Use of hpcal1 protein in enhancing cardiac function

Abstract

Provided is use of an HPCAL1 protein in enhancing cardiac function. The HPCAL1 protein can specifically bind to a recombinant β3-adrenergic receptor, and can be used as a β3-adrenergic receptor antagonist protein. It is found in animal experiments that by intravenously injecting recombinant mouse HPCAL1 with a certain concentration into an ischemia-reperfusion mouse model, the cardiac function of the mice can be effectively improved, and the ejection fraction of the mice is enhanced, indicating that the HPCAL1 protein can be used for enhancing the cardiac function and has potential medicinal value for treating cardiovascular diseases with cardiac function decline. Moreover, the HPCAL1 protein is a class of biomacromolecules with low toxicity, which solves the problem of high hepatic and renal toxicity of small molecule chemical drugs. Therefore, the HPCAL1 protein has relatively high application value.

Description

Application of HPCAL1 protein in enhancing cardiac function Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of HPCAL1 protein in enhancing cardiac function. Background Art

[0002] Heart failure refers to a condition in which the heart's systolic and diastolic functions are impaired, resulting in an inability to adequately discharge venous return blood from the heart. This leads to blood congestion in the venous system and insufficient blood perfusion in the arterial system, which in turn causes a syndrome of cardiac circulatory disorders. This syndrome is mainly manifested as pulmonary congestion and vena cava congestion. Heart failure is not an independent disease, but rather the terminal stage of heart disease. Almost all cardiovascular diseases will eventually lead to heart failure. Myocardial damage caused by any reason, such as myocardial infarction, cardiomyopathy, hemodynamic overload, inflammation, etc., can cause changes in myocardial structure and function, ultimately leading to poor ventricular pumping and / or filling function.

[0003] Factors influencing cardiovascular health include smoking, dietary nutrition, physical activity, overweight and obesity, and psychological factors. Risk factors for cardiovascular disease include hypertension, dyslipidemia, diabetes, chronic kidney disease, metabolic syndrome, and air pollution. Many cardiovascular diseases, such as coronary artery disease, heart failure, cor pulmonale, atrial fibrillation, rheumatic heart disease, and congenital heart disease, are associated with decreased heart function. Treatment of these conditions requires the use of cardiotonic agents. Currently, cardiotonic agents on the market primarily fall into two categories: cardiac glycosides and non-cardiac glycosides: Cardiotonic agents are cardiotonic glycosides, with commonly used agents in clinical practice including digitonin, digoxin, desacetyl scutellarin, and scutellarin K; while non-cardiac glycosides include β-adrenergic receptor agonists, such as dobutamine and dopamine isobutyl ester, and phosphodiesterase inhibitors such as amipirone. In recent years, it has been discovered that β3-adrenergic receptor (β3-AR) expression is elevated in the heart in various cardiovascular diseases, and that β3-AR blockers may have a role in cardiovascular diseases associated with heart failure. Therefore, the development of specific β3-AR blockers or antagonists has become a new focus in the treatment of cardiovascular diseases. However, small molecule drugs generally have high hepatotoxicity and renal toxicity, and their toxicity cannot be ignored.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to use HPCAL1 protein or related biomaterials in the preparation of drugs for strengthening the heart and treating cardiovascular diseases such as heart failure.

[0006] The technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of HPCAL1 protein or related biomaterials in any of the following:

[0008] (A1) preparing a β3 adrenergic receptor antagonist;

[0009] (A2) non-therapeutic inhibition of β3 adrenergic receptor activation;

[0010] (A3) preparing a medicament for treating cardiovascular disease;

[0011] (A4) Preparation of a cardiotonic agent.

[0012] In some embodiments of the present invention, the β3 adrenergic receptor antagonist can be used to prepare a drug for treating cardiovascular diseases; preferably, the drug for treating cardiovascular diseases is a cardiotonic drug.

[0013] In some embodiments of the present invention, the cardiovascular disease includes a heart failure disease.

[0014] In some embodiments of the present invention, the heart function decline disease includes at least one of myocardial infarction, heart disease, myocardial ischemia-reperfusion and heart failure.

[0015] In some embodiments of the present invention, the heart disease comprises a hereditary heart disease, a drug-induced heart disease, or an infection-induced heart disease.

[0016] In some embodiments of the present invention, the HPCAL1 protein is HPCAL1 protein derived from shrimp, human, or mouse.

[0017] In some embodiments of the present invention, the HPCAL1 protein is a protein shown in any one of the following B1-B4:

[0018] (B1) a protein having an amino acid sequence of any one of SEQ ID NOs: 1-3;

[0019] (B2) a protein having the same function as any one of SEQ ID NOs: 1-3 by substitution and / or deletion and / or addition of one or more amino acid residues;

[0020] (B3) a protein having at least 99%, 98%, 97%, 95%, 90%, 85% or 80% homology to the amino acid sequence defined in any one of (B1) or (B2) and having the same function; or

[0021] (B4) Modifying the N-terminus, C-terminus, amino acid backbone and / or amino acid side chain groups of the protein defined in any one of (B1) to (B3) to obtain a polypeptide derivative with the same function.

[0022] Preferably, the amino acid sequence shown in SEQ ID NO: 3 is substituted with one or more amino acid residues, and the amino acid sequence of the protein mutant having the same function is as shown in SEQ ID NO: 4.

[0023] Preferably, the addition in (B2) is the addition of one or more amino acid residues to the N-terminus and / or C-terminus of the amino acid sequence.

[0024] In some embodiments of the present invention, the adding further comprises connecting a tag sequence to the N-terminus and / or C-terminus of the protein defined in any one of B1-B4.

[0025] Preferably, the protein further comprises a fusion protein obtained by connecting a tag sequence to the N-terminus and / or C-terminus of the protein defined in any one of items B1-B4.

[0026] In some embodiments of the present invention, the tag sequence includes at least one of a signal peptide, a targeting peptide, a tag peptide, a fluorescent protein, and a transmembrane peptide.

[0027] "Homology" as used herein refers to the relatedness between two amino acid sequences, described by the parameter "identity"; the at least 95% homology may be at least 96%, 97%, 98%, or 99% identity. The at least 90% homology may be at least 91%, 92%, 93%, or 94% identity. The at least 85% homology may be at least 86%, 87%, 88%, or 89% identity. The at least 80% homology may be at least 81%, 82%, 83%, or 84% identity. Furthermore, the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (e.g., a protein identical to the human HPCAL1 protein can bind to the β3 adrenergic receptor and downregulate β3 adrenergic receptor activation).

[0028] In some embodiments of the present invention, the HPCAL1 protein is a protein having the same function as the amino acid sequence of any one of SEQ ID NOs: 1-3, with one or more amino acid residues substituted, deleted, and / or added; preferably, the substitutions are conservative substitutions. More preferably, the deletions, insertions, and / or substitutions are changes of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues in the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments of the present invention, the modification in (B4) comprises one or more combinations of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol (PEG) modification, multimeric antigen peptide (MAP), prenylation and cyclization, acetylation, amidation, fatty acid, cyclization, or other modifications acceptable in the field of polypeptides.

[0029] In some embodiments of the present invention, the relevant biological material is any one of the following:

[0030] (C1) a nucleic acid molecule encoding the HPCAL1 protein;

[0031] (C2) an expression cassette containing the nucleic acid molecule described in (C1);

[0032] (C3) a recombinant vector containing the nucleic acid molecule described in (C1), or a recombinant vector containing the expression cassette described in (C2);

[0033] (C4) a recombinant cell containing the nucleic acid molecule described in (C1), or a recombinant cell containing the expression cassette described in (C2), or a recombinant cell containing the recombinant vector described in C3);

[0034] (C5) a recombinant tissue containing the nucleic acid molecule described in (C1), or a recombinant tissue containing the expression cassette described in (C2), or a recombinant tissue containing the recombinant vector described in (C3);

[0035] (C6) a recombinant organ containing the nucleic acid molecule described in (C1), a recombinant organ containing the expression cassette described in (C2), or a recombinant organ containing the recombinant vector described in (C3);

[0036] (C7) A recombinant microorganism containing the nucleic acid molecule described in (C1), or a recombinant microorganism containing the expression cassette described in (C2), or a recombinant microorganism containing the recombinant vector described in (C3).

[0037] In some embodiments of the present invention, the vector is well known to those skilled in the art, including but not limited to: a plasmid, a phage, a cosmid, a Ti plasmid or a viral vector.

[0038] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0039] In the above-mentioned biological materials, the recombinant cells include prokaryotic cells or eukaryotic cells. The cells do not involve new varieties of plants or animals.

[0040] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other prokaryotic cells well known in the art that can be used to express target proteins.

[0041] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell, and an insect cell.

[0042] The recombinant tissue may be recombinant insect tissue and / or recombinant plant tissue and / or recombinant animal tissue.

[0043] The recombinant organ can be a recombinant insect organ and / or a recombinant plant organ and / or a recombinant animal organ.

[0044] The second aspect of the present invention provides a drug comprising the HPCAL1 protein or its related biological materials according to the first aspect of the present invention.

[0045] In some embodiments of the present invention, the medicament further comprises an active ingredient for enhancing cardiac function and / or treating cardiovascular diseases.

[0046] In some embodiments of the present invention, the medicament contains an effective dose of HPCAL1 protein or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, or a buffer.

[0049] In some embodiments of the present invention, the dosage form of the drug includes at least one of injection, aerosol or drops.

[0050] In some embodiments of the present invention, the administration route of the drug includes at least one of intravenous injection, intravenous drip, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal administration or aerosol administration.

[0051] A third aspect of the present invention provides a method for preventing or treating cardiovascular disease, comprising administering to a subject in need thereof an effective amount of HPCAL1 protein or a pharmaceutically acceptable salt thereof, or administering a pharmaceutical composition comprising the aforementioned HPCAL1 protein and a pharmaceutically acceptable excipient.

[0052] In some embodiments of the present invention, the cardiovascular disease includes a heart failure disease. Preferably, the heart failure disease includes at least one of myocardial infarction, heart disease, heart failure, or myocardial ischemia-reperfusion.

[0053] In some embodiments of the present invention, the heart disease comprises a hereditary heart disease, a drug-induced heart disease, or an infection-induced heart disease.

[0054] In some embodiments of the present invention, the HPCAL1 protein or a pharmaceutically acceptable salt thereof in the drug can be combined with other ingredients useful for preventing and / or treating cardiovascular disease in various ways. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other ingredients useful for preventing and / or treating cardiovascular disease can be present in separate drug products and administered as a combined package or combination drug. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other ingredients useful for preventing and / or treating cardiovascular disease can be present in the same drug product and administered as a compound drug.

[0055] In the present invention, the appropriate daily dosage range of HPCAL1 protein or its pharmaceutically acceptable salt is 0.1 μg / kg-1 g / kg body weight; the above dosage can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience and the dosage regimen including the use of other treatment means.

[0056] In the present invention, when administered to animals, the daily dosage of the recombinant HPCAL1 protein or a pharmaceutically acceptable salt thereof is 0.5 μg / kg-1 mg / kg mouse; and / or the administration frequency is 1 to 3 times.

[0057] In the present invention, the effective dose can be reasonably adjusted according to the actual situation and the judgment of the clinician. For different species, it can be reasonably adjusted based on the drug dose conversion formula or ratio between different species in the art.

[0058] Definition of terms:

[0059] The term "HPCAL1 protein" as used herein refers to hippocampal calcium-binding protein-like protein 1, a neuron-specific calcium-binding member of the recoverin family found in the retina and brain. Preliminary research has investigated the functional mechanisms of HPCAL1 in humans, showing its involvement in calcium-dependent regulation of rhodopsin phosphorylation and potential involvement in neuronal signaling in the central nervous system. In particular, research exploring its mechanisms in cancer and other related diseases has shown that it significantly inhibits the development of liver cancer and promotes the proliferation of glioblastoma.

[0060] The term "β3-adrenergic receptor" refers to a G-protein-coupled tissue receptor that mediates the effects of catecholamines. β3-adrenergic receptors are primarily distributed on muscle cells and are upregulated following myocardial injury. Stimulation of β3 receptors can produce a negative inotropic effect. This negative inotropic effect is mediated by inhibitory G proteins and may also be mediated through the nitric oxide pathway.

[0061] The term "β3 adrenergic receptor antagonist" refers to a molecule that binds to the β3 adrenergic receptor and blocks the effects of receptor agonists such as epinephrine and octopamine. The term "octopamine receptor" refers to a protein receptor unique to the G protein-coupled receptor superfamily in invertebrates.

[0062] The term "heart failure disease" refers to a type of disease in which the heart is unable to meet the body's demand for oxygen and nutrients, resulting in impaired body function. Late-stage heart failure is also called heart failure. Heart failure diseases include myocardial infarction, hereditary heart disease, drug-induced heart disease, infection-induced heart disease, myocardial ischemia-reperfusion, and heart failure.

[0063] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0064] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art.

[0065] The present invention has at least one of the following beneficial effects:

[0066] The present invention discovered that the HPCAL1 protein can bind to the β3 adrenergic receptor and function as a β3 adrenergic receptor antagonist; and further discovered that the HPCAL1 protein has the effect of treating cardiac dysfunction and can be used to prepare cardiotonic drugs or drugs for treating cardiac dysfunction, especially drugs for treating cardiac dysfunction caused by ischemia.

[0067] The present invention experimentally discovered that injecting the protein HPCAL1 into shrimp can inhibit the phosphorylation level of the blood cell transcription factor STAT; the protein binds to the shrimp octopamine receptor and blocks the effect of octopamine in the shrimp serum; it is also found that the human HPCAL1 protein can effectively block the activation of overexpressed β3-adrenergic receptors by epinephrine and octopamine in 293T cells, and in vitro protein-protein interaction analysis experiments further demonstrate that the human HPCAL1 protein can specifically bind to the recombinant human β3-adrenergic receptor; in animal experiments, it was found that intravenous injection of a certain concentration of recombinant mouse HPCAL1 in an ischemia-reperfusion mouse model can effectively improve the mouse's cardiac function and enhance its ejection fraction.

[0068] Experiments in this study demonstrate that the HPCAL1 protein can be used to enhance cardiac function and has potential medicinal value for the preparation of drugs to treat cardiovascular diseases associated with heart failure. Furthermore, the HPCAL1 protein is a biomacromolecule with low toxicity, addressing the high hepato-renal toxicity associated with small molecule drugs, thus possessing high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 shows the sequence homology analysis of HPCAL1 proteins from different species.

[0070] Figure 2 shows the prokaryotic expression of recombinant enhanced green fluorescent protein (rEGFP) and shrimp HPCAL1 protein (rPvHPCAL1).

[0071] Figure 3 shows the changes in phosphorylated STAT (signal transducer and activator of transcription) in shrimp hemocytes after injection of recombinant enhanced green fluorescent protein (rEGFP) and recombinant shrimp HPCAL1 (rPvHPCAL1) proteins, respectively. Figure 3A is the immunoblotting results repeated three times, and Figure 3B is a scatter plot of the ratio of the grayscale of the phosphorylated STAT band to the grayscale of the corresponding internal control tubulin band (three repeated experiments).

[0072] Figure 4 shows the results of the GST pull-down experiment.

[0073] Figure 5 shows the effects of recombinant HPCAL1 (rPvHPCAL1) injection on shrimp blood cell functional genes 3 hours after injection; PPO2: prophenoloxidase 2; STAT: signal transducer and activator of transcription protein; CRUL: crustacean antimicrobial peptide crustin-like; PPO1: prophenoloxidase 2; PPAF1: prophenoloxidase activating enzyme 1; PEN3: crustacean antimicrobial peptide Penaeidin-3.

[0074] Figure 6 shows the changes in STAT (signal transducer and activator of transcription) phosphorylation in shrimp primary hemocytes after treatment with octopamine + recombinant enhanced green fluorescent protein (rEGFP) or octopamine + recombinant shrimp HPCAL1 protein (rPvHPCAL1). Figure 6A is the immunoblotting results repeated three times, and Figure 6B is a scatter plot of the ratio of the grayscale of the phosphorylated STAT band to the grayscale of the corresponding internal control tubulin band, showing three repeated experiments.

[0075] Figure 7 shows the results of an in vitro interaction experiment between recombinant human β3 adrenergic receptor and recombinant human HPCAL1. Figure 7A shows prokaryotically expressed recombinant human HPCAL1 (rhsHPCAL1), recombinant human β3 adrenergic receptor (rHis-ADRB3), and purified human serum albumin (HSA). Figure 7B shows the concentration gradient binding curve of the ligand recombinant human β3 adrenergic receptor (rHis-ADRB3) and the analyte human serum albumin (HSA). Figure 7C shows the concentration gradient binding curve of the ligand recombinant human β3 adrenergic receptor (rHis-ADRB3) and the analyte human HPCAL1 (rhsHPCAL1). Figure 7D shows the binding constants of the ligand recombinant human β3 adrenergic receptor (rHis-ADRB3) with the analytes human serum albumin (HSA) and recombinant human HPCAL1 (rhsHPCAL1).

[0076] Figure 8 shows the immunoblotting results of phosphorylated ERK signal, internal reference GADPH signal and Flag-tag signal of transfected receptor in 293T cell overexpression experiment; Figure 8A shows the treatment of 293T cells transfected with β1-adrenergic receptor by adrenaline plus recombinant enhanced green fluorescent protein (rEGFP) and adrenaline plus recombinant human HPCAL1 protein (rhsHPCAL1); Figure 8B shows the treatment of 293T cells transfected with β2-adrenergic receptor by adrenaline plus recombinant enhanced green fluorescent protein (rEGFP) and adrenaline plus recombinant human HPCAL1 protein (rhsHPCAL1); Figure 8C shows the immunoblotting results of phosphorylated ERK signal, internal reference GADPH signal and Flag-tag signal of transfected receptor in 293T cell overexpression experiment; Figure 8A shows the treatment of 293T cells transfected with β1-adrenergic receptor by adrenaline plus recombinant enhanced green fluorescent protein (rEGFP) and adrenaline plus recombinant human HPCAL1 protein (rhsHPCAL1); Figure 8 Figure 8D shows 293T cells transfected with β3-adrenergic receptor treated with octopamine plus recombinant enhanced green fluorescent protein (rEGFP) and octopamine plus recombinant human HPCAL1 protein (rhsHPCAL1); Figure 8E shows prokaryotically expressed recombinant human HPCAL1 mutant protein (rhsHPCAL1_mut); Figure 8F shows 293T cells transfected with β3-adrenergic receptor treated with octopamine plus recombinant enhanced green fluorescent protein (rEGFP) and octopamine plus recombinant human HPCAL1 mutant protein (rhsHPCAL1_mut).

[0077] Figure 9 shows the effects of tail vein injection of recombinant mouse HPCAL1 on left ventricular ejection fraction and left ventricular fractional shortening in mice with myocardial ischemia-reperfusion model; Figure 9A shows eukaryotically expressed recombinant mouse HPCAL1 and purified mouse serum albumin (MSA); Figure 9B shows B-ultrasound images of mice with myocardial ischemia-reperfusion before and after tail vein injection of recombinant mouse HPCAL1 (rmsHPCAL1), as well as the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening; Figure 9C shows B-ultrasound images of mice with myocardial ischemia-reperfusion before and after tail vein injection of mouse serum albumin (MSA), as well as the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening. DETAILED DESCRIPTION

[0078] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0079] The abbreviations have the following meanings: “h” refers to hour, “min” refers to minute, “s” refers to second, “ms” refers to millisecond, “d” refers to day, “μL” refers to microliter, “mL” refers to milliliter, “L” refers to liter, “mM” refers to millimole, and “μM” refers to micromolar.

[0080] Example 1

[0081] The homology of HPCAL1 proteins from different animal sources was studied. The results are shown in Figure 1 , which show that HPCAL1 proteins are highly conserved in different species.

[0082] in:

[0083] The amino acid sequence of the shrimp HPCAL1 protein is shown in SEQ ID NO: 1; the amino acid sequence of the mouse HPCAL1 protein is shown in SEQ ID NO: 2; the amino acid sequence of the human HPCAL1 protein is shown in SEQ ID NO: 3, and the mutant of the human HPCAL1 protein is shown in SEQ ID NO: 4;

[0084] SEQ ID NO: 1:

[0085] >XP_027227598.1 hippocalcin-like protein 1[Penaeus vannamei]

[0086] SEQ ID NO: 2:

[0087] >NP_001348574.1 hippocalcin-like protein 1[Mus musculus]

[0088] SEQ ID NO:3:

[0089] >NP_001245286.1 hippocalcin-like protein 1[Homo sapiens]

[0090] SEQ ID NO:4:

[0091] >hippocalcin-like protein 1 mutant

[0092] The mutant (SEQ ID NO: 4) is prepared by mutating K at position 137 to R, K at position 163 to R, and R at position 171 of the amino acid sequence of human HPCAL1 protein.

[0093] Therefore, shrimp, mouse, human HPCAL1 proteins and mutants were used as experimental materials, and the function of the protein was tested in the mouse ischemia-reperfusion model.

[0094] Example 2

[0095] (1) Functional study of recombinant shrimp HPCAL1

[0096] 1.1 Prokaryotic expression and protein purification of recombinant shrimp HPCAL1

[0097] 1) The shrimp HPCAL1 sequence was cloned into the pET-20b plasmid (Pujian Biotechnology (Wuhan) Technology Co., Ltd.) and dissolved in ddH2O to 100 ng / μL;

[0098] 2) Add 1 μL of the plasmid to 50 μL of ArcticExpress (DE3) pRARE2 E. coli competent cells and mix thoroughly by gently pipetting.

[0099] 3) Place on ice for 30 min, bath in 42°C water for 90 s, and then bath on ice for 2 min;

[0100] 4) Add 950 μL of LB liquid medium in a clean bench and incubate at 37°C at 200 rpm for 1 hour;

[0101] 5) Centrifuge at 5000 rpm for 1 min, discard 900 μL of supernatant in a clean bench, and gently pipette to mix;

[0102] 6) Use a pipette to transfer the bacterial solution to a solid LB plate containing ampicillin. Heat a glass rod and cool it before applying it to the plate.

[0103] 7) Wait until the liquid on the plate is slightly dry, then invert and place in a 37°C bacterial incubator for overnight culture.

[0104] 8) Select a single clone and inoculate it into 5 mL of liquid LB medium containing Ampicillin (Amp) and activate it overnight at 37°C in a shaker.

[0105] 9) Add 3 mL of activated bacterial suspension to 50 mL of LB liquid medium containing ampicillin (Amp) and culture at 37°C until the OD600 is 0.6-1.0;

[0106] 10) Add IPTG (Isopropyl-β-D-thiogalactopyranoside) to a final concentration of 1 mM and induce at 37°C for 3 h. Take 20 μL of the bacterial culture before and after induction, add 5× SDS loading buffer, and boil in a 100°C water bath for 10 min.

[0107] 11) Centrifuge the induced bacterial solution at 8800 rpm for 8 minutes at 4°C and discard the supernatant.

[0108] 12) Resuspend the cells in 5 mL of pre-chilled PBS and centrifuge to remove the supernatant.

[0109] 13) Resuspend the pellet in 5 mL of pre-chilled PBS, add 100× PMSF, and sonicate at 60% power, 3 s on, 8 s off, for a total of 30 min.

[0110] 14) Centrifuge the disrupted solution at 9000 rpm for 8 min at 4°C and collect the supernatant for protein purification;

[0111] 15) Pipette 20 μL of supernatant and resuspended pellet into 200 μL EP tubes, add 5 μL of 5× SDS loading buffer, and boil at 100°C for 10 min.

[0112] 16) Take 10 μL of each sample and analyze it by polyacrylamide gel electrophoresis using 5% stacking gel and 15% separating gel, then stain with Coomassie Brilliant Blue for 1 hour, and analyze the protein expression after multiple decolorization treatments;

[0113] 17) Aspirate 25 μL Ni-Charged MagBeads magnetic beads were added to the supernatant of the disrupted solution in step 14 and incubated with rotation at 4°C for 30 min;

[0114] 18) Place the centrifuge tube close to the magnetic rack, remove the supernatant, add 1 mL of pre-chilled PBS to resuspend, transfer to a 1.5 mL enzyme-free EP tube, place the centrifuge tube close to the magnetic rack, and remove the supernatant;

[0115] 19) Add 1 mL of pre-cooled PBS containing 1% Triton X-114 and wash with rotation at 4°C for 8 min. Repeat this step 7 times to remove endotoxin contamination.

[0116] 20) Add 1 mL of pre-chilled PBS to the Ni column, mix thoroughly by inverting, and quickly adsorb the column material using a magnetic rack. Remove the supernatant and repeat this step three times.

[0117] 21) Add 100 μL of 20 mM, 50 mM, 100 mM, and 200 mM imidazole elution buffer in descending order of imidazole concentration, and elute on ice for 3 min. Elute twice at each imidazole concentration, and collect the elution buffer.

[0118] 22) Take 10 μL of each protein eluate in 21) and add 2.5 μL of 5× SDS loading buffer and boil in a 100°C water bath for 10 min;

[0119] 23) The samples were subjected to 5% stacking gel and 15% separating gel polyacrylamide gel electrophoresis analysis, followed by staining with Coomassie Brilliant Blue for 1 hour and multiple destaining to analyze protein expression;

[0120] 24) Boil the dialysis bag in a microwave for 3 minutes and place it in a clean bench to cool;

[0121] 25) In a clean bench, add the protein eluate with high purity to a sterile 1.5 mL EP tube. Cut off the cap, seal the tube with a dialysis bag, and reinforce the tube with a rubber band. Place the tube upside down in sterile PBS and dialyze with magnetic stirring at 4°C for 4 hours. Replace the sterile PBS every 4 hours until the imidazole content in the protein eluate is less than 1 μM.

[0122] 26) Detect the concentration of recombinant protein using BCA protein quantification method:

[0123] a) According to BCA protein quantitative detection kit instructions to perform the following operations:

[0124] i. Dilute the BSA standard sample (BSA standard sample concentration 2 mg / mL) to 80 μL 500 μg / mL BSA solution: 20 μL BSA solution + 60 μL ddH2O;

[0125] ii. Preparation of BCA working solution: Prepare the working solution in a ratio of solution A: solution B = 50:1;

[0126] b) Standard curve preparation is shown in Table 1.

[0127] Table 1

[0128] The measurement system of both the standard and sample is 20 μL. After adding the working solution each time, gently blow and mix, and incubate in a 37°C constant temperature incubator for 30 minutes; place the 96-well plate on a microplate reader to measure the absorbance value at a wavelength of 562 nm, make a standard curve, and calculate the protein concentration of the sample.

[0129] 1.2. rEGFP prokaryotic expression and protein purification

[0130] 1) Inoculate DH5α bacteria containing the pET-28a-EGFP plasmid into 5 mL of LB liquid medium containing kanamycin (kana) and activate overnight at 37°C in a shaking incubator.

[0131] 2) Add 3 mL of activated bacterial suspension to 50 mL of liquid LB medium containing kanamycin (kana) and incubate at 37°C in a shaking incubator until the OD600 is 0.6-1.0.

[0132] 3) Add IPTG to a final concentration of 1 mM and induce at 37°C for 3 h. Take 20 μL of the bacterial culture before and after induction, add 5× SDS loading buffer, and boil in a 100°C water bath for 15 min.

[0133] 4) Centrifuge the induced bacterial solution at 8800 rpm for 8 minutes at 4°C and discard the supernatant;

[0134] 5) Resuspend the cells in 5 mL of pre-chilled PBS and centrifuge to remove the supernatant.

[0135] 6) Resuspend the pellet in 5 mL of pre-chilled PBS, add 100× PMSF, and sonicate at 60% power, 3 seconds on, 8 seconds interval, for a total of 15 minutes.

[0136] 7) Centrifuge the disrupted solution at 9000 rpm for 8 min at 4°C and collect the supernatant for protein purification;

[0137] 8) Pipette 20 μL of supernatant and resuspended pellet into 200 μL centrifuge tubes, add 5 μL of 5× SDS loading buffer, and boil at 100°C for 10 min.

[0138] 9) 10 μL of each sample was analyzed by polyacrylamide gel electrophoresis using 5% stacking gel and 15% separating gel, followed by staining with Coomassie Brilliant Blue for 1 hour and multiple decolorization treatments to analyze protein expression;

[0139] 10) Aspirate 30 μL Ni-Charged MagBeads column material was used to collect the supernatant of the disruption solution in step 7) and incubated with rotation at 4°C for 30 min;

[0140] 11) Place the centrifuge tube close to the magnetic rack, remove the supernatant, and add 1 mL of 0.01 mM pre-chilled PBS to resuspend. Use a pipette to transfer the suspension to a 1.5 mL enzyme-free EP tube. Place the centrifuge tube close to the magnetic rack and remove the supernatant.

[0141] 12) Add 1 mL of pre-chilled PBS containing 1% Triton X-114 and wash with rotation at 4°C for 8 min. Repeat this step 7 times to remove endotoxin contamination.

[0142] 13) Add 1 mL of PBS to the Ni column, invert it, and quickly adsorb the column material using a magnetic rack. Remove the supernatant and repeat this step three times.

[0143] 14) Add 200 μL of 20 mM, 50 mM, 100 mM, and 200 mM imidazole elution buffer in descending order of imidazole concentration, invert and elute on ice for 3 min. Wash twice with each imidazole concentration, and collect the elution buffer;

[0144] 15) Take 10 μL of each protein eluate of 14) and add 2.5 μL of 5× SDS loading buffer and boil in a 100°C water bath for 10 min.

[0145] 16) The samples were subjected to 5% stacking gel and 15% separating gel polyacrylamide gel electrophoresis analysis, followed by staining with Coomassie Brilliant Blue for 1 hour and multiple destaining to analyze protein expression;

[0146] 17) Boil the dialysis bag in a microwave for 3 minutes and place it in a clean bench to cool;

[0147] 18) In a laminar flow hood, add the protein eluate with high purity to a sterile 1.5 mL EP tube. Remove the cap, seal the tube with a dialysis bag, and secure the tube with a rubber band. Place the tube on a float and invert it in sterile PBS. Dialyze the protein eluate with magnetic stirring at 4°C for 4 hours. Replace the sterile PBS every 4 hours until the imidazole content in the protein eluate is less than 1 μM.

[0148] 19) The concentration of recombinant enhanced green fluorescent protein (negative control protein) was detected using the 1.1.26 BCA protein quantification method.

[0149] 1.3 Identification of recombinant protein

[0150] 1) Take the prokaryotic expressed HPCAL1 protein sample from 1.1 and analyze it by polyacrylamide gel electrophoresis using 5% stacking gel and 12% separating gel;

[0151] 2) Cut a PVDF membrane slightly larger than the gel and soak it in methanol for 15 seconds. Then soak the sandwich with electrotransfer solution. Stack the electrotransfer sandwich in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, and negative electrode.

[0152] 3) The total protein was transferred to a PVDF membrane using a wet transfer electroporator (Bio-Rad) at a constant current of 300 mA for 1.5 h.

[0153] 4) Discard the gel and place the PVDF membrane in a blocking solution containing 5% skim milk powder at room temperature for 1 hour.

[0154] 5) Transfer the membrane to the blocking solution Incubate in rabbit anti-HPCAL1 polyclonal antibody (1:1000) at 4°C overnight;

[0155] 6) Wash three times with 1× TBST, 15 min each time;

[0156] 7) Transfer the membrane to the blocking solution Incubate in goat anti-rabbit IgG-HRP (1:5000) at room temperature for 1 h;

[0157] 8) Wash three times with 1× TBST, 15 min each time;

[0158] 9) Using Merck Immobilon Western Chemilum HRP Substrate was used as the color developing solution and the GE AI600 imaging system was used for ECL color development and imaging, and the images were saved; the results are shown in Figure 2.

[0159] 1.4 Immunoblotting analysis of hemocytes after injection of recombinant HPCAL1 into shrimp

[0160] 1) The concentrations of the recombinant shrimp HPCAL1 and rEGFP proteins purified in 1.1 and 1.2 were determined by BCA protein quantification method;

[0161] 2) Dilute the recombinant protein to 5 μg / mL with sterile PBS;

[0162] 3) Fifteen shrimps were taken from each experimental group and control group, and 100 μL of recombinant protein (injection dose: 0.1 μg / g) was injected into the junction of the second and third abdominal segments of each shrimp;

[0163] 4) After 3 h, hemolymph was extracted from the experimental and control groups at a ratio of 300 μL sterile anticoagulant to 100 μL hemolymph into 15 mL centrifuge tubes.

[0164] 5) Centrifuge at 900 rpm for 10 min at 4°C and remove the supernatant;

[0165] 6) Add 5 mL of sterile anticoagulant and resuspend, centrifuge at 900 rpm for 10 min at 4°C, and repeat this step three times;

[0166] 7) Resuspend the pellet in 20 μL PBS, add 5 μL 5× SDS loading buffer, and boil in a 100°C water bath for 20 min.

[0167] 8) Analyze the samples by polyacrylamide gel electrophoresis using 5% stacking gel and 12% separating gel;

[0168] 9) Cut a PVDF membrane slightly larger than the gel and soak it in methanol for 15 seconds. Then soak the sandwich with electrotransfer solution. Stack the electrotransfer sandwich in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, and negative electrode.

[0169] 10) The total protein was transferred to a PVDF membrane using a wet transfer electroporator (Bio-Rad) at a constant current of 300 mA for 1.5 h.

[0170] 11) Discard the gel and place the PVDF membrane in 5% BSA blocking solution at room temperature for 1 hour;

[0171] 12) Cut the PVDF membrane from the middle of 65-75kDa, and dilute the high molecular weight membrane and low molecular weight membrane with blocking solution. Rabbit anti-STAT5a polyclonal antibody (1:500) and Mouse anti-α-Tubulin monoclonal antibody (1:3000) was incubated overnight at 4°C;

[0172] 13) Wash three times with 1× TBST, 15 min each time;

[0173] 14) Diluted with blocking solution Goat anti-rabbit IgG-HRP (1:5000) and Goat anti-mouse IgG-HRP (1:5000) was incubated at room temperature for 1 h;

[0174] 15) Wash three times with 1× TBST, 15 min each time;

[0175] 16) Using Merck Immobilon Western Chemilum HRP Substrate was used as the color developing solution and GE AI600 imaging system was used for ECL color development and image preservation.

[0176] The results are shown in Figures 3, 5 and 6.

[0177] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were injected into shrimp, respectively. Three hours later, shrimp hemocytes were extracted for immunoblotting analysis of STAT phosphorylation. The results are shown in Figure 3. Figure 3A is the immunoblotting result repeated three times, and Figure 3B is a scatter plot of the ratio of the grayscale of the phosphorylated STAT band to the grayscale of the corresponding internal reference tubulin band. These results indicate that recombinant shrimp HPCAL1 protein can effectively inhibit STAT (signal transducer and activator of transcription) phosphorylation in shrimp hemocytes.

[0178] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were injected into shrimp, respectively. Three hours later, RNA was extracted from shrimp hemocytes for real-time fluorescence quantitative PCR (the experiment was repeated three times). The results are shown in Figure 5 , which show that recombinant shrimp HPCAL1 protein can effectively inhibit the expression of multiple immune genes in shrimp hemocytes, such as the immune-related transcription factor STAT, three enzymes involved in the production of oxygen free radicals PPO1, PPO2, and PPAF1, and two antimicrobial peptides PEN3 and CRUL.

[0179] Shrimp hemocytes were primary cultured and then treated in vitro with octopamine (0.5 μg / mL) plus recombinant enhanced green fluorescent protein (0.1 μg / mL) and octopamine (0.5 μg / mL) plus recombinant shrimp HPCAL1 (0.1 μg / mL), respectively. Changes in STAT phosphorylation in shrimp hemocytes were then detected by immunoblotting. The results are shown in Figure 6 . Figure 6A is the immunoblotting result repeated three times, and Figure 6B is a scatter plot of the ratio of the grayscale of the phosphorylated STAT band to the grayscale of the corresponding internal control tubulin band. These results indicate that recombinant shrimp HPCAL1 can inhibit the activation of STAT (signal transducer and activator of transcription) phosphorylation in shrimp hemocytes by octopamine in vitro.

[0180] In summary, shrimp HPCAL1 inhibits shrimp blood cell activity and STAT (signal transducer and activator of transcription) phosphorylation by binding to octopamine receptors.

[0181] 1.5 Prokaryotic expression and purification of rGST-PvHPCAL1 protein

[0182] 1) Dissolve the constructed pGEX-6P-1-PvHPCAL1 plasmid in ddH2O to 100 ng / μL;

[0183] 2) Add 1 μL of the plasmid to 50 μL of BL21 (DE3) E. coli competent cells and mix thoroughly by gently pipetting.

[0184] 3) Place on ice for 30 min, bath in 42°C water for 90 s, and then bath on ice for 2 min;

[0185] 4) Add 950 μL of LB medium without antibiotics in a clean bench and incubate at 37°C at 200 rpm for 1 h.

[0186] 5) Centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant in a clean bench, and mix gently by pipetting;

[0187] 6) Use a pipette to transfer the mixture to a solid LB plate containing ampicillin. After the glass rod is burned and cooled, apply it to the plate.

[0188] 7) Wait until the liquid on the plate is slightly dry, then invert and place in a 37°C bacterial incubator for overnight culture.

[0189] 8) Pick a single clone and inoculate it into 5 mL of LB liquid medium containing Ampicillin (Amp) and activate it overnight at 37°C in a shaker.

[0190] 9) Add 3 mL of activated bacterial suspension to 50 mL of LB liquid medium containing ampicillin (Amp) and culture at 37°C until the OD600 is 0.6-1.0;

[0191] 10) Add IPTG to a final concentration of 1 mM and induce at 37°C for 3 h. Take 20 μL of the bacterial culture before and after induction, add 5× SDS loading buffer, and boil in a 100°C water bath for 10 min.

[0192] 11) Collect the cells by centrifugation at 8800 rpm for 10 min at 4°C and resuspend in 5 mL of pre-chilled PBS containing 1% Triton X-110 and 1% Tween-20. Place on ice for several minutes and add 1× PMSF to the suspension for sonication. The sonication conditions were: 3 s for 3 seconds, 8 s for 8 seconds, 30 min for 30 seconds, and 60% power.

[0193] 12) Centrifuge the disrupted bacterial solution at 9000 rpm for 8 min and collect the supernatant;

[0194] 13) Take 20 μL of the supernatant and precipitate after disruption, add 5 μL of 5× SDS loading buffer respectively, and boil in a 100°C water bath for 10 min;

[0195] 14) Detect the induced expression effect by polyacrylamide gel electrophoresis using 5% stacking gel and 12% separating gel;

[0196] 15) Take the supernatant collected in step 12, add 30 μL of Glutathione Sepharose 4B column, incubate at 4°C with rotation for 30 min, centrifuge at 1200 rpm at 4°C for 3 min, and discard the supernatant;

[0197] 16) Add 1 mL of pre-chilled PBS to resuspend the column, mix by inversion for 2 minutes, centrifuge at 1200 rpm for 3 minutes at 4°C, and discard the supernatant.

[0198] 17) Add 1 mL of pre-chilled PBS containing 1% Triton X-110 to resuspend the column, mix by inversion for 2 minutes, centrifuge at 1200 rpm for 3 minutes at 4°C, discard the supernatant, and repeat the wash seven times.

[0199] 18) The GST-PvHPCAL1 protein enriched column was subjected to 5% stacking gel and 12% separating gel polyacrylamide gel electrophoresis analysis.

[0200] 1.6 Prokaryotic expression and purification of rGST protein

[0201] 1) Inoculate the bacteria containing the pGEX-6P-1 plasmid into 5 mL of LB liquid medium containing Ampicillin (Amp) and activate overnight at 37°C in a shaking incubator.

[0202] 2) Add 3 mL of activated bacterial suspension to 50 mL of LB liquid medium containing ampicillin (Amp) and culture at 37°C until the OD600 is 0.6-1.0;

[0203] 3) Add IPTG to a final concentration of 1 mM and induce at 37°C for 3 h. Take 20 μL of each tube for induction;

[0204] 4) Add 5× SDS loading buffer to the induced bacterial solution and boil it in a 100°C water bath for 10 minutes.

[0205] 5) Collect the cells by centrifugation at 8800 rpm for 10 min at 4°C and resuspend in 5 mL of pre-chilled PBS containing 1% Triton X-110 and 1% Tween-20. Place on ice for 10 min, then add PMSF to the suspension at a final concentration of 1× and disrupt by sonication using the following conditions: 3 s for each cycle, 8 s for each cycle, 30 min for each cycle, and 60% power.

[0206] 6) Centrifuge the disrupted bacterial suspension at 8800 rpm for 10 min and collect the supernatant;

[0207] 7) Take 20 μL of the supernatant and inclusion bodies after disruption, add 5 μL of 5× SDS loading buffer and boil in a 100°C water bath for 10 min;

[0208] 8) Detect the induced expression effect by 5% stacking gel and 12% separation polyacrylamide gel electrophoresis;

[0209] 9) Take the supernatant collected in step 6, add 30 μL of Glutathione Sepharose 4B column, incubate at 4°C with rotation for 30 minutes, centrifuge at 1200 rpm at 4°C for 3 minutes, and discard the supernatant;

[0210] 10) Add 1 mL of pre-chilled PBS to resuspend the column, mix by inversion for 2 minutes, centrifuge at 1200 rpm at 4°C for 3 minutes, and discard the supernatant.

[0211] 11) Add 1 mL of pre-chilled PBS containing 1% Triton X-110 to resuspend the column, mix by inversion for 2 minutes, centrifuge at 1200 rpm for 3 minutes at 4°C, discard the supernatant, and repeat the wash seven times.

[0212] 12) The GST protein-enriched column was subjected to 5% stacking gel and 12% separation gel polyacrylamide gel electrophoresis analysis.

[0213] 1.7 GST pull-down in blood lymphocytes

[0214] 1) In Prepare 20 mL of sterile anticoagulant in a 50 mL centrifuge tube. Attach a 1 mL syringe to a 12-gauge needle. Draw a small amount of anticoagulant and aim the needle at the junction of the second and third muscle segments of the shrimp to extract hemolymph. Then, inject the mixture from the syringe into the centrifuge tube containing the anticoagulant. Repeat this process to extract hemolymph from approximately 200 shrimp, totaling approximately 40 mL of anticoagulant.

[0215] 2) Centrifuge at 1200 rpm for 25 min at 4°C to pellet the cells and discard the supernatant;

[0216] 3) Repeat washing three times with 15 mL of sterile anticoagulant, centrifuge at 1200 rpm for 5 minutes at 4°C, and discard the supernatant;

[0217] 4) Add 2 mL of pre-chilled cell lysis buffer to the cell pellet, pipette evenly, and place on ice.

[0218] 5) Disrupt the cells using an ultrasonic disruptor at 40% power, 2 seconds on, 8 seconds off, for 7 cycles. Stop disruption when the mixture becomes slightly clear.

[0219] 6) Centrifuge the mixture at 20,000 rpm for 20 min at 4°C. Collect the supernatant as the cell lysate and divide it into two tubes, 1 mL each.

[0220] 7) Take another 10 μL of cell lysate and add 5× SDS loading buffer, boil for 10 minutes, and save as input.

[0221] 8) Take 15 μL of the prepared GST-bound column and 20 μL of the rGST-PvHPCAL1-bound column, incubate each with 1 mL of cell lysate. After incubation at 4°C overnight, wash the columns with 1 mL of cell lysate each time. Repeat 7 times, centrifuging at 1200 rpm for 3 minutes each time and remove the supernatant.

[0222] 9) Add 60 μL PreScission Proteas (GE) digestion buffer and 1 μL PreScission Protease (GE) to each tube, incubate with rotation at 4°C for 4 h, and centrifuge at 1200 rpm for 3 min to collect the supernatant;

[0223] 10) Take 20 μL of the collected supernatant and 2 μL of cell lysate. Premixed protein marker (Low) was used as a protein molecular weight control and electrophoresis was performed on 5% stacking gel and 15% separating gel polyacrylamide gel.

[0224] 11) After electrophoresis, perform silver staining to identify differential bands:

[0225] a) Transfer the gel to 100 mL of fixative solution and shake on a shaker at room temperature for 1 h at 65 rpm.

[0226] b) Discard the fixative solution, add 100 mL of 30% ethanol, and shake on a shaker at room temperature for 10 minutes at 65 rpm.

[0227] c) Aspirate and discard 100 mL of ethanol, add 200 mL of Milli-Q grade water, and shake on a shaker at room temperature for 15 minutes at 65 rpm.

[0228] d) Discard the Milli-Q water and add 30 mL of freshly prepared silver stain sensitizer. Shake on a shaker at room temperature for 2 minutes at 65 rpm.

[0229] e) Discard the silver staining sensitizer solution, add 200 mL of Milli-Q grade water, and shake on a shaker at room temperature for 1 minute at 65 rpm.

[0230] f) repeat the above steps;

[0231] g) Discard the Milli-Q water, add 30 mL of the freshly prepared silver solution, and shake at room temperature for 10 minutes on a shaker at 65 rpm.

[0232] h) Discard the silver solution, add 100 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 1.5 minutes at a speed of 65 rpm;

[0233] i) Discard the Milli-Q water, add 30 mL of silver staining solution, and shake on a shaker at room temperature for 8 minutes at 65 rpm.

[0234] j) After the desired protein bands appear, add 20 mL of silver staining stop solution and shake at room temperature for 10 minutes on a shaker at 65 rpm.

[0235] k) Discard the silver staining stop solution and add 100 mL of Milli-Q grade pure water for storage.

[0236] 12) Cut the identified differential bands with a clean blade and place them in a 1.5 mL light-proof EP tube. Send them with an ice pack to Shanghai Houji Biotechnology Co., Ltd. for Q Excative mass spectrometry identification.

[0237] 13) Based on the mass spectrometry data returned by the company, the octopamine receptor beta-2R-like (LvOAR), a potential interacting protein with HPCAL1, was screened out.

[0238] The results are shown in FIG4 , which show that shrimp HPCAL1 can specifically bind to the octopamine receptor in shrimp blood cells.

[0239] (2) 293T cell overexpression experiment

[0240] 1) Prokaryotic expression of recombinant human HPCAL1 (strep-tag) was obtained from Wuhan Pujian Biotechnology Co., Ltd. The recombinant human HPCAL1 mutant (SEQ ID NO: 4, His-tag) plasmid was synthesized by BGI and purified in-house. The recombinant EGFP purification method is described in 1.2. Human β1, β2, and β3 adrenergic receptor plasmids were obtained from Genecopia (EX-Y5305-M35, EX-A4389-M35-B, and EX-U1168-M35).

[0241] 2) Human β1, β2, and β3 adrenergic receptor plasmids were transfected into 293T cells. 48 hours after transfection, cells were treated with 100 μM epinephrine plus 0.5 μg / mL recombinant EGFP; 100 μM epinephrine plus 0.5 μg / mL recombinant human HPCAL1; 100 μM octopamine plus 0.5 μg / mL recombinant human EGFP; and 100 μM octopamine plus 0.5 μg / mL recombinant human HPCAL1 for 0, 0.5, 1, and 2 hours, respectively.

[0242] 3) After collecting the samples, immunoblotting was used to detect the phosphorylated ERK signal, the internal reference GADPH (human phosphoglycoside dehydrogenase) signal and the Flag-tag signal of the transfected receptor.

[0243] The results are shown in Figure 8 . Adrenalin can effectively activate ERK phosphorylation in 293T cells overexpressing adrenergic receptor β1 (Figure 8A), β2 (Figure 8B), and β3 (Figure 8C). Compared with the negative control (recombinant enhanced green fluorescent protein), the addition of recombinant human HPCAL1 can inhibit adrenaline-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 (Figure 8C), but cannot inhibit adrenaline-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β1 and β2 (Figures 8A and 8B).

[0244] It is known to those skilled in the art that adrenergic receptor β3 is the primary receptor for octopamine in the human body. Therefore, the blocking effects of recombinant human HPCAL1 and its mutants on octopamine-activated β3-adrenergic receptor were examined. The results showed that the addition of recombinant human HPCAL1 inhibited octopamine-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 ( Figure 8D ). The addition of recombinant human HPCAL1 mutants also inhibited octopamine-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 ( Figure 8F ). Figure 8E shows the purified recombinant human HPCAL1 mutants.

[0245] The above results indicate that recombinant human HPCAL1 can effectively and selectively block the activation of β3-adrenergic receptor by epinephrine and octopamine; recombinant human HPCAL1 mutants can effectively and selectively block the activation of β3-adrenergic receptor by octopamine; recombinant human HPCAL1 and its mutants can serve as β3-adrenergic receptor antagonist proteins.

[0246] (3) In vitro interaction experiment between recombinant human β3 adrenergic receptor and recombinant human HPCAL1

[0247] 1) Recombinant human β3-adrenergic receptor (GENE ID: 155) was obtained from Wuhan Huamei Bioengineering Co., Ltd. (CSB-CF001393HU), human serum albumin was obtained from Shanghai Jizhi Biochemical Technology Co., Ltd. (ACMEC, A93920-100 mg), and recombinant human HPCAL1 (strep-tag) was obtained from Wuhan Pujian Biotechnology Co., Ltd.

[0248] 2) Biocore protein interaction experiments were performed by Wuhan Pujian Biotechnology Co., Ltd. The specific steps are as follows:

[0249] (a) Start the Biacore T200 instrument according to standard operating procedures.

[0250] (b) Prepare 500 mL of PBST (pH 7.4) buffer and deionized water (filtered with a 0.22 μm membrane) for rinsing the injection needle.

[0251] (c) Start chip installation and install the NTA chip according to the standard process.

[0252] (d) Prepare to start the formal experiment. The buffer solution will flush the flow system inside the entire system at a high flow rate.

[0253] (e) Select an appropriate procedure based on the sample size.

[0254] (f) The ligand capture time was set to 60 s and the flow rate was 10 μL / min. The analyte binding time was set to 120 s and the flow rate was 30 μL / min. The dissociation time was set to 300 s and the flow rate was 30 μL / min. The regeneration time was set to 30 s and the flow rate was 30 μL / min.

[0255] (g) Prepare the corresponding samples to be tested as required and start the automatic running program for testing.

[0256] (h) Result analysis: Based on the running results, data fitting analysis is performed to obtain the final affinity fitting KD value.

[0257] The results are shown in Figure 7, indicating that the recombinant human HPCAL1 protein can specifically bind to the recombinant human β3-adrenergic receptor in in vitro experiments.

[0258] (4) Recombinant HPCAL1 injection experiment in heart failure mice

[0259] 1. Myocardial ischemia-reperfusion model in mice

[0260] 1) Anesthesia: Anesthetize mice with an intraperitoneal injection of avertin (body weight x 0.015). Cut three long pieces of tape and six short pieces of tape (to secure the limbs, tail, and teeth). Disinfect with 75° alcohol and arrange the necessary surgical instruments.

[0261] 2) Fixation: Use forceps to clamp the mouse's hind legs with appropriate force to check whether anesthesia is complete. After confirming that anesthesia is complete, fix the mouse with tape and thread;

[0262] 3) Intubation: Shine a light on the mouse's throat, pull the mouse's tongue to the right, and gently lift it with a tongue depressor to locate the trachea (round hole, continuously scalable). Intubate with a 20 / 22G intravenous cannula. Remove the needle and observe whether the mouse's breathing is stable. Connect the ventilator to check for a consistent respiratory rate. Be sure to turn on the ventilator before connecting the mouse to avoid excessive airflow that could damage the mouse's lungs.

[0263] 4) Hair Removal: Use depilatory cream to remove hair on the left side of the mouse chest. After hair removal, clean the surgical site with saline.

[0264] 5) Thoracotomy: Adjust the mouse's position, disinfect the skin surface with iodine, and locate the spot on the left side of the mouse's chest where the heaving is greatest. Open the chest parallel to the right side of this spot. First, cut the skin, then the two layers of pectoral muscle. Then, bluntly open the intercostal space with the largest gap (do not use scissors, as this can easily sever the artery in the middle of the chest). Be careful not to injure the left lung.

[0265] 6) Locate the heart. The left anterior descending artery is located 1.5 mm below the left atrium. The ligature width is approximately 2-3 mm. After ligation, double-thread ligatures can be tightened. If the myocardium below the thread appears white, the ligature position is correct.

[0266] 7) Find the location and tie a small knot. Place the PE-10 catheter. Tie the knot tightly and tie another knot. Once the ligature is complete, time it for 60 minutes. Cover the area with a piece of gauze soaked in saline to prevent the skin from drying out.

[0267] 8) Remove the small tube and cut the string;

[0268] 9) Suture the intercostal space, pectoral muscles, and skin. Use interrupted sutures and three stitches for intercostal and pectoral muscle sutures, and continuous sutures for skin sutures.

[0269] 10) After suturing, disinfect with iodine.

[0270] 11) Disconnect the ventilator, remove the cannula, and observe the condition of the mouse after it slowly recovers. The mouse survived 5 days after modeling, but the ejection fraction was lower than that of healthy mice, and symptoms of heart failure appeared. The mice with successfully modeled myocardial damage were used for subsequent experiments.

[0271] 2. Ultrasound imaging monitoring of changes in cardiac function in mice before and after HPCAL1 injection

[0272] 1) Eukaryotic recombinant mouse HPCAL1 was obtained from Wuhan Pujian Biotechnology Co., Ltd., and mouse serum albumin was obtained from Wuhan FineTest Biotechnology Co., Ltd. (FineTest, P3125). The mice used were 8-12-week-old male C57BL / 6JNifdc mice. The relevant animal experiments were approved by the Experimental Animal Ethics and Use Committee of Shantou University.

[0273] 2) Pass The LAZR small animal in vivo multimodal imaging system was used to acquire echocardiograms of mice. The specific steps are as follows:

[0274] (a) Anesthesia and Fixation: Mice reperfused for 5 days were placed in an anesthesia induction box filled with 3% isoflurane gas to rapidly induce full anesthesia (slight squinting of the eyes and slight tremor indicate anesthesia). The mice were then quickly transferred to a 37°C thermostatted operating table, where the isoflurane concentration was adjusted to 0.5%-1% and anesthesia was maintained with a breathing mask. The mouse's limbs were secured to electrodes coated with coupling agent using medical tape to monitor vital signs such as heart rate and respiration.

[0275] (b) Data Acquisition: Tilt the operating table approximately 45° and adjust the MS-400 ultrasound probe so that its notch is perpendicular to the long axis of the heart. Apply coupling gel to the left chest of the mouse heart (hair removed before modeling). Gently press the probe down until it contacts the coupling gel but does not compress the heart. At a controlled heart rate of 420-440 beats / min, acquire dynamic videos of the mouse's parasternal short-axis section in B-Mode and M-Mode.

[0276] (C) Drug treatment: After collecting pre-drug data (Before), mice were injected via the tail vein with rmsHPCAL1 (20 μg / mouse, 0.39 mg / ml) or an equivalent dose of MSA (20 μg / mouse, 0.4 mg / mL). Echocardiograms were obtained 20-40 minutes after administration (After) as described in (B).

[0277] (D) Data processing: Vevo LAB 3.0.0 software was used to process data. Cardiac function parameters, including left ventricular anterior wall thickness, posterior wall thickness, and left ventricular internal diameter, were measured during diastole and systole for five consecutive cardiac cycles in each group. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were used to assess cardiac systolic function in mice: LVEF = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume; LVFS = (left ventricular end-diastolic diameter - left ventricular end-systolic diameter) / left ventricular end-diastolic diameter.

[0278] The results are shown in FIG9 , which show that the tail vein injection of recombinant mouse HPCAL1 into mice with myocardial ischemia-reperfusion for 5 days can effectively increase the left ventricular ejection fraction (about 20%) and left ventricular minor axis shortening (about 30%) of the mice.

[0279] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. Application of HPCAL1 protein or related biomaterials in any of the following: (A1) preparing a β3 adrenergic receptor antagonist; (A2) non-therapeutic inhibition of β3 adrenergic receptor activation; (A3) preparing a medicament for treating cardiovascular disease; (A4) Preparation of a cardiotonic agent.

2. The use according to claim 1, characterized in that The cardiovascular diseases include diseases of declining heart function.

3. The use according to claim 2, characterized in that The heart function decline disease includes at least one of myocardial infarction, heart disease, myocardial ischemia reperfusion and heart failure.

4. The use according to claim 1, characterized in that The HPCAL1 protein comprises the protein shown in any one of the following B1-B4: (B1) a protein having an amino acid sequence of any one of SEQ ID NOs: 1-3; (B2) a protein having the same function as any one of SEQ ID NOs: 1-3 by substitution and / or deletion and / or addition of one or more amino acid residues; (B3) a protein having at least 99%, 98%, 97%, 95%, 90%, 85% or 80% homology to the amino acid sequence defined in any one of (B1) or (B2) and having the same function; or (B4) Modifying the N-terminus, C-terminus, amino acid backbone and / or amino acid side chain groups of the protein defined in any one of (B1) to (B3) to obtain a polypeptide derivative with the same function.

5. The use according to claim 4, characterized in that The addition in (B2) is to add one or more amino acid residues to the N-terminus and / or C-terminus of the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

6. The use according to claim 4, characterized in that The one or several amino acid residues in (B2) include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

7. The use according to claim 4, characterized in that The adding includes adding a tag sequence.

8. The use according to claim 7, characterized in that The tag sequence includes at least one of a signal peptide, a targeting peptide, a tag peptide, a fluorescent protein, and a transmembrane peptide.

9. The use according to claim 4, characterized in that The modification comprises one or more of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol modification, multimeric antigen peptide, prenylation and cyclization, acetylation, amidation, fatty acid, and cyclization.

10. The use according to claim 4, characterized in that The amino acid sequence of the protein having the same function as that shown in SEQ ID NO: 3 is replaced by one or more amino acid residues as shown in SEQ ID NO:

4.

11. The use according to any one of claims 1 to 10, characterized in that: The relevant biological material is a biological material expressing HPCAL1 protein, preferably any one of the following: (C1) a nucleic acid molecule encoding the HPCAL1 protein; (C2) an expression cassette containing the nucleic acid molecule described in (C1); (C3) a recombinant vector containing the nucleic acid molecule described in (C1), or a recombinant vector containing the expression cassette described in (C2); (C4) a recombinant cell containing the nucleic acid molecule described in (C1), or a recombinant cell containing the expression cassette described in (C2), or a recombinant cell containing the recombinant vector described in (C3); (C5) a recombinant tissue containing the nucleic acid molecule described in (C1), or a recombinant tissue containing the expression cassette described in (C2), or a recombinant tissue containing the recombinant vector described in (C3); (C6) a recombinant organ containing the nucleic acid molecule described in (C1), a recombinant organ containing the expression cassette described in (C2), or a recombinant organ containing the recombinant vector described in (C3); (C7) A recombinant microorganism containing the nucleic acid molecule described in (C1), or a recombinant microorganism containing the expression cassette described in (C2), or a recombinant microorganism containing the recombinant vector described in (C3).

12. A medicine comprising the HPCAL1 protein or related biological materials according to any one of claims 1 to 11.

13. The drug according to claim 12, characterized in that The drug also includes pharmaceutically acceptable excipients.

14. The drug according to claim 12, characterized in that The dosage forms of the drug include injection, aerosol or drops.

15. Use of the drug according to any one of claims 12 to 14 in any one of the following: (A1) preparing a β3 adrenergic receptor antagonist; (A2) non-therapeutic inhibition of β3 adrenergic receptor activation; (A3) preparing a medicament for treating cardiovascular disease; (A4) Preparation of a cardiotonic agent.

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