Application of HPCAL1 protein in enhancing heart function

Abstract

The invention discloses an application of an HPCAL1 protein in enhancing a heart function. The invention finds that the HPCAL1 protein can be specifically combined with a recombinant beta3-adrenergic receptor and can be used as a beta3-adrenergic receptor antagonist protein; animal experiments find that by intravenous injection of a certain concentration of recombinant mouse HPCAL1 in an ischemia reperfusion mouse model, the mouse heart function can be effectively improved, and the ejection fraction of the mouse can be increased; therefore, the HPCAL1 protein can be used for enhancing the heart function and has a potential medicinal value for treating the heart function decline type cardiovascular diseases. The HPCAL1 protein is a biomacromolecule and is low in toxicity, so that the problem of high liver and kidney toxicity of chemical small molecule drugs is solved; and the method has a relatively high application value.

Description

Technical Field

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

[0002] Heart failure refers to the inability of the heart's contractile and diastolic functions to adequately discharge venous blood from the heart, resulting in 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 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, and ultimately lead to poor ventricular pumping and (or) filling function.

[0003] Factors affecting 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 are related to heart function decline, such as coronary heart disease, heart failure, cor pulmonale, atrial fibrillation, rheumatic heart disease, congenital heart disease, etc. The treatment of these related diseases requires the use of cardiotonic agents. Currently, the cardiotonic agents on the market mainly include two categories: cardiac glycosides and non-cardiac glycosides positive inotropic drugs: the former is a cardiotonic glycoside, and the commonly used clinical drugs include digitalis, digoxin, deacetyl scutellarin and scutellarin K, etc. The latter includes β-adrenergic receptor agonists, such as the commonly used dobutamine, dopamine isobutyl ester, phosphodiesterase inhibitors such as amipirone, etc. In recent years, it has been found that β3 adrenergic receptors are expressed more frequently in the heart in a variety of cardiovascular diseases, and β3 adrenergic receptor blockers may play a certain role in cardiovascular diseases such as heart failure. Therefore, the development of specific blockers or antagonists for β3 adrenergic receptors has become one of the new hot spots in the treatment of cardiovascular diseases in recent years. However, chemical small molecule drugs generally have high hepatotoxicity and renal toxicity, and their drug toxicity cannot be ignored. Summary of the invention

[0004] The purpose of the present invention is to use HPCAL1 protein or related biomaterials in preparing drugs for strengthening the heart and treating cardiovascular diseases such as heart failure.

[0005] The technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides the use of HPCAL1 protein or related biological materials in any of the following items;

[0007] (A1) preparing a β3 adrenergic receptor antagonist;

[0008] (A2) Inhibition of β3 adrenergic receptor activation for non-therapeutic purposes;

[0009] (A3) preparing a medicament for treating cardiovascular disease;

[0010] (A4) Preparation of cardiotonic agent.

[0011] 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.

[0012] In some embodiments of the present invention, the cardiovascular disease includes a heart function decline disease.

[0013] 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.

[0014] 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.

[0015] In some embodiments of the present invention, the HPCAL1 protein is HPCAL1 protein derived from shrimp, human, or mouse.

[0016] In some embodiments of the present invention, the HPCAL1 protein is any one of the following proteins B1-B4:

[0017] (B1) a protein having an amino acid sequence of any one of SEQ ID Nos. 1 to 3;

[0018] (B2) a protein having the same function as the amino acid sequence shown in any one of SEQ ID Nos. 1 to 3, with one or more amino acid residues substituted and / or deleted and / or added;

[0019] (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

[0020] (B4) Modifying the N-terminus, C-terminus, amino acid backbone and / or amino acid side chain groups of any of the proteins defined in (B1)-(B3) to obtain a polypeptide derivative having the same function.

[0021] Preferably, the amino acid sequence shown in SEQ ID No.3 is replaced by 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.

[0022] 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.

[0023] In some embodiments of the present invention, the adding further comprises connecting a tag sequence to the N-terminus and / or C-terminus of any protein defined in B1 to B4.

[0024] Preferably, the protein also includes a fusion protein obtained by connecting a tag sequence to the N-terminus and / or C-terminus of any protein defined in B1 to B4.

[0025] 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.

[0026] "Homology" herein refers to the correlation between two amino acid sequences, which is described by the parameter "Identity"; the at least 95% homology may be at least 96%, 97%, 98%, 99% identity. The at least 90% homology may be at least 91%, 92%, 93%, 94% identity. The at least 85% homology may be at least 86%, 87%, 88%, 89% identity. The at least 80% homology may 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, a protein having identity with human HPCAL1 protein can bind to the β3 adrenergic receptor and downregulate the ability of β3 adrenergic receptor activation).

[0027] In some embodiments of the present invention, the HPCAL1 protein is a protein having the same function as the amino acid sequence shown in any one of SEQ ID No. 1 to 3, wherein one or more amino acid residues are substituted and / or deleted and / or added; preferably, the substitution is a conservative substitution. More preferably, the deletion, insertion and / or substitution of 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 No. 1 to 3.

[0028] 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), isoprenylation and cyclization, acetylation, amidation, fatty acid, cyclization or other modifications acceptable in the polypeptide field.

[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 b3);

[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), or 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 a 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 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 a recombinant insect tissue and / or a recombinant plant tissue and / or a 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 solubilizer, 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] The third aspect of the present invention provides a method for preventing or treating cardiovascular diseases, comprising administering 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 to a subject in need thereof.

[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 its pharmaceutically acceptable salt in the drug can be combined with other ingredients that can be used to prevent and / or treat cardiovascular diseases in various ways. Preferably, the HPCAL1 protein or its pharmaceutically acceptable salt and other ingredients that can be used to prevent and / or treat cardiovascular diseases can be present in different drugs respectively, and administered in the form of combined packaging or combined medication. Preferably, the HPCAL1 protein or its pharmaceutically acceptable salt and other ingredients that can be used to prevent and / or treat cardiovascular diseases can be present in the same drug together, and administered in the form of 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-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 methods.

[0056] In the present invention, when administering medicine to animals, the daily dosage of the recombinant HPCAL1 protein or its pharmaceutically acceptable salt is 0.5 μg-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" referred to in the present invention refers to hippocampal calcium-binding protein-like protein 1, which is a neuron-specific calcium-binding member of the recoverin family found in the retina and brain. The functional mechanism of HPCAL1 in the human body has been preliminarily studied. It is involved in the calcium-dependent regulation of rhodopsin phosphorylation and may also be related to neuronal signals in the central nervous system. In particular, in the exploration of mechanisms related to cancer and other related diseases, it has been shown to have a significant inhibitory effect on the development of liver cancer and a proliferation-promoting effect on glioblastoma.

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

[0061] The term "β3 adrenergic receptor antagonist" refers to a molecule that can bind to the β3 adrenergic receptor and block the effects mediated by the receptor agonists such as adrenaline 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 function decline disease" refers to a type of disease in which the heart cannot meet the body's demand for oxygen and nutrients, resulting in impaired body function. Late-stage heart function decline is also called heart failure. Heart function decline 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 matching 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 being 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 a total of 6 positions match). Typically, two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by 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 MoI 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 Blossum62 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 a 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 finds that HPCAL1 protein can bind to β3 adrenergic receptor and function as a β3 adrenergic receptor antagonist; and further finds that HPCAL1 protein has the effect of treating cardiac function decline, and can be used to prepare cardiotonic drugs, or prepare drugs for treating cardiac function decline, especially to prepare drugs for cardiac function decline caused by ischemia.

[0067] The present invention has found through experiments that injecting protein HPCAL1 into shrimp can inhibit the phosphorylation level of blood cell transcription factor STAT; the protein binds to shrimp octopamine receptors and blocks the effect of octopamine in shrimp serum; it is also found that human HPCAL1 protein can effectively block the activation of overexpressed β3-adrenergic receptors by adrenaline and octopamine in 293T cells, and protein-protein interaction in vitro analysis experiments further prove that human HPCAL1 protein can specifically bind to recombinant human β3-adrenergic receptors; in animal experiments, it is found that intravenous injection of a certain concentration of recombinant mouse HPCAL1 in an ischemia-reperfusion mouse model can effectively improve the mouse heart function and enhance its ejection fraction.

[0068] The experiment of the present invention shows that the protein HPCAL1 can be used to enhance cardiac function and has potential medicinal value for preparing drugs for treating cardiovascular diseases such as cardiac function decline. Moreover, the HPCAL1 protein is a type of biological macromolecule with low toxicity, which solves the problem of high liver and kidney toxicity of chemical small molecule drugs and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Sequence homology analysis of HPCAL1 proteins from different species.

[0070] Figure 2 They are prokaryotically expressed recombinant enhanced green fluorescent protein (rEGFP) and shrimp HPCAL1 protein (rPvHPCAL1).

[0071] Figure 3 The changes of 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, Figure 3 A is the result of immunoblotting repeated three times. Figure 3 B 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 (triplicate experiments).

[0072] Figure 4 This is the result of GST pull down experiment.

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

[0074] Figure 6 The changes in STAT (signal transduction and transcription activator protein) phosphorylation in primary shrimp hemocytes after treatment with octopamine + recombinant enhanced green fluorescent protein (rEGFP) or octopamine + recombinant shrimp HPCAL1 protein (rPvHPCAL1) were shown in Figure 2. Figure 6 A is the result of immunoblotting repeated three times. Figure 6 B is the scatter plot of the ratio of the grayscale of the phosphorylated STAT band to the grayscale of the corresponding internal reference tubulin band Figure 3 Repeat the experiment.

[0075] Figure 7The results of the in vitro interaction experiment between recombinant human β3 adrenergic receptor and recombinant human HPCAL1; Figure 7 A is prokaryotically expressed recombinant human HPCAL1 (rhsHPCAL1), recombinant human β3 adrenergic receptor (rHis-ADRB3) and purified human serum albumin (HSA); Figure 7 B is the concentration gradient binding curve of the ligand recombinant human β3 adrenergic receptor (rHis-ADRB3) and the analyte human serum albumin (HSA); Figure 7 C is the concentration gradient binding curve of the ligand recombinant human β3 adrenergic receptor (rHis-ADRB3) and the analyte human HPCAL1 (rhsHPCAL1). Figure 7 D is the binding constant 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 The immunoblot results of phosphorylated ERK signal, internal reference GADPH signal and Flag-tag signal of transfected receptor in 293T cell overexpression experiment; Figure 8 A: Adrenalin plus recombinant enhanced green fluorescent protein (rEGFP) and adrenalin plus recombinant human HPCAL1 protein (rhsHPCAL1) were used to treat 293T cells transfected with β1-adrenergic receptor; Figure 8 B: Adrenalin plus recombinant enhanced green fluorescent protein (rEGFP) and adrenalin plus recombinant human HPCAL1 protein (rhsHPCAL1) were used to treat 293T cells transfected with β2-adrenergic receptor; Figure 8 C is the treatment of 293T cells transfected with β3-adrenergic receptor with adrenalin plus recombinant enhanced green fluorescent protein (rEGFP) and adrenalin plus recombinant human HPCAL1 protein (rhsHPCAL1); Figure 8 D is 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 8 E is the prokaryotic 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] Fig. 9 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; Fig. 9 A is eukaryotically expressed recombinant mouse HPCAL1 and purified mouse serum albumin (MSA); Fig. 9 B is the B-ultrasound image of myocardial ischemia-reperfusion mice 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 short axis shortening rate; Fig. 9 C is the B-ultrasound image 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 be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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 meanings of the abbreviations are as follows: “h” means hour, “min” means minute, “s” means second, “ms” means millisecond, “d” means day, “μL” means microliter, “mL” means milliliter, “L” means liter, “mM” means millimole and “μM” means micromolar.

[0080] Example 1

[0081] The homology of HPCAL1 proteins from different animal sources was studied. Figure 1 , the results showed that HPCAL1 protein is 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_027227598.1hippocalcin-like protein 1[Penaeus vannamei]

[0086] MGKQNSKLKPEVLEDLRSNTEFTDSEIQEWYKGFLKDCPTGHLSVEEFKKIYGNFFPYGDASKQFAEHVFRTFDANGDGTIDFREFLCALSVTSRGKLEQKLRWAFSMYDLDGNGYISRQEMLEIVTAIYKMVGSVMKMPEDESTPEKRTDKIFRQMDKNKDGKLSLEEFIEGAKSDPSIVRLLQCDPQSSQ;

[0087] SEQ ID NO.2:

[0088] >NP_001348574.1hippocalcin-like protein 1[Mus musculus]

[0089] MGKQNSKLRPEVLQDLREHTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNSDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTDKIFRQMDTNNDGKLSLEEFIKGAKSDPSIVRLLQCDPSSASQF;

[0090] SEQ ID NO.3:

[0091] >NP_001245286.1hippocalcin-like protein 1[Homo sapiens]

[0092] MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTDKIFRQMDTNNDGKLSLEEFIRGAKSDPSIVRLLQCDPSSASQF;

[0093] SEQ ID NO.4:

[0094] >hippocalcin-like protein 1mutant

[0095] MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMRMPEDESTPEKRTDKIFRQMDTNNDGRLSLEEFIKGAKSDPSIVRLLQCDPSSASQF;

[0096] The mutant (SEQ ID NO. 4) is prepared by mutating the 137th position of K to R, the 163rd position of K to R, and the 171st position of R to K in the amino acid sequence of human HPCAL1 protein.

[0097] Therefore, shrimp, mouse, human HPCAL1 proteins and mutants were used as experimental materials, and the function of the protein was tested in the ischemia-reperfusion model of mice.

[0098] Example 2

[0099] (1) Functional study of recombinant shrimp HPCAL1

[0100] 1.1 Prokaryotic expression and protein purification of recombinant shrimp HPCAL1

[0101] 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;

[0102] 2) Take 1 μL of the plasmid and add it to 50 μL ArcticExpress (DE3) pRARE2 E. coli competent cells; mix by gently pipetting;

[0103] 3) Place on ice for 30 min, bath in 42°C water for 90 s, and bath on ice for 2 min;

[0104] 4) Add 950 μL LB liquid culture medium in a clean bench and culture at 37°C, 200 rpm, on a shaker for 1 h;

[0105] 5) Centrifuge at 5000 rpm for 1 min, discard 900 μL of supernatant in a clean bench, and mix gently by pipetting;

[0106] 6) Use a pipette to transfer the bacterial solution to a solid LB plate with ampicillin, and apply the solution to the plate after the glass rod is burned and cooled;

[0107] 7) Wait until the liquid on the plate is slightly dry, then invert and place in a 37°C bacterial incubator for overnight culture;

[0108] 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 shaking incubator;

[0109] 9) Take 3 mL of the activated bacterial solution and add it to 50 mL of LB liquid medium containing Ampicillin (Amp), and culture at 37°C until the OD600 is 0.6-1.0;

[0110] 10) Add IPTG with a final concentration of 1 mM, induce at 37°C for 3 h, take 20 μL of the bacterial solution before and after induction, add 5×SDS loading buffer, heat and boil in a 100°C water bath for 10 min for later use;

[0111] 11) Centrifuge the induced bacterial solution at 8800 rpm for 8 min at 4°C and remove the supernatant;

[0112] 12) Resuspend the cells in 5 mL of pre-cooled PBS and centrifuge to remove the supernatant;

[0113] 13) Add 5 mL of pre-cooled PBS to resuspend the precipitate, add 100× PMSF, and set the ultrasonic disruption program to: 60% power, ultrasonic on for 3 seconds, interval of 8 seconds, total time of 30 minutes;

[0114] 14) The disrupted liquid was centrifuged at 9000 rpm for 8 min at 4°C and the supernatant was collected for protein purification;

[0115] 15) Pipette 20 μL of supernatant and resuspended precipitate into 200 μL EP tubes respectively, add 5 μL 5× SDS loading buffer, boil at 100°C for 10 min and set aside;

[0116] 16) Take 10 μL of the sample and analyze it by polyacrylamide gel electrophoresis using 5% concentrated gel and 15% separation gel, then stain it with Coomassie Brilliant Blue for 1 hour, and analyze its protein expression after multiple decolorization treatments;

[0117] 17) Pipette 25 μL Ni-Charged MagBeads magnetic beads were added to the supernatant of the disruption solution prepared in step 14 and incubated with rotation at 4°C for 30 min;

[0118] 18) Place the centrifuge tube close to the magnetic rack, remove the supernatant, add 1 mL of pre-cooled PBS to resuspend, and transfer it to a 1.5 mL enzyme-free EP tube. Place the centrifuge tube close to the magnetic rack and remove the supernatant;

[0119] 19) Add 1 mL of pre-cooled PBS containing 1% Triton X-114, rotate and wash at 4°C for 8 min, and repeat this step 7 times to remove endotoxin contamination;

[0120] 20) Add 1 mL of pre-cooled PBS to the Ni column, mix by inverting, quickly adsorb the column material with a magnetic rack, remove the supernatant, and repeat this step 3 times;

[0121] 21) Add 100 μL of 20 mM, 50 mM, 100 mM and 200 mM imidazole elution buffer respectively, invert and elute on ice for 3 min, elute in order from low to high imidazole concentrations, elute twice at each imidazole concentration, and collect the elution buffer;

[0122] 22) Take 10 μL of each concentration of protein eluate in 21), add 2.5 μL of 5×SDS loading buffer, heat and boil in a 100°C water bath for 10 min;

[0123] 23) The samples were subjected to 5% stacking gel and 15% separation gel polyacrylamide gel electrophoresis analysis, followed by staining with Coomassie Brilliant Blue for 1 hour, and then analyzed for protein expression after multiple decolorization;

[0124] 24) Boil the dialysis bag in a microwave oven for 3 minutes and cool it in a clean bench;

[0125] 25) In a clean bench, take the eluate with higher protein purity and add it to a sterile 1.5 mL EP tube. Cut off the cap, seal the tube mouth with a dialysis bag, and reinforce the tube mouth with a rubber rope. Invert it in sterile PBS and place it at 4°C for 4 hours with magnetic stirring. Replace with new sterile PBS every 4 hours until the imidazole content in the protein eluate is less than 1 μM.

[0126] 26) Detect the concentration of recombinant protein using BCA protein quantification method:

[0127] a) According to BCA protein quantitative detection kit instructions to perform the following operations:

[0128] 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;

[0129] ii. Preparation of BCA working solution: Prepare the working solution in the ratio of A solution: B solution = 50:1;

[0130] b) Standard curve preparation is shown in Table 1;

[0131] Table 1

[0132]

[0133] The measurement system of standards and samples 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 an enzyme reader to measure the absorbance at a wavelength of 562 nm, make a standard curve, and calculate the protein concentration of the sample.

[0134] 1.2. rEGFP prokaryotic expression and protein purification

[0135] 1) Inoculate DH5α bacteria containing pET-28a-EGFP plasmid into 5 mL LB liquid medium containing kanamycin (kana) and activate overnight at 37°C in a shaking incubator;

[0136] 2) Take 3 mL of the activated bacterial solution and add it to 50 mL of liquid LB medium containing kanamycin (kana), and culture it in a shaking incubator at 37°C until the OD600 is 0.6-1.0;

[0137] 3) Add IPTG with a final concentration of 1 mM, induce at 37°C for 3 h, take 20 μL of the bacterial solution before and after induction, add 5×SDS loading buffer, heat and boil in a 100°C water bath for 15 min for later use;

[0138] 4) Centrifuge the induced bacterial solution at 8800 rpm at 4°C for 8 min and remove the supernatant;

[0139] 5) Resuspend the cells in 5 mL of pre-cooled PBS and centrifuge to remove the supernatant;

[0140] 6) Add 5 mL of pre-cooled PBS to resuspend the precipitate, add 100× PMSF, and set the ultrasonic disruption program to: 60% power, ultrasonic on for 3 seconds, interval of 8 seconds, total time for 15 minutes;

[0141] 7) Centrifuge the disrupted liquid at 9000 rpm for 8 min at 4°C and collect the supernatant for protein purification;

[0142] 8) Pipette 20 μL of supernatant and resuspended precipitate into 200 μL centrifuge tubes respectively, add 5 μL of 5×SDS loading buffer, heat and boil at 100°C for 10 min for later use;

[0143] 9) Take 10 μL of the sample and analyze it by polyacrylamide gel electrophoresis using 5% stacking gel and 15% separation gel, then stain it with Coomassie Brilliant Blue for 1 hour, and analyze its protein expression after multiple decolorization treatments;

[0144] 10) Pipette 30 μL The supernatant of the disruption solution of Ni-Charged MagBeads column in 7) was incubated with rotation at 4°C for 30 min;

[0145] 11) Place the centrifuge tube close to the magnetic rack, remove the supernatant, add 1 mL of 0.01 mM pre-cooled PBS to resuspend, and transfer it to a 1.5 mL enzyme-free EP tube with a pipette. Place the centrifuge tube close to the magnetic rack and remove the supernatant;

[0146] 12) Add 1 mL of pre-cooled PBS containing 1% Triton X-114, rotate and wash at 4°C for 8 min, and repeat this step 7 times to remove endotoxin contamination;

[0147] 13) Add 1 mL of PBS to the Ni column, turn it upside down, quickly adsorb the column material with a magnetic rack, remove the supernatant, and repeat this step 3 times;

[0148] 14) Add 200 μL of 20 mM, 50 mM, 100 mM and 200 mM imidazole elution buffer respectively, invert and elute on ice for 3 min, elute in order from low to high imidazole concentrations, wash twice at each imidazole concentration, and collect the elution buffer;

[0149] 15) Take 10 μL of each concentration of protein eluate in 14), add 2.5 μL of 5×SDS loading buffer, heat and boil in a 100°C water bath for 10 min;

[0150] 16) The samples were subjected to 5% stacking gel and 15% separation gel polyacrylamide gel electrophoresis analysis, followed by staining with Coomassie Brilliant Blue for 1 hour, and protein expression was analyzed after multiple decolorization;

[0151] 17) Boil the dialysis bag in a microwave oven for 3 minutes and cool it in a clean bench;

[0152] 18) In a clean bench, take the eluate with higher protein purity and add it to a sterile 1.5 mL EP tube. Cut off the cap, seal the tube mouth with a dialysis bag, and reinforce the tube mouth with a rubber rope. Place it on a buoy, invert it in sterile PBS, and place it at 4°C for 4 hours with magnetic stirring. Replace with new sterile PBS every 4 hours until the imidazole content in the protein eluate is less than 1 μM.

[0153] 19) Use 1.1.26 BCA protein quantification method to detect the concentration of recombinant enhanced green fluorescent protein (negative control protein).

[0154] 1.3 Identification of recombinant protein

[0155] 1) Take the HPCAL1 protein sample expressed in prokaryotic cells in 1.1 and analyze it by polyacrylamide gel electrophoresis using 5% stacking gel and 12% separation gel;

[0156] 2) Cut a PVDF membrane slightly larger than the gel, soak it in methanol for 15 seconds, and 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;

[0157] 3) The protein gel was electroporated at a constant current of 300 mA for 1.5 h using a wet electroporator (Bio-Rad) to transfer the total protein to a PVDF membrane;

[0158] 4) Discard the gel and place the PVDF membrane in a blocking solution containing 5% skimmed milk powder for 1 hour at room temperature;

[0159] 5) Transfer the membrane to the dilution of blocking solution Rabbit anti-HPCAL1 polyclonal antibody (1:1000) was incubated at 4°C overnight;

[0160] 6) Wash with 1×TBST 3 times, 15 min each time;

[0161] 7) Transfer the membrane to the dilution of blocking solution Incubate in goat anti-rabbit IgG-HRP (1:5000) at room temperature for 1 h;

[0162] 8) Wash three times with 1× TBST, 15 min each time;

[0163] 9) Use Merck Immobilon Western Chemilum HRP Substrate and GE AI600 imaging system were used for ECL color development and image preservation. Figure 2 .

[0164] 1.4 Immunoblotting analysis of hemocytes after injection of recombinant HPCAL1 into shrimp

[0165] 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;

[0166] 2) Dilute the recombinant protein to 5 μg / mL with sterile PBS;

[0167] 3) 15 shrimps were taken from each experimental group and control group, and 100 μL of recombinant protein (injection dose was 0.1 μg / g) was injected into the junction of the second and third abdominal segments of each shrimp;

[0168] 4) After 3 hours, extract the hemolymph of the experimental group and the control group into 15 mL centrifuge tubes according to the ratio of 300 μL sterile anticoagulant: 100 μL hemolymph;

[0169] 5) Centrifuge at 900 rpm for 10 min at 4°C and remove the supernatant;

[0170] 6) Add 5 mL of sterile anticoagulant to resuspend, centrifuge at 900 rpm for 10 min at 4°C, and repeat this step 3 times;

[0171] 7) Resuspend the pellet with 20 μL PBS, add 5 μL 5× SDS loading buffer, and boil in a 100°C water bath for 20 min;

[0172] 8) Perform polyacrylamide gel electrophoresis analysis on the samples using 5% stacking gel and 12% separation gel;

[0173] 9) Cut a PVDF membrane slightly larger than the gel, soak it in methanol for 15 seconds, and 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;

[0174] 10) The protein gel was electroporated at a constant current of 300 mA for 1.5 h using a wet electroporator (Bio-Rad) to transfer the total protein to a PVDF membrane;

[0175] 11) Discard the gel and place the PVDF membrane in 5% BSA blocking solution for 1 hour at room temperature;

[0176] 12) Cut the PVDF membrane from 65-75 kDa, 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;

[0177] 13) Wash with 1×TBST 3 times, 15 min each time;

[0178] 14) Diluted with blocking buffer Goat anti-rabbit IgG-HRP (1:5000) and Goat anti-mouse IgG-HRP (1:5000) was incubated at room temperature for 1 h;

[0179] 15) Wash three times with 1× TBST, 15 min each time;

[0180] 16) Use 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.

[0181] The results are as follows Figure 3 , 5 and 6;

[0182] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were injected into shrimps, and then shrimp blood cells were extracted 3 hours later for immunoblotting detection of STAT phosphorylation. The results are shown in Figure 3 , Figure 3 A is the result of immunoblotting repeated three times. Figure 3 B 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. The results show that the recombinant shrimp HPCAL1 protein can effectively inhibit the phosphorylation of STAT (signal transduction and transcription activator protein) in the shrimp's blood cells;

[0183] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were injected into shrimps, and then the shrimp blood cell RNA was extracted 3 hours later for real-time fluorescence quantitative PCR detection (the experiment was repeated 3 times); the results are shown in Figure 5 The results showed that the recombinant shrimp HPCAL1 protein can effectively inhibit the expression of multiple immune genes in shrimp blood cells, such as the immune-related transcription factor STAT, three enzymes involved in the production of oxygen free radicals PPO1, PPO2, PPAF1, and two antimicrobial peptides PEN3 and CRUL;

[0184] The shrimp blood cells were cultured in primary culture, and then treated with octopamine (0.5 μg / ml) + recombinant enhanced green fluorescent protein (0.1 μg / ml) and octopamine (0.5 μg / ml) + recombinant shrimp HPCAL1 (0.1 μg / ml) in vitro, and then the STAT phosphorylation changes of shrimp blood cells were detected by immunoblotting; the results are shown in Figure 6 , Figure 6 A is the result of immunoblotting repeated three times. Figure 6B 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. The results show that the recombinant shrimp HPCAL1 can inhibit the activation of STAT (signal transduction and transcription activator protein) phosphorylation in shrimp blood cells by octopamine in vitro.

[0185] In summary, shrimp HPCAL1 inhibits shrimp blood cell activity and STAT (signal transducer and activator of transcription) phosphorylation by binding to octopamine receptors.

[0186] 1.5 Prokaryotic expression and purification of rGST-PvHPCAL1 protein

[0187] 1) Add the constructed pGEX-6P-1-PvHPCAL1 plasmid to ddH2O and dissolve it to 100 ng / μL;

[0188] 2) Take 1 μL of the plasmid and add it to 50 μL of BL21 (DE3) E. coli competent cells. Mix by gently pipetting.

[0189] 3) Place on ice for 30 min, bath in 42°C water for 90 s, and bath on ice for 2 min;

[0190] 4) Add 950 μL LB antibiotic-free liquid culture medium in a clean bench and culture at 37°C, 200 rpm, on a shaker for 1 h;

[0191] 5) Centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant in a clean bench, and mix gently by pipetting;

[0192] 6) Use a pipette to transfer the mixture to a solid LB plate with ampicillin, and apply the glass rod to the plate after it is cooled by burning;

[0193] 7) Wait until the liquid on the plate is slightly dry, then invert and place in a 37°C bacterial incubator for overnight culture;

[0194] 8) Pick a single clone and inoculate it into 5 mL of LB liquid medium containing Ampicillin (Amp) and activate it in a shaking incubator at 37°C overnight;

[0195] 9) Take 3 mL of the activated bacterial solution and add it to 50 mL of LB liquid medium containing Ampicillin (Amp), and culture at 37°C until the OD600 is 0.6-1.0;

[0196] 10) Add IPTG with a final concentration of 1 mM, induce at 37°C for 3 h, take 20 μL of the bacterial solution before and after induction, add 5×SDS loading buffer, heat and boil in a 100°C water bath for 10 min for later use;

[0197] 11) Collect the cells by centrifugation at 8800 rpm for 10 min at 4°C, resuspend the cells in 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20, place on ice for a few minutes, add 1×PMSF to the cell suspension, and perform ultrasonic disruption. The ultrasonic disruption conditions are as follows: disruption for 3 s, interval for 8 s, total time for 30 min, and power of 60%;

[0198] 12) Centrifuge the disrupted bacterial solution at 9000 rpm for 8 min and collect the supernatant;

[0199] 13) Take 20 μL of the supernatant and precipitate after disruption, add 5 μL of 5×SDS loading buffer respectively, and heat to boil in a 100°C water bath for 10 min;

[0200] 14) Detect the induced expression effect by 5% concentrated gel and 12% separating gel polyacrylamide gel electrophoresis;

[0201] 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;

[0202] 16) Add 1 mL of pre-cooled PBS to resuspend the column, mix by inversion for 2 min, centrifuge at 1200 rpm for 3 min at 4°C, and discard the supernatant;

[0203] 17) Add 1 mL of pre-cooled PBS containing 1% Triton X-110 to resuspend the column, mix by inversion for 2 min, centrifuge at 1200 rpm at 4°C for 3 min, discard the supernatant, and repeat the washing process 7 times;

[0204] 18) The column material enriched with GST-PvHPCAL1 protein was subjected to 5% stacking gel and 12% separation gel polyacrylamide gel electrophoresis analysis;

[0205] 1.6 Prokaryotic expression and purification of rGST protein

[0206] 1) Inoculate the bacteria containing the pGEX-6P-1 plasmid into 5 mL of LB liquid medium containing Ampicillin (Amp) and activate it overnight at 37°C in a shaking incubator;

[0207] 2) Take 3 mL of the activated bacterial solution and add it to 50 mL of LB liquid medium containing Ampicillin (Amp), and culture at 37°C until the OD600 is 0.6-1.0;

[0208] 3) Add IPTG with a final concentration of 1 mM, induce at 37°C for 3 h, and take 20 μL of each for induction;

[0209] 4) Add 5×SDS loading buffer to the bacterial solution and heat and boil it in a 100°C water bath for 10 min for later use;

[0210] 5) Collect the cells by centrifugation at 8800 rpm for 10 min at 4°C, resuspend the cells in 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20, place on ice for 10 min, add PMSF at a final concentration of 1× to the suspension, and perform ultrasonic disruption. The ultrasonic disruption conditions were set as follows: disruption for 3 s, interval for 8 s, total time for 30 min, and 60% power.

[0211] 6) Centrifuge the disrupted bacterial suspension at 8800 rpm for 10 min and collect the supernatant;

[0212] 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;

[0213] 8) 5% concentrated gel and 12% separated polyacrylamide gel electrophoresis to detect the induced expression effect;

[0214] 9) Take the supernatant collected in step 6, 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;

[0215] 10) Add 1 mL of pre-cooled PBS to resuspend the column, mix by inversion for 2 min, centrifuge at 1200 rpm for 3 min at 4°C, and discard the supernatant;

[0216] 11) Add 1 mL of pre-cooled PBS containing 1% Triton X-110 to resuspend the column, mix by inversion for 2 min, centrifuge at 1200 rpm at 4°C for 3 min, discard the supernatant, and repeat the washing process 7 times;

[0217] 12) Take the column material enriched with GST protein and perform electrophoresis analysis on 5% stacking gel and 12% separation gel polyacrylamide gel.

[0218] 1.7 GST pull down in blood lymphocytes

[0219] 1) In Prepare 20mL of sterile anticoagulant in a 50mL centrifuge tube in advance, equip a 1mL syringe with a No. 12 needle, draw a small amount of anticoagulant, aim the needle at the junction of the second and third muscle segments of the shrimp to extract hemolymph, and then inject the mixed liquid in the syringe into the centrifuge tube containing anticoagulant. Repeat this operation to extract hemolymph from about 200 shrimps, containing about 40mL of anticoagulant in total;

[0220] 2) Centrifuge at 1200 rpm for 25 min at 4°C to pellet the cells and remove the supernatant;

[0221] 3) Repeat washing 3 times with 15 mL of sterile anticoagulant, centrifuge at 1200 rpm for 5 minutes at 4°C, and discard the supernatant;

[0222] 4) Add 2 mL of pre-cooled cell lysis solution to the cell pellet, pipette evenly and place on ice;

[0223] 5) Use an ultrasonic disruptor to disrupt the cells, at 40% power, for 2 seconds, with an interval of 8 seconds, for a total of 7 cycles. Stop the disruption when the mixture becomes slightly clear.

[0224] 6) Centrifuge the mixture at 20,000 rpm at 4°C for 20 min. Collect the supernatant as the cell lysate and divide it into 2 tubes, 1 mL in each tube.

[0225] 7) Take another 10 μL of cell lysate, add 5×SDS loading buffer, boil for 10 min, and save as input for later use;

[0226] 8) Take 15 μL of the prepared GST-bound column and 20 μL of the rGST-PvHPCAL1-bound column, incubate with 1 mL of cell lysate, incubate at 4°C overnight, take the cell lysate solvent to wash the column, 1 mL each time, repeat 7 times, centrifuge at 1200 rpm for 3 min each time to remove the supernatant;

[0227] 9) Add 60 μL PreScission Proteas (e GE) digestion buffer and 1 μL PreScission Protease (GE) to both tubes, incubate at 4°C for 4 h, centrifuge at 1200 rpm for 3 min, and collect the supernatant;

[0228] 10) Take 20 μL of the collected supernatant and 2 μL of cell lysis buffer. Premixed protein marker (Low) was used as a control for protein molecular weight, and 5% stacking gel and 15% separation gel polyacrylamide gel electrophoresis were performed;

[0229] 11) After electrophoresis, silver staining is performed to identify differential bands:

[0230] a) Transfer the gel to 100 mL of fixative solution and shake on a shaker at room temperature for 1 h at 65 rpm;

[0231] b) Discard the fixative solution, add 100 mL of 30% ethanol, and shake on a shaker at room temperature for 10 min at a speed of 65 rpm;

[0232] c) Aspirate and discard 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;

[0233] d) Discard the Milli-Q grade pure water, add 30 mL of freshly prepared silver staining sensitizer, and shake on a shaker at room temperature for 2 minutes at a shaking speed of 65 rpm;

[0234] e) Discard the silver staining sensitization solution, add 200 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 1 min at a shaking speed of 65 rpm;

[0235] f) repeat the above steps;

[0236] g) Discard the Milli-Q grade pure water, add 30 mL of the freshly prepared silver solution, and shake at room temperature for 10 min on a shaker at a shaking speed of 65 rpm;

[0237] 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;

[0238] i) Discard the Milli-Q grade pure water, add 30 mL of silver staining solution, and shake on a shaker at room temperature for 8 minutes at a shaking speed of 65 rpm;

[0239] j) After the desired protein bands appeared, add 20 mL of silver staining stop solution and shake at room temperature for 10 min on a shaker at 65 rpm;

[0240] k) Discard the silver staining stop solution and add 100 mL of Milli-Q grade pure water for storage.

[0241] 12) Cut the identified differential bands with a clean blade and place them in a 1.5 mL light-proof EP tube, and send them to Shanghai Houji Biotechnology Co., Ltd. with an ice pack for Q Excative mass spectrometry identification;

[0242] 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.

[0243] The results are as follows Figure 4 The results showed that shrimp HPCAL1 can specifically bind to the octopamine receptors in shrimp blood cells.

[0244] (2) 293T cell overexpression experiment

[0245] 1) 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 by the laboratory. The purification method of recombinant EGFP is shown in 1.2. The plasmids of human β1, β2, and β3 adrenergic receptors were obtained from Genecopia, USA (EX-Y5305-M35, EX-A4389-M35-B, EX-U1168-M35).

[0246] 2) Human β1, β2, and β3 adrenergic receptor plasmids were transfected into 293T cells, respectively. 48 hours after transfection, the cells were treated with 100 μM epinephrine + 0.5 μg / ml recombinant EGFP; 100 μM epinephrine + 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 hour, 0.5 hour, 1 hour, and 2 hours, respectively.

[0247] 3) After collecting the samples, the phosphorylated ERK signal, the internal reference GADPH (human phosphoinositide dehydrogenase) signal and the Flag-tag signal of the transfected receptor were detected by immunoblotting.

[0248] The results are as follows Figure 8 As shown, adrenalin can effectively activate cells that overexpress adrenergic receptor β1 ( Figure 8 A),β2( Figure 8 B),β3( Figure 8 Compared with the negative control (recombinant enhanced green fluorescent protein), the addition of recombinant human HPCAL1 can inhibit the phosphorylation of ERK in 293T cells overexpressing adrenergic receptor β3 activated by adrenaline ( Figure 8 C), but failed to inhibit adrenaline-activated ERK phosphorylation in 293T cells overexpressing adrenaline receptor β1 and β2 ( Figure 8 A, Figure 8 B).

[0249] It is known that adrenergic receptor β3 is the main receptor of octopamine in the human body. We then tested the blocking effect of recombinant human HPCAL1 and its mutants on octopamine-activated β3-adrenergic receptor. The results showed that the addition of recombinant human HPCAL1 inhibited octopamine-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3. Figure 8 D), the addition of recombinant human HPCAL1 mutants can also inhibit octopamine-activated ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 ( Figure 8 F), where Figure 8 E is the purified recombinant human HPCAL1 mutant.

[0250] The above results indicate that recombinant human HPCAL1 can effectively and selectively block the activation of β3-adrenergic receptor by adrenaline 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.

[0251] (3) In vitro interaction experiment between recombinant human β3 adrenergic receptor and recombinant human HPCAL1

[0252] 1) Recombinant human β3 adrenergic receptor (GENE ID: 155) was from Wuhan Huamei Bioengineering Co., Ltd. (CSB-CF001393HU), human serum albumin was from Shanghai Jizhi Biochemical Technology Co., Ltd. (ACMEC, A93920-100 mg), and recombinant human HPCAL1 (strep-tag) was from Wuhan Pujian Biotechnology Co., Ltd.

[0253] 2) Biocore protein interaction experiments were performed by Wuhan Pujian Biotechnology Co., Ltd. The specific steps are as follows:

[0254] (a) Start up the Biacore T200 instrument according to standard operating procedures.

[0255] (b) Prepare PBST (pH 7.4) buffer and 500 ml of deionized water (filtered through a 0.22 μm membrane) for cleaning the injection needle.

[0256] (c) Start chip installation and install the NTA chip according to the standard process.

[0257] (d) Prepare to start the formal experiment. The buffer will flush the flow system inside the entire system at a high flow rate.

[0258] (e) Select the appropriate procedure based on the sample size.

[0259] (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.

[0260] (g) Prepare the corresponding samples to be tested as required and start the automatic running program for testing.

[0261] (h) Result analysis: Based on the running results, data fitting analysis is performed to obtain the final affinity fitting KD value.

[0262] The results are as follows Figure 7 , indicating that recombinant human HPCAL1 protein can specifically bind to recombinant human β3-adrenergic receptor in in vitro experiments.

[0263] (4) Recombinant HPCAL1 injection experiment in heart failure mice

[0264] 1. Myocardial ischemia-reperfusion model in mice

[0265] 1) Anesthesia: Intraperitoneally inject Avertin (body weight*0.015) to anesthetize the mouse; cut three long tapes and six short tapes (to fix the limbs, tail and teeth), disinfect with 75° alcohol and place the required surgical instruments;

[0266] 2) Fixation: Use forceps to clamp the mouse's hind legs with appropriate force to check whether the anesthesia is complete. After confirming that the anesthesia is complete, fix the mouse with tape and thread;

[0267] 3) 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 find the trachea (round hole, zooming in and out), and intubate with an intravenous needle (20 / 22G). After completion, remove the inner needle, observe whether the mouse's breathing is stable, connect the ventilator to see if the respiratory rate is consistent, and be careful to turn on the ventilator before connecting the mouse to avoid excessive airflow after turning on the machine, which may cause lung damage to the mouse;

[0268] 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;

[0269] 5) Thoracotomy: Adjust the body position, disinfect the skin surface with iodine, find the position on the left side of the mouse chest with the highest frequency of ups and downs, and make an incision parallel to the right side of this point. First, cut the skin, cut the two layers of pectoral muscles separately, and then bluntly open the intercostal space with the largest gap (do not use scissors, which may easily cut the artery in the middle of the chest), and be careful not to injure the left lung;

[0270] 6) Find the heart. The left anterior descending branch is located 1.5mm below the left atrium. The ligature width is about 2-3mm. After ligation, the two lines can be crossed and tightened. If the myocardium under the observation line is white, it proves that the ligature position is correct.

[0271] 7) Find the position and tie a knot into a small circle, place the PE-10 catheter, then tie it tightly and tie another knot. After the ligation is completed, time it for 60 minutes and cover it with a piece of gauze soaked in saline to prevent the skin from drying out;

[0272] 8) Take out the small tube and cut off the rope;

[0273] 9) Suture the intercostal space, pectoral muscles, and skin. Use intermittent sutures and three stitches for suture of intercostal space and pectoral muscles, and continuous sutures for skin suture;

[0274] 10) After suturing, disinfect with iodine.

[0275] 11) The ventilator was disconnected, the cannula was removed, and the mice were observed after they slowly recovered. The mice survived 5 days after modeling, but their ejection fraction was lower than that of healthy mice, and they showed symptoms of heart failure. The mice with myocardial damage in the successful modeling were used for subsequent experiments.

[0276] 2. Ultrasound imaging monitoring of changes in cardiac function in mice before and after HPCAL1 injection

[0277] 1) Eukaryotic recombinant mouse HPCAL1 was obtained from Wuhan Pujian Biotechnology Co., Ltd., and mouse serum albumin was obtained from Wuhan Fine Biotechnology Co., Ltd. (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.

[0278] 2) By The LAZR small animal in vivo multimodal imaging system was used to collect echocardiograms of mice. The specific steps are as follows:

[0279] (a) Anesthesia and fixation: The mice reperfused for 5 days were placed in an anesthesia induction box filled with 3% isoflurane gas to quickly enter a state of complete anesthesia (slight squinting of the eyes and slight trembling indicate anesthesia). The mice were quickly transferred to a 37°C constant temperature operating table, the isoflurane concentration was adjusted to 0.5%-1% and a breathing mask was connected to maintain anesthesia. The limbs of the mice were fixed to the electrodes coated with coupling agent with medical tape to transmit their heart rate, respiration and other vital signs.

[0280] (b) Data acquisition: Tilt the operating table by about 45° and adjust the direction of the MS-400 ultrasound probe so that its notch is perpendicular to the long axis of the heart. Apply the coupling agent to the heart of the left chest of the mouse (the hair has been removed before modeling), and gently press the probe down until it contacts the coupling agent but does not squeeze the heart. Under the condition of controlling the heart rate to 420-440 times / min, collect dynamic videos of the mouse parasternal short axis section in B-Mode and -M-Mode.

[0281] (C) Drug treatment: After collecting the data before administration (Before), mice were injected with rmsHPCAL1 (20 μg / mouse, concentration of 0.39 mg / ml) or MSA (20 μg / mouse, concentration of 0.4 mg / ml) at the same dose through the tail vein. Echocardiograms were collected within 20-40 minutes after administration (After) as described in (b).

[0282] (D) Data processing: Vevo LAB 3.0.0 software was used to process the data, and cardiac function-related parameters such as left ventricular anterior wall thickness, posterior wall thickness and left ventricular internal diameter were measured in each group during diastole and systole for 5 consecutive cardiac cycles. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were used to evaluate the cardiac contractile function of 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).

[0283] The results are as follows Fig. 9 As shown, the results showed that after the tail vein injection of recombinant mouse HPCAL1 into mice with myocardial ischemia reperfusion for 5 days, the left ventricular ejection fraction (about 20%) and the left ventricular short-axis shortening rate (about 30%) of the mice were effectively increased.

[0284] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of HPCAL1 protein or its related biomaterials in any of the following; (A1) preparing a β3 adrenergic receptor antagonist; (A2) Inhibition of β3 adrenergic receptor activation for non-therapeutic purposes; (A3) preparing a medicament for treating cardiovascular disease; (A4) Preparation of 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 any one of the following proteins B1-B4: (B1) a protein having an amino acid sequence of any one of SEQ ID Nos. 1 to 3; (B2) a protein having the same function as the amino acid sequence shown in any one of SEQ ID Nos. 1 to 3, with one or more amino acid residues substituted and / or deleted and / or added; (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 any of the proteins defined in (B1)-(B3) to obtain a polypeptide derivative having 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 to 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 cell-penetrating 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, isoprenylation 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 related 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), 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 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 of the following: (A1) preparing a β3 adrenergic receptor antagonist; (A2) Inhibition of β3 adrenergic receptor activation for non-therapeutic purposes; (A3) preparing a medicament for treating cardiovascular disease; (A4) Preparation of cardiotonic agent.

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