Monoclonal antibody targeting PGLYRP1 as well as preparation method and application thereof

Abstract

The invention discloses a monoclonal antibody targeting PGLYRP1 as well as a preparation method and application of the monoclonal antibody. According to the invention, the monoclonal antibody capable of accurately identifying the PGLYRP1 is constructed, and the conserved structural domain of the PGLYRP1 can be stably identified. The monoclonal antibody provided by the invention can significantly reduce cross reaction and improve the accuracy of experimental results, and is especially suitable for signal detection in complex tissues. Besides, the monoclonal antibody is derived from stable cell cloning and can be cultured and amplified for a long time, so that the quality stability of the product is ensured, and the problems that the polyclonal antibody is large in batch difference, cannot be duplicated and the like are solved.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and immunology, and particularly relates to a monoclonal antibody targeting PGLYRP1 and a preparation method and application thereof. Background Art

[0002] PGLYRP1 is a member of the peptidoglycan recognition protein (PGRP) family, which is highly conserved between insects and mammals. Mammalian PGLYRP1 is approximately 200 amino acids long, has a molecular weight of 18-20 kDa, and possesses a signal peptide. PGLYRP1 is expressed along the digestive tract, particularly in the granules of neutrophils and eosinophils, with lower expression in non-immune cells. PGLYRP1 is a key effector molecule in the innate immune system, with its core function being to recognize and bind to peptidoglycan, a major component of the bacterial cell wall.

[0003] Currently, most PGLYRP1 tests use polyclonal antibodies. Polyclonal antibodies are derived from different B cell clones, so they recognize multiple epitopes of the antigen, which results in low antibody specificity. Polyclonal antibodies bind to different parts of the target antigen or cross-react with similar molecules, resulting in nonspecific binding. In complex samples (such as serum or cell lysates), nonspecific binding may interfere with the interpretation of results and affect the accuracy and reproducibility of the experiment.

[0004] Therefore, there is an urgent need to develop a monoclonal antibody that can target PGLYRP1.

[0005] The information in the background technology is only intended to illustrate the general background of the invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to a person skilled in the art. Summary of the Invention

[0006] To address at least some of the technical problems in the prior art, the present invention provides a monoclonal antibody targeting PGLYRP1, a preparation method thereof, and applications thereof. Specifically, the present invention includes the following contents.

[0007] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can target peptidoglycan recognition protein 1, and the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO. 7 and / or the light chain variable region sequence shown in SEQ ID NO. 8; (II) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence of (I) or (II) and having the same function.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention comprises a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment comprises a Fab, Fab', F(ab')2, scFv or scFv Fc fragment.

[0010] The second aspect of the present invention provides a nucleic acid molecule comprising the nucleotide sequence of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0011] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule according to the second aspect of the present invention.

[0012] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect of the present invention or the vector molecule described in the third aspect of the present invention.

[0013] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, wherein the antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0014] In a sixth aspect, the present invention provides a detection product for peptidoglycan recognition protein 1, comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0015] In certain embodiments, the detection product according to the present invention comprises a kit, a test paper or a protein chip.

[0016] The seventh aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention in preparing a product for detecting peptidoglycan recognition protein 1.

[0017] The present invention constructs a monoclonal antibody that can accurately recognize PGLYRP1, ensuring that it can stably recognize the conserved domain of PGLYRP1, and its binding effect is significantly better than that of polyclonal antibodies that recognize multiple non-specific epitopes. The monoclonal antibody of the present invention can significantly reduce cross-reactions and improve the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. In addition, the monoclonal antibody of the present invention is derived from a stable cell clone and can be cultured and expanded over a long period of time, ensuring the quality stability of the product and overcoming the problems of large batch variability and non-reproducibility of polyclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the results of plasmid enzyme digestion verification after the target gene is constructed, where M is a DNA marker, 1 is the plasmid digested with XbaI and HindIII, and 2 is plasmid DNA.

[0019] Figure 2 The results of antigen protein expression and purification (Coomassie Brilliant Blue staining) are shown, wherein A is a purification test graph, B is a final concentration graph, M is a protein marker, IN is the original sample, FT is the flow-through, W is the washing solution, and E is the eluent.

[0020] Figure 3 The antibody expression and purification results (Coomassie Brilliant Blue staining) are shown, wherein A is a purification test graph, B is a final concentration graph, M is a protein marker, IN is the original sample, FT is the flow-through, W is the washing solution, and E is the eluent. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0024] Antibodies or antigen-binding fragments thereof In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can bind to peptidoglycan recognition protein 1 with high affinity and targeting. Without being bound by any theory, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6, wherein the amino acid sequences of the heavy chain CDR1-3 are GFSLSSY (SEQ ID NO.1), GNYGN (SEQ ID NO.2) and GDYYIYGYSQNGFDP (SEQ ID NO.3), respectively; the amino acid sequences of the light chain CDR1-3 are QASQSIGSYLN (SEQ ID NO.4), SASTLAS (SEQ ID NO.5) and QQGYSSTDIANA (SEQ ID NO.6), respectively.

[0025] In a specific embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO. 7 and / or the light chain variable region sequence shown in SEQ ID NO. 8; (II) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence of (I) or (II) and having the same function.

[0026] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0027] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region interspersed with three "complementarity determining regions (CDRs)." The framework region is used to coordinate the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues primarily responsible for antigen binding in the antibody. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0028] As used herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for comparison, including any algorithm required for achieving optimal comparison in the full-length sequence being compared.

[0029] Herein, the antibody sequences obtained by modification also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0030] In the present invention, the antibody includes a chimeric antibody, a humanized antibody or a murine antibody.

[0031] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoclonal antibody" or Ab, refers to an immunoglobulin obtained from a pure cell line, having the same structure and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies directed against different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not contaminated with other immunoglobulins. This characteristic contrasts with polyclonal antibody preparations, which generally include antibodies directed against different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0032] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequences in another species. For example, the variable regions of the light and heavy chains are both derived from the variable regions of an antibody from one animal species (such as a mouse, rat, etc.), while the constant portion is homologous to the antibody sequence from another species (such as a human). For example, to obtain a chimeric antibody, a non-human B cell or hybridoma cell can be used to produce the variable region, while the constant region combined with it is from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined with it. At the same time, since the constant region of a chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody using a non-human constant region.

[0033] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while retaining the antibody's full antigen binding affinity and specificity.

[0034] As used herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, murine antibodies may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced by random or point mutations in vitro or by somatic mutation in vivo.

[0035] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab', or F(ab')2.

[0036] In the present invention, the terms "directed against," "binding," "immunobinding," "specific binding," and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific for the immunoglobulin. The strength or affinity of the immunological binding interaction can be expressed in terms of a dissociation constant (Kd), where a smaller Kd represents a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the art and described herein.

[0037] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. An "isolated" antibody or fragment thereof thus includes an antibody or fragment thereof purified by standard purification methods. The term also includes antibodies or fragments thereof prepared by recombinant expression in a host cell and chemically synthesized antibodies or fragments thereof.

[0038] Nucleic acid molecules In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0039] As used herein, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, and / or their analogs, including DNA, RNA, and DNA / RNA hybrids, including DNA or RNA analogs such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases. Thus, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, and the like.

[0040] Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities using recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then isolate it from the propagated host cells by conventional methods.

[0041] Carrier molecules One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0042] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0043] host cells In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule of the present invention.

[0044] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. Host cells include any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0045] Preparation method One aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention. The preparation method is not particularly limited and includes preparation by artificial synthesis or genetic engineering.

[0046] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention or antigen-binding fragments thereof can be obtained by direct amino acid synthesis.

[0047] In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems used for genetic engineering expression include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems. In a preferred embodiment, the antibodies of the present invention can be prepared by the following steps: (1) constructing a recombinant vector expressing the antibody heavy chain with the amino acid sequence shown in SEQ ID NO. 7 and / or a recombinant vector expressing the antibody light chain with the amino acid sequence shown in SEQ ID NO. 8; (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression; (3) Collect and purify the antibodies.

[0048] In another preferred embodiment, the antibody of the present invention is prepared by immunization with an antigen, wherein the antigen has the amino acid sequence shown in SEQ ID No.9.

[0049] Testing products The present invention further provides a detection product for detecting (or quantifying) peptidoglycan recognition protein 1, which comprises the antibody or antigen-binding fragment thereof described in the present invention, and instructions on how to implement the detection method of the present invention on peptidoglycan recognition protein 1.

[0050] In a preferred embodiment, the detection product includes a kit, a test paper or a protein chip.

[0051] As used herein, the term "test kit" refers to a combination of reagents and other materials. The test kit is expected to include reagents such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or their antigen-binding fragments, control proteins, and test containers (e.g., microtiter plates, etc.). The term "test kit" is not limited to a specific combination of reagents and / or other materials. For example, the test kit may also include instructions for using the reagents. The test kit may be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for detecting the instructions. The test kit may be prepared by various methods known in the art.

[0052] In certain embodiments, the kit may further include at least one of a cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. A known substrate may be used for the substrate solution, and examples thereof include, but are not limited to, chromogenic substrates, fluorescent substrates, and luminescent substrates. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, BSA solution, trehalose solution, and animal serum.

[0053] application The present invention further provides use of the antibody or antigen-binding fragment thereof described herein in the preparation of a product for detecting peptidoglycan recognition protein 1, wherein the product comprises a kit, a test strip, or a protein chip. The product is used for the prediction, diagnosis, and / or prognosis of diseases associated with peptidoglycan recognition protein 1.

[0054] Example This example illustrates the preparation and detection process of monoclonal antibodies targeting PGLYRP1.

[0055] 1. Antigen Design and Preparation 1.1 Gene synthesis and vector construction PCR amplification was performed using the amino acid sequence of M40505-PGLYRP1 as the antigen sequence (as shown in SEQ ID NO. 9). The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2. Electrophoresis detection of the amplified fragment and recovery steps were performed according to the instructions of the commercially available kit.

[0056] Table 1 PCR amplification reaction system Table 2 PCR amplification reaction conditions The vector and target gene were digested at 37°C for 1-2 h. The digestion reaction system was shown in Table 3. The digestion products were detected by electrophoresis and recovered.

[0057] Table 3 Enzyme digestion reaction system of vector and target gene The vector and target gene were connected, and the reaction system was shown in Table 4.

[0058] Table 4 Ligation reaction system of vector and target gene Add the DNA fragment to be transformed to a tube containing TOP10 competent cells (25 ng of DNA is required for 50 μl of competent cells). The volume should not exceed 5% of the competent cells. Gently swirl the contents several times to mix thoroughly, and place on ice for 30 minutes. Place the centrifuge tube mixture in circulating water warmed to 42°C and heat shock for 90 seconds without shaking the tube. Quickly transfer the tube to an ice bath and allow the cells to cool for 1-2 minutes. Add 200 μl of SOC liquid medium to each tube and warm the medium to 37°C in a water bath. Then transfer the tube to a shaker set at 37°C and incubate at 220 rpm for 45 minutes to allow the cells to recover and express the resistance marker gene encoded by the plasmid. Transfer an appropriate volume (200 μl per 90 mm plate) of the transformed competent cells to LB medium containing the corresponding antibiotic. Invert the plate and incubate at 37°C. Plaques should appear after 12-16 hours.

[0059] After colonies grow on the plate, randomly pick a few and perform colony PCR verification to detect transformants. Positive clones are verified by sequencing and enzyme digestion verification results are as follows Figure 1 shown.

[0060] 1.2 Protein expression and purification The expression test was performed using the mammalian HEK293 cell expression system. The target gene was transfected into HEK293 cells for protein expression and purification test.

[0061] The expression conditions are as follows: HEK293 cell density: 1.5-2.0×10 6 cells / ml; culture temperature: 37°C; culture speed: 130 rpm / min; culture and sampling time: 6 days.

[0062] Specific experimental process: (1) According to 0.5×10 6 Inoculate cells into 300 ml of culture medium in a 1 L shake flask.

[0063] (2) Incubate in a shaking incubator at 37°C, 120 rpm, and 5% carbon dioxide for 24 h until the cell density reaches 1×10 6 cells / ml (cells must be able to double every 24 hours).

[0064] (3) Pipette 300 μg of DNA (sterilized by filtration) and add it to 30 ml of PBS. Then vortex for 3 seconds to mix thoroughly.

[0065] (4) Add 1.2 ml of filter-sterilized PEI solution (0.5 mg / ml) to the PBS / DNA mixture.

[0066] (5) Let the PEI-DNA mixture stand at room temperature for 20 min.

[0067] (6) Add the DNA / PEI mixture to the cells. The cell density must reach 1.5-2.0×10 6 cells / ml.

[0068] (7) After transfection, culture the cells in a shaking incubator at 37°C, 120 rpm, and 5% CO2 for 6 days.

[0069] (8) Centrifuge at 3000 g for 5 min to collect the culture supernatant and cell pellet, and store at -80°C or use for subsequent purification.

[0070] M40505 collected 1 L of culture medium supernatant, centrifuged at high speed, passed through a 0.22 μm filter membrane, and bound to resin to purify the target protein. The resin was Protein A resin, the binding and equilibration solution was PBS at pH = 7.4, the washing solution was PBS at pH = 7.4, the elution solution was 0.1 M glycine at pH = 3.0, and the neutralization solution was 1 M Tris-HCl at pH = 8.5. The expression and purification results are shown in Figure 2. Figure 2 As shown, the calculated theoretical size of the protein is 21.5 kDa, which is consistent with the size of the SDS-PAGE result, and the protein can be used for immunization.

[0071] 2. Animal immunization 2.1 Immunity Healthy female New Zealand white rabbits, 4 months old and weighing 2.1 kg, were selected. The primary immunization antigen consisted of a recombinant protein mixed and emulsified with an equal volume of Freund's complete adjuvant. The secondary, tertiary, and quadruple immunization antigens consisted of a recombinant protein mixed and emulsified with an equal volume of Freund's incomplete adjuvant.

[0072] First immunization: On the first day, the immunization antigen was Freund's complete adjuvant + recombinant protein; Second immunization: On the 14th day, the immunization antigen was Freund's incomplete adjuvant + recombinant protein; Three immunizations: On the 28th day, the immunization antigen was Freund's incomplete adjuvant + recombinant protein; Blood collection after three immunizations: On the 35th day, 1 ml of blood was collected from the ear vein and the antiserum titer was tested by ELISA; Four immunizations: On day 42, the immunization antigen was Freund's incomplete adjuvant + recombinant protein; Final bloodletting: On the 49th day, the antiserum titer detected by ELISA reached the requirement, and whole blood was collected from the carotid artery.

[0073] 2.2 Indirect ELISA Antigen was diluted to 6 μg / ml in 0.05 mol / L carbonate (pH 9.6), with 100 μl / well added, and incubated overnight at 4°C. The wells were washed three times with 0.05% Tween-20 (PBST), each for 3 minutes. 150 μl of blocking buffer (5% skim milk powder (PBST)) was added to each well and blocked at 37°C for 60 minutes. The wells were washed three times with 0.05% Tween-20 (PBST), each for 3 minutes. Antiserum was diluted 1:1000 and serially diluted, and incubated at 37°C for 1 hour. The wells were washed three times with 0.05% Tween-20 (PBST), each for 3 minutes. Horseradish enzyme-conjugated goat anti-rabbit IgG (H+L) (Cat. No. 116154, Jackson) was diluted 1:8000 and incubated at 37°C for 45 minutes. Remove the plate and wash five times with 0.05% Tween-20 (PBST), 3 minutes each time. Add 100 μl / well of substrate solution (TMB) and incubate for 5-10 minutes. Finally, add 100 μl of 2 mol / L sulfuric acid to terminate the reaction. Measure the OD value at 450 nm using a microplate reader (Kehua ST-360).

[0074] 2.3 Antibody affinity purification Place an appropriate amount of affinity purification magnetic beads into a purification column. Wash three times with 10 mM HCl (approximately 30 ml). Wash any residual hydrochloric acid with binding buffer. Add the purified antigen dissolved in PBS to the beads. After adding the buffer, incubate overnight at 4°C. Wash three times with the acid, water, and base used for the column, and finally rinse with PBS. Locate the frozen antiserum and corresponding magnetic beads according to the antibody purification protocol. Keep a record. Thaw the serum and transfer the magnetic beads to the purification column. Dispense the required amount of serum, returning any excess. Centrifuge the serum at 3400 rpm for 20 minutes at 4-8°C. Use a pipette to remove surface lipids from the centrifuged serum and retain a small sample for ELISA testing. Incubate the centrifuged serum with the magnetic beads for 2 hours at room temperature (or in a 37°C incubator if room temperature is low) or overnight at 4°C. After incubation, collect the flow-through (FT) and wash the beads three times with PBS, 10 column volumes each time. Wash the purification column with 5 ml of pre-cooled pH 5.0 pre-wash solution. Elute with pre-cooled pH 2.5 eluent, collecting 1 ml each time. Add 50 μl of neutralizing solution to the EP tube in advance. Use Coomassie to detect the collection peak of the collected liquid. Add 10 μl of collection liquid to 100 μl of Coomassie, and determine the required antibody based on the collection peak. Store at 4 degrees. Stop the collection when there is no color in the Coomassie test, and wash with 10 ml of PBS. Incubate the flow cytosol with the magnetic beads again and repeat the above steps. Concentrate the collected antibodies with PEG. Add 50% glycerol and 0.02% sodium azide according to the volume of the antibody, mix well, and store for later use.

[0075] 2.4 Potency testing The ELISA test results of the M40505 project antibody are shown in Table 5. The titers of the purified antibodies are: A ≥ 1024K, B ≥ 1024K. Among them, rabbit A has the best titer, so rabbit A was selected for subsequent serum collection and B cell enrichment screening.

[0076] Table 5 Antibody ELISA test results After sorting and culturing the enriched B cells from rabbit A, the supernatant was assayed by ELISA, and candidate positive clones 1G3, 1B1, 1E4, 1E11, and 2E5 were screened (Table 6). Among them, clone 2E5 was selected for subsequent antibody heavy and light chain gene amplification and vector construction for recombinant expression.

[0077] Table 6 Antibody titer test results The results of purification of the recombinantly expressed antibody from the 2E5 positive clone are as follows Figure 3 shown.

[0078] The results of the titer detection of the recombinantly expressed antibody of the 2E5 positive clone are shown in Table 7.

[0079] Table 7 2E5 antibody titer test results 3. Sequencing The recombinantly expressed antibody of the 2E5 clone was subsequently sequenced, and the sequence is shown in Table 8: Table 8 Amino acid sequence of 2E5 monoclonal antibody This example constructs a monoclonal antibody that can accurately recognize PGLYRP1 by screening specific epitopes and verifying their binding sites, ensuring that it can stably recognize the conserved domain of PGLYRP1, and the binding effect is significantly better than that of a polyclonal antibody that recognizes multiple non-specific epitopes. The monoclonal antibody of the present invention can significantly reduce cross-reactions and improve the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. In addition, the monoclonal antibody of the present invention is derived from a stable cell clone and can be cultured and amplified for a long time, ensuring stable and consistent product quality, overcoming the limitations of large differences and non-replicability between polyclonal antibody batches.

[0080] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof, characterized in that: It can target peptidoglycan recognition protein 1, and the antibody or its antigen-binding fragment includes heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III): (I) the heavy chain variable region sequence shown in SEQ ID NO. 7 and / or the light chain variable region sequence shown in SEQ ID NO. 8; (II) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (I) and has the same function; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence of (I) or (II) and having the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibodies include humanized antibodies or murine antibodies, and the antigen-binding fragments include Fab, Fab', F(ab')2, scFv or scFv Fc fragments.

4. A nucleic acid molecule, characterized in that It comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4 or the vector molecule according to claim 5.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: Prepared by artificial synthesis or genetic engineering.

8. A detection product for peptidoglycan recognition protein 1, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. The detection product according to claim 8, characterized in that: The detection products include test kits, test strips or protein chips.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a product for detecting peptidoglycan recognition protein 1.

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