusion protein are present in the pharmaceutical composition in a therapeutically effective amount.

13. The pharmaceutical composition according to claim 12, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

14. A method for preparing the fusion protein according to claim 1, characterized in that: The preparation method comprises culturing the recombinant host cell according to any one of claims 5 to 9 so as to enable the recombinant host cell to express the fusion protein according to claim 1.

15. A method for preparing the recombinant host cell according to any one of claims 5 to 9, characterized in that: The preparation method comprises introducing the nucleic acid molecule according to any one of claims 2 to 3 or the vector according to claim 4 into a host cell.

16. A method for promoting cell tube formation in vitro, characterized in that: The method comprises the following steps: contacting cells to be formed into tubes with the fusion protein of claim 1, the nucleic acid molecule of any one of claims 2-3, the vector of claim 4, the recombinant host cell of any one of claims 5-9, the multimeric fusion protein of claim 10, and the pharmaceutical composition of any one of claims 12-13; and detecting the tube formation status, wherein the cells are HUVEC cells.

17. Use of the fusion protein of claim 1, the nucleic acid molecule of any one of claims 2-3, the vector of claim 4, the recombinant host cell of any one of claims 5-9, the multimeric fusion protein of claim 10, or the pharmaceutical composition of any one of claims 12-13 in the preparation of a drug for treating myocardial infarction and myocardial ischemia-reperfusion injury.

18. Use of the fusion protein of claim 1, the nucleic acid molecule of any one of claims 2-3, the vector of claim 4, the recombinant host cell of any one of claims 5-9, the multimeric fusion protein of claim 10, or the pharmaceutical composition of any one of claims 12-13 in promoting cell tube formation in vitro, wherein the cells are HUVEC cells.

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