CN117417450A

CN117417450A - Myeloperoxidase binding proteins, methods of preparation and uses

Info

Publication number: CN117417450A
Application number: CN202311356197.8A
Authority: CN
Inventors: 张晓晴; 徐亮; 曾敏霞
Original assignee: Zhuhai Lihe Medical Diagnosis Products Co ltd
Current assignee: Zhuhai Lihe Medical Diagnosis Products Co ltd
Priority date: 2023-10-18
Filing date: 2023-10-18
Publication date: 2024-01-19

Abstract

The invention provides a myeloperoxidase binding protein, a preparation method and application thereof, and relates to the technical field of antibodies. The myeloperoxidase binding protein comprises a light chain variable region and a heavy chain variable region, wherein the VH-CDR1 is shown as SEQ ID NO.1, the VH-CDR2 is shown as SEQ ID NO.2, and the VH-CDR3 is shown as SEQ ID NO. 3; VL-CDR1 is shown as SEQ ID NO.4, the amino acid sequence of VL-CDR2 is YAS, and VL-CDR3 is shown as SEQ ID NO. 5. The binding protein has good specific binding capacity with myeloperoxidase, and can be used for identifying myeloperoxidase antigen and performing or assisting in performing diagnosis and detection of cardiovascular diseases.

Description

Myeloperoxidase binding proteins, methods of preparation and uses

Technical Field

The invention relates to the technical field of antibodies, in particular to a myeloperoxidase binding protein, a preparation method and application.

Background

Myeloperoxidase (MPO), a heme protease, is present in the eosinophil of myeloid cells (mainly neutrophils and monocytes). External stimuli can cause neutrophils to aggregate, releasing myeloperoxidase. MPO has a relative molecular weight of 150kDa and is a tetramer formed by covalent bonding of two subunits, each of which is composed of a heavy chain alpha (relative molecular weight 60 kDa) and a light chain beta (relative molecular weight 15 kDa).

MPO can kill microorganisms in phagocytes by catalyzing and oxidizing chloride ions to generate hypochlorous acid, destroy various target substances, and play roles in various aspects such as organism generation and regulation of inflammatory reaction. More importantly, oxidative modification of Low Density Lipoproteins (LDL) can cause atherosclerosis, and thus MPO is believed to be involved in the occurrence of cardiovascular disease.

Currently, MPO is considered the most promising cardiovascular marker, and elevated MPO levels in the body are predictive of risk of arteriosclerosis and coronary heart disease, an early warning of myocardial infarction, more sensitive than other indicators such as troponin T, CK-MB and CRP, and earlier diagnosis and risk assessment.

In addition, most of anti-MPO antibodies in the conventional detection kit are human positive serum, but the serum sources are difficult, and the batch-to-batch differences are large; in addition, there are rabbit or mouse monoclonal antibodies produced by hybridoma technology; and purifying polyclonal antibody in immune animal positive serum, coupling human IgG, and has complex operation, long time consumption and high cost.

Therefore, the development of the high-affinity anti-MPO recombinant monoclonal antibody which is simple to obtain, can be produced in a large scale and is widely applied, and particularly can be used in a clinical detection kit, thereby having important significance and value.

In view of this, the present invention has been made.

Disclosure of Invention

The object of the present invention is to provide a myeloperoxidase binding protein, which has a good specific binding ability to myeloperoxidase. Based on the myeloperoxidase binding proteins provided by the invention, the invention also aims at providing applications thereof. It is another object of the present invention to develop myeloperoxidase binding proteins with good binding activity in a more simple manner to operate, which can be used for the identification of myeloperoxidase antigens and to aid in the diagnostic detection of cardiovascular diseases.

In order to solve the technical problems, the invention adopts the following technical scheme:

noun definition:

the term "binding protein" as used herein refers to a protein that binds to a particular antigen, and broadly refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). The protein or protein fragment may be an antibody, "antibody" particularly referring to a full-length antibody. The terms "antibody" and "full length antibody" include polyclonal antibodies and monoclonal antibodies. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the terms "antibodies" and "immunoglobulins" are used interchangeably.

Proteins and protein fragments may also be antigen binding fragments comprising a portion or all of the CDRs of an antibody that lack at least some of the amino acids present in the full-length chain but are still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from but not limited to F (ab') ₂ Fab', fab, fv (consisting of VH and VL), scFv (single chain antibody, wherein VH and VL are linked by a linker peptide), dsFv (disulfide stabilized Fv antibody (disulfide stabilized Fv fragments, dsFv)), bispecific antibody, nanobody, and antibody minimal recognition unit. In addition to the functional fragments described above, any fragment whose half-life has been increased is included.

"variable region" or "variable domain" of a binding protein refers to the domain of an antibody that recognizes and binds an antigen at the amino terminus of the heavy or light chain, the composition and arrangement of the amino acids of the segment determining the specificity of an antibody for recognizing an antigen. The heavy chain variable domain may be referred to as a "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The variable regions of the heavy and light chains each consist of 3 complementarity-determining region (CDRs) (also known as hypervariable regions) linked by 4 Framework Regions (FR). The framework regions and the ranges of the CDRs have been precisely defined, for example in Kabat (see sequence of immunologically important proteins ((Sequences of Proteins of Immunological Interest), E.Kabat et al) and Chothia), any of the CDR determination methods well known in the art, including combinations of methods, can identify the CDRs of the variable domain the CDRs in each chain are held closely together by the FRs to form the variable region, and in general the variable regions VL/VH of the heavy and light chains can be obtained by ligating the CDRs numbered from the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combined arrangement.

The term "constant region" or "constant domain" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination. The heavy chain of an antibody has one variable domain (VH) followed by a number of constant domains or regions, such as one or more of the hinges, CH1, CH2, CH3, and CH4, the CH1 domain being adjacent to the VH domain and amino-terminal to the hinge region of the heavy chain of the antibody, and not forming part of the Fc region of the antibody; the hinge region includes a portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is typically a CH3 domain, which CH3 domain typically forms the C-terminal portion of an antibody, and in some antibody types, the constant region in IgM and IgE, for example, also includes a CH4 domain. The constant regions of antibodies may be derived from IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, as well as subclasses and mutated forms thereof.

The invention is not limited in the manner in which the myeloperoxidase binding proteins are obtained. In some alternative embodiments, the corresponding antibody is obtained upon expression in a cell after ligation to the vector using a polynucleotide encoding a myeloperoxidase binding protein. The above vectors may be introduced into eukaryotic cells, particularly mammalian cells, and constructed to express the myeloperoxidase binding proteins. In other alternative embodiments, the binding protein may also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as an automated peptide synthesizer (e.g., an automated peptide synthesizer sold by Applied BioSystems, etc.); the antigen binding fragments are also optionally produced by enzymatic cleavage of antigen binding molecules (including intact antibodies), such as pepsin or papain cleavage; or chemical cleavage, e.g., by chemical reduction cleavage of disulfide bonds.

The terms "specifically recognizes," "selectively binds," and "specifically binds" or the like refer to the binding of a binding protein to an epitope on a predetermined antigen. Typically, the binding protein is less than about 10 ^-5 M, e.g. less than about 10 ^-5 M、10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M or 10 ^-10 M or less K _d The values are combined. The K of an antibody can be determined using methods well established in the art _d Values. Other standard assays for evaluating the binding capacity of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, western blot, RIA, and flow cytometry analysis.

The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide encodes the MPO binding protein described above, optionally the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. Offspring may differ from primary cells morphologically and/or in genomic DNA by natural, accidental, or deliberate mutations that are not necessarily identical to primary cells. "transformant" and "transformed cell" include primary test cells and cultures derived therefrom.

Herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes polypeptides or nucleic acids that are removed from their original environment, e.g., if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some protein or other cellular component that is normally associated therewith or that is normally admixed therewith or in solution. Isolated polypeptides include naturally produced said polypeptides contained in cell lysates, purified or partially purified forms of said polypeptides, recombinant polypeptides, said polypeptides expressed or secreted by cells, and said polypeptides in heterologous host cells or cultures. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

In order to solve the technical problems, the following technical scheme is provided:

in a first aspect, there is provided a myeloperoxidase binding protein, also referred to herein simply as MPO binding protein, comprising a light chain variable region and a heavy chain variable region;

the heavy chain variable region comprises complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region comprises complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3;

the amino acid sequence of VH-CDR1 is GYTFTSYW (SEQ ID NO. 1);

the amino acid sequence of VH-CDR2 is IHPSDSYT (SEQ ID NO. 2);

the amino acid sequence of VH-CDR3 is TRSTMITSFAMDY (SEQ ID NO. 3);

the amino acid sequence of VL-CDR1 is QSVSND (SEQ ID NO. 4);

the amino acid sequence of VL-CDR2 is YAS;

the amino acid sequence of VL-CDR3 is QQYNSYPYT (SEQ ID NO. 5).

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the MPO binding protein is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 7.

In alternative embodiments, the MPO binding protein is an intact antibody, F (ab') ₂ One of Fab', fab, fv, scFv, dsFv, bispecific antibodies and antibody minimal recognition units.

In alternative embodiments, the species from which the remainder of the sequence of the antibody is derived includes one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

In alternative embodiments, the MPO binding protein is an antibody or antigen binding fragment comprising a constant region.

In alternative embodiments, at least a portion of the constant region sequence of the MPO binding protein is a human consensus constant region sequence.

In alternative embodiments, the constant region of the MPO binding protein may be selected from the group consisting of the sequence of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD, and subclasses and mutant forms thereof. The constant regions may be of a species such as, but not limited to, bovine, equine, dairy cow, porcine, ovine, caprine, rat, mouse, guinea pig, hamster, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.

In alternative embodiments, the MPO binding protein comprises a heavy chain constant region and a light chain constant region;

in alternative embodiments, the heavy chain constant region in the MPO binding protein is of human origin, and further preferably the constant region of human IgG1, including CH1-CH2-CH3. The amino acid sequence of the heavy chain constant region preferably comprises the sequence shown as SEQ ID NO. 8.

In alternative embodiments, the light chain constant region in the MPO binding protein is of murine origin, and the amino acid sequence of the murine light chain constant region preferably comprises the sequence set forth in SEQ ID No. 9.

In alternative embodiments, the MPO binding protein is a human murine chimeric antibody; the heavy chain comprises a heavy chain constant region with an amino acid sequence shown as SEQ ID NO.10 and derived from human, and the light chain comprises a mouse light chain constant region with an amino acid sequence shown as SEQ ID NO. 11.

In a second aspect, there is also provided a biological material comprising a polynucleotide, a vector, a cell; wherein the polynucleotide encodes the MPO binding protein; the vector carries the polynucleotide; the cells carry the polynucleotide, or contain the vector or are capable of expressing the MPO binding protein.

After the polynucleotide encoding the MPO-binding protein is ligated to a vector, the vector may be introduced into eukaryotic cells, particularly mammalian cells, and a cell line capable of expressing the MPO-binding protein is constructed, and the corresponding protein is obtained by expression in the cells.

In some alternative embodiments, the host cell used to express the MPO binding protein is a 293 cell (human kidney epithelial cell line), preferably a 293F cell.

In some alternative embodiments, the host cell used to express the MPO binding protein is a CHO cell (chinese hamster ovary cell).

In a third aspect, there is also provided a method of preparing an MPO binding protein comprising culturing a cell capable of expressing the MPO binding protein.

In an alternative embodiment, the method of preparing further comprises transforming a polynucleotide encoding a protein comprising said MPO binding protein into a host cell and expressing, and obtaining the MPO binding protein by purification.

In alternative embodiments, the MPO binding protein may be obtained by synthesizing a polynucleotide comprising the gene encoding the MPO binding protein, if desired, and/or preparing a suitable expression vector, transforming the expression vector into a desired host cell and expressing the same, and purifying the same, if desired.

In an alternative embodiment, the polynucleotide of the MPO binding protein comprises a heavy chain expression plasmid and a light chain expression plasmid, the heavy chain expression plasmid and the light chain expression plasmid are co-transformed into a host cell, and the MPO binding protein is obtained by purification.

In an alternative embodiment, a complete IgG1 heavy chain expression plasmid is constructed comprising fusing the C-terminal heavy chain variable region to a constant region fragment of human IgG 1.

In alternative embodiments, the constant region segments of human IgG1 include CH1, CH2, and CH3.

In alternative embodiments, the host cell is preferably a eukaryotic cell, more preferably a mammalian cell, and even more preferably a 293 cell or CHO cell.

In alternative embodiments, the 293 cells comprise 293F cells.

In a fourth aspect, there is also provided the use of the MPO binding protein of the first aspect, the biomaterial of the second aspect, in any one of (a) to (d) as follows;

(a) Preparing a reagent or a kit for detecting myeloperoxidase or detecting an autoantibody of myeloperoxidase;

(b) Preparing a reagent or a kit for assisting in diagnosing a disease;

(c) Detection of myeloperoxidase or myeloperoxidase autoantibodies for non-diagnostic and therapeutic purposes;

(d) For purifying myeloperoxidase;

in alternative embodiments, the disease comprises cardiovascular disease and/or a tumor.

In alternative embodiments, the disease includes atherosclerosis, coronary heart disease, myocardial infarction, acute coronary syndrome, lung cancer, and Alzheimer's disease.

In alternative embodiments, the agent is any one of a quality control, a standard, a conjugate, and a labeled composition.

The conjugate comprises the myeloperoxidase binding protein and a solid support, wherein the solid support comprises microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or lateral flow devices; more specifically, for example, but not limited to, an enzyme-labeled well, an immunochromatographic strip, or a magnetic bead. The chromatography matrix may be selected from any chromatography matrix known to be acceptable in the art, including but not limited to polystyrene, polysaccharide polymers, silica gel, or the like. In an alternative embodiment, the chromatography matrix comprises gel particles.

The tagged composition comprises the myeloperoxidase binding protein and a label comprising one or more of an enzyme, a luminescent label, a fluorescent microsphere, a colored microsphere, a latex microsphere, colloidal gold, a quantum dot, biotin, streptavidin, a radionuclide, a radiocontrast agent, a paramagnetic ion, a metal, and a photosensitizer.

In a fifth aspect, there is provided an agent comprising the above MPO binding protein; and/or the above biological material.

The conjugate comprises a conjugate of an MPO binding protein of the first aspect and a solid support comprising a microtube, column, microparticle, nitrocellulose membrane, chromatography matrix, or lateral flow device; more specifically, for example, but not limited to, an enzyme-labeled well, an immunochromatographic strip, or a magnetic bead. The chromatography matrix may be selected from any chromatography matrix known to be acceptable in the art, including but not limited to polystyrene, polysaccharide polymers, silica gel, or the like. In an alternative embodiment, the chromatography matrix comprises gel particles.

The tagged composition comprises an MPO binding protein of the first aspect and a label; the label comprises one or more of enzyme, luminescent label, fluorescent microsphere, color microsphere, latex microsphere, colloidal gold, quantum dot, biotin, streptavidin, radionuclide, radiocontrast agent, paramagnetic ion, metal and photosensitizer.

The MPO binding protein and label in the tagged compositions of the fourth and fifth aspects described above may or may not be linked; when the connection is not needed, the connection is carried out when the connection is needed. The attachment may be, but is not limited to, chemical attachment or physical adsorption.

In alternative embodiments, examples of enzymes in the markers of the fourth and fifth aspects described above may be, for example but not limited to, alkaline phosphatase or horseradish peroxidase, luminescent markers may be, for example but not limited to, fluorescent proteins, synthetic small molecules or polymer dyes, etc., specific examples include, but are not limited to, alexa 350, alexa 405, alexa 430, alexa 488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxy fluorescein, 5-carboxy-2 ',4',5',7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascades Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl violet, light cresyl Blue, para-aminobenzoic acid, erythrosin, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, europium tripyridyl diamine, europium cryptate, or chelate, diamine, procyanidins, and mixtures thereof,La Jolla blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine, and texas red. The fluorescent microspheres, the colored microspheres and the latex microspheres are each independently selected from the group of products acceptable in the art, such as from commercially available products. Radionuclides include, but are not limited to ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ⁹⁴ mTc、 ⁹⁴ Tc、 ⁹⁹ mTc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ⁵² mMn、 ⁵⁵ Co、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ⁸² mRb and is provided with ⁸³ One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

In a sixth aspect, there is also provided a kit comprising the reagents of the fifth aspect.

In alternative embodiments, the kit comprises a detection kit for detecting a myeloperoxidase, comprising a solid support to which the myeloperoxidase-binding protein is coupled, or a tracer-tagged myeloperoxidase-binding protein.

In alternative embodiments, the kit comprises a detection kit for detecting the myeloperoxidase autoantibody, which may comprise a quality control, a standard, a quality control for a kit or a quantification of the myeloperoxidase autoantibody.

In alternative embodiments, the kit comprises a kit for purifying a myeloperoxidase, which may comprise a chromatography matrix to which the myeloperoxidase-binding protein is coupled.

It will be appreciated that the kits provided by the present invention also optionally include reagents and/or consumables well known to those skilled in the art for detecting reactions, such as, but not limited to, one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, wash solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality controls and labels.

In alternative embodiments, the reagents or kits of any of the above aspects may be used in a general immunoassay method acceptable in the art, including but not limited to immunoblotting, immunohistochemistry, ELISA, immunochromatography or immunomagnetic beads, and those skilled in the art may formulate the reagents or other reagents in the kit according to the corresponding detection means, which is not limited in the present invention.

The invention discloses an MPO binding protein capable of specifically recognizing and binding an MPO antigen, and further preparing a human-mouse chimeric recombinant monoclonal antibody. Compared with the prior art, the invention has the following beneficial effects:

(1) The MPO binding proteins disclosed herein can bind MPO efficiently with an EC50 value of about 1nM; can be coupled to a solid phase or a label, and can be used for detecting MPO antigen.

(2) The preparation process of the method is simple to operate, the time consumption is short, the monoclonal antibody sequence can be expressed at any time through cells, the expression quantity is high, the production process is controllable, and the product batch-to-batch difference is small.

(3) The MPO binding protein disclosed by the invention can be used as a quality control product or a standard product in an MPO autoantibody detection kit, can relieve the problems of complicated operation of immune polyclonal antibody and low subsequent coupling efficiency, reduces the production cost, stabilizes the product quality, and can obviously improve the reaction value; on the other hand, compared with the direct use of human serum, the method can also avoid the problems of difficult sample sources and high cost.

(4) The MPO binding protein can be coupled with a chromatography matrix to carry out immunoaffinity chromatography purification of MPO antigen, and the purity of the purified protein is high.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a nucleic acid electrophoretogram of heavy (Fd) and light (L) chains used to construct a library in one embodiment of the invention;

FIG. 2 is a nucleic acid electrophoretogram of Fab gene fragments used in one embodiment of the invention to construct a library;

FIG. 3 is an SDS-PAGE protein electrophoresis of an anti-MPO recombinant monoclonal antibody according to an embodiment of the present invention, MPO-Ab-non-reducing: about 150kDa in size; MPO-Ab-reduction: the heavy and light chains are 50kDa and 25kDa, respectively;

FIG. 4 is an ELISA assay for binding of recombinant mab to MPO protein, ab-MPO is the result of binding of recombinant mab to MPO antigen, and Ab-BSA is the result of non-specific binding of recombinant mab to control protein BSA.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 screening of MPO binding proteins

Preparation of phage display library

(1) Spleen was taken from mice immunized with MPO antigen, and lymphocytes were isolated using a mouse lymphocyte isolate.

(2) Extracting RNA: taking 1×10 ⁶ And extracting total RNA of the lymphocyte by the cell.

(3) Reverse transcription: and (3) carrying out reverse transcription on the extracted total RNA to synthesize cDNA.

(4) Antibody gene fragment amplification: the VH-CH1 (Fd) regions of the kappa, lambda light and heavy chains of the antibody were amplified by specific amplification primers using cDNA as template.

20. Mu.L of reaction system: 1 μL cDNA,0.8 μL LPrime F,0.8 μL LPrime R,10 μL2× phanta max master mix,7.4 μL LNuclease-Free Water; the reaction procedure: melting 95 ℃ for 30s, annealing 55 ℃ for 30sec, extension 72 ℃ for 45s,30 cycles.

After the reaction is finished, loading buffer is added into the system, 1% agarose gel electrophoresis identification is carried out, an electrophoresis diagram is shown in figure 1, and the target band is about 750bp.

The target band was excised, and antibody heavy chain (Fd) gene fragment and light chain gene fragment (κ, λ) were recovered, respectively.

(5) The antibody light and heavy chain gene fragments were combined into complete Fab gene fragments by overlapping PCR.

25 μl reaction system: light chain 30ng,linker 4.3ng,30ng heavy chain, 0.5. Mu.LsfiF (upstream primer), 0.5. Mu.LsfiR (downstream primer), 12.5. Mu.L 2X phanta max master mix, nucleic-Free Water was added to the total system of 25. Mu.L.

The reaction procedure: melting 95 ℃ for 30s, annealing 55 ℃ for 30sec, extension 72 ℃ for 90s,30 cycles.

After the reaction is finished, loading buffer is added into the system, 1% agarose gel electrophoresis identification is carried out, an electrophoresis diagram is shown in figure 2, and the target band is about 1500bp. The target band was excised and the antibody Fab fragment was recovered.

The upstream primer sfi if: 5'> GAGCAGGAGCATAGGAGGATCGGGCGGCGGCC <3' (SEQ ID NO. 12);

downstream primer sfi ir: 5'> CCATGGCAATGGTGATTCTGCTGCGCGGCCTGGCC <3' (SEQ ID NO. 13);

(6) Constructing a plasmid: the antibody Fab fragment and pComb3xSS plasmid were digested with sfi I, respectively, and the digested fragments were mixed in a molar ratio of 3:1, and T4 DNAligenase was added thereto, followed by ligation overnight at 16 ℃.

(7) Library construction: recovering the ligation product from the previous step. Taking 1.5 mu l of recovered product, adding TG1 competence, uniformly mixing, transferring to an electric rotating cup for electric rotating, and carrying out parameter selection: bac-Ec1. After completion of the electrotransformation, the culture was activated at 37℃for 1 hour, and the activated bacterial liquid was transferred to 200mL of a 2 XYT medium, and 1/1000 of ampicillin and a glucose solution having a final concentration of 2% were added thereto, followed by culturing at 37℃and 220rpm until OD600 = 0.6. Helper phage M13K07 was added in 20-fold bacterial counts. Mixing, standing in shaking table at 37deg.C for 45min. Centrifugation is carried out for 15min, and 200mL of new 2 XYT+Amp+Kana culture medium is used for resuspension precipitation, 30 ℃ and 220rpm shaking is carried out for 14h for phage amplification. The following day the supernatant was collected by centrifugation, phage were settled by adding 1/4 of the volume of 20% PEG6000 and resuspended in PBS to give phage display library.

(II) panning of anti-MPO antibodies Using phage display libraries

(1) The MPO protein coupled with biotin is used as a target antigen, and after the MPO protein is incubated for 1h together with SA magnetic beads, 1 multiplied by 10 is added into a reaction system ¹² The phage obtained in example 1 were incubated for 1h, and specific phage were captured by antigen. Wash 10 times with PBST (PBS+0.05% Tween-20).

(2) The antigen-binding magnetic beads were added to TG1 bacteria solution, allowed to stand at 37℃for 45min for infection, and then subjected to activation culture at 220rpm at 37℃for 1h. And (3) taking a proper amount of bacterial liquid subjected to standing infection, carrying out gradient dilution, coating an ampicillin plate, adding glucose (the final concentration is 2%) and 1/1000 of ampicillin antibiotics into the rest bacterial liquid, and shaking at 37 ℃ and 220rpm for 3 hours.

(3) 10 mu LM13K07 is added into the bacterial liquid, and the bacterial liquid is kept stand at 37 ℃ for infection for 45min.

(4) 6000 Xg, centrifugal 10min, with 10mL2 XYT+Amp+kana medium heavy suspension, 30 degrees, 220rpm, shaking 14h amplification phage. The following day the supernatant was collected by centrifugation, 1/4 of the volume of 20% PEG6000 settled phage were added and resuspended in PBS to give phage for the first round of screening.

(5) And (3) repeating the steps 1-4 by using the phage obtained by the first round of screening, and carrying out the 2 nd round of screening.

(6) Monoclonal identification: the second round of screening coated plates was used to select for single clones in 96-well deep well plates and phage were amplified overnight by shaking.

And (3) wrapping the plate: MPO antigen coated ELISA plates with BSA as control protein coated plate, overnight at 4 ℃.

Closing: the next day the coating was discarded, the plates were washed 3 times with PBST, patted dry, 3% milk added and blocked at 37℃for 2h.

Preparation of an antibody: and (3) centrifuging the 96-well deep hole plate, diluting the supernatant in a final concentration of 1% milk, and uniformly mixing to serve as a primary antibody for later use.

Incubation resistance: the blocking solution was discarded, the plates were washed 3 times with PBST, the plates were dried by pipetting, and primary antibodies were added and incubated for 2h at 37 ℃.

Secondary antibody incubation: the primary Antibody was discarded, the plates were washed 5 times with PBST, and 1:5000 dilution of Anti-M13 Antibody (HRP) secondary Antibody was added and incubated for 1h at 37 ℃. Discarding the secondary antibody, washing the plate for 5 times by PBST, beating, adding a chromogenic substrate for color development, adding a stop solution after 15min to stop the reaction, and reading by an enzyme-labeling instrument.

(7) Clones with high read (MPO antigen well OD > 1) and low non-specific binding (BSA control protein well OD < 0.5) were selected for sequencing.

Light chain sequencing primer: bomp:5'> GTGTGGAATTGTGAGCGG <3' (SEQ ID NO. 14);

heavy chain sequencing primer: PELB:5'> ACCTATTGCCTACGGCAGCCG <3' (SEQ ID NO. 15);

sequencing to obtain an MPO binding protein sequence comprising an antigen binding domain, wherein the heavy chain variable region sequence is shown as SEQ ID NO.6, and the light chain variable region sequence is shown as SEQ ID NO. 7:

SEQ ID NO.6：

QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIHPSDSYTYYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSTMITSFAMDYWGQGTSVTVSS。

SEQ ID NO.7：

DIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAEYFCQQYNSYPYTFGGGTKLEIK。

further analysis resulted in MPO binding proteins having the following complementarity determining regions (i.e., the underlined portions of the sequences described above):

heavy chain:

VH-CDR1：GYTFTSYW(SEQ ID NO.1)；

VH-CDR2：IHPSDSYT(SEQ ID NO.2)；

VH-CDR3：TRSTMITSFAMDY(SEQ ID NO.3)；

light chain:

VL-CDR1：QSVSND(SEQ ID NO.4)；

VL-CDR2：YAS；

VL-CDR3：QQYNSYPYT(SEQ ID NO.5)。

EXAMPLE 2 expression purification of anti-MPO recombinant monoclonal antibody

Sequencing to obtain the Fab region sequence of the candidate antibody, and then synthesizing the gene:

the vector selects PTT5 plasmid, takes EcoRI and BamHI as cloning sites, and inserts light chain gene fragments. The constant region sequence of the light chain is shown as SEQ ID NO.9 and is a murine sequence; the complete sequence of the light chain is shown as SEQ ID NO. 11.

The CH1 of the heavy chain was replaced with CH1 of human IgG1, and the Fc of human IgG1 was fused at the C-terminus, and the constructed complete heavy chain fragment was inserted into the PTT5 plasmid with EcoRI+BamHI as cloning site. The constant region sequence of the heavy chain is shown as SEQ ID NO.8, the sequence of the human source is shown as SEQ ID NO. 10.

Expression was performed using a mammalian cell 293F expression system.

(1) Plasmid extraction: the plasmids synthesized by the genes are respectively transformed into TOP10 competence, activated for 1h, transferred into 200mL LB culture medium, cultured overnight at 37 ℃, and extracted by using an endotoxin-free plasmid extraction kit the next day to obtain the corresponding heavy chain plasmids and light chain plasmids.

(2) 293F cells were inoculated 1 day prior to transfection into 500mL suspension cell culture flasks and the cell density was controlled at 1X 10 ⁶ And each mL.

(3) The next day 40. Mu.g of heavy chain plasmid and 80. Mu.g of light chain plasmid were diluted and gently mixed in 6mL transfection buffer, 480. Mu.L PEI was added, gently mixed, and incubated at room temperature for 20 minutes. Dropwise adding into cells, placing cells into an incubator at 98rpm at 37deg.C with 5% CO ₂ And (5) suspension culture.

(4) After 6 days, the culture supernatant was collected, igG was purified with rProteinA as a filler, eluted with 0.1M Glycine (pH 3.0), and neutralized with 1M Tris (pH 8.0). After the completion of elution, the ultrafiltration tube was replaced with PBS buffer and concentrated, and the protein concentration was measured, and the purity was confirmed by SDS-PAGE. As in fig. 3.

EXAMPLE 3ELISA determination of the binding Activity of recombinant monoclonal antibody to MPO antigen

(1) MPO protein (2. Mu.g/mL) was coated on ELISA plates, while BSA was coated as a non-specific binding control, and incubated overnight at 4 ℃.

(2) The coating was discarded, washed 3 times with PBST, patted dry, 3% milk added and blocked for 2h at 37 ℃.

(3) Blocking solution was discarded, PBST was washed 3 times, patted dry, and 125nM of the initial 2-fold gradient diluted antibody was added and incubated for 1.5h at 37 ℃.

(4) The primary antibody was discarded, PBST was washed 5 times, patted dry, and horseradish peroxidase (HRP) -labeled murine anti-human IgG secondary antibody was added and incubated for 1h at 37 ℃.

(5) Discarding the secondary antibody, washing with PBST for 5 times, drying, adding a chromogenic substrate for color development, adding a stop solution after 15min to stop the reaction, and measuring the OD value by an enzyme-labeled instrument.

(6) Nonlinear fitting was performed with OD values on the ordinate and the logarithm of the molar concentration of antibody on the abscissa. As shown in FIG. 4, the EC50 value of the antibody was about 1nM.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A myeloperoxidase binding protein, comprising a light chain variable region and a heavy chain variable region;

the amino acid sequence of the VH-CDR1 is shown as SEQ ID NO.1, the amino acid sequence of the VH-CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the VH-CDR3 is shown as SEQ ID NO. 3;

the amino acid sequence of the VL-CDR1 is shown as SEQ ID NO.4, the amino acid sequence of the VL-CDR2 is YAS, and the amino acid sequence of the VL-CDR3 is shown as SEQ ID NO. 5.

2. The myeloperoxidase binding protein according to claim 1, wherein the heavy chain variable region amino acid sequence of the myeloperoxidase binding protein is shown in SEQ ID NO.6, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 7.

3. The myeloperoxidase binding protein according to claim 1 or 2, wherein the myeloperoxidase binding protein is an intact antibody, F (ab') ₂ One of Fab', fab, fv, scFv, dsFv, bispecific antibody and antibody minimal recognition unit;

preferably, the species from which the remainder of the sequences of the antibodies are derived include one or more of mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans.

4. The myeloperoxidase binding protein according to claim 1 or 2, further comprising a constant region;

preferably, at least a portion of the constant region sequence is a human consensus constant region sequence;

preferably, the constant region sequence is selected from the sequence of part or all of the constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD;

preferably, the myeloperoxidase binding protein comprises a heavy chain constant region and a light chain constant region;

preferably, the heavy chain constant region in the myeloperoxidase binding protein is derived from human, further preferably from the constant region of human IgG 1;

preferably, the amino acid sequence of the human derived heavy chain constant region is shown in SEQ ID NO. 8.

5. The myeloperoxidase binding protein according to claim 4, wherein the myeloperoxidase binding protein is a human murine chimeric antibody;

the heavy chain amino acid sequence of the myeloperoxidase binding protein is shown as SEQ ID NO.10, and the light chain amino acid sequence is shown as SEQ ID NO. 11.

6. A biological material comprising a polynucleotide, vector, or cell;

the polynucleotide encoding the myeloperoxidase binding protein according to any one of claims 1 to 5;

the vector carries the polynucleotide;

the cell carrying the polynucleotide or containing the vector or being capable of expressing the myeloperoxidase binding protein according to any one of claims 1 to 5.

7. A method for preparing a myeloperoxidase-binding protein, comprising culturing a cell according to claim 6;

preferably, the cell is produced by transforming a host cell with a polynucleotide encoding a myeloperoxidase binding protein comprising the myeloperoxidase binding protein according to any one of claims 1-5, wherein the polynucleotide for myeloperoxidase binding protein comprises a heavy chain expression plasmid and a light chain expression plasmid, and wherein the cell expressing the myeloperoxidase binding protein is produced by co-transforming the heavy chain expression plasmid and the light chain expression plasmid into a host cell;

preferably, the host cell is a eukaryotic cell, preferably a mammalian cell;

preferably, the mammalian cells comprise 293 cells or CHO cells, further preferably 293F cells;

preferably, the construction of the heavy chain expression plasmid containing the complete IgG1 heavy chain comprises fusing a fragment of the constant region of human IgG1 at the C-terminus of the heavy chain variable region.

8. Use of the myeloperoxidase binding protein according to any one of claims 1-5, or the biomaterial according to claim 6, in any one of the following (a) - (d);

(b) Preparing a reagent or a kit for assisting in diagnosing a disease;

(d) For purifying myeloperoxidase;

preferably, the disease comprises cardiovascular disease and/or tumor;

preferably, the agent is any one of a quality control, a standard, a conjugate, and a labeled composition;

the conjugate comprising the myeloperoxidase binding protein according to any one of claims 1-5 and a solid support, which comprises a microtube, a column, a microparticle, a nitrocellulose membrane, a chromatography matrix, or a lateral flow device;

the tagged composition comprises the myeloperoxidase binding protein according to any one of claims 1-5 and a label comprising one or more of an enzyme, a luminescent label, a fluorescent microsphere, a color microsphere, a latex microsphere, colloidal gold, a quantum dot, biotin, streptavidin, a radionuclide, a radiocontrast agent, a paramagnetic ion, a metal, and a photosensitizer.

9. An agent comprising the myeloperoxidase binding protein according to any one of claims 1 to 5; and/or the biomaterial of claim 6;

the conjugate comprising the myeloperoxidase binding protein according to any one of claims 1-5 and a solid support comprising a microtube, a column, a microparticle, a nitrocellulose membrane, a chromatography matrix, or a lateral flow device;

the tagged composition comprising the myeloperoxidase binding protein according to any one of claims 1-5 and a marker; the label comprises one or more of enzyme, luminescent label, fluorescent microsphere, color microsphere, latex microsphere, colloidal gold, quantum dot, biotin, streptavidin, radionuclide, radiocontrast agent, paramagnetic ion, metal and photosensitizer.

10. A kit comprising the reagent of claim 9.