Application of HPCAL1 protein in enhancing cardiac function

Abstract

The present invention discloses the application of HPCAL1 protein in enhancing cardiac function; the present invention discovers that the HPCAL1 protein can specifically bind to the recombinant β3 - adrenergic receptor and can serve as a β3 - adrenergic receptor antagonist protein; and 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 cardiac function of mice and enhance their ejection fraction; indicating that the HPCAL1 protein can be used to enhance cardiac function and has potential medicinal value for treating cardiovascular diseases such as cardiac function decline. Moreover, the HPCAL1 protein is a type of biological macromolecule with low toxicity, solving the problem of high liver and kidney toxicity of chemical small - molecule drugs; it has high application value.

Description

Technical Field

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

[0002] Heart failure refers to the syndrome caused by the impairment of the systolic and diastolic functions of the heart, which cannot fully discharge the venous return blood volume from the heart, resulting in blood stasis in the venous system and insufficient blood perfusion in the arterial system. This syndrome is mainly manifested as pulmonary congestion and vena cava congestion. Heart failure is not an independent disease, but the terminal stage of the development of heart diseases. Almost all cardiovascular diseases will eventually lead to the occurrence of heart failure. Myocardial injury caused by any reason such as myocardial infarction, cardiomyopathy, hemodynamic overload, and inflammation can cause changes in myocardial structure and function, and finally lead to low ventricular pumping and / or filling function.

[0003] Cardiovascular health influencing factors include smoking, dietary nutrition, physical activity, overweight and obesity, and psychological factors, etc. Risk factors for cardiovascular diseases include hypertension, dyslipidemia, diabetes, chronic kidney disease, metabolic syndrome, and air pollution. Among them, 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 requires the use of cardiotonic agents. Currently, the cardiotonic agents on the market mainly include two categories: cardiac glycosides and non-cardiac glycoside inotropic agents. The former is a strong cardiac glycoside, and commonly used clinically are digitalis glycosides, digoxin, deslanoside, and strophanthin K, etc. The latter includes β-adrenergic receptor agonists such as commonly used dobutamine, dopamine isobutyrate, and phosphodiesterase inhibitors such as amrinone, etc. In recent years, it has been found that the expression of β3-adrenergic receptor increases in the heart in various cardiovascular diseases, and β3-adrenergic receptor blockers may play a certain role in cardiovascular diseases with heart function decline. Therefore, the research and development of specific blockers or antagonists for β3-adrenergic receptor have become one of the new hotspots in the treatment of cardiovascular diseases recently. However, chemical small molecule drugs generally have relatively high liver and kidney toxicity, and their drug toxicity cannot be ignored. Summary of the Invention

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

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

[0006] In the first aspect of the present invention, there is provided the application of HPCAL1 protein or its related biological materials in any one of the following;

[0007] (A1) Preparation of β3 adrenergic receptor antagonists;

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

[0009] (A3) Preparation of drugs for treating cardiovascular diseases;

[0010] (A4) Preparation of cardiac stimulants.

[0011] In some embodiments of the present invention, the β3 adrenergic receptor antagonist can be used to prepare drugs for treating cardiovascular diseases; preferably, the drug for treating cardiovascular diseases is a cardiac drug.

[0012] In some embodiments of the present invention, the cardiovascular diseases include heart function decline diseases.

[0013] In some embodiments of the present invention, the heart function decline diseases include 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 includes genetic heart disease, drug-induced heart disease, or infection-induced heart disease.

[0015] In some embodiments of the present invention, the HPCAL1 protein is the 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 shown as B1 - B4:

[0017] (B1) A protein with an amino acid sequence of any one of SEQ ID No.1 - 3;

[0018] (B2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in any one of SEQ ID No.1 - 3 and having the same function;

[0019] (B3) A protein having at least 99%, 98%, 97%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined in any one of (B1) or (B2) and having the same function; or

[0020] (B4) A polypeptide derivative obtained by modifying the N-terminal, C-terminal, amino acid backbone, and / or amino acid side chain groups of the protein defined in any one of (B1) - (B3) and having the same function.

[0021] Preferably, the amino acid sequence shown in SEQ ID No. 3 is substituted by one or several amino acid residues, and the amino acid sequence of the protein mutant with the same function is shown in SEQ ID No. 4.

[0022] Preferably, the addition in (B2) is to add one or several amino acid residues at the N-terminus and / or C-terminus of the amino acid sequence.

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

[0024] Preferably, the protein further includes a fusion protein obtained by connecting a tag sequence to the N-terminus and / or C-terminus of the protein defined by any one of 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 homology of at least 95% can be identity of at least 96%, 97%, 98%, 99%. The homology of at least 90% can be identity of at least 91%, 92%, 93%, 94%. The homology of at least 85% can be identity of at least 86%, 87%, 88%, 89%. The homology of at least 80% can be identity of at least 81%, 82%, 83%, 84%. And the sequence basically retains at least one biological activity of the amino acid sequence from which it is derived (for example, a protein having identity with the human HPCAL1 protein can bind to the β3 adrenergic receptor and down-regulate the ability of the β3 adrenergic receptor to be activated).

[0027] In some embodiments of the present invention, the HPCAL1 protein is: a protein in which the amino acid sequence shown in any one of SEQ ID No. 1 to 3 is substituted and / or deleted and / or added with one or several amino acid residues and has the same function; preferably, the substitution is a conservative substitution. More preferably, the deletion, insertion and / or substitution of the amino acid residues are changes of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues on 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) includes one or a combination of more than one of glycosylation, phosphorylation, N-methylation, myristoylation, palmitoylation, biotinylation, fluorescent labeling, polyethylene glycol (PEG) modification, multiple antigenic peptide (MAP), isoprenylation cyclization, acetylation, amidation, fatty acids, cyclization, or other modifications acceptable in the polypeptide field.

[0029] In some embodiments of the present invention, the related 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 in (C1);

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

[0033] (C4) A recombinant cell containing the nucleic acid molecule in (C1), or a recombinant cell containing the expression cassette in (C2), or a recombinant cell containing the recombinant vector in (b3);

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

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

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

[0037] In some embodiments of the present invention, the vector is well-known to those skilled in the art and includes, but is not limited to: plasmids, phages, cosmids, Ti plasmids, or viral vectors.

[0038] In some embodiments of the present invention, the expression cassette refers to DNA that can express the gene in a host cell, and this DNA may not only include a promoter that initiates gene transcription but also a terminator that terminates gene transcription. Further, the expression cassette may also include an enhancer sequence.

[0039] Among 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 prokaryotic cells well known in the art such as Escherichia coli, Streptomyces, Bacillus subtilis, etc. that can be used to express target proteins.

[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 recombinant insect organ and / or recombinant plant organ and / or recombinant animal organ.

[0044] In a second aspect of the present invention, there is provided a drug comprising the HPCAL1 protein or its related biomaterials in the first aspect of the present invention.

[0045] In some embodiments of the present invention, the drug further includes active ingredients for enhancing cardiac function and / or treating cardiovascular diseases.

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

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

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, solubilizers, preservatives, pH regulators, osmotic pressure regulators, 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 injections, aerosols, or drops.

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

[0051] In a third aspect of the present invention, there is provided a method for preventing or treating cardiovascular diseases, including administering an effective amount of the HPCAL1 protein or a pharmaceutically acceptable salt thereof to a subject in need, or administering a pharmaceutical composition comprising the aforementioned HPCAL1 protein and pharmaceutically acceptable excipients.

[0052] In some embodiments of the present invention, the cardiovascular disease includes heart function decline diseases. Preferably, the heart function decline diseases include 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 includes genetic 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 drug can be combined with other components useful for preventing and / or treating cardiovascular diseases in various ways. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other components useful for preventing and / or treating cardiovascular diseases can be present in different drugs respectively and administered in a combined package or in a combined medication manner. Preferably, the HPCAL1 protein or a pharmaceutically acceptable salt thereof and other components useful for preventing and / or treating 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 dose range of the HPCAL1 protein or a pharmaceutically acceptable salt thereof is 0.1 μg - 1 g / kg body weight; the above dose can be administered in one dose unit or divided into several dose units, depending on the doctor's clinical experience and the dosing regimen including the use of other treatment means.

[0056] In the present invention, when medicating animals, the daily dosing dose of the recombinant HPCAL1 protein or a pharmaceutically acceptable salt thereof is 0.5 μg - 1 mg / kg mouse; and / or the number of dosing times is 1 - 3 times.

[0057] In the present invention, the effective dose can be reasonably adjusted according to the actual situation and the judgment of clinicians. 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" involved 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 the brain. The functional mechanism of HPCAL1 in the human body has been preliminarily studied. It participates in the calcium-dependent regulation of rhodopsin phosphorylation and may also be related to neuronal signals in the central nervous system. Especially in the exploration of the mechanisms of related diseases such as cancer, it has been proven to have an obvious inhibitory effect on the development of liver cancer and a promoting effect on the proliferation of glioblastoma.

[0060] The term "β3 - adrenergic receptor" is a tissue receptor that mediates the action of catecholamines and is of the G - protein - coupled type. The β3 - adrenergic receptor is mainly distributed on muscle cells and its expression is up - regulated after myocardial injury. After activation, the β3 receptor can produce a negative inotropic effect. The β3 receptor causes a negative inotropic effect 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 can bind to the β3 - adrenergic receptor and block the actions mediated by agonists of this receptor such as adrenaline and octopamine. The term "octopamine receptor" refers to a specific protein receptor of the G - protein - coupled receptor superfamily in invertebrates.

[0062] The term "cardiac function decline disease" refers to a class of diseases in which the heart is unable to meet the body's demand for oxygen and nutrients, resulting in impaired body functions. Advanced cardiac function decline is also called heart failure. Cardiac function decline diseases include myocardial infarction, genetic heart diseases, drug - induced heart diseases, infection - induced heart diseases, myocardial ischemia - reperfusion, and heart failure, etc.

[0063] The term "identity" is used to refer to the sequence match between two polypeptides or between two nucleic acids. When a position in both of the two sequences being compared 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), 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 × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For instance, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of a total of 6 positions match). Generally, comparison is made by aligning the two sequences to yield maximum identity. Such alignment can be achieved by using, e.g., the method of Needleman et al. (1970) J. Mol. Biol. 48:443 - 453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11 - 17 (1988)), incorporated into the ALIGN program (version 2.0), can also be used, with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J MoI Biol. 48:444 - 453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used, with a Blossum62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0064] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing 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 of an amino acid residue with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having 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). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art.

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

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

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

[0068] Experiments of the present invention show that the protein HPCAL1 can be used to enhance cardiac function and has potential medicinal value for the preparation of cardiovascular drugs for treating heart 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 small chemical drugs, and has high application value. Description of the Drawings

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

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

[0071] Figure 3 They are the changes of phosphorylated STAT (signal transducer and activator of transcription) in penaeid shrimp hemocytes after injection of recombinant enhanced green fluorescent protein (rEGFP) and recombinant penaeid shrimp HPCAL1 (rPvHPCAL1) protein respectively. Figure 3 A is the immunoblotting result repeated 3 times. Figure 3 B is the scatter plot of the ratio of the band gray value of phosphorylated STAT to the band gray value of the corresponding internal reference tubulin band (three repeated experiments).

[0072] Figure 4 They are the results of GST pull down experiments.

[0073] Figure 5 They are the effects on the functional genes of penaeid shrimp hemocytes 3 hours after injection of recombinant penaeid shrimp HPCAL1 (rPvHPCAL1); among them, PPO2: prophenoloxidase 2; STAT: signal transducer and activator of transcription; CRUL: crustin-like antimicrobial peptide; PPO1: prophenoloxidase 2; PPAF1: prophenoloxidase activating enzyme 1; PEN3: penaeidin-3 antimicrobial peptide.

[0074] Figure 6 They are the changes of STAT (signal transducer and activator of transcription) phosphorylation after treating primary penaeid shrimp hemocytes with octopamine + recombinant enhanced green fluorescent protein (rEGFP) or octopamine + recombinant penaeid shrimp HPCAL1 protein (rPvHPCAL1). Figure 6 A is the immunoblotting result repeated 3 times. Figure 6 B is the scatter plot of the ratio of the band gray value of phosphorylated STAT to the band gray value of the corresponding internal reference tubulin band Figure 3 Three repeated experiments.

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

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

[0077] Figure 9 Effect of injecting recombinant mouse HPCAL1 into the tail vein of mice with myocardial ischemia - reperfusion model on the left ventricular ejection fraction and left ventricular fractional shortening rate of mice; Figure 9 A is recombinant mouse HPCAL1 expressed eukaryotically and purified mouse serum albumin (MSA); Figure 9 B is the echocardiogram of mice with myocardial ischemia - reperfusion before and after injecting recombinant mouse HPCAL1 (rmsHPCAL1) into the tail vein, and the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening rate; Figure 9 C is the echocardiogram of mice with myocardial ischemia - reperfusion before and after injecting mouse serum albumin (MSA) into the tail vein, and the corresponding changes in left ventricular ejection fraction and left ventricular fractional shortening rate. Detailed implementation manners

[0078] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0079] The meanings of the abbreviations are as follows: "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, "μM" refers to micromole.

[0080] Example 1

[0081] Study the homology of HPCAL1 proteins from different animal sources, and the results are as Figure 1 , and the results show that the HPCAL1 protein is highly conserved in different species.

[0082] Among them:

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

[0084] SEQ ID NO.1:

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

[0086] MGKQNSKLKPEVLEDLRSNTEFTDSEIQEWYKGFLKDCPTGHLSVEEFKKIYGNFFPYGDASKQFAEHVFRTFDANGDGTIDFREFLCALSVTSRGKLEQKLRWAFSMYDLDGNGYISRQEMLEIVTAIYKMVGSVMKMPEDESTPEKRTDKIFRQMDKNKDGKLSLEEFIEGAKSDPSIVRLLQCDPQSSQ;

[0087] SEQ ID NO.2:

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

[0089] MGKQNSKLRPEVLQDLREHTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNSDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTDKIFRQMDTNNDGKLSLEEFIKGAKSDPSIVRLLQCDPSSASQF;

[0090] SEQ ID NO.3:

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

[0092] MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMKMPEDESTPEKRTDKIFRQMDTNNDGKLSLEEFIRGAKSDPSIVRLLQCDPSSASQF;

[0093] SEQ ID NO.4:

[0094] >hippocalcin-like protein 1mutant

[0095] MGKQNSKLRPEVLQDLRENTEFTDHELQEWYKGFLKDCPTGHLTVDEFKKIYANFFPYGDASKFAEHVFRTFDTNGDGTIDFREFIIALSVTSRGKLEQKLKWAFSMYDLDGNGYISRSEMLEIVQAIYKMVSSVMRMPEDESTPEKRTDKIFRQMDTNNDGRLSLEEFIKGAKSDPSIVRLLQCDPSSASQF;

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

[0097] Therefore, subsequently, shrimp, mice, human HPCAL1 protein and the mutant were used as experimental materials, and the function of this protein was detected 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) Clone the shrimp HPCAL1 sequence into the pET-20b plasmid (PuJian Bio (Wuhan) Technology Co., Ltd.) and dissolve it in ddH2O to 100 ng / μL;

[0102] 2) Take 1 μL of this plasmid into 50 μL of ArcticExpress(DE3)pRARE2 E.coli competent cells; gently pipette and mix well;

[0103] 3) Let it stand on ice for 30 min, water bath at 42 °C for 90 s, and then ice bath for 2 min;

[0104] 4) Add 950 μL of LB liquid medium in a laminar flow hood, and culture it in a shaker at 37 °C and 200 rpm for 1 h;

[0105] 5) Centrifuge at 5000 rpm for 1 min, discard 900 μL of the supernatant in a laminar flow hood, and gently pipette and mix;

[0106] 6) Use a pipette to aspirate the bacterial solution onto an LB solid plate containing ampicillin, and spread the plate after the glass rod is burned and cooled;

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

[0108] 8) Pick monoclonal colonies and inoculate them into 5 mL of liquid LB medium containing Ampicillin (Amp), and activate them overnight in a shaker at 37 °C;

[0109] 9) Take 3 mL of the activated bacterial solution and transfer it into 50 mL of LB liquid medium containing Ampicillin (Amp), and expand the culture at 37 °C until the OD600 reaches 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 respectively, add 5×SDS loading buffer, and heat and boil in a 100 °C water bath for 10 min for standby;

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

[0112] 12) Resuspend the bacterial cells with 5 mL of pre-cooled PBS, and centrifuge to discard the supernatant;

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

[0114] 14) Centrifuge the disrupted solution at 4 °C and 9000 rpm for 8 min, and collect the supernatant for protein purification;

[0115] 15) Respectively aspirate 20 μL of the supernatant and the resuspended precipitate into a 200 μL EP tube, add 5 μL of 5×SDS loading buffer, and boil at 100 °C for 10 min for standby;

[0116] 16) 10 μL of the sample was taken respectively and subjected to polyacrylamide gel electrophoresis analysis using 5% stacking gel and 15% separating gel. Subsequently, it was stained with Coomassie Brilliant Blue for 1 h, and after multiple decolorization treatments, its protein expression was analyzed;

[0117] 17) Pipette 25 μL of Ni-Charged MagBeads magnetic beads into the supernatant of the lysate in 14), and incubate with rotation at 4 °C for 30 min;

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

[0119] 19) Add 1 mL of pre-cooled PBS containing 1% Triton X-114, wash by rotation 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 up and down, quickly adsorb the column material with the magnetic stand, 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, elute by inverting on ice for 3 min, elute in sequence according to the imidazole concentration from low to high, elute 2 times for each imidazole concentration, and collect the elution buffer;

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

[0123] 23) Perform polyacrylamide gel electrophoresis analysis on the above samples using 5% stacking gel and 15% separating gel. Subsequently, stain with Coomassie Brilliant Blue for 1 h, and after multiple decolorizations, analyze its protein expression;

[0124] 24) Boil the dialysis bag in the microwave oven for 3 min and place it in the laminar flow hood to cool;

[0125] 25) In the laminar flow hood, take the eluate with higher protein purity and add it to a sterile 1.5 mL EP tube. Cut off the lid, seal the tube opening with a dialysis bag, and reinforce the tube opening with a rubber band. Invert it in sterile PBS and place it in a magnetic stirrer for dialysis at 4 °C for 4 h. Replace the fresh sterile PBS every 4 h until the imidazole content in the protein eluate is lower than 1 μM;

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

[0127] a) According to Perform the following operations according to the instruction manual of the BCA Protein Quantification Detection Kit:

[0128] i. Dilute the BSA standard sample (BSA standard sample concentration: 2 mg / mL) to an 80 μL BSA solution of 500 μg / mL: 20 μL BSA solution + 60 μL ddH2O;

[0129] ii. Prepare the BCA working solution: Prepare the working solution according to the ratio of Solution A: Solution B = 50:1;

[0130] b) Prepare the standard curve as shown in Table 1;

[0131] Table 1

[0132]

[0133] The measurement systems for both the standard products and samples are 20 μL. After adding the working solution each time, gently pipette to mix evenly, and incubate in a 37°C constant temperature incubator for 30 min; Place the 96-well plate on the microplate reader to measure the absorbance at a wavelength of 562 nm, prepare the standard curve, and calculate the protein concentration of the sample.

[0134] 1.2. Prokaryotic expression and protein purification of rEGFP

[0135] 1) Inoculate the DH5α strain containing the pET-28a-EGFP plasmid into 5 mL of LB liquid medium containing kanamycin (kana), and activate it overnight on a shaker at 37°C;

[0136] 2) Take 3 mL of the activated bacterial solution and transfer it to 50 mL of liquid LB medium containing kanamycin (kana), and expand the culture on a shaker at 37°C until the OD600 reaches 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 respectively, add 5×SDS loading buffer, and heat it in a 100°C water bath for 15 min for standby;

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

[0139] 5) Resuspend the bacterial cells with 5 mL of pre-cooled PBS, and centrifuge to discard the supernatant;

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

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

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

[0143] 9) Take 10 μL of each sample and perform polyacrylamide gel electrophoresis analysis using a 5% stacking gel and a 15% separating gel. Then stain with Coomassie Brilliant Blue for 1 h and analyze the protein expression after multiple decolorization treatments.

[0144] 10) Pipette 30 μL of Ni-Charged MagBeads column material into the supernatant of the lysate in 7), and incubate at 4 °C with rotation for 30 min.

[0145] 11) Place the centrifuge tube close to the magnetic stand, aspirate the supernatant, add 1 mL of pre-cooled 0.01 mM PBS for resuspension, and transfer it to a 1.5 mL enzyme-free EP tube using a pipette. Then place the centrifuge tube close to the magnetic stand and aspirate the supernatant.

[0146] 12) Add 1 mL of pre-cooled PBS containing 1% Triton X-114, wash by rotation 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, invert it up and down, quickly adsorb the column material with the magnetic stand, and remove the supernatant. 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 sequence from low to high concentration of imidazole, wash twice with each imidazole concentration, and collect the elution buffer.

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

[0150] 16) Perform polyacrylamide gel electrophoresis analysis on the above samples using a 5% stacking gel and a 15% separating gel. Then stain with Coomassie Brilliant Blue for 1 h and analyze the protein expression after multiple decolorizations.

[0151] 17) Boil the dialysis bag in a microwave oven for 3 min and place it in the laminar flow hood to cool.

[0152] 18) In a laminar flow hood, take the eluate with a relatively high protein purity and add it to a sterile 1.5 mL EP tube. Cut off the lid, seal the tube mouth with a dialysis bag, and reinforce the tube mouth with a rubber band. Place it on a floating buoy, invert it in sterile PBS, and place it in a magnetic stirrer at 4°C for dialysis for 4 h. Replace the fresh sterile PBS every 4 h until the imidazole content in the protein eluate is lower than 1 μM;

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

[0154] 1.3 Recombinant protein identification

[0155] 1) Take the prokaryotic-expressed HPCAL1 protein sample in 1.1 and perform polyacrylamide gel electrophoresis analysis on it using a 5% stacking gel and a 12% separating gel;

[0156] 2) Cut a PVDF membrane slightly larger than the gel, soak it in methanol for 15 s, and soak the sandwich in electrotransfer buffer. Stack the electrotransfer sandwich in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, and negative electrode;

[0157] 3) Use a wet transfer electrotransfer instrument (Bio-Rad) to perform electrotransfer of the protein gel at a constant current of 300 mA for 1.5 h to transfer the total protein to the PVDF membrane;

[0158] 4) Discard the gel, put the PVDF membrane into a blocking solution containing 5% skim milk powder, and block it at room temperature for 1 h;

[0159] 5) Transfer the membrane to the rabbit anti-HPCAL1 polyclonal antibody (1:1000) diluted in the blocking solution and incubate it overnight at 4°C;

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

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

[0162] 8) Wash it 3 times with 1×TBST, 15 min each time;

[0163] 9) Use Merck chromogenic solution Immobilon Western Chemilum HRP Substrate and GE AI600 imaging system to perform ECL chromogenic imaging and save the pictures; The results are shown in Figure 2 .

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

[0165] 1) Determine the concentrations of the purified recombinant penaeid HPCAL1 and rEGFP proteins in 1.1 and 1.2 by the BCA protein quantification method;

[0166] 2) Dilute the recombinant proteins to 5 μg / mL with sterilized PBS respectively;

[0167] 3) Take 15 shrimps each from the experimental group and the control group, and inject 100 μL of the recombinant protein (injection dose: 0.1 μg / g) at the joint of the second and third abdominal segments of each shrimp;

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

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

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

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

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

[0173] 9) Cut a PVDF membrane slightly larger than the gel, soak it in methanol for 15 s, soak the sandwich clamp in the electrotransfer solution, and stack the electrotransfer sandwich clamp in the order of positive electrode, thick filter paper, PVDF membrane, gel, thick filter paper, negative electrode;

[0174] 10) Use a wet transfer electrotransfer instrument (Bio-Rad) to electrotransfer the protein gel at a constant current of 300 mA for 1.5 h to transfer the total protein to the PVDF membrane;

[0175] 11) Discard the gel, put the PVDF membrane into a 5% BSA blocking solution, and block it at room temperature for 1 h;

[0176] 12) Cut the PVDF membrane in the middle of 65 - 75 kDa. The high-molecular-weight membrane and the low-molecular-weight membrane are respectively incubated overnight at 4°C with rabbit anti-STAT5a polyclonal antibody (1:500) and mouse anti-α-Tubulin monoclonal antibody (1:3000) diluted with the blocking solution;

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

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

[0179] 15) Wash 3 times with 1×TBST, 15 min each time;

[0180] 16) Use Merck Color development solution Immobilon Western Chemilum HRP Substrate and GE AI600 imaging system were used for ECL color development imaging and the pictures were saved.

[0181] The results were as Figure 3 、 5 and 6;

[0182] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were respectively injected into shrimp. After 3 h, shrimp hemocytes were collected for immunoblotting detection of STAT phosphorylation. The results are shown in Figure 3 , Figure 3 A is the immunoblotting result repeated 3 times, Figure 3 B is the scatter plot of the ratio of the band gray value of phosphorylated STAT to the band gray value of the corresponding internal reference tubulin. The results indicate that recombinant shrimp HPCAL1 protein can effectively inhibit STAT (signal transducer and activator of transcription) phosphorylation in shrimp hemocytes;

[0183] Recombinant enhanced green fluorescent protein (0.1 μg / g) and recombinant shrimp HPCAL1 (0.1 μg / g) were respectively injected into shrimp. After 3 h, shrimp hemocyte RNA was collected for real-time fluorescence quantitative PCR detection (this experiment was repeated 3 times); the results are shown in Figure 5 ,which indicates that recombinant shrimp HPCAL1 protein can effectively inhibit the expression of multiple immune genes in shrimp hemocytes, such as the immune-related transcription factor STAT, three enzymes PPO1, PPO2, PPAF1 involved in oxygen free radical production, and two antimicrobial peptides PEN3 and CRUL;

[0184] Shrimp hemocytes were primary cultured, and then 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 HPCAL1 (0.1 μg / ml), respectively. Then, immunoblotting was used to detect the change of STAT phosphorylation in shrimp hemocytes; the results are shown in Figure 6 , Figure 6 A is the immunoblotting result repeated 3 times, 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 reference tubulin band. The results indicate that recombinant shrimp HPCAL1 can inhibit the activation of octopamine-induced STAT (signal transducer and activator of transcription) phosphorylation in shrimp hemocytes in vitro.

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

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

[0187] 1) Dissolve the constructed pGEX-6P-1-PvHPCAL1 plasmid in ddH2O to a concentration of 100 ng / μL.

[0188] 2) Take 1 μL of this plasmid and add it to 50 μL of BL21(DE3) E. coli competent cells, and gently pipette to mix.

[0189] 3) Incubate on ice for 30 min, heat in a 42 °C water bath for 90 s, and then incubate on ice for 2 min.

[0190] 4) Add 950 μL of LB liquid medium without antibiotics in a laminar flow hood, and culture in a shaker at 37 °C and 200 rpm for 1 h.

[0191] 5) Centrifuge at 5000 rpm for 3 min, discard 900 μL of the supernatant in a laminar flow hood, and gently pipette to mix.

[0192] 6) Transfer the mixture to an LB solid plate containing ampicillin using a pipette, and spread the plate after cooling the glass rod by burning.

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

[0194] 8) Pick a single colony and inoculate it into 5 mL of LB liquid medium containing Ampicillin (Amp), and activate it overnight in a shaker at 37 °C.

[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 reaches 0.6 - 1.0.

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

[0197] 11) Centrifuge the bacterial cells at 8800 rpm for 10 min at 4℃ to collect the cells, resuspend the cells in 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20, place on ice for several minutes, and add PMSF at a final concentration of 1× to the bacterial suspension. Then perform ultrasonic disruption, and the ultrasonic disruption conditions are set as follows: disrupt for 3 s, interval for 8 s, total time for 30 min, and 60% power;

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

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

[0200] 14) Use 5% stacking gel and 12% separating gel for SDS-PAGE to detect the induction expression effect;

[0201] 15) Take the supernatant collected in step 12, add 30 μL of Glutathione Sepharose 4B column material, place at 4℃, rotate and incubate for 30 min, centrifuge at 1200 rpm at 4℃ for 3 min, and discard the supernatant;

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

[0203] 17) Add 1 mL of pre-cooled PBS containing 1% Triton X-110 to resuspend the column material, invert and mix well for 2 min, centrifuge at 1200 rpm at 4℃ for 3 min, and discard the supernatant. Repeat the washing 7 times;

[0204] 18) Take the column material enriched with GST-PvHPCAL1 protein for SDS-PAGE analysis using 5% stacking gel and 12% separating gel;

[0205] 1.6 r GST protein prokaryotic expression and purification

[0206] 1) Inoculate the bacterial strain containing pGEX-6P-1 plasmid into 5 mL of LB liquid medium containing Ampicillin (Amp), and activate it overnight in a shaker at 37℃;

[0207] 2) Take 3 mL of the activated bacterial suspension and transfer it into 50 mL of LB liquid medium containing Ampicillin (Amp), and culture it at 37℃ until the OD600 reaches 0.6 - 1.0;

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

[0209] 4) Add the bacterial liquid after the treatment to 5×SDS loading buffer, heat it in a boiling water bath at 100°C for 10 min, and set aside for later use;

[0210] 5) Centrifuge at 8800 rpm for 10 min at 4°C to collect the bacteria. Resuspend the bacteria with 5 mL of pre-cooled PBS containing 1% Triton X-110 and 1% Tween-20, place it on ice for 10 min, and add PMSF with a final concentration of 1× to the suspension. Then perform ultrasonic disruption. The ultrasonic disruption conditions are set as follows: disrupt for 3 s, interval for 8 s, total time for 30 min, and power of 60%;

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

[0212] 7) Take 20 μL of the disrupted supernatant and inclusion bodies, add 5 μL of 5×SDS loading buffer, and boil in a boiling water bath at 100°C for 10 min;

[0213] 8) Use 5% stacking gel and 12% separating polyacrylamide gel electrophoresis to detect the induction expression effect;

[0214] 9) Take the supernatant collected in step 6, add 30 μL of Glutathione Sepharose 4B column material, place it at 4°C, rotate and incubate for 30 min, centrifuge at 1200 rpm for 3 min at 4°C, and discard the supernatant;

[0215] 10) Add 1 mL of pre-cooled PBS to resuspend the column material, invert and mix well 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 material, invert and mix well for 2 min, centrifuge at 1200 rpm for 3 min at 4°C, and discard the supernatant. Repeat the washing 7 times;

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

[0218] 1.7 GST pull-down of hemolymph cells

[0219] 1) In Prepare 20 mL of sterile anticoagulant in a 50 mL centrifuge tube in advance. Attach a 12-gauge needle to a 1 mL syringe, draw a small amount of anticoagulant, then align the needle with the muscle connection between the second and third 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 the hemolymph of about 200 shrimps, with a total of about 40 mL containing anticoagulant;

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

[0221] 3) Wash 3 times repeatedly 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 solvent to the cell pellet, pipette to mix evenly, and place on ice.

[0223] 5) Lyse the cells using an ultrasonic cell disruptor at 40% power, run for 2 s, pause for 8 s, and repeat the cycle 7 times. Stop the disruption when the mixture becomes slightly clearer.

[0224] 6) Centrifuge the mixture at 20000 rpm for 20 min at 4°C. The collected supernatant is the cell lysate, which is divided into 2 tubes, 1 mL per tube.

[0225] 7) Separately, take 10 μL of the 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-conjugated column material and 20 μL of the rGST-PvHPCAL1-conjugated column material, incubate separately with 1 mL of the cell lysate overnight at 4°C. After incubation, pipette the cell lysate solvent to wash the column material, 1 mL each time, repeat 7 times, and centrifuge at 1200 rpm for 3 min each time to discard the supernatant.

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

[0228] 10) Take 20 μL of the collected supernatant and 2 μL of the cell lysate, and use Premixed protein Marker (Low) as the protein molecular weight control, and perform SDS-PAGE using a 5% stacking gel and a 15% separating gel.

[0229] 11) After electrophoresis, perform silver staining to identify the differential bands:

[0230] a) Transfer the gel to 100 mL of fixing solution, shake on a shaker at room temperature for 1 h at a shaking speed of 65 rpm.

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

[0232] c) Aspirate and discard 100 mL of ethanol, add 200 mL of Milli-Q grade pure water, shake on a shaker at room temperature for 15 min at a shaking speed of 65 rpm.

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

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

[0235] f) Repeat the above steps;

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

[0237] h) Discard the silver solution, add 100 mL of Milli-Q grade pure water, shake at room temperature on a shaker for 1.5 min at a shaking speed of 65 rpm;

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

[0239] j) After an ideal protein band appears, add 20 mL of silver staining stopping solution, shake at room temperature on a shaker with a shaking speed of 65 rpm for 10 min;

[0240] k) Discard the silver staining stopping solution, add 100 mL of Milli-Q grade pure water for preservation.

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

[0242] 13) According to the mass spectrometry data returned by the company, screen out the potential interacting protein octopamine receptor (Octopamine receptor beta-2R-like, LvOAR) with HPCAL1.

[0243] The results are as Figure 4 , and the results show that shrimp HPCAL1 can specifically bind to the octopamine receptor in shrimp blood cells.

[0244] (2) 293T cell overexpression experiment

[0245] 1) The prokaryotic expression recombinant human HPCAL1 (strep-tag) was from Wuhan PuJian Biotechnology Co., Ltd. The plasmid of the mutant of recombinant human HPCAL1 (SEQ ID NO.4, His-tag) was synthesized by BGI and then purified independently in the laboratory. The purification method of recombinant EGFP is shown in 1.2. The plasmids of human β1, β2, and β3 adrenergic receptors were from GeneCopoeia, Inc. (EX-Y5305-M35, EX-A4389-M35-B, EX-U1168-M35).

[0246] 2) The plasmids of human β1, β2, and β3 adrenergic receptors were transfected into 293T cells respectively. After 48 hours of transfection, the 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, 100 μM octopamine + 0.5 μg / ml recombinant human HPCAL1 for 0 hour, 0.5 hour, 1 hour, and 2 hours.

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

[0248] The results are as Figure 8 shown. Adrenalin can effectively activate the ERK phosphorylation in 293T cells overexpressing adrenergic receptor β1 ( Figure 8 A), β2 ( Figure 8 B), β3 ( Figure 8 C). Compared with the negative control (recombinant enhanced green fluorescent protein), the addition of recombinant human HPCAL1 can inhibit the ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 ( Figure 8 C) activated by adrenaline, but cannot inhibit the ERK phosphorylation in 293T cells overexpressing adrenergic receptors β1 and β2 ( Figure 8 A, Figure 8 B) activated by adrenaline.

[0249] It is known that adrenergic receptor β3 is the main receptor of octopamine in the human body. Then, the blocking effect of recombinant human HPCAL1 and its mutant on the activation of β3-adrenergic receptor by octopamine was detected. The results showed that the addition of recombinant human HPCAL1 inhibited the ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 activated by octopamine ( Figure 8 D), and the addition of the mutant of recombinant human HPCAL1 also inhibited the ERK phosphorylation in 293T cells overexpressing adrenergic receptor β3 activated by octopamine ( Figure 8 F), among whichFigure 8 E is a 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 mutant can effectively and selectively block the activation of β3 - adrenergic receptor by octopamine; recombinant human HPCAL1 and its mutant can be used 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) is from Wuhan Huamei Bio - engineering Co., Ltd. (CSB - CF001393HU), human serum albumin is from Shanghai Jizhi Biochemical Technology Co., Ltd. (ACMEC, A93920 - 100mg), and recombinant human HPCAL1 (strep - tag) is from Wuhan Pujian Biotechnology Co., Ltd.

[0253] 2) The Biocore protein interaction experiment was specifically completed by Wuhan Pujian Biotechnology Co., Ltd. The specific steps are as follows:

[0254] (a) Turn on the machine according to the standard operation of the Biacore T200 instrument.

[0255] (b) Prepare buffer PBST (PH7.4) and 500 ml of deionized water for cleaning the injection needle (already filtered through a 0.22μm membrane).

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

[0257] (d) Prepare to start the formal experiment, and the buffer will flush the internal flow path system of the whole system at a relatively high flow rate.

[0258] (e) Select the appropriate program according to the sample volume.

[0259] (f) Set the ligand capture time to 60 s, the flow rate to 10 μl / min, the analyte binding time to 120 s, the flow rate to 30 μl / min; the dissociation time to 300 s, the flow rate to 30 μl / min; the regeneration time to 30 s, the flow rate to 30 μl / min.

[0260] (g) Prepare the corresponding samples to be detected as required and start the automatic operation of the program for detection.

[0261] (h) Result analysis: According to the operation results, perform data fitting analysis to obtain the final affinity fitting KD value.

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

[0263] (4) Injection experiment of recombinant HPCAL1 in heart failure mice

[0264] 1. Modeling of myocardial ischemia-reperfusion in mice

[0265] 1) Anesthesia: Anesthetize the mice by intraperitoneal injection of avertin (body weight * 0.015); cut three long tapes and six short tapes (to fix the limbs, tail and teeth), disinfect the required surgical instruments with 75° alcohol and place them properly;

[0266] 2) Fixation: Use forceps to gently clamp the hind feet of the mice to see if they are fully anesthetized. After confirming that the anesthesia is complete, fix the mice with tapes and threads;

[0267] 3) Tracheal intubation: Shine the light on the throat position of the mice, pull the tongue of the mice to the right, then gently lift the tongue with a tongue depressor to find the trachea position (round hole shape, constantly expanding and contracting), insert a venous indwelling needle (20 / 22G), remove the inner needle after completion, observe whether the breathing of the mice is stable, connect the ventilator to see if the breathing frequency is consistent, and note that the ventilator should be turned on after connecting the mice to avoid lung damage to the mice caused by excessive air flow after starting the machine;

[0268] 4) Depilation: Use depilatory cream to depilate the left side of the chest of the mice. After depilation, wash the incision site with normal saline;

[0269] 5) Thoracotomy: Adjust the body position, disinfect the skin surface with iodophor, find the position on the left side of the chest of the mice with the highest undulation frequency, make an incision on the right side parallel to this point, first cut the skin, then cut the two layers of pectoral muscles respectively, and then bluntly expand 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), and note not to damage the left lung;

[0270] 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 two threads can be crossed and tightened. If the myocardium under the thread shows white, it proves that the ligation position is correct;

[0271] 7) Find the position, first tie a small loop, place a PE-10 catheter, then tie it tightly and tie another knot. After the ligation is completed, time for 60 minutes, and cover it with a piece of gauze soaked in normal saline to avoid the skin and flesh from getting too dry;

[0272] 8) Remove the small tube and cut the rope;

[0273] 9) Suture the intercostal space, pectoral muscles and skin. Interrupted sutures are used for suturing the intercostal space and pectoral muscles, sewing three stitches, and continuous suture is used for skin suture;

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

[0275] 11) Disconnect the ventilator, remove the intubation. After the mice recovered slowly, observe their status. The mice survived 5 days after modeling, but the ejection fraction was lower than that of healthy mice, showing symptoms of heart failure. The mice with myocardial injury successfully modeled were used for subsequent experiments.

[0276] 2. Monitoring the changes of cardiac function in mice before and after injecting HPCAL1 by ultrasonic imaging

[0277] 1) The eukaryotic expression recombinant mouse HPCAL1 was from Wuhan PuJian Biotechnology Co., Ltd., and mouse serum albumin was from Wuhan FineTest 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 Laboratory Animal Ethics and Use Committee of Shantou University.

[0278] 2) Through 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: Put the mice reperfused for 5 days into an anesthesia induction box with 3% isoflurane gas to quickly enter a fully anesthetized state (slightly squinted eyes and slightly trembling represent anesthesia). Quickly transfer the mice to a 37°C constant temperature operating table, adjust the isoflurane concentration to 0.5% - 1% and connect a breathing mask to maintain anesthesia. Fix the four limbs of the mice on the electrode pads coated with coupling agent to conduct physical sign parameters such as their heart rate and respiration.

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

[0281] (C) Drug treatment: After the mice collected the data before administration (Before), inject rmsHPCAL1 (20 μg / mouse, concentration of 0.39 mg / ml) or the same dose of MSA (20 μg / mouse, concentration of 0.4 mg / ml) into the tail vein respectively. According to the method described in (b), collect the echocardiograms within 20 - 40 min after administration (After).

[0282] (D) Data processing: The data was processed using Vevo LAB 3.0.0 software, and cardiac function-related parameters such as the thickness of the anterior wall and posterior wall of the left ventricle and the left ventricular internal diameter in the diastolic and systolic phases were measured in 5 consecutive cardiac cycles of each group. The cardiac systolic function of the mice was evaluated using left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), where: LVEF = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume; LVFS = (left ventricular end-diastolic internal diameter - left ventricular end-systolic internal diameter) / left ventricular end-diastolic internal diameter).

[0283] The results are as Figure 9 shown. The results showed that after intravenous injection of recombinant mouse HPCAL1 into the tail vein of mice with myocardial ischemia-reperfusion for 5 days of modeling, it could effectively increase the left ventricular ejection fraction (about 20%) and the left ventricular fractional shortening rate (about 30%) of the mice.

[0284] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of HPCAL1 protein or related biomaterials in the preparation of a drug for treating heart failure, wherein the HPCAL1 protein is a protein as shown in any one of the following B1 or B2: (B1) a protein having an amino acid sequence of SEQ ID NO: 3; (B2) a protein whose amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 4; The related biological material is a biological material expressing the HPCAL1 protein, which is any 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 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).

2. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include injection, aerosol or drops.

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