Anti-myoglobin nanobodies and uses thereof

Abstract

The application provides an anti-myoglobin nanobody and application thereof, relates to a nanobody, a polypeptide containing the nanobody and application thereof and the like, a variable region in an amino acid sequence of the nanobody comprises three complementarity determining regions CDR and a framework region FR, the complementarity determining regions CDR comprise a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the most important site participating in antigen recognition and combination is located at TKILSYGKP of CDR3, especially K96, L98, S99, Y100 and K102, the nanobody and the polypeptide thereof of the application have very high affinity and activity, can specifically recognize and combine myoglobin, and can be used for adsorption, removal and detection of myoglobin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-myoglobin nanobody, a polypeptide containing the nanobody, its preparation method, and its application. Background Technology

[0002] Myoglobin (Mb) is an iron-containing porphyrin protein with a molecular weight of approximately 17.8 kDa, primarily found in cardiac and skeletal muscle cells, and in smaller quantities in smooth muscle cells. Its core function is to store and transport oxygen, maintaining normal energy metabolism in muscle tissue. Under physiological conditions, the level of myoglobin in the blood is extremely low; the serum myoglobin concentration in healthy adults is typically 10-70 ng / mL. However, when the membrane integrity of cardiac or skeletal muscle cells is disrupted due to factors such as ischemia (e.g., acute myocardial infarction), mechanical trauma (e.g., crush injuries, burns), or drug-induced damage (e.g., drug-induced myonecrosis), a large amount of intracellular myoglobin is released into the blood, causing a significant increase in blood concentration within a short period. In acute myocardial infarction, myoglobin levels can rise within 0.5-1 hour of onset, reaching peak concentration (5-20 times the upper limit of normal) within 2-4 hours. In rhabdomyolysis, its concentration can exceed the upper limit of normal by more than 100 times within a few hours, with some severely ill patients even exceeding 5000 ng / mL.

[0003] The core danger of elevated blood myoglobin levels is acute kidney injury. When large amounts of myoglobin exceed the kidneys' metabolic capacity, they easily denature and polymerize in acidic urine, forming casts that block renal tubules. The released iron ions also generate reactive oxygen species that damage renal tubular epithelial cells, while constricting renal blood vessels and causing renal ischemia. In severe cases, this can lead to acute renal failure. Furthermore, high concentrations of myoglobin can affect cardiovascular function, increasing the risk of thrombosis or arrhythmias. If it originates from skeletal muscle necrosis, it can also cause local inflammation. Without timely intervention, symptoms can progress from tea-colored urine and muscle aches to electrolyte imbalances and multiple organ dysfunction, with a mortality rate of 10%-20%.

[0004] However, there are still significant technical bottlenecks in the detection and removal of myoglobin, making it difficult to fully meet the core requirements of "accurate diagnosis + efficient and timely intervention".

[0005] First, there are significant limitations in detection capabilities: while conventional immunochromatography (colloidal gold method) is convenient to operate, its detection limit is only 0.5-1 ng / mL, easily missing early cases; enzyme-linked immunosorbent assay (ELISA) has slightly better sensitivity, but requires multiple incubation steps (total time 1-2 hours), making it unsuitable for point-of-care testing (POCT) in emergency situations; chemiluminescent immunoassay, although achieving pg-level detection limits, relies on large-scale equipment and specialized operation, limiting its application to tertiary hospital central laboratories and making it difficult to cover primary healthcare institutions and emergency sites. Most importantly, existing detection technologies rely on traditional monoclonal antibodies, which suffer from large molecular weight (150 kDa), poor tissue penetration, and susceptibility to sample interference (such as hemolysis leading to false positives), further restricting improvements in detection performance.

[0006] Secondly, there is a lack of targeted solutions for myoglobin removal: current treatments mainly rely on non-specific blood purification therapies. Conventional hemodialysis (HD) only removes 10%-20% of myoglobin, and can only remove free small molecule toxins. Even with high-flux hemodiafiltration (HDF), the removal rate is only 40%. Furthermore, while removing myoglobin, high-flux filters also simultaneously filter albumin, clotting factors, electrolytes, etc., leading to hypoproteinemia, coagulation dysfunction, or electrolyte disturbances (such as hypocalcemia and hypokalemia) in patients, increasing the difficulty of clinical management.

[0007] Therefore, there is an urgent need to make breakthroughs in the detection and removal of myoglobin in order to achieve "more sensitive, faster, more convenient, and more accurate" methods in order to overcome the current clinical technical bottlenecks. Summary of the Invention

[0008] The purpose of this invention is to provide nanobodies with specific structural amino acid sequences, peptides containing such nanobodies, and their applications, in order to solve the problems of insufficient sensitivity, weak anti-interference ability, low specificity, and poor efficiency in existing methods for myoglobin enrichment, purification, detection, and removal.

[0009] To achieve the above technical objectives, the technical solution adopted in this application is as follows:

[0010] In a first aspect, according to the anti-myoglobin nanobody in some embodiments of this application, the complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2 and CDR3 sequences: (I): (1) the amino acid sequence of CDR1 is shown in SEQ ID NO. 7, (2) the amino acid sequence of CDR2 is shown in SEQ ID NO. 15; and (3) the amino acid sequence of CDR3 is shown in SEQ ID NO. 22; or (II): an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences (1), (2) and (3) of (I), and having the same function as the amino acid sequence of (I).

[0011] According to some embodiments of the present application, the anti-myoglobin nanobody, wherein: (II) is an amino acid sequence obtained by substituting one or more amino acids of the amino acid sequence described in (1), (2), and (3) of (I), including (II1): the amino acid sequence of CDR1 as shown in SEQ ID NO. 8, the amino acid sequence of CDR2 as shown in SEQ ID NO. 16, and the amino acid sequence of CDR3 as shown in SEQ ID NO. 22; or (II2): the amino acid sequence of CDR1 as shown in SEQ ID NO. 9, the amino acid sequence of CDR2 as shown in SEQ ID NO. 16, and the amino acid sequence of CDR3 as shown in SEQ ID NO. 22; or (II3): the amino acid sequence of CDR1 as shown in SEQ ID NO. 10, the amino acid sequence of CDR2 as shown in SEQ ID NO. 17, and the amino acid sequence of CDR3 as shown in SEQ ID NO. 22.

[0012] According to some embodiments of the present application, the anti-myoglobin nanobody includes: (III) the framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.6, (2) the amino acid sequence of FR2 is as shown in SEQ ID NO.11, (3) the amino acid sequence of FR3 is as shown in SEQ ID NO.18, and (4) the amino acid sequence of FR4 is as shown in SEQ ID NO.23; or (IV) an amino acid sequence having more than 50% homology with the amino acid sequences of (1), (2), (3) and (4) of (III).

[0013] According to the anti-myoglobin nanobodies in some embodiments of this application, (IV) of the amino acid sequences described in (1), (2), (3), and (4) of (III) have more than 50% homology, including (IV-1): the amino acid sequence of FR1 is as shown in SEQ ID NO.6, the amino acid sequence of FR2 is as shown in SEQ ID NO.12, the amino acid sequence of FR3 is as shown in SEQ ID NO.19, and the amino acid sequence of FR4 is as shown in SEQ ID NO.23; or (IV-2): the amino acid sequence of FR1 is as shown in SEQ ID NO.6, the amino acid sequence of FR2 is as shown in SEQ ID NO.13, the amino acid sequence of FR3 is as shown in SEQ ID NO.20, and the amino acid sequence of FR4 is as shown in SEQ ID NO.24; or (IV-3): the amino acid sequence of FR1 is as shown in SEQ ID NO.6, the amino acid sequence of FR2 is as shown in SEQ ID NO.14, the amino acid sequence of FR3 is as shown in SEQ ID NO.20, and the amino acid sequence of FR4 is as shown in SEQ ID NO.24. As shown in NO.24; or (IV-4): the amino acid sequence of FR1 is shown in SEQ ID NO.6, the amino acid sequence of FR2 is shown in SEQ ID NO.13, the amino acid sequence of FR3 is shown in SEQ ID NO.21, and the amino acid sequence of FR4 is shown in SEQ ID NO.24.

[0014] According to some embodiments of the anti-myoglobin nanobody of this application, the nanobody has (V) an amino acid sequence as shown in SEQ ID NO. 1; or (VI) an amino acid sequence obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequence of (V), and having the same function as the amino acid sequence of (I). According to some embodiments of the anti-myoglobin nanobody of this application, wherein the amino acid sequence of the nanobody of (VI) is as shown in any one of SEQ ID NO. 2 to SEQ ID NO. 5.

[0015] In a second aspect, according to some embodiments of the present application, the humanized nanobody (VII): the amino acid sequence of the humanized nanobody is shown in any one of SEQ ID NO.25 to SEQ ID NO.37.

[0016] On a third-party level, the polypeptides in some embodiments of this application include any of the nanobodies described herein.

[0017] In a fourth aspect, nucleic acid molecules encoding any one of the nanobodies according to some embodiments of this application.

[0018] In a fifth aspect, the expression vector according to some embodiments of this application includes the nucleic acid molecule.

[0019] In a sixth aspect, the host cells of the expression vector described herein are transformed or transfected according to some embodiments of this application.

[0020] In a seventh aspect, the conjugates or conjugates according to some embodiments of this application include nanobodies described in any one of the chemically labeled or biologically labeled methods.

[0021] In an eighth aspect, the adsorbents according to some embodiments of this application include any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the couplings, and the carriers.

[0022] In a ninth aspect, the kit according to some embodiments of this application includes any of the nanobodies; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the conjugates; or the adsorbents, as well as adjuvants acceptable for detection.

[0023] In a tenth aspect, the apparatus according to some embodiments of this application is used to capture, adsorb, and / or detect myoglobin, including any of the nanobodies described; or the polypeptides; or the nucleic acid molecules; or the expression vectors; or the host cells; or the conjugates; or the coupling agents; or the adsorbents; or the kits described.

[0024] In the eleventh aspect, the use of the nanobody; or the polypeptide; or the nucleic acid molecule; or the expression vector; or the host cell; or the conjugate; or the coupling compound; or the adsorbent; or the kit; or the device according to some embodiments of this application in the preparation of preparations for the specific capture, adsorption, and / or detection of myoglobin; in the preparation of cell preparations for the specific capture, adsorption, and / or detection of myoglobin, for the enrichment and / or purification of myoglobin; or in the preparation of immunofluorescence or immunohistochemical reagents for the specific capture, adsorption, and / or detection of myoglobin.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The nanobody of the present invention is an anti-myoglobin nanobody with a novel amino acid sequence discovered through screening. This nanobody and its polypeptide have high affinity and activity, and can specifically recognize and bind to myoglobin. It can be used for myoglobin capture and detection, and can be applied to immunofluorescence analysis or immunohistochemical analysis through appropriate antibody labeling technology. Attached Figure Description

[0027] Figure 1 This is a diagram showing the monoclonal specificity identification results of the nanobody screening process in Example 2;

[0028] Figure 2 This is a kinetic binding curve of the nanobody with myoglobin (Mb) from different sources in Example 4;

[0029] Figure 3 This is a molecular docking diagram of nanobody B01 and Mb protein in Example 4, where a is the overall effect, b is a local magnification from the first angle, and c is a local magnification from the second angle.

[0030] Figure 4 This is a molecular docking diagram of nanobody BO2 and Mb protein in Example 4, where a is the overall effect, b is a local magnification from the first angle, and c is a local magnification from the second angle.

[0031] Figure 5 This is a molecular docking diagram of nanobody B03 and Mb protein in Example 4, where a is the overall effect, b is a local magnification from the first angle, and c is a local magnification from the second angle.

[0032] Figure 6 This is a molecular docking diagram of nanobody B04 and Mb protein in Example 4, where a is the overall effect, b is a local magnification from the first angle, and c is a local magnification from the second angle.

[0033] Figure 7 This is a molecular docking diagram of nanobody B05 and Mb protein in Example 4, where a is the overall effect, b is a local magnification from the first angle, and c is a local magnification from the second angle.

[0034] Figure 8 This is a schematic diagram of the antigen-antibody molecular interaction between the nanobody and myoglobin (Mb) in Example 4;

[0035] Figure 9 These are superimposed comparison diagrams of the molecular structures of the target nanobodies in Example 4, where a is the overall superimposed effect of the 5 nanobodies, and b is the overall superimposed diagram from another perspective;

[0036] Figure 10 This is a competitive ELISA standard curve of HRP-labeled nanobody B05 (B05-HRP) in Example 8;

[0037] Figure 11 This is a graph showing the immunohistochemical detection results of the HRP-labeled nanobodies of this invention;

[0038] Figure 12 This describes the dynamic adsorption effect of the nano-antibody adsorbent of the present invention;

[0039] Figure 13 This is an SDS-PAGE electrophoresis verification image of the nano-antibody adsorbent of the present invention after purification of myoglobin (Mb). Detailed Implementation

[0040] The foregoing and other aspects of the present invention will be further described below, wherein:

[0041] (1) Unless otherwise specified, the term “sequence” is used herein (as in similar terms such as “antibody sequence”, “variable region sequence”, “V”). HH In the term "sequence" or "protein sequence," it should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said amino acid sequence, unless the context requires a narrower interpretation.

[0042] (2) Unless otherwise specified, all methods, steps, techniques and operations not specifically described are known and well known to those skilled in the art. For example, references are still made to the general background art cited above and other references cited therein.

[0043] (3) The term "specificity" refers to the ability of a specific antigen-binding molecule (such as the nanobodies or peptides of the present invention) to bind to different types of antigens or antigenic determinants. The specificity of an antigen-binding molecule can be determined based on its affinity and / or activity. Affinity is expressed as the dissociation equilibrium constant (K0) between the antigen and the antigen-binding molecule. D K is a measure of the binding strength between antigens and antigen-binding molecules. D The smaller the K value, the stronger the binding strength between the antigen and the antigen-binding molecule; conversely, the larger the K value, the stronger the binding strength between the antigen and the antigen-binding molecule. D The higher the value, the weaker the binding strength between the antigen and the antigen-binding molecule. a K represents the associative constant. a The larger the value, the faster the binding. a The smaller the value, the slower the binding; K d K represents the dissociation constant. d The larger the value, the faster the dissociation. d The smaller the value of K, the slower the dissociation; and K D = K d / K a .

[0044] (4) The encoding of single-domain antibodies (i.e., nanobodies) is mainly based on their V domains. HHThe amino acid sequences of the domains are standardized and numbered to distinguish between the framework region (FR), which is responsible for maintaining spatial structural stability, and the complementarity-determining region (CDR), which mediates antigen-specific binding. Commonly used coding methods in this field include IMGT, Kabat, and Chothia. This application adopts the IMGT coding method to define FR and CDR. IMGT coding is a universal standard recognized by the International Federation of Immunological Societies (IFIS) of the World Health Organization. It is based on sequence alignment using a complete reference gene database of the entire immunoglobulin superfamily and is a consensus coding system in the global immunoglobulin and single-domain antibody research field. It ensures the comparability and interoperability of different research data and cross-species single-domain antibody sequences. For nanobodies, residues are counted consecutively from 1 to 128. If a residue is missing, the corresponding number is left blank without complex additional annotations, avoiding problems such as numbering confusion and inconsistent labeling of inserted residues that are prone to occur with other coding methods. IMGT coding has its own FR-IMGT and CDR-IMGT definitions, with clear boundaries and a high degree of fit with the structure and function of single-domain antibodies. It can accurately correspond to the conserved backbone region and the antigen-binding variable region, providing reliable support for subsequent sequence analysis and epitope identification.

[0045] (5) The term “load capacity” refers to the total amount of ligands coupled to a unit volume of affinity medium (adsorbent).

[0046] (6) The term “homogeneous nanobody” refers to a class of nanobodies that are derived from the germline of the variable region gene of the same natural heavy chain antibody, produced by high-frequency mutation in somatic cells, have highly homologous backbone regions (FR homology > 90%), differ only in the complementarity-determining region (CDR), and target the same or similar antigenic epitopes.

[0047] Amino acid substitution can generally be described as the substitution of amino acid residues by amino acids with similar or dissimilar chemical structures, as long as it has little or no effect on the function, activity, or other biological properties of the polypeptide. Preferably, the amino acid residues can be substituted by amino acids with similar chemical structures.

[0048] For the above-mentioned substitution methods, examples disclosed in documents WO04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300 can be listed, but are not limited to these. In addition, (preferred) types and / or combinations of the substitution can be selected based on relevant information from other references cited in WO 04 / 037999 and WO 06 / 122786.

[0049] The amino acid substitutions of the present invention can be listed, but are not limited to, the following substitution methods, in which one amino acid in the following groups (a) to (e) is replaced by another amino acid in the same group: (a) Ala, Ser, Thr, Pro and Gly; (b) Asp, Asn, Glu and Gln; (c) His, Lys and Arg; (d) Met, Leu, Ile, Val and Cys; (e) Phe, Tyr and Trp.

[0050] Preferred amino acid substitutions may include, but are not limited to, the following: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser or Thr; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Glu, or Gln; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Tyr, or Leu; Ser is substituted with Thr; Thr is substituted with Ser; Tyr is substituted with Trp; Trp is substituted with Tyr.

[0051] The framework region is more conserved compared to the complementarity-determining region. Those skilled in the art will rationally screen the sequence structure of the framework region based on the actual application and function of the nanobody. For the amino acid sequence of the framework region, an amino acid sequence with homology of 50% or more is preferred, further preferred is an amino acid sequence with homology of 70% or more, and even more preferably an amino acid sequence with homology of 95% or more. Tables 1 and 2 show the framework region sequence information of each antibody in Example 2:

[0052] Table 1. Comparison of Nanobody Serial Numbers and FR Sequence Information - 1

[0053]

[0054] Table 2 Comparison of Nanobody Serial Numbers and FR Sequence Information - 2

[0055]

[0056] The framework region contributes relatively little to affinity; therefore, amino acid substitutions in the framework region generally do not affect the affinity of nanobodies. As long as they can exist in a soluble form, the amino acid substitution methods described above also apply to the framework region. Humanization is a typical example of amino acid substitution in the framework region. In this invention, SEQ ID No. 25 to SEQ ID No. 37 represent humanized forms of nanobodies, none of which affected the affinity of the original sequence.

[0057] Furthermore, the total number of residues in nanobodies can be in the range of 110-120. However, the portions, fragments, or analogs of nanobodies are not particularly limited in their length and / or size, provided that such portions, fragments, or analogs meet the further requirements listed below and are also suitable for the purposes described herein.

[0058] The nanobodies in this invention belong to the same family of nanobodies, with the same total length of amino acid sequences, the same length of each frame region (FR) and antigen-binding region (CDR), high sequence identity, similar structure, and basically equivalent antigen-binding capacity.

[0059] The preparation method of "nanobody" is, in its broadest sense, not limited to specific biological resources or specific preparation methods. For example, the nanobody of the present invention can be obtained as follows: (1) by isolating V of naturally occurring heavy chain antibodies. HH Structural domain; (2) Encoding the naturally occurring V through expression HH The nucleotide sequence of the domain; (3) by using naturally occurring V HH The structural domain is "humanized" (as described below) or the humanized V is expressed by encoding the expression. HH (4) preparing a protein, polypeptide or other amino acid sequence by means of synthetic or semi-synthetic techniques; (5) preparing a nucleic acid encoding a nanobody by means of nucleic acid synthesis techniques and then expressing the nucleic acid thus obtained; and / or (6) by any of the foregoing combinations.

[0060] Furthermore, a variant of the nanobody based on the present invention also includes having a similarity to naturally occurring V. HH Nanobodies with corresponding but humanized amino acid sequences for their structural domains. Humanization refers to the use of V-chain antibodies derived from conventional 4-chain antibodies from humans. H One or more amino acid residues at the corresponding positions in the domain replace the naturally occurring V. HH One or more amino acid residues in a domain sequence.

[0061] According to a non-limiting embodiment of the present invention, the above-mentioned polypeptide is substantially composed of nanobodies. "Substantially composed of" means that the amino acid sequence of the polypeptide of the present invention is exactly the same as or corresponds to the amino acid sequence of the nanobodies, wherein a limited number of amino acid residues, such as 1 to 10 amino acid residues, and preferably 1 to 6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, are added to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the nanobodies or polypeptides.

[0062] The aforementioned amino acid residues may not alter the biological properties of the nanobody, and may even impart other functionalities to the nanobody. For example, the amino acid residues may:

[0063] 'a' is a purification tag, i.e., an amino acid sequence or residue that facilitates the purification of the nanobody, for example, by using affinity techniques targeting the sequence or residue for purification. Some preferred, but not limited, examples of such residues are multiple sets of His-tags (His6 or His8), GST-tags, MBP-tags, Myc-tags, Strep-tags, Flag-tags, HA-tags, V5-tags, S-tags, and E-tags;

[0064] b is a soluble tag, which is a tag that increases the solubility of nanobodies, such as SUMO;

[0065] c is an N-terminal amino acid residue, such as Met, Ala, Gln or MetAlaGln, AlaGln, which can be expressed in a heterologous host cell or in a host organism.

[0066] d is a C-terminal Cys residue, which can, for example, react with -SH on a ligand or with the Au surface;

[0067] e is a hinge to provide a link or spacer between the nanobody and other groups, such as a combination of GlySer, an IgG hinge, an IgA hinge, or other synthetic hinges.

[0068] f is provided in a known manner with one or more amino acid residues that have functional groups and / or have been functionalized, for example, as is known in the art, amino acid residues such as lysine or cysteine ​​allow PEG groups to attach.

[0069] The polypeptide of the present invention may also include two or more of the nanobodies, also known as multivalent polypeptides.

[0070] A bivalent polypeptide comprises two nanobodies, optionally linked by one hinge sequence; a trivalent polypeptide comprises three nanobodies, optionally linked by two hinge sequences; and a tetravalent polypeptide comprises four nanobodies, optionally linked by three hinge sequences. Multivalent polypeptides can bind to the same antigenic epitope or different antigen-binding epitopes; the latter are also called multispecific polypeptides.

[0071] Regarding containing one or more V HH For information on multivalent and multispecific polypeptides with structural domains and their preparation, please refer to EP0822985.

[0072] Hinges for multivalent and multispecific peptides should be well known to those skilled in the art, including, for example, Gly-Ser, such as (Gly4Ser)3 or (Gly3Ser2)3 as described in WO 99 / 42077; or naturally occurring heavy chain antibody hinge regions or portions thereof. For other suitable hinges, reference may also be made to the comprehensive background art cited above.

[0073] In addition to the one or more nanobodies mentioned above, the polypeptides of the present invention may also contain functional groups, portions or residues, such as therapeutically active substances, and / or tags, such as fluorescent labels, isotope labels, biotin labels and enzyme catalytic tags.

[0074] Furthermore, the dissociation equilibrium constant (K0) of the nanoantibody or polypeptide of the present invention with myoglobin D ) is 10 -9 ~10 -10 Moles per liter (M). The dissociation equilibrium constant of this invention was determined using plasmon resonance technology.

[0075] The specific binding between the aforementioned antigen and antigen-binding molecules can be determined by any suitable known method, including Sercatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), as well as other novel methods known in the art, such as plasma resonance (SPR) and / or biomembrane interference (BLI) techniques. Furthermore, those skilled in the art should recognize that affinity parameters measured using different methods can vary considerably, even by two to three orders of magnitude.

[0076] The nanobodies, peptides, and nucleic acids encoding the present invention can be prepared in known ways, as will become clear to those skilled in the art from the further description herein. A particularly useful method for preparing the nanobodies, peptides, and nucleic acids typically includes the following steps:

[0077] (1) Expressing nucleic acid encoding the nanobody or polypeptide of the present invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by;

[0078] (2) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0079] Alternatively, other methods can be used, including the following steps:

[0080] (3) Cultivate and / or maintain the host of the present invention under certain conditions, so that the host of the present invention expresses and / or produces a nanobody and / or polypeptide of the present invention; optionally, proceed thereafter;

[0081] (4) Isolate and / or purify the nanobodies or polypeptides of the present invention thus obtained.

[0082] One advantage of using nanobodies is that the peptides based on them can be expressed and prepared in prokaryotic systems, and suitable prokaryotic expression systems, vectors, and host cells are well known to those skilled in the art, as cited in the references above. However, it should be noted that this invention is not limited, in its broadest sense, to expression in bacterial systems.

[0083] Preferably, in this invention, the nanobodies or peptides are produced in bacterial cells, particularly in bacterial cells suitable for large-scale drug production.

[0084] When the nanobodies or peptides of the present invention are expressed in cells, the nanobodies or peptides of the present invention may be generated intracellularly (e.g., in the cytoplasm or periplasmic space), then isolated from the host cell, and optionally further purified; or they may be generated extracellularly (i.e., secreted expression), then isolated from the culture medium, and optionally further purified.

[0085] Some preferred but non-limiting vectors for use with these host cells include vectors for expression in mammalian cells—pMANneo (Clonetech), pUCTtag (ATCC37460), and pMClneo (Stratagene); vectors for expression in bacterial cells—pET vector (Novagen) and pQE vector (Qiagen); expression vectors for expression in yeast or other fungal cells—pYES2 (Invitrogen) and Pichia pastoris expression vector (Invitrogen); expression vectors for expression in insect cells—pBlueBacⅡ (Invitrogen) and other baculovirus vectors; and so on.

[0086] The corresponding techniques for transforming the host or host cells of the present invention are well known to those skilled in the art.

[0087] After transformation, it is possible to detect and select those hosts that have been successfully transformed with the nucleotide sequence / genetic construct of the present invention. The transformed host cells (which may be in the form of stable cell lines) or host organisms (which may be in the form of stable mutant lines or strains) form another aspect of the present invention.

[0088] The amino acid sequence of the present invention can then be isolated from the host cell / host organism and / or from the culture medium in which the host cell or host organism is cultured, using essentially known protein separation and / or purification techniques, such as (preparative) chromatography and / or electrophoresis, differential precipitation, affinity techniques (e.g., using a specific / cleavable amino acid sequence fused with the amino acid sequence of the present invention) and / or preparative immunological techniques (i.e., using antibodies against the amino acid sequence to be isolated).

[0089] The nanobodies or peptides of the present invention can specifically bind to the antigen myoglobin. Therefore, a preferred but non-limiting application of the present invention is as a myoglobin adsorbent, wherein the adsorbent comprises a carrier matrix and the nanobodies or peptides.

[0090] The aforementioned myoglobin includes myoglobin from different species, and the nanobody of the present invention has the ability to adsorb myoglobin from different species.

[0091] The aforementioned carrier matrix can be a porous material, such as agarose gel microspheres, cellulose spheres, magnetic beads, silica microspheres, activated carbon, or resin microspheres.

[0092] The carriers used for the aforementioned adsorbents are commercially available, such as agarose gel Sepharose CL-6B (GE Healthcare, US) and resin microspheres Nanomicro series (Suzhou Nanomicro Technology Co., Ltd.), but are not limited to these products.

[0093] When using the above-mentioned carrier, it is preferable that the carrier be activated. This activation method may include, but is not limited to, the following: first, epoxy activation; second, diaminopropylimine (DADPA) activation; and finally, iodoacetic acid activation, etc.

[0094] The aforementioned adsorbent is obtained by coupling nanobodies or peptides to an activated carrier. The specific method is not particularly limited. For example, the final adsorbent can be obtained by mixing a purified nanobodies or peptide solution with the carrier, separating by centrifugation, and finally washing / filtering the gel.

[0095] The adsorbent of the present invention can be used to specifically recognize myoglobin.

[0096] The nanobodies, peptides, or adsorbents of this invention can be used to purify myoglobin and to prepare kits for detecting myoglobin.

[0097] This invention provides nanobodies with specific amino acid sequences, peptides containing these nanobodies, and their applications, to address existing problems in myoglobin enrichment, purification, detection, and removal, such as insufficient sensitivity, weak anti-interference ability, low specificity, and poor efficiency. Specifically, in a first aspect, this invention provides a myoglobin-binding nanobody, wherein the variable region in the amino acid sequence of the nanobody includes a complementarity-determining region (CDR) and a framework region (FR). The CDR includes CDR1, CDR2, and CDR3, wherein the most important sites involved in antigen recognition and binding are K96, L98, S99, Y100, and K102 on CDR3.

[0098] Preferably, the amino acid sequence of the complementarity-determining region CDR1 includes SEQ ID No. 7 to SEQ ID No. 10, the amino acid sequence of the complementarity-determining region CDR2 includes SEQ ID No. 15 to SEQ ID No. 17, and the amino acid sequence of the complementarity-determining region CDR3 includes SEQ ID No. 22 and a sequence with more than 75% homology to it.

[0099] Preferably, the amino acid sequence of the nanobody includes: SEQ ID No.1 ~ SEQ ID No.5.

[0100] Preferably, the nanobody is a humanized nanobody, and more preferably, the humanized nanobody includes: SEQ ID No. 25 ~ SEQ ID No. 37.

[0101] In a second aspect, the present invention provides a polypeptide obtained by modifying the amino acids at the N-terminus and / or C-terminus of the aforementioned nanobody.

[0102] Preferably, the N-terminal and / or C-terminal amino acid modification of the nanobody includes:

[0103] Method 1: Tagging the N-terminal and / or C-terminal amino acids of the nanobody;

[0104] Method 2: After tagging the N-terminal and / or C-terminal amino acids of the nanobody, the tag is further connected to protect the amino acids via a hinge;

[0105] Preferably, the tag includes at least one of His-tag, GST-tag, Myc-tag, SUMO-tag, Strep-tag, and Flag-tag; the hinge includes at least one of GS hinge, IgG hinge, IgA hinge, and PEG; and the protected amino acid includes Ala, Gln, Glu, Met, or any combination of two or more of the aforementioned amino acids.

[0106] Thirdly, the present invention provides a polypeptide obtained by multivalent synthesis of the aforementioned nanobody.

[0107] Fourthly, the present invention provides a nucleic acid that encodes the aforementioned nanobody or the aforementioned polypeptide.

[0108] Fifthly, the present invention provides an expression vector comprising the expression frame of the nucleic acid described above.

[0109] In a sixth aspect, the present invention provides a host cell containing the expression vector described above.

[0110] In a seventh aspect, the present invention provides the application of the nanobodies and / or the peptides described herein in immunoassay, enrichment and / or purification.

[0111] Preferably, the nanobody and / or the polypeptide are used in the preparation of myoglobin adsorbents, myoglobin purification kits, and myoglobin detection kits.

[0112] Example

[0113] The following examples illustrate specific implementations of the present invention. However, the implementation of the present invention is not limited to these examples, and any selections and modifications can be made within the scope of the technical effects to be achieved by the present invention.

[0114] Example 1: Construction of an anti-myoglobin nanobody library.

[0115] The phage display library used in this invention is an immune library based on T7 phage, and the establishment steps are as follows:

[0116] (1) Alpacas (numbered 2508-1 and 2508-2) were immunized with human myoglobin. After four immunizations, jugular vein blood was collected from the two alpacas, peripheral blood lymphocytes were isolated, and total RNA (PuerLink) was extracted. TM RNA Mini Kit, Life Technologies: 12183018A);

[0117] (3) Total RNA was reverse transcribed into cDNA, and V was amplified using two rounds of nested PCR. HH Gene;

[0118] The first round of PCR used cDNA as a template, with UP primer1 and DOWN primer1 as upstream and downstream primers, respectively. After amplification, a band of 650-750 bp was recovered. This band was then used as the template for the second round of PCR, with UP primer2 and DOWN primer2 as upstream and downstream primers, respectively. A PCR product of 450-500 bp was recovered.

[0119] UP primer1:CTTGGTGGTCCTGGCTGCTCT,

[0120] DOWN primer1:GGTACGTGCTGTTGAACTGTTCC,

[0121] UP primer2:TATCTAGTCGAATTCCGCCCAGGTGCAGCTC,

[0122] DOWN primer2: AGCGACTAAGCTTTGAGGAGACGGTGAC;

[0123] (3) The PCR product was digested with EcoRI and HindIII and subjected to agarose gel electrophoresis. The gene band of 350-500 bp was recovered, which is V. HH Gene fragments;

[0124] (4) Ligate the T7 vector (T7Serelect® 10-3 Cloning Kit, MeterckMetillipore Novagen®: 70550-3) and V using T4 ligase. HH Gene fragments;

[0125] (5) The ligation product is mixed with the packaging protein to form a complete T7 phage. The mixture is then amplified to obtain the original phage library.

[0126] (6) The titer of the original library was found to be 8.65 × 10⁻⁶. 9 pfu / mL, diversity was 6.4 × 10 6 .

[0127] Example 2: Screening and sequencing of nanobodies.

[0128] Pigs, dogs, and other animals are often used as medical experimental models because their myoglobin structure is highly similar to that of humans. In order to obtain nanobodies that can bind to all three species simultaneously, this invention uses myoglobin from three species (human, pig, and dog) – Mb-human, Mb-pig, and Mb-dog – for three rounds of antibody screening.

[0129] First, the antigen (Mb-human) was diluted to 10 μg / mL with TBS, and 100 μL was added to a 96-well plate and incubated at 4°C for 12 h. The antigen dilution was aspirated from the wells, the plate was washed 3 times with TBS, blotted dry, and 300 μL of 1% protein-free blocking buffer (purchased from Sangon Biotech Co., Ltd.) was added to each well. The plate was incubated at room temperature for 2 h (1% protein-free blocking buffer and 1% BSA were used alternately during screening). The blocking agent was aspirated from the wells, the plate was washed 6 times with TBST, blotted dry, and 100 μL of amplified phage was added to each well. The plate was incubated at room temperature for 30 min. The plate was washed 10 times with TBST, and the phage was eluted with T7 elution buffer (1% SDS). The plate was incubated at room temperature for 30 min, and the elution buffer was amplified for the next round of screening.

[0130] The antigen (Mb-pig) was then diluted and coated in the same way, and the above elution solution was incubated, panned, and amplified again for the next round of screening.

[0131] The antigen (Mb-dog) was diluted and coated in the same way, and the above eluent was incubated, panned, and amplified again.

[0132] After three rounds of screening, the screening eluent was subjected to solid amplification. Forty-eight phage plaques were randomly selected and inoculated into a deep-well plate containing 1 mL of host bacteria for phage amplification (numbered according to position). The amplification supernatant containing phages was collected by centrifugation.

[0133] Three antigens (Mb-human, Mb-pig, and Mb-dog) at a hydrophobic concentration of 1 µg / mL, along with a negative control (uncoated), were hydrophobically coated onto ELISA plates. The plates were blocked with 3% BSA, and 100 µL of phage-containing amplification supernatant was added to each. After incubation, the plates were washed 10 times with TBST, and anti-V antibodies were added. HH HRP IgG was incubated, washed 5 times with TBST, and then ELISA working solution was added for color development. OD was measured. 450 The ratio of the test wells to the negative control wells is calculated. Generally, a ratio >10 is considered positive (indicating significant binding to the corresponding antigen). A larger ratio qualitatively reflects stronger binding ability or higher affinity. Results are as follows... Figure 1 As shown.

[0134] Figure 1 The results showed that five of the monoclonal antibodies, C1, B2, E2, D5, and B6, strongly bound to Mb-human, Mb-pig, and Mb-dog (ratio > 30); while the remaining clones showed positive binding to only one or two of the antigens, or no obvious binding to any of the three antigens.

[0135] Clones exhibiting significant binding (positive ratio >10) among Mb-human, Mb-pig, and Mb-dog will be sequenced using the following method:

[0136] Using plaque amplification solution as a template, and UP primer3 and DOWN primer3 as upstream and downstream primers, PCR amplification was performed.

[0137] UP primer3:TTCCTTAACATATGGCCCAGGTGCAGCTCGT,

[0138] DOWN primer3: TTAAGGAACTCGAGCACGGTGACCAGGGTC;

[0139] A portion of the PCR products were sequenced externally to obtain the nanobody sequence information. Analysis of the sequencing results revealed that the CDR3 sequences of monoclonal antibodies C1, B2, E2, D5, and B6 were identical, while the remaining regions showed high homology but were not entirely identical, conforming to the definition of homologous antibodies in this field. These were renamed B01-B05. The nanobody naming and CDR sequence number information are shown in Table 3. B01 and B02 have the same CDR sequence, differing only in their FR sequences.

[0140] Table 3. Comparison of Nanobody Nomenclature and CDR Serial Number Information

[0141]

[0142] SEQ ID No. 1:

[0143] QLQESGGGSVQPGGSLRLSCAAS-GFTTSDYG-MNWFRQAPGKQREFVSS-ITRGGDWT-TYADSVKGRSTISRNDAKNTMYLQLNSLKTEDTAMYYC-TKILSYGKP-WGQGTQVTVSS

[0144] SEQ ID No. 2:

[0145] QLQESGGGSVQPGGSLRLSCAAS-GFTTSDYG-MNWFRQAPGKQREFVAT-ITRGGDWT-TYADSVKGRSTISRDNAKNTMYLQLNSLKTEDTAMYYC-TKILSYGKP-WGQGTQVTVSS

[0146] SEQ ID No. 3:

[0147] QLQESGGGSVQPGGSLRLSCAAS-GFTFSDYG-MNWFRQAPGKQREFVST-INNVGDWT-TYADSVKGRFTISRDNAKNMLYLQLNSLKTEDTAMYYC-TKILSYGKP-PGQGTQVTVSS

[0148] SEQ ID No.4:

[0149] QLQESGGGSVQPGGSLRLSCAAS-GTFFSEYG-MNWFRQAPGKQREWVST-INNVGDWT-TYADSVKGRFTISRDNAKNMLYLQLNSLKTEDTAMYYC-TKILSYGKP-PGQGTQVTVSS

[0150] SEQ ID No. 5:

[0151] QLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWFRQAPGKQREFVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYC-TKILSYGKP-PGQGTQVTVSS

[0152] Example 3: Construction of genetically engineered bacteria and preparation of antibodies.

[0153] (a) Construction method of genetically engineered bacteria: Another part of the PCR product was double digested with NdeI and XhoI, and the digested products were recovered. At the same time, the digested products and vector were recovered by the same method. The digested products and vector were ligated with T4 ligase, and the ligation product was transformed into Escherichia coli to obtain genetically engineered bacteria expressing myoglobin-specific nanobodies.

[0154] (II) Antibody preparation methods:

[0155] (1) The basic culture medium for nanobodies is TB medium. The inoculum is 5% and cultured at 37℃ for 3-5 h. The inducing agent galactoside (IPTG) (final concentration 0.25 mM, the same below) is added for overnight induction.

[0156] (2) After induction, centrifuge at 4000 rpm for 20 min to obtain wet bacteria containing nanobodies;

[0157] (3) Add lysis buffer (10mM imidazole, 500mM NaCl, pH7.4 0.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700bar high-pressure homogenizer to disrupt the cells;

[0158] (4) Centrifuge at 4℃ and 10000 rpm for 20 min and collect the supernatant;

[0159] (5) The supernatant was filtered through a 0.45 μm filter and then purified by affinity chromatography (GE Healthcare, US) to separate Mb nanobodies. The packing material of the affinity chromatography column was Ni Sepharose High Performance.

[0160] (6) The nanobody purified by affinity chromatography was subjected to SDS-PAGE electrophoresis to determine its purity and molecular weight, and the protein concentration was determined by BCA method.

[0161] Example 4: Analysis of the binding ability of nanobodies to Mb using SPR technology.

[0162] Mb was amino-coupled to the CM5 sensor chip at a density of 500–800 RU. Nanobodies were injected at seven different concentrations ranging from 1–100 nM, with a flow rate of 45 μL / min in all experiments. Chip regeneration conditions were glycine-HCl pH 1.5. The kinetic parameter K was calculated using binding curves obtained at different nanobodies concentrations. a K d and K D . Figure 2 The curves, from top to bottom, represent the response curves of the nanobody at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. The colored lines represent the response curves at different concentrations, and the black line is the fitted line. The kinetic parameters, as shown in Table 4, were calculated using equation fitting. The nanobody exhibits high affinity for Mb-human, Mb-pig, and Mb-dog. K D The range is all within 10 -9 (M).

[0163] Table 4 Kinetic parameters of nanobodies for different Mb

[0164]

[0165] Example 5: Calculation of key amino acids.

[0166] Molecular docking was performed with sequences B01-B05 and Mb (human) protein to predict V on Mb. HH The antigen-binding epitopes and key amino acids involved in the interaction were identified. Furthermore, molecules B01-B05 were spatially stacked to verify their structural similarity.

[0167] First, obtain the Mb structure from the database, and then combine the Mb sequence with V. HH After antibody sequence assembly, Alohafold2multimer v3 was used for complex structure prediction and search. Following the generation of the complex structure, Amber was used to perform a Relax Process to optimize the side chain structure. AutoDock Tools were used to check hydrogen atoms and calculate potentials, and Ligplot+ was used to analyze V. HH The hydrogen bonding and hydrophobic interaction network at the CDR region amino acid-Mb binding interface was examined, and the atomic contacts between structural domains were investigated. A reasonable conformation meeting the above conditions was selected as V. HH Structural model of the antibody-Mb protein complex.

[0168] V was calculated through molecular docking. HH The binding epitopes of Mb: B01, B02, B03, B04, and B05 all involve antigen recognition and binding via K96, L98, S99, Y100, and K102 on CDR3. Their recognition epitopes are E109, D126, N132, K133, and E136 of Mb. Specifically, K96 and L98 recognize and bind to Mb's E109; L98 and S99 jointly bind and recognize Mb's N132; Y100 recognizes and binds to Mb's E136 and K133 respectively; and K102 recognizes and binds to Mb's D126.

[0169] Molecular docking results as follows Figure 3-7 As shown, where, Figure 3 Image a shows the molecular docking diagram and a partial magnified view of the nanobody BO1 and Mb protein in Example 4 of this invention. To further clarify each point and the docking relationship, Figure 3 Figures b and c show magnified portions of the molecular docking diagram from different angles; Figure 4 Image a shows the molecular docking diagram and a magnified view of the nanobody BO2 with the Mb protein. Figure 4 Figures b and c show magnified portions of the molecular docking diagram from different angles; Figure 5 Image a shows the molecular docking diagram and a magnified view of the nanobody BO3 with the Mb protein. Figure 5 Figures b and c show magnified portions of the molecular docking diagram from different angles; Figure 6 Image a shows the molecular docking diagram and a magnified view of the nanobody B04 with the Mb protein. Figure 6 Figures b and c show magnified portions of the molecular docking diagram from different angles; Figure 7 Image a shows the molecular docking diagram and a magnified view of the nanobody B05 with the Mb protein. Figure 7 Figures b and c show magnified portions of the molecular docking diagram from different angles.

[0170] V is visible HHThe main amino acid residues involved in antigen recognition and binding on (purple) are K96, L98, S99, Y100 and K102 on CDR3 (blue), which bind to E109, D126, N132, K133 and E136 on Mb (gray) through electrostatic interactions and hydrogen bonding interactions; CDR1 (pink) and CDR2 (green) of nanobodies B01-B05 do not participate in antigen recognition.

[0171] Figure 8 This is a schematic diagram of the interaction between antigen and antibody molecules. Red represents electrostatic interaction, and blue represents hydrogen bonding.

[0172] The interactions on the interface can be divided into three cores, specifically including:

[0173] 1) K96 and L98 on nanobody CDR3 recognize and bind to E109 of Mb, and L98 and S99 on nanobody CDR3 jointly bind to and recognize N132 of Mb.

[0174] 2) Y100 on the nanobody CDR3 recognizes and binds to E136 and K133 of Mb, respectively;

[0175] 3) K102 on the nanobody CDR3 recognizes and binds to D126 of Mb.

[0176] To verify the structural similarity of the five nanobodies, spatial superposition analysis was performed. First, the three-dimensional structures of five candidate sequences (B01-B05) were predicted using AlphaFold 3 (AF3). Then, using B01 as a reference template, the remaining four structures were sequence aligned and spatially superimposed. The results showed that the frame regions (FRs) of the five nanobodies, as well as the complementarity-determining regions CDR1, CDR2, and CDR3, which are mainly involved in antigen binding, exhibited extremely high overlap. Only a small angular difference was observed in some flexible, freely rotatable loop regions, indicating that the overall three-dimensional structures of these five nanobodies were highly consistent.

[0177] The result of molecular superposition is as follows Figure 9 As shown, a) is an overall superposition diagram of the five nanobodies, showing that the three-dimensional structures of each molecule are almost completely overlapping. It is clearly shown that only the flexible loop regions of CDR1 and CDR2 have slight angular changes, and the core conformation trends remain consistent, while CDR3 is almost completely overlapping. b) is an overall superposition diagram from another perspective, which also clearly shows that the CDR3 region is almost completely overlapping, and the CDR1 and CDR2 regions have slight angular changes only in the flexible loop regions.

[0178] The analysis results show that the overall three-dimensional structures of the five nanobodies B01-B05 are highly homologous and their core conformations are highly consistent. The conformational conservation of their framework region and key antigen-binding regions (CDR1, CDR2, CDR3) indicates that the five nanobodies have similar structural bases and only have minor non-functional structural differences due to molecular flexibility. This suggests that the five nanobodies are likely to have similar antigen-binding modes and biological functional characteristics.

[0179] The molecular superposition results, combined with the kinetic and molecular docking results in this embodiment, show that the five nanobodies B01-B05, belonging to the same Mb group, have the same affinity, K D The range is all within 10 -9 (M); The five nanobodies B01-B05 all participate in antigen recognition and binding via K96, L98, S99, Y100, and K102 on CDR3. This indicates that the five nanobodies B01-B05 are homologous antibodies, sharing the same antigenic epitopes, similar structures, and comparable affinity.

[0180] Example 6: Humanization of nanobodies and determination of affinity.

[0181] 1. Sequence Design

[0182] (1) FR site-directed mutation

[0183] V HH It shares high homology with the VH domain of human IgG. The core difference between the two lies in the presence of four amino acid mutations in the FR2 domain (according to the Kabat numbering system: V37F, G44E, L45R, W47G). In this embodiment, re-plucking these four mutation sites back into the VH domain-preferred residues of human IgG can significantly reduce V... HH Immunogenicity. The mutated sequences were renamed HB01-HB05, with sequence numbers as shown in SEQ ID No. 25 to SEQ ID No. 29.

[0184] (2) CDR region transplantation

[0185] In addition to the site-directed mutagenesis method mentioned above, conventional humanized nanobody backbones or highly stable nanobody backbones in this field can also be used to complete the humanization and stability modification of nanobodies through CDR region transplantation.

[0186] This invention selects two nanobody backbones for modification, namely the ah and com backbones. Wherein:

[0187] The ah backbone is a universal, fully humanized backbone, derived from the literature (Chi, XJ et al. Humanized single domain antibodies neutralize SARS-CoV-2 by targeting the spike receptor binding domain. Nature Communications 11, doi:10.1038 / s41467-020-18387-8 (2020).).

[0188] com is a highly stable and highly expressive general-purpose scaffold, as described in the literature (Ferrari, D., Garrapa, V., Locatelli, M. & Bolchi, A. A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries. Molecular Biotechnology 62, 43-55, doi:10.1007 / s12033-019-00224-z (2020).).

[0189] The sequences modified based on the ah skeleton were renamed ah01, ah03, ah04, and ah05, as shown in SEQ ID No. 30 to SEQ ID No. 33; the sequences modified based on the com skeleton were renamed com01, com03, com04, and com05, as shown in SEQ ID No. 34 to SEQ ID No. 37.

[0190] 2. Nanobody Preparation

[0191] The above-mentioned humanized nanobody sequence was synthesized outsourced, and the process of constructing genetically engineered bacteria and preparing antibodies was as described in Example 3.

[0192] 3. Nanobody Affinity Assay

[0193] The binding affinity of the humanized nanobody to Mb was analyzed using SPR technology, and the experimental procedure was as described in Example 4. The kinetic parameters, as shown in Table 5, were calculated through equation fitting. Compared with the original nanobody sequence, the affinity of the humanized nanobody to the target antigen Mb was not significantly reduced, and its binding activity to this antigen remained on the same order of magnitude (10⁻⁶) as the original sequence. -9 It maintains good antigen-binding ability.

[0194] Table 5 Kinetic parameters of humanized nanobodies for Mb

[0195]

[0196] SEQ ID No. 25:

[0197] QLQESGGGSVQPGGSLRLSCAAS-GFTTSDYG-MNWVRQAPGKGLEWVSS-ITRGGDWT-TYADSVKGRSTISRNDAKNTMYLQLNSLKTEDTAMYYC-TKILSYGKP-WGQGTQVTVSS;

[0198] SEQ ID No. 26:

[0199] QLQESGGGSVQPGGSLRLSCAAS-GFTTSDYG-MNWVRQAPGKGLEWVAT-ITRGGDWT-TYADSVKGRSTISRDNAKNTMYLQLNSLKTEDTAMYYC-TKILSYGKP-WGQGTQVTVSS;

[0200] SEQ ID No. 27:

[0201] QLQESGGGSVQPGGSLRLSCAAS-GFTFSDYG-MNWVRQAPGKGLEWVST-INNVGDWT-TYADSVKGRFTISRDNAKNMLYLQLNSLKTEDTAMYYC-TKILSYGKP-PGQGTQVTVSS;

[0202] SEQ ID No. 28:

[0203] QLQESGGGSVQPGGSLRLSCAAS-GFTFSEYG-MNWVRQAPGKGLEWVST-INNVGDWT-TYADSVKGRFTISRDNAKNMLYLQLNSLKTEDTAMYYC-TKILSYGKP-PGQGTQVTVSS;

[0204] SEQ ID No. 29:

[0205] QLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWVRQAPGKGLEWVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYC-TKILSYGKP-PGQGTQVTVSS;

[0206] SEQ ID No.30:

[0207] QLVESGGGLVQPGGSLRLSCAAS-GFTTSDYG-GWFRQAPGKGLEAVAA-ITRGGDWT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-TKILSYGKP-WGQGTLVTVSS;

[0208] SEQ ID No.31:

[0209] QLVESGGGLVQPGGSLRLSCAAS-GFTFSDYG-GWFRQAPGKGLEAVAA-INNVGDWT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-TKILSYGKP-WGQGTLVTVSS;

[0210] SEQ ID No.32:

[0211] QLVESGGGLVQPGGSLRLSCAAS-GFTFSEYG-GWFRQAPGKGLEAVAA-INNVGDWT-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-TKILSYGKP-WGQGTLVTVSS;

[0212] SEQ ID No.33:

[0213] QLVESGGGLVQPGGSLRLSCAAS-GFTTSNTY-GWFRQAPGKGLEAVAA-ITRGGEST-YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-TKILSYGKP-WGQGTLVTVSS;

[0214] SEQ ID No.34:

[0215] QLQESGGGLVQAGGSLRLSCAAS-GFTTSDYG-GWFRQAPGKEREFVAA-ITRGGDWT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-TKILSYGKP-WGQGTQVTVSS;

[0216] SEQ ID No. 35:

[0217] QLQESGGGLVQAGGSLRLSCAAS-GFTFSDYG-GWFRQAPGKEREFVAA-INNVGDWT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-TKILSYGKP-WGQGTQVTVSS;

[0218] SEQ ID No. 36:

[0219] QLQESGGGLVQAGGSLRLSCAAS-GTFFSEYG-GWFRQAPGKEREFVAA-INNVGDWT-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-TKILSYGKP-WGQGTQVTVSS;

[0220] SEQ ID No. 37:

[0221] QLQESGGGLVQAGGSLRLSCAAS-GFTTSNTY-GWFRQAPGKEREFVAA-ITRGGEST-YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC-TKILSYGKP-WGQGTQVTVSS;

[0222] Note: 1. The “” between FR and CDR is a connector used to distinguish between FR and CDR, and there is no vacancy site.

[0223] Example 7: Modification of multivalent antibodies and determination of affinity.

[0224] In antigen detection or affinity adsorption applications, the higher the binding affinity between an antibody and the target antigen, the better its detection sensitivity, adsorption specificity, and practical application performance. Preparing multivalent nanobodies is an effective technique for enhancing antibody binding affinity. Multivalent nanobodies can be constructed based on the same or different epitopes, including identical nanobodies targeting the same antigen epitope, different nanobodies targeting the same antigen epitope, and nanobodies targeting different epitopes of the antigen. Preparation methods include intracellular or extracellular chemical conjugation, and nanobodies can also be expressed in tandem through genetic engineering to construct bivalent, trivalent, and higher valence states of multivalent nanobodies.

[0225] This embodiment uses the B05 nanobody as a base and selects two conventional flexible short peptide linkers in the art to construct two bivalent nanobodies targeting the same antigenic epitope through genetic engineering. These nanobodies are named bNb-01 and bNb-02, respectively, and their sequences are shown in SEQ ID No. 38 and SEQ ID No. 39.

[0226] SEQ ID No. 38:

[0227] QLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWFRQAPGKQREFVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYC-TKILSYGKP-PGQGTQVTVSSGGGGSGG GGSGGGGSAQLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWFRQAPGKQREFVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYCTKILSYGKP-PGQGTQVTVSS

[0228] SEQ ID No. 39:

[0229] QLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWFRQAPGKQREFVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYC-TKILSYGKP-PGQGTQVTVSSGSAGSAA GSAAGSGEFQLQESGGGSVQPGGSLRLSCAAS-GFTTSNTY-MNWFRQAPGKQREFVST-ITRGGEST-DYADSVKGRSTISRDNVKNMLYLQLNSLKPEDTAVYYCTKILSYGKP-PGQGTQVTVSS

[0230] The DNA sequences of these bivalent peptides were artificially synthesized, and the DNA fragments were ligated into the pET23a vector to construct bivalent peptide plasmids, which were then transformed into E. coli to obtain bivalent peptide engineered bacteria.

[0231] The bivalent peptide was then expressed and purified.

[0232] (1) The basic culture medium for the divalent polypeptide is TB medium. The inoculum is 5% by volume. The culture is carried out at 37℃ for 3-5 hours. The induction agent galactoside (IPTG) (final concentration 0.25 mM) is added for overnight induction.

[0233] (2) After induction, centrifuge at 4000 rpm for 20 min to obtain wet bacteria containing divalent polypeptides;

[0234] (3) Add lysis buffer (10mM imidazole, 500mM NaCl, pH7.4 0.02M PB) to the obtained wet bacteria at a ratio of 1:10, and use a 700bar high-pressure homogenizer to disrupt the cells;

[0235] (4) Centrifuge at 4℃ and 10000 rpm for 20 min and collect the supernatant;

[0236] (5) The supernatant was filtered through a 0.45 μm filter and then purified by affinity chromatography (GE Healthcare, US) using a Ni Sepharose High Performance column.

[0237] (6) The purity of the nanobody purified by affinity chromatography was determined by SDS-PAGE electrophoresis. The protein concentration was determined by BCA method for protein solutions with high purity.

[0238] The binding affinity of divalent peptides to Mb was analyzed using SPR technology: Mb was amino-coupled to a CM5 sensor chip at a density of 500-800 RU, and nanobodies were injected at five different concentrations ranging from 1-50 nM, with a flow rate of 45 μL / min in all experiments. Chip regeneration conditions were glycine-HCl pH 1.5. The binding curves obtained at different nanobodies concentrations were used to calculate the kinetic parameters Ka, Kd, ​​and KD. Specific data are shown in Table 6.

[0239] Table 6 Kinetic parameters of bivalent nanobodies against Mb

[0240]

[0241] Therefore, compared with monovalent nanobodies, the affinity of bivalent nanobodies to antigen Mb is increased by an order of magnitude. This is mainly manifested in the fact that the binding constant does not change significantly, but the dissociation constant is greatly reduced. This indicates that the bivalent antibody does not affect antigen recognition and capture, but can significantly slow down antigen dissociation, ultimately increasing the overall affinity by an order of magnitude.

[0242] Example 8: End modification of nanobody.

[0243] In practical applications, antibodies or antibody fragments can not only modify their own structure, but also be coupled with other functional elements through fusion / tandem expression, post-translational modification or chemical modification, thereby expanding their functional applications.

[0244] The above-mentioned modification and expression strategies include, but are not limited to: fusion or tandem expression of purification tags such as His6 tags; fusion or tandem expression of biotinylated tags and biotin labeling through intracellular or in vitro post-translational modifications; and biotinylation and HRP labeling through chemical modifications.

[0245] The method for end-modifying the nanobodies in this invention is as follows:

[0246] Method 1: Biotinylation labeling via tandem expression of a biotinylated tag. A biotinylated tag (amino acid sequence GLNDIFEAQKIEWHE) is tandemly attached to one end (preferably the C-terminus) of the antibody to be labeled. A linker peptide of appropriate length (preferably GGGGS) can be optionally inserted between the antibody and the tag. After codon optimization of the recombinant gene, the recombinant gene is prepared using a whole-gene synthesis method, an expression strain is constructed, and the antibody carrying the biotinylated tag is purified to obtain the antibody to be labeled. Subsequently, the antibody is biotinylated using the Avi-tagged protein biotinylation kit (BirA method) (Beyotime, catalog number P0630M) to obtain the target labeled antibody. The labeled antibody is ultrafiltered to replace 1×PBS buffer, the protein concentration is adjusted to 2 mg / mL, an equal volume of glycerol is added and mixed, and then stored at -20℃.

[0247] Method 2: Biotinylation labeling of nanobodies via chemical modification. The antibody to be labeled was replaced with 10 mM NaHCO3 buffer, and the protein concentration was adjusted to 2 mg / mL. Five molar amounts of biotin NHS ester were added, and the mixture was gently pipetted to mix. The reaction system was incubated at 37°C in the dark for 30 min. Subsequently, the product was ultrafiltered to 1×PBS buffer, the protein concentration was adjusted to 2 mg / mL, an equal volume of glycerol was added, and the mixture was stored at -20°C.

[0248] Method 3: HRP labeling of nanobodies via chemical modification. Labeling was performed according to the instructions of the HRP rapid labeling kit (GA method) (Beyotime, catalog number P1311S).

[0249] Example 9: HRP-labeled antibody competitive ELISA method for detecting human myoglobin.

[0250] The labeled antibody used in this method can be any anti-human myoglobin (Mb) antibody, including the nanobody described in this invention. In this embodiment, nanobody B05 is selected and labeled according to the HRP chemical modification labeling method described in Example 8. The labeled antibody is designated as B05-HRP and aliquoted for storage.

[0251] (1) Antigen coating: Dilute human myoglobin (Mb) to 5 µg / mL with 1×PBS buffer, add 100 µL of diluted antigen solution to each well of a highly absorbent transparent microplate, and incubate overnight at 4°C; the next day, discard the coating solution in the wells, wash the microplate 3 times with 1×PBST washing solution, and pat dry the residual liquid at the bottom of the plate after each wash.

[0252] (2) Blocking: Add 300 µL of 3% BSA blocking solution (prepared with 1×PBS buffer) to each well of the microplate and incubate at room temperature for 2 hours; discard the blocking solution, wash 3 times with 1×PBST washing solution, and pat dry for later use.

[0253] (3) Competitive binding incubation: B05-HRP was diluted to 1 µg / mL with 1×PBS; human myoglobin was diluted to 800 ng / mL with 1×PBS, and then serially diluted 2-fold, for a total of 7 gradients as standard curve points; 100 µL of human myoglobin at each concentration gradient was taken and gently mixed with an equal volume of 1 µg / mL B05-HRP by pipetting, and incubated at room temperature for 10 min. The test sample was diluted to a suitable concentration with 1×PBS in advance, and an equal volume of 1 µg / mL B05-HRP was added, and gently mixed by pipetting, and incubated at room temperature for 10 min. 100 µL of human myoglobin or the test sample at different concentration gradients after incubation was added to each well, and incubated at room temperature for 1 hour.

[0254] (4) Washing and removing impurities: Discard the reaction solution in the wells, wash the microplate three times with 1×PBST washing solution, pat dry the residual liquid, and remove unbound free proteins and impurities.

[0255] (5) Enzymatic colorimetric reaction: Add 100 µL of TMB working solution and incubate at 37°C for 30 min.

[0256] (6) Termination of reaction: Add the stop solution (such as 2M H2SO4) to each well, gently shake to mix, and terminate the enzymatic colorimetric reaction.

[0257] (7) Signal detection and result analysis: The absorbance (OD) of each well at a wavelength of 450 nm was measured using a microplate reader. 450 Based on the negative correlation between OD value and antibody concentration, a standard curve is plotted, and the concentration of the sample to be tested is calculated.

[0258] Standard curve such as Figure 10 As shown, the results indicate that the standard curve fits well, with the equation y=(AD) / [1+(x / C)^B]+D, where A=2.63921, B=1.26332, C=154.66450, D=0.07226, and the coefficient of determination R0 is 1.26332. 2 The value is >0.99, and the method limit of quantitation is higher than 12.5 ng / mL, which meets the detection requirements.

[0259] Example 10: Immunohistochemical analysis of HRP-labeled antibody.

[0260] A crush syndrome model was constructed using experimental pigs. The pigs were sacrificed 48 hours after modeling, and kidney tissue from the corticomedullary junction was collected as the test sample. The tissue samples were embedded in paraffin and then prepared into serial sections of 1cm×1cm. HE staining and HRP-labeled B05 anti-myoglobin nanobody (B05-HRP) immunohistochemical staining were performed. The expression and localization of myoglobin in the kidney tissue were observed under an optical microscope.

[0261] Experimental results are as follows Figure 11 As shown: Microscopic examination reveals numerous casts within the renal tubules. Brownish-yellow to brownish-red positive and strongly positive casts are visible within the distal convoluted tubules and collecting ducts, identified as positive myoglobin casts. This demonstrates that the HRP-labeled nanobody of the present invention can specifically bind to myoglobin in tissue samples, with clear colorimetric signals and low background interference, making it suitable for immunohistochemical detection.

[0262] Example 11: Preparation and evaluation of anti-myoglobin nanobody adsorbent.

[0263] In patients with rhabdomyolysis, crush syndrome, and other similar conditions, the concentration of myoglobin in the blood can rise sharply, soaring from normal levels (<100 µg / L) to >10 mg / L. This massive influx of myoglobin into the bloodstream exceeds the reabsorption threshold of the kidneys, forming casts within the renal tubules and directly causing tubular obstruction. Simultaneously, the breakdown of myoglobin in the acidic urine environment generates free radicals, which can directly damage renal tubular epithelial cells, triggering acute tubular necrosis and leading to a rapid deterioration of renal function (i.e., myoglobinuric nephropathy).

[0264] For these patients, emergency dialysis is necessary to rapidly remove excess myoglobin from the bloodstream in order to protect renal function. The following is a brief example of a method for blood purification in patients with hypermyoglobinemia by conjugating anti-myoglobin antibodies to the matrix surface:

[0265] (1) Activation of agarose gel.

[0266] Take several agarose gel microspheres, wash them thoroughly with purified water to remove ethanol and other substances, remove excess water, and filter them into a wet cake for later use. Weigh 10 mL of the washed agarose gel microspheres, add 15 mL of sodium hydroxide solution and 8 mL of 1,4-butanediol diglycidyl ether, and stir the reaction for at least 60 min. After the activation reaction is complete, wash the solid material with plenty of purified water until it is clean, remove excess water, and filter it into a wet cake for later use.

[0267] (2) Immobilization of nanobodies.

[0268] 10 mL of epoxy-activated agarose gel microspheres were added to 30 mL of nanobody (BO5) solution, and the mixture was stirred at 37°C and 150 rpm for at least 24 h. After the reaction, the solid material was washed thoroughly with purified water until clean, thus completing the preparation of the myoglobin adsorbent. The nanobody loading was calculated to be 9.8 mg / mL gel using the differential method.

[0269] The novel dynamic adsorption evaluation method for the prepared myoglobin adsorbent is as follows:

[0270] (1) Preparation of simulated samples: human myoglobin (Mb) was dissolved in bovine serum to prepare simulated high myoglobin serum samples with final concentrations of 1 mg / L, 10 mg / L, 50 mg / L and 100 mg / L.

[0271] (2) Adsorption system setup: Weigh 1 g of myoglobin adsorbent and fill it into the adsorption column shell; take 100 mL of the above-mentioned simulated serum samples of various concentrations and place them in beakers respectively, and maintain the homogeneity of the samples by magnetic stirring.

[0272] (3) Dynamic adsorption experiment: A peristaltic pump was used to drive the simulated serum sample through the adsorption column at a flow rate of 20 mL / min, and the effluent was returned to the original stirred beaker (this flow rate corresponds to one cycle of serum every 5 minutes). Samples were taken from the beaker at 0 cycles (initial state), 4 cycles, and 8 cycles, respectively, and the concentration of residual myoglobin in the sample was measured and the myoglobin removal rate was calculated.

[0273] (4) Blank control setting: Blank agarose gel microspheres without anti-myoglobin antibody were used as the control adsorbent. Dynamic adsorption experiments were carried out on simulated serum samples with a concentration of 100 mg / L under the same conditions as above, and the removal rate was measured and calculated simultaneously.

[0274] Experimental results are as follows Figure 12 As shown, even under extreme conditions of a high myoglobin concentration of 100 mg / L, the myoglobin adsorbent prepared in this invention can achieve an 80% myoglobin removal rate after 4 cycles; while the myoglobin removal rate of the blank control adsorbent is only 12%.

[0275] The above results indicate that the significant decrease in myoglobin concentration in simulated serum is due to the specific and efficient adsorption of myoglobin by the target adsorbent, rather than the non-specific adsorption by the matrix material. In summary, the myoglobin adsorbent described in this invention possesses excellent dynamic adsorption performance and can effectively remove high concentrations of myoglobin, making it suitable for blood purification treatment of patients with hypermyoglobinemia caused by diseases such as rhabdomyolysis and crush syndrome.

[0276] Example 12: Myoglobin adsorbent for Mb affinity purification.

[0277] The myoglobin adsorbent of the present invention can also be used for affinity purification of human, porcine, canine myoglobin or recombinant myoglobin.

[0278] The adsorbent was prepared as described in Example 11. The method for preparing recombinant human myoglobin is as follows:

[0279] Based on the codon preference of Escherichia coli, the human myoglobin gene was codon optimized. An NdeI restriction endonuclease site was added to the 5' end of the optimized gene, and a stop codon TAA and an XhoI restriction endonuclease site were added to the 3' end. The modified human myoglobin gene DNA fragment was synthesized by total chemical synthesis.

[0280] The chemically synthesized DNA fragment and pET21a plasmid were digested with NdeⅠ and XhoⅠ, and the digestion products were ligated to construct a recombinant expression plasmid. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformed bacterial culture was plated on LB solid medium plates containing ampicillin (Amp) resistance. After overnight incubation at 37°C, single colonies were picked from the plates to obtain the recombinant human myoglobin Escherichia coli expression strain.

[0281] Pick a single colony and inoculate it into 20 mL of LB liquid medium. Incubate at 37°C and 180 rpm for 12 h using a shaker. Transfer 2 mL of the culture to 200 mL of LB liquid medium and continue incubating at 37°C and 180 rpm for 6 h using a shaker. Add IPTG to the culture system to a final concentration of 5 mM and continue induction culture at 37°C and 180 rpm for 6 h. After incubation, centrifuge to collect the bacterial cells and freeze for later use.

[0282] Purification of recombinant human myoglobin:

[0283] The cells were resuspended in 10 volumes of 1×PBS buffer and the suspension was disrupted by sonication. The disrupted solution was centrifuged at 1000×g and the supernatant was collected, which is the crude extract of recombinant human myoglobin.

[0284] 1 g of adsorbent was packed into a chromatography column, and the crude extract was loaded at a flow rate of 1 mL / min. After loading, the column was equilibrated by washing with 10 volumes of 1×PBS buffer. Elution was performed using 20 mM glycine-hydrochloric acid (Gly-HCl, pH 3.0) buffer, and the eluted fraction was collected to obtain the recombinant human myoglobin sample. The results are as follows: Figure 13 As shown in the elution results 1-3, this adsorbent can purify recombinant human myoglobin with a purity >95% from crude extract in one step.

[0285] Industrial availability

[0286] The nanobody of the present invention is an anti-myoglobin nanobody with a novel amino acid sequence discovered through screening of a phage library. This nanobody and its polypeptide have high affinity and activity, and can specifically recognize and bind to myoglobin. The nanobody prepared by the present invention can be used for the capture and adsorption of myoglobin, and can be applied to immunofluorescence analysis or immunohistochemical analysis through appropriate antibody labeling technology.

[0287] The above description represents a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An anti-myoglobin nanobody, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.15, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

22.

2. The nanobody according to claim 1, characterized in that, The framework region FR of the nanobody includes FR1, FR2, FR3 and FR4 sequences, wherein: the amino acid sequence of FR1 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 6, the amino acid sequence of FR2 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 11, the amino acid sequence of FR3 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 18, and the amino acid sequence of FR4 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO.

23.

3. The nanobody according to claim 2, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO.6, the amino acid sequence of FR2 is shown in SEQ ID NO.11, the amino acid sequence of FR3 is shown in SEQ ID NO.18, and the amino acid sequence of FR4 is shown in SEQ ID NO.

23.

4. The nanobody according to claim 1, characterized in that, The framework region FR of the nanobody includes sequences FR1, FR2, FR3, and FR4, wherein: the amino acid sequence of FR1 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 6; the amino acid sequence of FR2 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 12; the amino acid sequence of FR3 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 19; and the amino acid sequence of FR4 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO.

23.

5. The nanobody according to claim 4, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO.6, the amino acid sequence of FR2 is shown in SEQ ID NO.12, the amino acid sequence of FR3 is shown in SEQ ID NO.19, and the amino acid sequence of FR4 is shown in SEQ ID NO.

23.

6. An anti-myoglobin nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

1.

7. An anti-myoglobin nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

2.

8. An anti-myoglobin nanobody, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.8, the amino acid sequence of CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

22.

9. The nanobody according to claim 8, characterized in that, The framework region FR of the nanobody includes sequences FR1, FR2, FR3, and FR4, wherein: the amino acid sequence of FR1 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 6; the amino acid sequence of FR2 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 13; the amino acid sequence of FR3 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 20; and the amino acid sequence of FR4 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO.

24.

10. The nanobody according to claim 9, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO. 6, the amino acid sequence of FR2 is shown in SEQ ID NO. 13, the amino acid sequence of FR3 is shown in SEQ ID NO. 20, and the amino acid sequence of FR4 is shown in SEQ ID NO.

24.

11. An anti-myoglobin nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

3.

12. An anti-myoglobin nanobody, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

22.

13. The nanobody according to claim 12, characterized in that, The framework region FR of the nanobody includes sequences FR1, FR2, FR3, and FR4, wherein: the amino acid sequence of FR1 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 6; the amino acid sequence of FR2 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 14; the amino acid sequence of FR3 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 20; and the amino acid sequence of FR4 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO.

24.

14. The nanobody according to claim 13, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO. 6, the amino acid sequence of FR2 is shown in SEQ ID NO. 14, the amino acid sequence of FR3 is shown in SEQ ID NO. 20, and the amino acid sequence of FR4 is shown in SEQ ID NO.

24.

15. An anti-myoglobin nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

4.

16. An anti-myoglobin nanobody, characterized in that, The complementarity-determining region (CDR) of the nanobody includes CDR1, CDR2, and CDR3 sequences: The amino acid sequence of CDR1 is shown in SEQ ID NO.10, the amino acid sequence of CDR2 is shown in SEQ ID NO.17, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

22.

17. The nanobody according to claim 16, characterized in that, The framework region FR of the nanobody includes sequences FR1, FR2, FR3, and FR4, wherein: the amino acid sequence of FR1 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 6; the amino acid sequence of FR2 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 13; the amino acid sequence of FR3 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO. 21; and the amino acid sequence of FR4 is an amino acid sequence with more than 50% homology to the amino acid sequence shown in SEQ ID NO.

24.

18. The nanobody according to claim 17, characterized in that, The amino acid sequence of FR1 is shown in SEQ ID NO. 6, the amino acid sequence of FR2 is shown in SEQ ID NO. 13, the amino acid sequence of FR3 is shown in SEQ ID NO. 21, and the amino acid sequence of FR4 is shown in SEQ ID NO.

24.

19. An anti-myoglobin nanobody, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO.

5.

20. A humanized nanobody, characterized in that, The amino acid sequence of the humanized nanobody is shown in any one of SEQ ID NO. 25 to SEQ ID NO.

37.

21. A polypeptide, characterized in that, Includes the nanobody described in any one of claims 1 to 19.

22. A nucleic acid molecule encoding the nanobody according to any one of claims 1 to 19.

23. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 22.

24. A host cell that transforms or transfects the expression vector of claim 23.

25. A combination or coupling, characterized in that, Including nanobodies as described in any one of claims 1 to 19 that have been chemically or biologically labeled.

26. An adsorbent, characterized in that, Including the nanobody according to any one of claims 1 to 19, or the polypeptide according to claim 21, Or the nucleic acid molecule as described in claim 22, or the expression vector as described in claim 23, or the host cell as described in claim 24, Or the combination or coupling as described in claim 25, and the carrier.

27. A reagent kit, characterized in that, Including the nanobody according to any one of claims 1 to 19, or the polypeptide according to claim 21, Or the nucleic acid molecule as described in claim 22, or the expression vector as described in claim 23, or the host cell as described in claim 24, Or the combination or coupling described in claim 25, Or the adsorbent as described in claim 26, and an auxiliary agent acceptable in the detection.

28. A device, characterized in that, For capturing, adsorbing, and / or detecting myoglobin, including nanobodies according to any one of claims 1 to 19, or the polypeptide according to claim 21. Or the nucleic acid molecule as described in claim 22, or the expression vector as described in claim 23, or the host cell as described in claim 24, Or the combination or coupling described in claim 25, Or the adsorbent of claim 26, or the kit of claim 27.

29. The nanobody according to any one of claims 1 to 19, or the polypeptide according to claim 21. Or the nucleic acid molecule as described in claim 22, or the expression vector as described in claim 23, or the host cell as described in claim 24, Or the combination or coupling described in claim 25, Or the adsorbent of claim 26, or the kit of claim 27. Applications in the preparation of formulations for the specific capture, adsorption, and / or detection of myoglobin; or Application in the preparation of cell preparations for the specific capture, adsorption and / or detection of myoglobin; or Applications in the preparation of immunofluorescence or immunohistochemical reagents for the specific capture, adsorption, and / or detection of myoglobin.

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