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 heart function TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of HPCAL1 protein in enhancing heart function. BACKGROUND

[0002] Heart failure refers to that due to obstruction of the systolic function and diastolic function of the heart, the heart cannot fully discharge the blood volume returned from veins, leading to blood stasis in the venous system and insufficient blood perfusion in the arterial system, thereby causing a heart circulation disorder syndrome, and the syndrome is mainly manifested as pulmonary congestion and venous congestion. Heart failure is not an independent disease, but a terminal stage of heart disease. Almost all cardiovascular diseases eventually lead to the occurrence of heart failure. Myocardial infarction, cardiomyopathy, excessive hemodynamic load, inflammation and other causes of myocardial damage can all cause changes in myocardial structure and function, and finally lead to low ventricular pumping and / or filling function.

[0003] The influencing factors of cardiovascular health include smoking, dietary nutrition, physical activity, overweight and obesity, and psychological factors. The risk factors of cardiovascular disease include hypertension, dyslipidemia, diabetes, chronic kidney disease, metabolic syndrome and air pollution. Many cardiovascular diseases are related to heart function decline, such as coronary heart disease, heart failure, pulmonary heart disease, atrial fibrillation, rheumatic heart disease, congenital heart disease, etc. The treatment of these related diseases needs to use cardiac agents. The cardiac agents on the market mainly include cardiac glycosides and non-cardiac glycosides: the former is a cardiac glycoside, and the commonly used ones in clinical practice are digitalis glycosides, digoxin, desacetyllanatoside C and strophanthin K, etc. The latter includes beta-adrenergic receptor agonists, such as dobutamine, dopamine isobutyl ester, phosphodiesterase inhibitors such as aminpyrine, etc. In recent years, it has been found that beta3-adrenergic receptors are expressed in the heart in various cardiovascular diseases, and beta3-adrenergic receptor blockers may have a certain effect on heart function decline in cardiovascular diseases. Therefore, the development of specific blockers or antagonists of beta3-adrenergic receptors has become one of the new hotspots in the treatment of cardiovascular diseases in recent years. However, chemical small molecule drugs generally have high liver and kidney toxicity, and their drug toxicity cannot be ignored.

[0004] SUMMARY

[0005] The purpose of the present application is the use of HPCAL1 protein or its related biomaterials in the preparation of cardiac agents for treating heart failure and other cardiovascular diseases.

[0006] The technical scheme adopted by the present application is:

[0007] In a first aspect of the present application, there is provided use of HPCAL1 protein or a related biological material thereof in any of the following:

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

[0009] (A2) inhibiting activation of β3 adrenergic receptor for non-therapeutic purposes;

[0010] (A3) preparing a drug for treating cardiovascular diseases;

[0011] (A4) preparing a cardiotonic agent.

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

[0013] In some embodiments of the present application, the cardiovascular diseases include a heart function decline disease.

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

[0015] In some embodiments of the present application, the heart disease includes genetic heart disease, drug-induced heart disease, or infection-induced heart disease.

[0016] In some embodiments of the present application, the HPCAL1 protein is HPCAL1 protein derived from Penaues monodon, human, or mouse.

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

[0018] (B1) a protein with an amino acid sequence as shown in any of SEQ ID NOs: 1-3;

[0019] (B2) a protein with an amino acid sequence as shown in any of SEQ ID NOs: 1-3, which has been subjected to substitution and / or deletion and / or addition of one or several amino acid residues and has the same function;

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

[0021] (B4) a polypeptide derivative obtained by modifying the protein defined in any of (B1)-(B3) at the N-terminus, C-terminus, amino acid backbone, and / or amino acid side chain group, which has the same function.

[0022] Preferably, the amino acid sequence of the protein mutant of SEQ ID NO: 3 with the same function after substitution of one or several amino acid residues is shown as SEQ ID NO: 4.

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

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

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

[0027] The "homology" herein refers to the correlation between two amino acid sequences, which is described by the parameter "identity"; the at least 95% homology can be at least 96%, 97%, 98%, 99% identity. The at least 90% homology can be at least 91%, 92%, 93%, 94% identity. The at least 85% homology can be at least 86%, 87%, 88%, 89% identity. The at least 80% homology can be at least 81%, 82%, 83%, 84% identity. And the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (for example, the protein with the same identity as human HPCAL1 protein can bind to β3 adrenergic receptor and down-regulate the activation of β3 adrenergic receptor).

[0028] In some embodiments of the present application, the HPCAL1 protein is a protein having the same function as the amino acid sequence shown in any one of SEQ ID NOs: 1-3, with substitution and / or deletion and / or addition of one or several amino acid residues. Preferably, the substitution is a conservative substitution. More preferably, the deletion, insertion and / or substitution of the amino acid residues is a change of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs: 1-3. In some embodiments of the present application, the modification in (B4) comprises a combination of one or more of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol (PEG) modification, multiple antigenic peptide (MAP), isoprenylated cyclization, acetylation, amidation, fatty acid, cyclization or other modification acceptable in the art of polypeptides.

[0029] In some embodiments of the present application, 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 of (C1);

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

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

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

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

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

[0037] In some embodiments of the present application, the vector is well known to those skilled in the art, including but not limited to: a plasmid, a bacteriophage, 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. This DNA may include not only a promoter to initiate gene transcription but also a terminator to terminate gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0039] The recombinant cells in the aforementioned biological materials include prokaryotic or eukaryotic cells. These cells do not involve new plant or animal varieties.

[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 the target protein.

[0041] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

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

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

[0044] A second aspect of the present invention provides a medicament comprising the HPCA1 protein or related biological material as described in the first aspect of the present invention.

[0045] In some embodiments of the present invention, the medicament further includes active ingredients that enhance cardiac function and / or treat cardiovascular diseases.

[0046] In some embodiments of the present invention, the drug contains an effective dose of HPCA1 protein or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, or buffers.

[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 route of administration of the drug includes at least one of intravenous injection, intravenous drip, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal administration, or nebulized administration.

[0051] A third aspect of the present invention provides a method for preventing or treating cardiovascular disease, comprising administering to a desired subject an effective amount of HPCA1 protein or a pharmaceutically acceptable salt thereof, or administering a pharmaceutical composition comprising the aforementioned HPCA1 protein and a pharmaceutically acceptable excipient.

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

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

[0054] In some embodiments of the present invention, the HPCAL1 protein or a pharmaceutically acceptable salt thereof in the medicament may be combined in various ways with other ingredients that can be used to prevent and / or treat cardiovascular diseases. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other ingredients that can be used to prevent and / or treat cardiovascular diseases may be present in different medicaments and administered in combination or as a co-packaged or combined medication. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other ingredients that can be used to prevent and / or treat cardiovascular diseases may coexist in the same medicament and be administered as a compound medication.

[0055] In this invention, the appropriate daily dose range of HPCA1 protein or a pharmaceutically acceptable salt thereof is 0.1 μg / kg to 1 g / kg body weight; the above dose may be administered as a single dose unit or divided into several dose units, depending on the physician’s clinical experience and the dosing regimen, including the use of other treatment methods.

[0056] In this invention, when used on animals, the daily dose of the recombinant HPCA1 protein or a pharmaceutically acceptable salt thereof is 0.5 μg / kg to 1 mg / kg mice; and / or, the number of administrations is 1 to 3 times.

[0057] In this invention, the effective dose can be reasonably adjusted according to the actual situation and the judgment of the clinician. When dealing with different species, the dose can be reasonably adjusted based on the drug dose conversion formula or ratio between different species in the field.

[0058] Definition of terminology:

[0059] The term "HPCAL1 protein" used in this invention 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 studies have been conducted on the functional mechanisms of HPCAL1 in humans; it participates in the calcium-dependent regulation of rhodopsin phosphorylation and may also be related to neuronal signaling in the central nervous system. Particularly in the exploration of its mechanisms related to cancer and other diseases, it has been shown to have a significant inhibitory effect on the development of liver cancer and a proliferative effect on glioblastoma.

[0060] The term "β3-adrenergic receptor" refers to a tissue receptor that mediates the action of catecholamines; it is a G-protein-coupled receptor. β3-adrenergic receptors are mainly distributed on muscle cells and their expression is upregulated after myocardial injury. Activation of β3 receptors can produce negative inotropic effects. β3 receptors induce negative inotropic effects through 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 β3-adrenergic receptors and blocks the effects mediated by receptor agonists such as adrenaline and octopamine. The term "octopamine receptor" refers to a protein receptor specific to the G protein-coupled receptor superfamily in invertebrates.

[0062] The term "heart failure" refers to a group of diseases in which the heart is unable to meet the body's needs for oxygen and nutrients, resulting in impaired bodily functions. Advanced heart failure is also known as heart failure. Heart failure includes 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 sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage 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 × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights 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 alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace amino acid residues 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 or hydrogen bonds). 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art.

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

[0066] This invention discovers that the HPCA1 protein can bind to β3-adrenergic receptors, thus functioning as a β3-adrenergic receptor antagonist; and further discovers that the HPCA1 protein has the effect of treating heart failure, and can be used to prepare drugs for strengthening the heart, or drugs for treating heart failure, especially drugs for heart failure caused by ischemia.

[0067] This invention has shown through experiments that injecting the protein HPCA1 into shrimp can inhibit the phosphorylation level of the transcription factor STAT in blood cells; the protein binds to the shrimp octopamine receptor, blocking the effect of octopamine in shrimp serum; it has also been found that the human HPCA1 protein can effectively block the activation of overexpressed β3-adrenergic receptors by adrenaline and octopamine in 293T cells, and in vitro protein-protein interaction analysis further proves that the human HPCA1 protein can specifically bind to the recombinant human β3-adrenergic receptor; in animal experiments, it has been found that intravenous injection of a certain concentration of recombinant mouse HPCA1 in a mouse model of ischemia-reperfusion can effectively improve cardiac function and enhance ejection fraction in mice.

[0068] The experiments of this invention show that the protein HPCA1 can be used to enhance cardiac function and has potential pharmaceutical value for the treatment of cardiovascular diseases such as heart failure. Furthermore, HPCA1 protein is a biomacromolecule with low toxicity, solving the problem of high hepatotoxicity and nephrotoxicity associated with small chemical molecule drugs; thus, it has high application value. Attached Figure Description

[0069] Figure 1 shows the sequence homology analysis of HPCA1 proteins from different species.

[0070] Figure 2 shows the recombinant enhanced green fluorescent protein (rEGFP) and shrimp HPCAL1 protein (rPvHPCAL1) expressed in prokaryotes.

[0071] Figure 3 shows the changes in phosphorylated STAT (signal transduction and transcription activator protein) in shrimp hemocytes after injection of recombinant enhanced green fluorescent protein (rEGFP) and recombinant shrimp HPCAL1 (rPvHPCAL1) protein, respectively. Figure 3A shows the results of three repeated immunoblotting experiments, and Figure 3B is a scatter plot of the ratio of the gray value of the phosphorylated STAT band to the gray value 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 effect of injecting recombinant shrimp HPCAL1 (rPvHPCAL1) 3 hours later on the functional genes of shrimp hemocytes; where PPO2: prophenoloxidase 2; STAT: signal transduction and transcription activator protein; CRUL: crustin-like antimicrobial peptide; PPO1: prophenoloxidase 2; PPAF1: prophenoloxidase activator enzyme 1; PEN3: penaeidin-3 antimicrobial peptide.

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

[0075] Figure 7 shows the in vitro interaction results between recombinant human β3-adrenergic receptor and recombinant human HPCAL1; Figure 7A shows the 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 curves of the ligand recombinant human β3-adrenergic receptor (rHis-ADRB3) and the analyte human serum albumin (HSA); Figure 7C shows the concentration gradient binding curves 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 analyte human serum albumin (HSA) and recombinant human HPCAL1 (rhsHPCAL1).

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

[0077] Figure 9 shows the effect 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 ultrasound images of myocardial ischemia-reperfusion mice before and after tail vein injection of recombinant mouse HPCAL1 (rmsHPCAL1), and the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening. Figure 9C shows ultrasound images of myocardial ischemia-reperfusion mice before and after tail vein injection of mouse serum albumin (MSA), and the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening. Detailed Implementation

[0078] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort 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 micromole.

[0080] Example 1

[0081] The homology of HPCAL1 protein from different animal sources was studied, and the results are shown in Figure 1. The results show that HPCAL1 protein is highly conserved across different species.

[0082] in:

[0083] The amino acid sequence of shrimp HPCAL1 protein is shown in SEQ ID NO:1; the amino acid sequence of mouse HPCAL1 protein is shown in SEQ ID NO:2; the amino acid sequence of human HPCAL1 protein is shown in SEQ ID NO:3; and the mutant of 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] This mutant (SEQ ID NO:4) is a mutant prepared by mutating K to R at position 137, K to R at position 163, and R to K at position 171 of the amino acid sequence of human HPCA1 protein.

[0093] Therefore, shrimp, mice, human HPCA1 protein and mutants were used as experimental materials, and the function of this protein was tested in a mouse ischemia-reperfusion model.

[0094] Example 2

[0095] (1) Functional study of recombinant shrimp HPCA1

[0096] 1.1 Prokaryotic expression and protein purification of recombinant shrimp HPCA1

[0097] 1) The shrimp HPCA1 sequence was cloned into the pET-20b plasmid (Pujian Biotechnology (Wuhan) Co., Ltd.) and dissolved in ddH2O to a concentration of 100 ng / μL;

[0098] 2) Take 1 μL of the plasmid and add it to 50 μL of ArcticExpress(DE3)pRARE2 E.coli competent cells; gently pipette to mix.

[0099] 3) Let stand on ice for 30 minutes, then in a 42℃ water bath for 90 seconds, followed by an ice bath for 2 minutes;

[0100] 4) Add 950 μL of LB liquid culture medium to the clean bench and incubate at 37°C and 200 rpm for 1 h.

[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 culture onto an LB agar plate containing ampicillin, ignite a glass rod, cool it, and then spread it onto the plate;

[0103] 7) Once the liquid in the agar plate has slightly dried, invert it and incubate it overnight in a 37°C bacterial incubator.

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

[0105] 9) Take 3 mL of activated bacterial culture and add it to 50 mL of LB liquid medium containing Ampicillin (Amp). Incubate 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, induce at 37℃ for 3 h, and take 20 μL of bacterial culture before and after induction, add 5×SDS loading buffer, heat and boil in a 100℃ water bath for 10 min for later use.

[0107] 11) Centrifuge the induced bacterial culture at 4℃ and 8800 rpm for 8 min, and discard the supernatant;

[0108] 12) Resuspend the bacterial cells in 5 mL of pre-cooled PBS, centrifuge and discard the supernatant;

[0109] 13) Add 5 mL of pre-cooled PBS to resuspend the precipitate, add 100×PMSF, and set the sonication program to: 60% power, sonication on for 3 seconds, interval for 8 seconds, total time 30 min;

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

[0111] 15) Take 20 μL of supernatant and resuspended precipitate into 200 μL EP tubes, add 5 μL of 5×SDS loading buffer, boil at 100℃ for 10 min and set aside.

[0112] 16) Take 10 μL of each sample and perform polyacrylamide gel electrophoresis analysis using 5% stacking gel and 15% separating gel. Then, stain with Coomassie brilliant blue for 1 h and perform multiple destaining treatments to analyze protein expression.

[0113] 17) Take 25 μL Ni-Charged MagBeads were incubated in the supernatant of the lysate in 14) at 4°C for 30 min by rotation.

[0114] 18) Place the centrifuge tube firmly against the magnetic rack, remove the supernatant, add 1 mL of pre-cooled PBS for resuspending, and transfer it to a 1.5 mL enzyme-free EP tube. Place the centrifuge tube firmly against the magnetic rack and remove the supernatant.

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

[0116] 20) Add 1 mL of pre-cooled PBS to the Ni column, invert to mix, quickly use a magnetic rack to adsorb the column material, remove the supernatant, and repeat this step 3 times.

[0117] 21) Add 100 μL of 20 mM, 50 mM, 100 mM and 200 mM imidazole elution buffer sequentially from low to high concentration of imidazole, elute on ice for 3 min by inversion, elute twice for each imidazole concentration, and collect the elution buffer.

[0118] 22) Take 10 μL of each concentration of protein elution buffer from 21) and add it to 2.5 μL of 5×SDS loading buffer. Heat and boil in a 100℃ water bath for 10 min.

[0119] 23) The above samples were analyzed by 5% stacking gel and 15% separating gel polyacrylamide gel electrophoresis, followed by Coomassie brilliant blue staining for 1 hour, and protein expression was analyzed after multiple destaining.

[0120] 24) Boil the dialysis bags in a microwave oven for 3 minutes, then place them in a clean bench to cool.

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

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

[0123] a) According to Follow the instructions in the BCA protein quantification kit manual to perform the following operations:

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

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

[0126] b) The preparation of the standard curve is shown in Table 1.

[0127] Table 1

[0128] The determination system for both standards and samples is 20 μL. After each addition of working solution, the mixture should be gently blown to mix, and then incubated at 37°C for 30 min. The absorbance of the 96-well plate at 562 nm is measured on a microplate reader to create 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 pET-28a-EGFP plasmid into 5 mL of LB liquid medium containing kanamycin (kana) and activate overnight at 37°C in a shaker.

[0131] 2) Take 3 mL of activated bacterial culture and add it to 50 mL of liquid LB medium containing kanamycin (kana). Incubate at 37°C in a shaker until the OD600 is 0.6-1.0.

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

[0133] 4) Centrifuge the induced bacterial culture at 8800 rpm for 8 min at 4℃ and discard the supernatant;

[0134] 5) Resuspend the bacterial cells in 5 mL of pre-cooled PBS, centrifuge to remove the supernatant;

[0135] 6) Add 5 mL of pre-cooled PBS to resuspend the precipitate, add 100×PMSF, and set the sonication program to: 60% power, sonication on for 3 seconds, interval for 8 seconds, total time 15 min.

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

[0137] 8) Take 20 μL of supernatant and resuspended precipitate into 200 μL centrifuge tubes, add 5 μL of 5×SDS loading buffer, heat at 100℃ and boil for 10 min before use.

[0138] 9) Take 10 μL of each sample and perform polyacrylamide gel electrophoresis analysis using 5% stacking gel and 15% separating gel. Then, stain with Coomassie brilliant blue for 1 h and perform multiple destaining treatments to analyze protein expression.

[0139] 10) Take 30 μL Ni-Charged MagBeads column supernatant in 7) was incubated at 4°C for 30 min by rotation in 4°C.

[0140] 11) Place the centrifuge tube firmly against the magnetic rack, aspirate the supernatant, add 1 mL of 0.01 mM pre-cooled PBS for resuspending, and transfer it to a 1.5 mL enzyme-free EP tube using a pipette. Place the centrifuge tube firmly against the magnetic rack and aspirate the supernatant.

[0141] 12) Add 1 mL of pre-cooled PBS containing 1% Triton X-114, and wash by 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, quickly use a magnetic rack to adsorb the column material, remove the supernatant, and repeat this step 3 times;

[0143] 14) Add 200 μL of 20 mM, 50 mM, 100 mM and 200 mM imidazole elution buffer sequentially according to the low to high concentration of imidazole. Elute on ice for 3 min by inversion. Wash twice for each imidazole concentration and collect the elution buffer.

[0144] 15) Take 10 μL of each concentration of protein elution buffer from 14) and add it to 2.5 μL of 5×SDS loading buffer. Heat and boil in a 100℃ water bath for 10 min.

[0145] 16) The above samples were analyzed by 5% stacking gel and 15% separating gel polyacrylamide gel electrophoresis, and then stained with Coomassie brilliant blue for 1 hour. After multiple destaining, the protein expression was analyzed.

[0146] 17) Boil the dialysis bags in a microwave oven for 3 minutes, then place them in a clean bench to cool.

[0147] 18) In a clean bench, take the eluent with high protein purity and add it to a sterile 1.5 mL EP tube. Cut off the cap, seal the tube opening with a dialysis bag, and reinforce the opening with a rubber band. Place it on a float, invert it in sterile PBS, and dialyze it at 4°C with magnetic stirring for 4 hours. Replace the sterile PBS every 4 hours until the imidazole content in the protein eluent 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 Proteins

[0150] 1) Take the HPCA1 protein sample expressed in prokaryotes from 1.1 and perform polyacrylamide gel electrophoresis analysis on it using a 5% stacking gel and a 12% separating gel;

[0151] 2) Cut the PVDF membrane to be slightly larger than the gel, immerse it in methanol for 15 seconds, and immerse the sandwich clip in the electrotransfer solution. Stack the discharge transfer sandwich clip in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, and negative electrode.

[0152] 3) The protein gel was electrospun for 1.5 hours using a wet transfer electrospinning apparatus (Bio-Rad) at a constant current of 300mA to transfer the total protein to a PVDF membrane;

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

[0154] 5) Transfer membrane to blocking solution for dilution Rabbit anti-HPCAL1 polyclonal antibody (1:1000) was incubated overnight at 4°C;

[0155] 6) Wash 3 times with 1×TBST, 15 minutes each time;

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

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

[0158] 9) Using Merck ECL colorimetric imaging was performed using Immobilon Western Chemilum HRP Substrate and a GE AI600 imaging system, and the images were saved; the results are shown in Figure 2.

[0159] 1.4 Immunoblot analysis of hematopoietic cells after injection of recombinant shrimp HPCA1

[0160] 1) The concentrations of recombinant shrimp HPCA1 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) Take 15 shrimp from each of the experimental group and the control group, and inject 100 μL of recombinant protein (0.1 μg / g) into the junction of the second and third abdominal segments of each shrimp;

[0163] 4) After 3 hours, blood and lymph were drawn from the experimental group and the control group into 15 mL centrifuge tubes at a ratio of 300 μL sterile anticoagulant to 100 μL blood and lymph.

[0164] 5) Centrifuge at 4℃ and 900 rpm for 10 min, then discard the supernatant;

[0165] 6) Add 5 mL of sterile anticoagulant and resuspend, centrifuge at 4℃ and 900 rpm for 10 min, and repeat this step 3 times;

[0166] 7) Resuspend the precipitate with 20 μL PBS, add 5 μL of 5×SDS loading buffer, and heat in a 100℃ water bath for 20 min.

[0167] 8) The samples were analyzed by polyacrylamide gel electrophoresis using a 5% stacking gel and a 12% separating gel;

[0168] 9) Cut the PVDF membrane to be slightly larger than the gel, immerse it in methanol for 15 seconds, and immerse the sandwich clip in the electrotransfer solution. Stack the discharge transfer sandwich clip in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, and negative electrode.

[0169] 10) The protein gel was electrospun for 1.5 hours using a wet transfer electrospinning apparatus (Bio-Rad) at a constant current of 300mA to transfer the total protein to a PVDF membrane;

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

[0171] 12) Cut the PVDF membrane at the midpoint between 65-75 kDa. Dilute the high molecular weight membrane and the low molecular weight membrane separately with the 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 3 times with 1×TBST, 15 minutes each time;

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

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

[0175] 16) Using Merck ECL colorimetric imaging was performed using Immobilon Western Chemilum HRP Substrate and a GE AI600 imaging system, and the images were saved.

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

[0177] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCA1 (0.1 μg / g) were injected into shrimp. Three hours later, shrimp hemocytocytes were extracted for immunoblotting detection of STAT phosphorylation. The results are shown in Figure 3. Figure 3A shows the results of three repeated immunoblottings. Figure 3B is a scatter plot of the ratio of the gray value of the phosphorylated STAT band to the gray value of the corresponding internal control tubulin band. The results indicate that recombinant shrimp HPCA1 protein can effectively inhibit STAT (signal transduction and transcription activator protein) phosphorylation in shrimp hemocytocytes.

[0178] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCA1 (0.1 μg / g) were injected into shrimp three hours later, and then the RNA from shrimp hemolymph cells was extracted for real-time quantitative PCR detection (this experiment was repeated three times). The results are shown in Figure 5. The results indicate that recombinant shrimp HPCA1 protein can effectively inhibit the expression of multiple immune genes in shrimp hemolymph cells, such as the immune-related transcription factor STAT, three enzymes involved in the production of oxygen free radicals PPO1, PPO2, and PPAF1, as well as two antimicrobial peptides PEN3 and CRUL.

[0179] Primary cultures of shrimp hemocytes were then performed. The primary cultures were treated in vitro with octopamine (0.5 μg / mL) + recombinant enhanced green fluorescent protein (0.1 μg / mL) and octopamine (0.5 μg / mL) + recombinant shrimp HPCA1 (0.1 μg / mL), respectively. The changes in STAT phosphorylation in shrimp hemocytes were then detected using Western blotting. The results are shown in Figure 6. Figure 6A shows the results of three replicates of Western blotting, and Figure 6B is a scatter plot of the ratio of the grayscale value of the phosphorylated STAT band to the grayscale value of the corresponding internal control tubulin band. These results indicate that recombinant shrimp HPCA1 can inhibit the activation of STAT (signal transduction and transcription activator protein) phosphorylation in shrimp hemocytes by octopamine in vitro.

[0180] In summary, shrimp HPCA1 inhibits shrimp hemocyte activity and STAT (signal transduction and transcription activator protein) phosphorylation by binding to octopamine receptors.

[0181] 1.5rGST-PvHPCAL1 protein prokaryotic expression and purification

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

[0183] 2) Take 1 μL of the plasmid and add it to 50 μL of BL21(DE3)E.coli competent cells, and gently pipette to mix.

[0184] 3) Let stand on ice for 30 minutes, then in a 42℃ water bath for 90 seconds, followed by an ice bath for 2 minutes;

[0185] 4) Add 950 μL of antibiotic-free liquid culture medium to the clean bench and incubate at 37°C and 200 rpm for 1 h.

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

[0187] 6) Use a pipette to transfer the mixture to an LB agar plate containing ampicillin, ignite and cool the glass rod, and then spread it onto the plate;

[0188] 7) Once the liquid in the agar plate has slightly dried, invert it and incubate it overnight in a 37°C bacterial incubator.

[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) Take 3 mL of activated bacterial culture and add it to 50 mL of LB liquid medium containing Ampicillin (Amp). Incubate at 37°C until the OD600 is 0.6-1.0.

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

[0192] 11) Collect bacterial cells by centrifugation at 8800 rpm for 10 min at 4℃. Resuspend the bacterial cells in 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20. Place on ice for a few minutes and add 1×PMSF to the bacterial solution for sonication. The sonication conditions are set as follows: 3 s of disruption, 8 s interval, total time 30 min, 60% power.

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

[0194] 13) Take 20 μL of the supernatant and precipitate after disruption and add 5 μL of 5×SDS loading buffer to each. Heat in a 100℃ water bath and boil for 10 min.

[0195] 14) 5% stacking gel and 12% separating gel polyacrylamide gel electrophoresis were used to detect the induced expression effect;

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

[0197] 16) Add 1 mL of pre-cooled PBS to resuspend the column, invert and mix for 2 min, centrifuge at 4℃ and 1200 rpm for 3 min and discard the supernatant;

[0198] 17) Add 1 mL of pre-cooled PBS containing 1% Triton X-110 to resuspend the column, invert and mix for 2 min, centrifuge at 1200 rpm for 3 min at 4 °C and discard the supernatant. Repeat the washing 7 times.

[0199] 18) The column enriched with GST-PvHPCAL1 protein was analyzed by 5% stacking gel and 12% separating gel polyacrylamide gel electrophoresis.

[0200] 1.6rGST protein prokaryotic expression and purification

[0201] 1) Inoculate the bacterial strain containing pGEX-6P-1 plasmid into 5 mL of LB liquid medium containing Ampicillin (Amp) and activate it overnight at 37°C in a shaker;

[0202] 2) Take 3 mL of activated bacterial culture and add it to 50 mL of LB liquid medium containing Ampicillin (Amp). Incubate at 37°C until the OD600 is 0.6-1.0.

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

[0204] 4) After induction, add 5×SDS loading buffer to the bacterial culture and heat in a 100℃ water bath for 10 minutes to boil for later use.

[0205] 5) Collect bacterial cells by centrifugation at 8800 rpm for 10 min at 4℃. Resuspend the bacterial cells in 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20. Place on ice for 10 min. Add 1×PMSF to the suspension and perform ultrasonic disruption. The ultrasonic disruption conditions are set as follows: disruption for 3 s, interval for 8 s, total time for 30 min, and 60% power.

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

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

[0208] 8) 5% stacking gel and 12% separating polyacrylamide gel electrophoresis were used to detect the induced expression effect;

[0209] 9) Take the supernatant collected in step 6, add 30 μL of Glutathione Sepharose 4B column, place at 4℃, rotate and incubate for 30 min, centrifuge at 1200 rpm at 4℃ for 3 min, and discard the supernatant;

[0210] 10) Add 1 mL of pre-cooled PBS to resuspend the column, invert and mix for 2 min, centrifuge at 4℃ and 1200 rpm for 3 min and discard the supernatant;

[0211] 11) Add 1 mL of pre-cooled PBS containing 1% Triton X-110 to resuspend the column, invert and mix for 2 min, centrifuge at 1200 rpm for 3 min at 4 °C and discard the supernatant. Repeat the washing 7 times.

[0212] 12) Take the column enriched with GST protein and perform 5% stacking gel and 12% separating gel polyacrylamide gel electrophoresis analysis.

[0213] 1.7 Blood lymphocytes GST pull down

[0214] 1) In Prepare 20 mL of sterile anticoagulant in a 50 mL centrifuge tube in advance. Fit a 1 mL syringe with a No. 12 needle, draw a small amount of anticoagulant, and then draw hemolymph from the junction of the second and third muscle segments of the shrimp. Then inject the mixture from the syringe into the centrifuge tube containing the anticoagulant. Repeat this operation to draw hemolymph from about 200 shrimp, with a total of about 40 mL of anticoagulant.

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

[0216] 3) Wash 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-cooled cell lysis buffer to the cell pellet, mix well by pipetting, and place on ice.

[0218] 5) Use an ultrasonic cell disruptor to disrupt the cells at 40% power, for 2 seconds on, 8 seconds off, for a total of 7 cycles. Stop disrupting when the mixture becomes slightly clear.

[0219] 6) Centrifuge the mixture at 20,000 rpm for 20 min at 4 °C. The collected supernatant is the cell lysis buffer. Divide it into two tubes, 1 mL each.

[0220] 7) In addition, take 10 μL of cell lysis buffer, add it to 5×SDS loading buffer, boil for 10 min, and store it as input for later use;

[0221] 8) Take 15 μL of the prepared GST-bound column and 20 μL of the prepared rGST-PvHPCAL1-bound column, incubate with 1 mL of cell lysis buffer, and incubate overnight at 4°C. Then, take 1 mL of cell lysis buffer to wash the column, repeating 7 times. Centrifuge at 1200 rpm for 3 min each time to remove the supernatant.

[0222] 9) Add 60 μL of PreScission Proteas (GE) enzyme digestion buffer and 1 μL of PreScission Protease (GE) to each tube and incubate at 4 °C for 4 h by rotation. Centrifuge at 1200 rpm for 3 min and collect the supernatant.

[0223] 10) Take 20 μL of the collected supernatant and 2 μL of cell lysis buffer, and... The premixed protein marker (Low) served as a molecular weight control for the protein. 5% stacking gel and 15% separating gel polyacrylamide gel electrophoresis were performed.

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

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

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

[0227] c) Remove 100 mL of ethanol, add 200 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 15 min at a shaking speed of 65 rpm.

[0228] d) Discard Milli-Q grade pure water, add 30 mL of freshly prepared silver staining sensitizing solution, and shake on a shaker at room temperature for 2 min at a shaking speed of 65 rpm;

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

[0230] f) Repeat the above steps;

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

[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 min at a shaking speed of 65 rpm;

[0233] i) Discard Milli-Q grade pure water, add 30 mL of silver staining developing solution, and shake on a shaker at room temperature for 8 min at a shaking speed of 65 rpm.

[0234] j) After the ideal protein band appears, add 20 mL of silver staining stop solution and shake at room temperature for 10 min on a shaker with a shaking speed of 65 rpm.

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

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

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

[0238] The results are shown in Figure 4, which indicate that shrimp HPCA1 can specifically bind to the octopamine receptor in shrimp hemocytes.

[0239] (2) 293T cell overexpression experiment

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

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

[0242] 3) After receiving the samples, the phosphorylated ERK signal, the internal control GADPH (human phosphooleoyl dehydrogenase) signal, and the Flag-tag signal of the transfected receptor were detected by immunoblotting.

[0243] As shown in Figure 8, adrenaline effectively activated ERK phosphorylation in 293T cells overexpressing adrenergic receptors 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 HPCA1 inhibited adrenaline-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 (Figure 8C), but could not inhibit adrenaline-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptors β1 and β2 (Figures 8A and 8B).

[0244] Those skilled in the art know that adrenaline receptor β3 is the main receptor for octopamine in the human body. Therefore, the blocking effect of recombinant human HPCAL1 and its mutant on octopamine activation of β3-adrenergic receptor was investigated. The results showed that the addition of recombinant human HPCAL1 inhibited ERK phosphorylation in 293T cells overexpressing adrenaline receptor β3 activated by octopamine (Figure 8D). The addition of the mutant of recombinant human HPCAL1 also inhibited ERK phosphorylation in 293T cells overexpressing adrenaline receptor β3 activated by octopamine (Figure 8F). Figure 8E shows the purified recombinant human HPCAL1 mutant.

[0245] The above results indicate that recombinant human HPCAL1 can effectively and selectively block the activation of β3-adrenergic receptors by adrenaline and octopamine; recombinant human HPCAL1 mutants can effectively and selectively block the activation of β3-adrenergic receptors by octopamine; and recombinant human HPCAL1 and its mutants can act 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 Biotechnology Co., Ltd. (CSB-CF001393HU), human serum albumin was obtained from Shanghai Jizhi Biochemical Technology Co., Ltd. (ACMEC, A93920-100mg), and recombinant human HPCAL1 (strep-tag) was obtained from Wuhan Pujian Biotechnology Co., Ltd.

[0248] 2) The Biocore protein-protein interaction experiment was specifically performed by Wuhan Pujian Biotechnology Co., Ltd., and the specific steps are as follows:

[0249] (a) Power on the Biacore T200 instrument according to the standard operating procedure.

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

[0251] (c) Begin chip installation, following the standard procedure to install the NTA chip.

[0252] (d) To prepare for the formal experiment, the buffer solution will flush the entire internal flow path system at a high flow rate.

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

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

[0255] (g) Prepare the corresponding samples to be tested as required, and start the automatic program to perform the test.

[0256] (h) Results analysis: Based on the results, perform data fitting analysis to obtain the final affinity fitting KD value.

[0257] The results, as shown in Figure 7, indicate that recombinant human HPCA1 protein can specifically bind to recombinant human β3-adrenergic receptors in vitro.

[0258] (4) Injection experiment of recombinant HPCA1 in heart failure mice

[0259] 1. Mouse model of myocardial ischemia-reperfusion

[0260] 1) Anesthesia: Mice were anesthetized by intraperitoneal injection of afodin (body weight × 0.015); three long strips of tape and six short strips of tape were cut (to fix the limbs, tail and teeth), and the necessary surgical instruments were disinfected with 75° alcohol and placed in place;

[0261] 2) Fixation: Use tweezers to hold the mouse's hind legs with appropriate force to check if the anesthesia is complete. Once the anesthesia is confirmed, fix the mouse with tape and thread.

[0262] 3) Endotracheal intubation: Shine a light on the mouse's throat, pull the mouse's tongue to the right, and then gently lift the tongue with a tongue depressor to locate the trachea (round hole, constantly expanding and contracting). Insert an intravenous catheter (20 / 22G). After completion, remove the catheter and observe whether the mouse's breathing is stable. Connect the ventilator to check if the respiratory rate is consistent. Note that the ventilator should be turned on before connecting the mouse to avoid excessive airflow that could damage the mouse's lungs.

[0263] 4) Hair removal: Hair removal cream was used to remove hair on the left side of the mouse's chest. After the hair removal was completed, the incision site was cleaned with saline solution.

[0264] 5) Chest opening: Adjust the position, disinfect the skin surface with iodine, find the position on the left side of the mouse's chest where the frequency of undulation is the greatest, and make an incision on the right side parallel to this point. First, cut the skin, then cut the two layers of pectoral muscles separately, and then bluntly open the intercostal space with the largest gap (do not use scissors, as it is easy to cut the artery in the middle of the chest). Be careful not to damage the left lung.

[0265] 6) Locate the heart. The left anterior descending branch is located 1.5 mm below the left atrium. The ligation width is about 2-3 mm. After ligation, the double lines can be crossed and tightened. If the myocardium below the line appears white, it proves that the ligation position is correct.

[0266] 7) Locate the position, tie a knot to form a small loop, place the PE-10 catheter, then tighten it and tie another knot. Once the ligation is complete, time it for 60 minutes. Cover the area with a gauze soaked in saline to prevent the skin from drying out.

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

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

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

[0270] 11) Disconnect the ventilator and remove the intubation tube. After the mice slowly recover, observe their condition. The mice survived 5 days after the model was established, but their ejection fraction was lower than that of healthy mice, and they showed symptoms of heart failure. The mice with myocardial damage that were successfully modeled were used for subsequent experiments.

[0271] 2. Ultrasound imaging to monitor changes in cardiac function in mice before and after HPCA1 injection.

[0272] 1) The eukaryotic expression recombinant mouse HPCA1 was obtained from Wuhan Pujian Biotechnology Co., Ltd., and the mouse serum albumin was obtained from Wuhan FineTest, P3125. The mice used were 8-12 week old male C57BL / 6JNifdc mice. The relevant animal experiments have been approved by the Experimental Animal Ethics and Use Committee of Shantou University.

[0273] 2) Through 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 quickly induce complete anesthesia (slightly squinting eyes and trembling indicate anesthesia). The mice were then quickly transferred to a 37°C constant-temperature operating table, and the isoflurane concentration was adjusted to 0.5%-1% and a breathing mask was connected to maintain anesthesia. The mice's limbs were fixed to electrodes coated with coupling agent using medical tape to transmit 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 and heart area of ​​the mouse (hair was removed before modeling). Gently press the probe down until it contacts the coupling gel but does not compress the heart. Under a heart rate controlled at 420-440 beats / min, acquire dynamic videos of the short-axis parasternal section of the mouse in B-Mode and -M-Mode.

[0276] (C) Drug treatment: After data collection before drug administration, mice were injected via tail vein with either rmsHPCAL1 (20 μg / mouse, concentration 0.39 mg / mL) or an equivalent dose of MSA (20 μg / mouse, concentration 0.4 mg / mL). Echocardiography was collected 20–40 min after drug administration as described in (b).

[0277] (D) Data Processing: Data were processed using Vevo LAB 3.0.0 software. Cardiac function-related parameters, such as left ventricular anterior wall thickness, posterior wall thickness, and left ventricular 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 evaluate cardiac systolic function in mice, where: 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 Figure 9. The results show that after injecting recombinant mouse HPCA1 into mice with myocardial ischemia-reperfusion model for 5 days, it can effectively increase the left ventricular ejection fraction (about 20%) and the left ventricular short axis shortening rate (about 30%).

[0279] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Application of HPCA1 protein or related biological materials in any of the following: (A1) Preparation of β3-adrenergic receptor antagonists; (A2) Non-therapeutic inhibition of β3 adrenergic receptor activation; (A3) Preparation of drugs for treating cardiovascular diseases; (A4) Preparation of cardiac stimulants.

2. The application according to claim 1, characterized in that, The cardiovascular diseases mentioned include heart failure.

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

4. The application according to claim 1, characterized in that, The HPCA1 protein comprises any one of the proteins shown in B1-B4: (B1) A protein whose amino acid sequence is any one of SEQ ID NOs:1-3; (B2) A protein having the same function as the amino acid sequence shown in 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 with and having the same function as the amino acid sequence defined by any one of (B1) or (B2); or (B4) Modify the N-terminus, C-terminus, amino acid backbone and / or amino acid side chain groups of the protein defined in any of (B1)-(B3) to obtain a polypeptide derivative with the same function.

5. The application according to claim 4, characterized in that, 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 shown in any of SEQ ID NOs:1-3.

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

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

8. The application according to claim 7, characterized in that, The tag sequence includes at least one of a signal peptide, a target peptide, a tag peptide, a fluorescent protein, and a membrane-penetrating peptide.

9. The application according to claim 4, characterized in that, The modifications include one or more of the following: glycosylation, phosphorylation, N-methylation, myristylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol modification, polyantigenic peptide, isoprene cyclization, acetylation, amidation, fatty acid, and cyclization.

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

4.

11. The application according to any one of claims 1-10, characterized in that, The relevant biomaterial is a biomaterial expressing the HPCAL1 protein, preferably any one of the following: (C1) The nucleic acid molecule encoding the HPCA1 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) Recombinant cells containing the nucleic acid molecule described in (C1), or recombinant cells containing the expression cassette described in (C2), or recombinant cells 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), or 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 medicament comprising the HPCA1 protein or related biological material as described in any one of claims 1-11.

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

14. The medicament according to claim 12, characterized in that, The dosage forms of the drug include injections, aerosols, or drops.

15. The use of the medicament according to any one of claims 12-14 in any of the following: (A1) Preparation of β3-adrenergic receptor antagonists; (A2) Non-therapeutic inhibition of β3 adrenergic receptor activation; (A3) Preparation of drugs for treating cardiovascular diseases; (A4) Preparation of cardiac stimulants.

Images