Myoglobin mutants, kits and uses thereof

Abstract

The application provides a myoglobin mutant, a kit and application thereof. The myoglobin mutant comprises: (a) a protein which is mutated based on wild-type myoglobin shown in SEQ ID NO: 1, and the mutation comprises a mutation at a G75 site; and (b) a protein which has more than 70 % homology with the amino acid sequence defined in (a) and has myoglobin activity. The application can solve the problem of poor stability of myoglobin in the prior art and is suitable for the field of myoglobin modification.

Description

Technical Field

[0001] This invention relates to the field of myoglobin modification, and more specifically, to a myoglobin mutant, a kit, and its application. Background Technology

[0002] Myoglobin is an oxygen-binding hemoglobin found in the cytoplasm of cardiac and skeletal muscle cells. This hemoglobin assists in the entry of oxygen into cells and stores some oxygen. In patients with acute myocardial infarction (AMI), myoglobin levels become abnormal within two hours, peak at 6-9 hours, and then return to normal levels 24-36 hours after the infarction. According to IFCC and NACB standards, myoglobin can serve as an early biomarker of myocardial injury. Current techniques typically involve directly extracting myoglobin from natural sources or obtaining it through recombinant expression. However, when wild-type myoglobin is used clinically as a biomarker, its stability is poor, easily leading to inaccurate test results. Summary of the Invention

[0003] The main objective of this invention is to provide a myoglobin mutant, a kit, and its application to solve the problem of poor stability of myoglobin in the prior art.

[0004] To achieve the above objectives, according to a first aspect of the present invention, a myoglobin mutant is provided, comprising (a) a protein mutated based on the wild-type myoglobin shown in SEQ ID NO: 1, the mutation including a mutation occurring at the G75 site; and (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having myoglobin activity.

[0005] Furthermore, in (a), the type of amino acid substituted at the G75 site includes G75C; where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid.

[0006] Furthermore, myoglobin mutations include any of the following amino acid mutation combinations: G75C+E86C or G74C+G75C; wherein G75C+E86C is the protein shown in SEQ ID NO: 2, and G74C+G75C is the protein shown in SEQ ID NO: 3; myoglobin mutants include proteins that have more than 70% homology with the proteins shown in SEQ ID NO: 2 or SEQ ID NO: 3 and possess myoglobin activity.

[0007] Furthermore, the myoglobin mutant includes a protein that has 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and has myoglobin activity.

[0008] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes a myoglobin mutant of any of the above-mentioned types.

[0009] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, which is linked to the aforementioned DNA molecule.

[0010] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided containing the aforementioned DNA molecule or the aforementioned recombinant plasmid.

[0011] Furthermore, the host cells mentioned above include prokaryotic cells or eukaryotic cells; preferably, the prokaryotic cells include Escherichia coli; preferably, the Escherichia coli includes Rosetta strain.

[0012] To achieve the above objectives, according to a fifth aspect of the present invention, a myoglobin assay kit is provided, the myoglobin assay kit comprising a calibrator, the calibrator comprising any of the aforementioned myoglobin mutants; the myoglobin assay kit further comprising at least one of the following: microspheres, a label, and a buffer; the microspheres comprising microspheres coated with myoglobin antibodies; the label comprising a myoglobin antibody label, preferably labeling the myoglobin antibody with any of the following: a luminescent label, an affinity label, a fluorescent dye, or a labeling enzyme; preferably, the luminescent label comprises ABEI; wherein the myoglobin antibody coated with the microspheres is different from the myoglobin antibody with the label.

[0013] To achieve the above objectives, according to a sixth aspect of the present invention, the use of any of the aforementioned myoglobin mutants, DNA molecules, recombinant plasmids, or host cells in the preparation of a detection product for detecting myocardial injury is provided.

[0014] By applying the technical solution of this invention, site-directed mutation is performed on the G75 site of wild-type myoglobin to obtain a myoglobin mutant, which has higher stability than wild-type myoglobin and can be used as a standard in myoglobin assay kits to improve the accuracy of the test results. Detailed Implementation

[0015] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0016] As mentioned in the background section, the wild-type myoglobin extracted in the prior art or the myoglobin obtained through recombinant expression has poor stability and is not sensitive enough when used as a marker in myoglobin assay kits, resulting in inaccurate final detection results. Therefore, in this application, the inventors attempted to develop a myoglobin mutant and its application, and applied it to the preparation of a calibrator in a myoglobin assay kit to make it more stable. Based on this, a series of protection schemes of this application are proposed.

[0017] In a first typical embodiment of this application, a myoglobin mutant is provided, comprising: (a) a protein mutated based on the wild-type myoglobin shown in SEQ ID NO: 1, the mutation including a mutation occurring at the G75 site; and (b) a protein having more than 70% homology with the amino acid sequence defined in (a) and having myoglobin activity.

[0018] Myoglobin obtained through artificial extraction or recombinant expression in existing technologies often suffers from poor stability. This invention uses molecular simulation and modern genetic engineering techniques to mutate the G75 site in the amino acid sequence of wild-type myoglobin. The mutated G75 site can form spatial disulfide bonds with other sites on myoglobin, improving the overall spatial conformational stability of the protein. After recombinant expression, mutant myoglobin is obtained, which has better stability than wild-type myoglobin.

[0019] SEQ ID NO: 1:

[0020] MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMK ASEDLKKHGATVLTALGGILKKKGHHEAEEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHP GDFGADAQGAMNKALELFRKDMASNYKELGFQG.

[0021] In a preferred embodiment, in (a) above, the type of amino acid substituted at the G75 site includes G75C; wherein the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid. The mutant G75C can bind with amino acids at other sites on myoglobin, preferably those at a distance from the G75 site. The amino acids (preferably cysteine) in the myoglobin form disulfide bonds, which improves the stability of myoglobin.

[0022] In a preferred embodiment, the above mutation includes any one of the following amino acid mutations: G75C+E86C or G74C+G75C; wherein G75C+E86C is the protein shown in SEQ ID NO: 2, and G74C+G75C is the protein shown in SEQ ID NO: 3; the above myoglobin mutant includes a protein that has more than 70% homology with the protein shown in SEQ ID NO: 2 or SEQ ID NO: 3 and has myoglobin activity.

[0023] SEQ ID NO: 2:

[0024] MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMK ASEDLKKHGATVLTALGCILKKKGHHEACIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHP GDFGADAQGAMNKALELFRKDMASNYKELGFQG.

[0025] SEQ ID NO: 3:

[0026] MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMK ASEDLKKHGATVLTALCCILKKKGHHEAEEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHP GDFGADAQGAMNKALELFRKDMASNYKELGFQG.

[0027] A disulfide bond is a covalent bond formed by the connection of two cysteine ​​residues. Its bonding strength is greater than that of ordinary hydrogen bonds and van der Waals forces, thus providing stronger cohesive forces to stabilize the spatial structure of a protein. Cysteine ​​residues are usually located inside proteins. When a disulfide bond forms between two cysteine ​​residues, in addition to enhancing spatial stability, it also promotes protein folding and assembly, guiding the protein to correctly fold into its specific three-dimensional structure. Therefore, in this application, glycine at position 75 of the myoglobin amino acid sequence is combined with either glutamic acid at position 86 or glycine at position 74 to undergo site-directed mutation to cysteine. A disulfide bond is then formed between the two sites, namely G75C+E86C or G74C+G75C, making the spatial conformation of the protein more stable and yielding a highly stable and less degradable myoglobin mutant.

[0028] In a preferred embodiment, any of the above-mentioned myoglobin mutants includes proteins that have 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and have myoglobin activity.

[0029] All the aforementioned amino acid mutations were experimentally investigated in the embodiments of this application, and compared with the parent protein having the amino acid sequence shown in SEQ ID NO: 1, they all exhibit myoglobin activity. All the above mutation sites are mutations performed around the active amino acid sites, which can improve the stability of the protein's spatial conformation. Mutations far from the active site have less impact on enhancing protein stability. Therefore, proteins with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology to the above amino acid sequences and possessing the same ability to enhance stability can be obtained.

[0030] In this specification, homology refers to the "identity" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0031] The proteins described above that share 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%) homology with the proteins provided by sequence (a) and have the same function, have active sites, active pockets, active mechanisms, and protein structures that are highly likely to be the same as those provided by sequence (a). These are homologous proteins obtained through amino acid mutation.

[0032] Obtaining sequences with the aforementioned homology can be achieved through amino acid substitution. Generally, substitutions between amino acids with similar properties produce similar effects. For ease of description, the abbreviations of the amino acid residues are listed below: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0033] Amino acid substitutions or replacements, for example, can occur in the aforementioned homologous proteins, where conserved amino acid substitutions may take place. "Conserved amino acid substitutions" include, but are not limited to:

[0034] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0035] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0036] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0037] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0038] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0039] In a second typical embodiment of this application, a DNA molecule is provided that encodes any of the aforementioned myoglobin mutants.

[0040] The aforementioned DNA can encode the aforementioned myoglobin mutant and can be ligated to a recombinant plasmid to form a circular DNA. Both the aforementioned DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc., to obtain the aforementioned myoglobin mutant.

[0041] In a third typical embodiment of this application, a recombinant plasmid is provided that links the aforementioned DNA molecule.

[0042] In a fourth typical embodiment of this application, a host cell is provided, which contains the aforementioned DNA molecule or the aforementioned recombinant plasmid.

[0043] In a preferred embodiment, the host cell includes a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, the Escherichia coli includes Rosetta strain.

[0044] Using the aforementioned host cells, recombinant plasmids can be replicated within the host cells, and the DNA molecules carried on the recombinant plasmids can be transcribed and translated to obtain a large number of myoglobin mutants. Myoglobin mutants can also be obtained by using existing technologies to disrupt the host cells, purify the proteins after disruption, or through other methods. The host cells used must be of non-plant origin.

[0045] The Escherichia coli expression system is a prokaryotic expression system that can express a variety of exogenous genes in Escherichia coli cells, including genes from fungi, plants, bacteria, and viruses.

[0046] The advantages of E. coli expression systems include:

[0047] (1) Compared with other expression systems, Escherichia coli grows rapidly and has a shorter expression cycle;

[0048] (2) Escherichia coli cells are easy to grow, easy to expand culture, and have high yield, which is conducive to large-scale expression of recombinant proteins;

[0049] (3) Escherichia coli expression conditions are simple, low cost and easy to scale up production.

[0050] The Rosetta strain used in this application belongs to one of the host cells of the Escherichia coli expression system. Rosetta is derived from the Origami series of host bacteria, which contains the original TrxB and Gor mutant genes of the Origami strain, as well as rare eukaryotic codons in the Rosetta series. This characteristic can improve the correct folding rate of disulfide bonds in cells, resulting in efficient protein expression. When applied to protein expression and preparation purification, it has the advantages of being economical, rapid, high-yield, safer, and widely applicable.

[0051] In a fifth typical embodiment of this application, a myoglobin assay kit is provided, comprising a calibrator selected from any of the aforementioned myoglobin mutants; preferably, the myoglobin assay kit further comprises at least one of the following: microspheres, a label, and a buffer; the microspheres comprise myoglobin antibody-coated microspheres; the label comprises a myoglobin antibody label, preferably any of the following labels labeling myoglobin antibodies: the label comprises a luminescent label, an affinity label, a fluorescent dye, or a labeling enzyme; preferably, the luminescent label comprises ABEI; wherein the myoglobin antibody coated on the microspheres is different from the myoglobin antibody labeled.

[0052] A luminescent label is a substance that labels biomolecules through a luminescent reaction. ABEI (Aminobenzyl-EDTA-Isoluminol) is an isoluminol derivative that can be used to label myoglobin antibodies. When the antibody labeled with the luminescent label binds to myoglobin, the presence of myoglobin can be detected by chemiluminescent immunoassay.

[0053] Affinity markers are biomolecule-binding markers that bind to specific biomolecules for their detection and quantification. These affinity markers can bind to myoglobin antibodies or form complexes with enzymes (such as horseradish peroxidase) or fluorescent dyes that bind to them, allowing for the detection of myoglobin via enzyme labeling or fluorescent dye labeling methods. Common affinity markers include TM05 (Tumor marker) or biotin.

[0054] Fluorescent dyes are compounds with fluorescent properties that can bind to biomolecules and be detected and quantified using methods such as fluorescence microscopy or flow cytometry. Using fluorescent dyes as markers for myoglobin antibodies allows for the detection of myoglobin through fluorescence labeling. The distribution and expression levels of myoglobin can also be observed under a fluorescence microscope or in flow cytometry using fluorescent dye-labeled antibodies. Commonly used fluorescent dyes include FITC, Cy3, and Cy5.

[0055] Labeled enzymes are enzyme molecules that can bind to specific antibodies or affinity small molecules for the detection and quantification of target molecules. Labeling myoglobin antibodies with enzymes allows for the detection of myoglobin using enzyme labeling methods. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP), which react with substrates to produce colorimetric or fluorescent signals for the quantitative or qualitative detection of the presence of myoglobin.

[0056] The preferred method for measuring myoglobin in this application is chemiluminescent immunoassay, a commonly used immunoassay method based on chemiluminescent and chemiimmunoassay reactions. A chemiluminescent reaction refers to the process by which chemical substances produce visible light under certain conditions. Chemiluminescent immunoassay mainly includes the following steps:

[0057] 1. Target molecule capture: Antibodies, such as magnetic microspheres, are immobilized in the test tube or on the surface of a solid support, enabling them to specifically bind to the target molecule to be detected, such as an antigen.

[0058] 2. Sandwich reaction: The sample to be tested is added, which may contain the target molecule. If the target molecule is present in the sample, it will specifically bind to the antibody immobilized on the solid support, forming an antigen-antibody complex.

[0059] 3. Binding of the label: Add a secondary antibody (or a small affinity molecule compound) carrying a chemiluminescent label. These labels can bind to the antigen or antibody in the complex. The label can be a chemiluminescent substance (such as ABEI or TM50) or other substances (such as affinity labels, labeling enzymes, or fluorescent dyes).

[0060] 4. Washing: The washing step removes unbound substances, leaving only the specifically bound complexes on the solid support.

[0061] 5. Luminescence detection: Under appropriate conditions, a chemiluminescent substrate is added, causing it to react chemically with the labeled substance and produce luminescence. The intensity of the luminescence is related to the concentration of the target molecule to be detected.

[0062] In a preferred embodiment of the present invention, the myoglobin assay kit includes the aforementioned myoglobin mutant, myoglobin antibody A marker ABEI, and magnetic microspheres coated with myoglobin antibody B protein. In the kit, the myoglobin mutant serves as the calibrator, the myoglobin antibody A marker ABEI serves as the marker, and the magnetic microspheres coated with myoglobin antibody B protein serve as the microspheres. In actual testing at 37°C, the myoglobin in the sample, the ABEI-labeled myoglobin antibody A, and the myoglobin antibody B protein coated on the magnetic microspheres undergo an immune reaction, forming a "sandwich" immune complex.

[0063] Myoglobin antibody A, linked to ABEI, and myoglobin antibody B, linked to magnetic microspheres, specifically bind to myoglobin, forming a sandwich-like complex. Through magnetic separation and washing steps, unbound antibodies, residual sample, and other impurities are removed, yielding a purified conjugate. Furthermore, by detecting the chemiluminescence of the conjugate, qualitative or quantitative detection can be achieved to determine the presence and concentration of myoglobin. In this application, the "sandwich" immune complex formed by the above three components is detected. After obtaining the relative light intensity (RLU), the myoglobin mutant described in this application is used as a calibration material to establish a detection curve. This curve is then compared with the relative light intensity obtained from the sample measurement to obtain the final detection result.

[0064] In a sixth typical embodiment of this application, the application of any of the above-mentioned myoglobin mutants, DNA molecules, recombinant plasmids, or any of the host cells is provided in the preparation of a detection product for detecting myocardial injury.

[0065] The beneficial effects of this application will be further explained in detail below with reference to specific embodiments. It should be noted that all reagents and consumables in the following embodiments, unless otherwise specified, were purchased from TransGen Biotech, and the specific models are shown in the embodiments.

[0066] Example 1

[0067] 1. Site-directed mutagenesis of myoglobin

[0068] Mutation site: G75C+E86C.

[0069] Design and synthesize forward and reverse mutation primers containing mutation sites:

[0070] Forward primer: Primer 1 (SEQ ID NO: 4): CGCCATATGATGGGGCTATCAGATGGAGAATG GCAA.

[0071] Reverse primer: Primer 2 (SEQ ID NO: 5): CCGCTCGAGGCCCTGAAAACCCAGTTCTTTAT AATTGC.

[0072] PCR amplification was performed using a plasmid containing the wild-type myoglobin gene (synthesized by GenScript). The amplification system using Pfu polymerase (Full Gold, AP221-13) is shown in Table 1.

[0073] Table 1

[0074]

[0075] The PCR reaction conditions are shown in Table 2.

[0076] Table 2

[0077]

[0078] After the PCR reaction was completed, all PCR products were subjected to 1% agarose gel electrophoresis. The gel block containing the target fragment was cut off and the target fragment was recovered using a gel extraction kit (Tiangen Biotech, MAT:4994007). The purification and recovery system after double digestion with NdeI / XhoI (Thermo Fisher, ER1701) is shown in Table 3.

[0079] Table 3

[0080] carrier 2μg NdeI endonuclease 1μL XhoI endonuclease 1μL 10× buffer solution 5μL sterile water Add to 50μL Total volume 50μL

[0081] The prokaryotic expression vector pET28a(+) (Ubibio, VT1207) was prepared, and the enzyme digestion system is shown in Table 4.

[0082] Table 4

[0083] Target gene Determined based on the recycling situation NdeI endonuclease (Thermo Fisher, ER1701) 1μL XhoI endonuclease (Thermo Fisher, ER1701) 1μL 10×buffer (Thermo Fisher, ER1701) 5μL sterile water Add to 50μL Total volume 50μL

[0084] The target fragment was recovered and ligated to the prokaryotic expression vector pET28a(+) (Ubibio, VT1207) using T4 DNA ligase, as shown in Table 5.

[0085] Table 5

[0086] gene fragments 1μL pET28a(+) vector (Ubibio, VT1207) 3μL <![CDATA[5×T4 DNA Ligase Buffer (Thermo Scientific TM , EL0011)]]> 2μL <![CDATA[T4 DNA Ligase (Thermo Scientific TM , EL0011)]]> 1μL sterile water 3μL Total volume 10μL

[0087] After ligation, the cells were transformed into strain JM109 (Weidi Bio, CAT#: EC1070) for amplification culture. The plasmid was extracted and transformed into strain Rosetta (Novagen / Millipore, ST1011) for low-temperature induced expression.

[0088] 2. Application of site-directed mutagenesis-mediated myoglobin in myoglobin assay kits

[0089] (1) Experimental apparatus

[0090] Shenzhen New Industries Biomedical Engineering Co., Ltd., MAGLUMI X3 Fully Automated Spectrophotometer SN: 0101010033012200180.

[0091] (2) The site-mutated myoglobin and wild-type myoglobin obtained were used as calibrators for this kit to establish a detection curve.

[0092] Wild-type and mutant myoglobin were diluted to 0.1 mg / mL and subjected to gradient dilutions of 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold, 640-fold, 1280-fold, and 12800-fold. Each value was measured three times and the average value was taken.

[0093] (3) Add myoglobin antibody A conjugated with ABEI and myoglobin antibody B conjugated with magnetic microspheres.

[0094] 10 μL sample + 100 μL buffer (Snibe, PBS-97) + 100 μL ABEI (Medix, 7001) conjugated with myoglobin antibody A + 20 μL magnetic microspheres (Snibe, 6643-1) conjugated with myoglobin antibody B, incubate at 37 °C for 5 min, wash, measure, and obtain the corresponding relative light intensity (RLU).

[0095] The results showed that the titer of the mutant was within 10% of that of the wild type, and the sensitivity analysis of the protein was comparable, meeting the requirements for use.

[0096] 3. Stability test verification

[0097] Wild-type and mutant myoglobin were diluted to the same concentration using the same diluent for stability testing. The diluted myoglobin samples were placed in a refrigerator at 2-8℃ and an oven at 40℃, respectively, and stability tests were performed on day 0, day 1, day 3, and day 7.

[0098] Testing standards:

[0099] 1) Decrease = (Light intensity value on day N / Light intensity value on day zero - 1) × 100%

[0100] 2) The temperature drop within 7 days at 2-8℃ and 40℃ was within 10%.

[0101] result:

[0102] 1) The mutant myoglobin regimen showed a decrease of less than 10% in the accelerated thermal stability effect at 2-8℃ and 40℃ for 7 days, which is significantly better than the stability of wild-type myoglobin.

[0103] 2) Through site-directed mutagenesis, myoglobin with better stability was obtained through recombinant expression.

[0104] The results are shown in Table 6.

[0105] Table 6

[0106]

[0107] Example 2

[0108] 1. Site-directed mutagenesis of myoglobin

[0109] The difference from Example 1 is that the mutation site in this example is G74C+G75C.

[0110] Design and synthesize forward and reverse mutation primers containing mutation sites:

[0111] Forward primer: Primer 3 (SEQ ID NO: 6): ACCGTTTTGACCGCACTGTGCTGCATTTTGAA GAAGGG.

[0112] Reverse primer: Primer 4 (SEQ ID NO: 7): CCCTTCTTCTTCAAAATGCAGCACAGTGCGGTCAAAACGGT.

[0113] The remaining steps are the same as in Example 1.

[0114] 2. Application of site-directed mutagenesis-mediated myoglobin in myoglobin assay kits

[0115] The experimental procedure is the same as in Example 1.

[0116] The results showed that the titer of the mutant was within 10% of that of the wild type, and the sensitivity analysis of the protein was comparable, meeting the requirements for use.

[0117] 3. Stability test verification

[0118] The experimental procedure is the same as in Example 1.

[0119] result:

[0120] 1) The mutant myoglobin regimen showed a decrease of less than 10% in the accelerated thermal stability effect at 2-8℃ and 40℃ for 7 days, which is significantly better than the stability of the wild type.

[0121] 2) Through site-directed mutagenesis, myoglobin with better stability was obtained through recombinant expression.

[0122] The results are shown in Table 7.

[0123] Table 7

[0124]

[0125] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present invention promotes the formation of spatial disulfide bonds within wild-type myoglobin by site-directed mutation of the G75 site and either E86 or G74, i.e., G75C+E86C or G74C+G75C, thereby improving the overall spatial conformational stability of the protein and obtaining a more stable myoglobin mutant. When the myoglobin mutant of this application is used as a standard in a myoglobin assay kit, it exhibits higher stability and comparable sensitivity compared to wild-type myoglobin, thus meeting the detection requirements, improving the accuracy of the detection results, and extending the shelf life of the kit.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A myoglobin mutant, characterized in that, The myoglobin mutant is a protein mutated based on the wild-type myoglobin shown in SEQ ID NO: 1, wherein the mutation is any combination of the following amino acid mutations: G75C+E86C or G74C+G75C; wherein G75C+E86C is the protein shown in SEQ ID NO: 2, and G74C+G75C is the protein shown in SEQ ID NO:

3.

2. A DNA molecule, characterized in that, The DNA molecule encodes the myoglobin mutant of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule of claim 2.

4. A host cell, characterized in that, The host cell contains the DNA molecule of claim 2 or the recombinant plasmid of claim 3.

5. The host cell according to claim 4, characterized in that, The host cells include prokaryotic cells or eukaryotic cells.

6. The host cell according to claim 5, characterized in that, The prokaryotic cells include Escherichia coli.

7. The host cell according to claim 6, characterized in that, The Escherichia coli includes the Rosetta strain.

8. A myoglobin assay kit, characterized in that, The myoglobin assay kit includes a calibrator, which includes the myoglobin mutant of claim 1; The myoglobin assay kit further includes at least one of the following: microspheres, a label, and a buffer solution; The microspheres include microspheres coated with myoglobin antibodies; The markers include myoglobin antibody markers; The myoglobin antibody coated on the microspheres is different from the myoglobin antibody with a marker.

9. The myoglobin assay kit according to claim 8, characterized in that, The marker includes any one of the following markers for myoglobin antibodies: luminescent marker, affinity marker, or marker enzyme.

10. The myoglobin assay kit according to claim 9, characterized in that, The luminescent marker includes a fluorescent dye.

11. The myoglobin assay kit according to claim 9, characterized in that, The luminescent marker includes ABEI.

12. The use of the myoglobin mutant of claim 1, the DNA molecule of claim 2, the recombinant plasmid of claim 3, or the host cell of any one of claims 4-7 as a standard in the preparation of a detection product for detecting myocardial injury.

Images