Preparation and application of anti-staphylococcus aureus IsdB antibody

Abstract

The invention discloses preparation and application of an anti-staphylococcus aureus IsdB antibody. The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 8; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO: 16. The invention also provides nucleic acid for coding the antibody, a vector containing the nucleic acid and a cell containing the vector. In addition, experiments prove that the antibody can inhibit systemic infection of MRSA and resist invasion of MRSA to pneumonia, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation and application of anti-Staphylococcus aureus IsdB antibody. Background Technology

[0002] Staphylococcus aureus (S. aureus), a representative of Gram-positive bacteria, is a type of pathogenic coccus widely found in nature and an important cause of hospital-acquired and community-acquired infections. S. aureus infections are characterized by acute, purulent lesions. Locally, they can cause persistent purulent infections of the skin and soft tissues; systemically, they can lead to serious infections and complications such as osteomyelitis, septic arthritis, endocarditis, pneumonia, and septicemia, with a mortality rate as high as 20%. Furthermore, the exotoxins of Staphylococcus aureus can cause fatal systemic infections such as food poisoning, scalded skin syndrome, and toxic shock syndrome.

[0003] Iron-regulated surface determinant B (IsdB) is distributed on the surface of Staphylococcus aureus cells. It is a hemoglobin receptor, maintains the growth of Staphylococcus aureus, and is closely related to the pathogenesis of MRSA infective endocarditis.

[0004] Currently, the treatment of Staphylococcus aureus infections typically involves antibiotics such as erythromycin, newer penicillins, gentamicin, vancomycin, or cephalosporin VI. However, due to the emergence of multidrug-resistant Staphylococcus aureus (MRSA), single-antibiotic treatment is becoming increasingly difficult. Clinical data shows that multidrug-resistant MRSA accounts for 60% of MRSA infections in the community and 80% in hospitals. Methicillin-resistant Staphylococcus aureus (MRSA) is particularly prominent. Infections caused by MRSA are difficult to cure with antibiotic therapy and have a high mortality rate. The bacteremia caused by invasive MRSA infections has a mortality rate exceeding 50% within 90 days. Therefore, since current mainstream antibiotic treatments are ineffective in controlling MRSA infections, antibody development is urgently needed. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide an antibody that specifically binds to Staphylococcus aureus iron ion surface determinant B and its application in the preparation of products for diagnosing or treating diseases related to Staphylococcus aureus infection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides an antibody against Staphylococcus aureus IsdB.

[0008] Furthermore, the antibody comprises a CDR region in the heavy chain variable region as shown in SEQ ID NO:8; and a CDR region in the light chain variable region as shown in SEQ ID NO:16.

[0009] Furthermore, the CDR region can be determined using any one of the following schemes: IMGT, Chothia, Kabat, AbM, or Contact.

[0010] Furthermore, the antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, or murine antibodies.

[0011] In this invention, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, provided that the antibody exhibits the desired biological activity. In a specific embodiment of the invention, the antibody is a monoclonal antibody. Antibodies may be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the uniformity of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies may be naked antibodies or bound to other molecules such as toxins, radioisotopes, etc.

[0012] A complete antibody typically consists of four polypeptides: two identical copies of the heavy (H) chain polypeptide and two identical copies of the light (L) chain polypeptide. Each of the heavy chains contains an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an N-terminal variable (VL (Kappa, Lambda)) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have the same general formula, wherein each region contains four frame regions whose sequences are relatively conserved. As used in this invention, the term "frame region" refers to a relatively conserved amino acid sequence located within a variable region between a hypervariable region or a complementarity-determining region (CDR). Four frame regions are present in each variable domain, referred to as FR1, FR2, FR3, and FR4. The framework region forms the β-sheet that provides the structural framework for the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY (2001)). Three CDRs, called CDR1, CDR2, and CDR3, form the “hypervariant region” of the antibody, which is responsible for antigen binding.

[0013] In this invention, the terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of the antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of the antibody.

[0014] In this invention, the CDR region is defined in various ways in the art, including but not limited to Kabat, Chothia, Abm, Contact, and IMGT. The IMGT definition used in this invention comes from IMGT (International Im Muno Gene Tics website imgt.org, Founder and Director: Marie-Paule Lefranc, Montpellier, France, see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Res., 27:209-212, both of which are incorporated herein by reference in their entirety). For the IMGT numbering system, (i) HCDR1 is typically located at heavy chain amino acid sites 26-33, (ii) HCDR2 is typically located at heavy chain amino acid sites 51-58, and (iii) HCDR3 is typically located at heavy chain amino acid sites 97-111. For the IMGT numbering system, (i) LCDR1 is usually located at light chain amino acid sites 27-32, (ii) LCDR2 is usually located at light chain amino acid sites 50-52, and (iii) LCDR3 is usually located at light chain amino acid sites 89-97.

[0015] In a specific embodiment of the present invention, the CDR regions of the heavy chain variable region are named HCDR1, HCDR2 and HCDR3, respectively, and their sequences are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the CDR regions of the light chain variable region are named LCDR1, LCDR2 and LCDR3, respectively, and their sequences are shown as SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0016] In this invention, the terms "constant region" or "constant domain" are used interchangeably and have their common meaning in the relevant field. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light and / or heavy chains that is not directly involved in antibody-antigen binding but can exhibit various effector functions (e.g., interaction with Fc receptors). The amino acid sequence of the constant region of an immunoglobulin molecule is generally more conserved than that of its variable domain.

[0017] In this invention, the term "heavy chain," when used in relation to antibodies, based on the amino acid sequence of a constant structural domain, can refer to any different type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mio (mu), which respectively generate the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art.

[0018] In this invention, the term "light chain," when used in relation to an antibody, can refer to any different type based on the amino acid sequence of a constant structural domain, such as kappa (κ) or lambda (λ). The amino acid sequence of the light chain is well known in the art.

[0019] Furthermore, the antibody has the heavy chain variable region sequence shown in SEQ ID NO: 8 and the light chain variable region sequence shown in SEQ ID NO: 16.

[0020] In some embodiments, the antibody comprises an amino acid sequence that has at least 90% homology with and has the same function as the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 16.

[0021] In some embodiments, the antibody further includes an amino acid sequence having the same function obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 16.

[0022] In this invention, the terms "identity" and "homology" mean at least 60%, preferably at least 70%, more preferably at least 80%, particularly at least 90%, further more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably higher percentages of sequence identity across the full length of the nucleotide sequence or the full length of the amino acid sequence. This invention therefore also includes all such modifications to all nucleic acid or amino acid sequences according to the invention having the aforementioned percentages of identity.

[0023] A second aspect of the present invention provides an antibody derivative.

[0024] Furthermore, the antibody derivative is a complex obtained by modifying the antibody described in the first aspect of the present invention; the modification includes conjugation modification using a detectable marker, conjugation modification using a therapeutic agent, and conjugation modification using an imaging agent.

[0025] Furthermore, the detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, or colloidal gold.

[0026] Furthermore, the therapeutic agents include cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0027] In this invention, the term "detectable marker" refers to a molecule or compound, or group of molecules or compounds, associated with a probe for identifying probes hybridized to genomic nucleic acids or reference nucleic acids. In some cases, a detectable marker can be detected directly. In other cases, a detectable marker may be part of a binding pair, which can then be detected. Signals from a detectable marker can be detected in a variety of ways and will depend on the properties of the detectable marker. Examples of ways to detect a detectable marker include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical methods, such as fluorescent dyes, colloidal gold, chemiluminescent catalysts, or chemiluminescent labels, or any other suitable method. The term "chemiluminescent label" refers to a label that, as a result of a chemical reaction, emits light (luminescence) with limited heat generation. Chemiluminescent labeling includes, but is not limited to, luminol and its derivatives, isoluminol and its derivatives, cyalume, pyrophenol, oxaloyl chloride, luciferin, TDAE (tetra(dimethylamino)ethylene), acridine esters and their derivatives, adamantane, rare earth elements, ruthenium bipyridine complexes, acridine ester, or dioxetane. Chemiluminescent catalysts include, but are not limited to, horseradish peroxidase and alkaline phosphatase.

[0028] As used in this application, the term "therapeutic agent" refers to a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need. Therapeutic agents include, but are not limited to, radionuclides, cytokines, gold nanoparticles, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes, and chemotherapeutic agents. The cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF, and M-CSF; the chemotherapeutic agents include, but are not limited to, cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxalate platinum, and oxaliplatin.

[0029] A third aspect of the present invention provides a nucleic acid molecule that encodes the antibody described in the first aspect of the present invention.

[0030] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the antibody according to the first aspect of the present invention is shown in SEQ ID NO:24.

[0031] Furthermore, the nucleotide sequence encoding the light chain variable region of the antibody according to the first aspect of the present invention is shown in SEQ ID NO:32.

[0032] In this invention, the term "nucleic acid molecule" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or modified nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides, ribonucleotides (RNA), or deoxyribonucleotides (DNA) of any length. These terms refer to the primary structure of the molecule and therefore include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. The terms include RNA or DNA analogs made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated and / or capped polynucleotides) as equivalents. Although many other bonds (e.g., thiophosphates, boron phosphates, etc.) are known in the art, nucleic acids are typically linked via phosphate ester bonds to form nucleic acid sequences or polynucleotides.

[0033] A fourth aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0034] Furthermore, the expression vector comprises a plasmid or a viral vector.

[0035] The term "expression vector" refers to a recombinant DNA construct designed to express a selected gene of interest (typically a protein) when correctly inserted into the expression vector. Non-limiting examples of expression vectors include plasmids or viral vectors. Suitable expression vectors may include regulatory elements such as promoters, operons, start codons, stop codons, polyadenylate signals, and enhancers, and can be prepared in various ways depending on the intended use. The promoter of the vector may be constitutive or inducible. Specifically, expression vectors can be prepared using plasmid vectors.

[0036] A fifth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the third aspect of the present invention or the expression vector described in the fourth aspect of the present invention.

[0037] Furthermore, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0038] In this invention, the host cell includes any suitable prokaryotic or eukaryotic cell. Cells that can be used include those that can grow easily and reliably, have a reasonably rapid growth rate, possess a well-characterized expression system, and can be easily and efficiently transformed or transfected. Eukaryotic cells are known in the art and include, but are not limited to, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in a mammalian cell. Many suitable mammalian host cells are known in the art. Examples of suitable mammalian cells include (but are not limited to) Chinese hamster ovary cells, CHO DHFR cells, human embryonic kidney (HEK) 293, HEK293E, HEK293-6E, HEK293F, or 293T cells (ATCC No. CRL1573) and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines include monkey COS-1 (ATCC CRL1650) and COS-7 (ATCC CRL1651), per.C6 cells, CV-1 (ATCC CCL70), myeloma cells, hybridoma cells, and NSO cells. Human cells are ideally chosen as the mammalian cell type. In a specific embodiment of the invention, the host cell is HEK293F cells. The nucleic acid sequence encoding any of the amino acids of the antibodies described herein can be introduced into the cells via transfection, transformation, or transduction.

[0039] The sixth aspect of the present invention provides a pharmaceutical composition.

[0040] Furthermore, the pharmaceutical composition comprises the antibody described in the first aspect of the present invention, the antibody derivative described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention.

[0041] In this invention, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof. As used herein, the term "pharmaceutical composition" refers to a formulation in which the biological activity of one or more active ingredients is effective and which does not contain other components that would have unacceptable toxicity to a subject to which the formulation is administered. Therefore, it is a composition suitable for pharmaceutical use in mammalian subjects (typically humans). Pharmaceutical compositions typically contain an effective amount of an active agent and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. Such formulations may be sterile.

[0042] The seventh aspect of this invention provides a product for detecting Staphylococcus aureus IsdB.

[0043] Furthermore, the product comprises the antibody described in the first aspect of the present invention, the antibody derivative described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention.

[0044] Furthermore, the product includes reagent kits and test strips.

[0045] The eighth aspect of the present invention provides the use of the antibody described in the first aspect of the present invention, the antibody derivative described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, the host cell described in the fifth aspect of the present invention, or the pharmaceutical composition described in the sixth aspect of the present invention in the preparation of products for diagnosing or treating diseases caused by Staphylococcus aureus infection.

[0046] Furthermore, diseases caused by Staphylococcus aureus infection include pneumonia, systemic infection, skin abscess, folliculitis, endocarditis, meningitis, otitis media, osteomyelitis, toxic shock syndrome, sepsis, or septicemia.

[0047] Furthermore, the diseases caused by Staphylococcus aureus infection include pneumonia and systemic infection.

[0048] In this invention, the term "treatment" means (1) preventing the development of symptoms or disease in a susceptible or unexhibited subject; (2) suppressing or preventing the development or recurrence of disease; or (3) improving or causing the remission of disease or symptom. As understood in the art, "treatment" is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this technology, a beneficial or desired outcome may include, but is not limited to, the reduction or improvement of one or more symptoms, the reduction of the severity of a symptom (including disease), the stabilization (i.e., non-deterioration) of a symptom (including disease), the delay or slowing of a symptom (including disease), the progression, improvement, or relief of a symptom (including disease), state, and remission (whether partial or complete), whether detectable or not.

[0049] The ninth aspect of this invention provides the use of the antibody described in the first aspect of this invention in the preparation of the antibody derivative described in the second aspect of this invention, the nucleic acid molecule described in the third aspect of this invention, the expression vector described in the fourth aspect of this invention, the host cell described in the fifth aspect of this invention, the pharmaceutical composition described in the sixth aspect of this invention, or the product for detecting Staphylococcus aureus IsdB described in the seventh aspect of this invention.

[0050] The tenth aspect of this invention provides the use of the antibody described in the first aspect of this invention, the antibody derivative described in the second aspect of this invention, the nucleic acid molecule described in the third aspect of this invention, the expression vector described in the fourth aspect of this invention, the host cell described in the fifth aspect of this invention, or the pharmaceutical composition described in the sixth aspect of this invention in the preparation of products that enhance the killing ability of phagocytes against Staphylococcus aureus.

[0051] In addition, the present invention also provides any of the following methods:

[0052] (1) A method for detecting Staphylococcus aureus IsdB in a sample for non-diagnostic purposes, the method comprising contacting the sample with the antibody described in the first aspect of the present invention and detecting the immune response of Staphylococcus aureus IsdB to the antibody;

[0053] (2) A method for preparing the host cell according to the fifth aspect of the present invention, the method comprising introducing the nucleic acid molecule according to the third aspect of the present invention or the expression vector according to the fourth aspect of the present invention into the cell;

[0054] (3) A method for generating the antibody according to the first aspect of the present invention, the method comprising culturing the host cell according to the fifth aspect of the present invention and isolating the antibody from the culture;

[0055] (4) A method for enhancing the ability of phagocytes to kill Staphylococcus aureus for non-therapeutic purposes, said method comprising applying the antibody described in the first aspect of the present invention;

[0056] (5) A method for specifically inhibiting the activity of Staphylococcus aureus IsdB, the method comprising using the antibody described in the first aspect of the present invention to inhibit the activity of Staphylococcus aureus IsdB or introducing the nucleic acid molecule described in the third aspect of the present invention into a biological somatic cell to inhibit the activity of Staphylococcus aureus IsdB by expressing the antibody described in the first aspect of the present invention.

[0057] In this invention, the term "introduction" refers to providing a vector to cells so that the vector becomes internalized within the cells. For example, the vector can be introduced into cells using transfection, transformation, and / or injection, and other methods known to those skilled in the art can also be used. Transfection reagents include, but are not limited to, liposome transfection reagents such as Lipofectamine 2000, Lipofectamine 3000, Turbiofect, and PEI, or chemical transfection reagents, or transfection by electroporation.

[0058] Advantages and beneficial effects of the present invention:

[0059] This invention provides an antibody that specifically binds to Staphylococcus aureus IsdB (named IsdB-H6). The IsdB-H6 antibody can inhibit the growth of Staphylococcus aureus in a low-iron environment and can resist the systemic invasion of MRSA and the invasion of pneumonia by MRSA, showing good prospects for clinical application. Attached Figure Description

[0060] Figure 1 IsdB- N2 Image of protein SDS-PAGE purity test results;

[0061] Figure 2 The image shows the expression and purification results of the IsdB-H6 monoclonal antibody.

[0062] Figure 3 The graph shows the results of the binding activity assay for the IsdB-H6 monoclonal antibody.

[0063] Figure 4 The epitope type identification results for the IsdB-H6 monoclonal antibody are shown in the figure.

[0064] Figure 5 Figure showing the results of IsdB-H6 monoclonal antibody inhibiting the growth of Staphylococcus aureus in a low-iron environment;

[0065] Figure 6 The image shows the opsonization and killing of Staphylococcus aureus by the IsdB-H6 monoclonal antibody; among them, Figure 6 In the diagram, A represents the phagocytosis result. Figure 6 B in the graph represents the kill rate.

[0066] Figure 7 The results of the IsdB-H6 monoclonal antibody in a systemic Staphylococcus aureus infection model are shown in the figure.

[0067] Figure 8 The image shows the results of the IsdB-H6 monoclonal antibody in a Staphylococcus aureus pneumonia model. Detailed Implementation

[0068] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0070] Example 1 Recombinant Staphylococcus aureus IsdB- N2 Antigen expression and purification

[0071] The full-length sequence of the IsdB gene was synthesized (Shanghai Jierui) and cloned into the expression vector pGEX-6P-2 using the restriction endonuclease recognition sites BamHI and SacI to construct the IsdB- gene. N2 protein prokaryotic expression vector pGEX-IsdB N2 / XL-1, after transfection and induction, makes IsdB- N2 The protein was expressed in E. coli, and IsdB- N2 The recombinant bacteria were inoculated into LB medium containing ampicillin and cultured overnight (37°C, 100 rpm). The next day, the culture was scaled up to OD at a ratio of 1:100 (V / V). 600 The pH value was 0.6-0.8. IPTG was added overnight to induce expression (4℃, 200 rpm). The cells were collected by centrifugation and lysed by sonication. The lysate was added to GST-4FF affinity packing material and vertically mixed overnight at 4℃. Glutathione was used as elution to obtain IsdB-. N2 -GST fusion protein, then Prescission protease is added to cleave the GST tag, and IsdB- is obtained by SPHP chromatography. N2 The protein was purified by replacing the buffer with PBS and then further concentrating and purifying it using ultrafiltration. The purity was assessed by SDS-PAGE gel electrophoresis. N2 The molecular weight and purity test results are as follows: Figure 1 As shown, the results indicate IsdB- N2 The protein purity is 100.0%.

[0072] Example 2: Isolation of PBMC cells

[0073] IsdB- based on Staphylococcus aureus vaccine N2 The protective antigen was approved by the ethics review committee of Jiangsu Provincial Center for Disease Control and Prevention, and peripheral blood lymphocyte samples of Phase Ia subjects were obtained from the Phase Ia clinical trial center of recombinant Staphylococcus aureus vaccine (Escherichia coli) and frozen in liquid nitrogen.

[0074] Example 3: Establishment of a human-specific anti-Staphylococcus aureus antibody library

[0075] 1. cDNA preparation:

[0076] Total RNA was extracted from peripheral blood lymphocytes isolated in Example 2, and then cDNA was synthesized by reverse transcription using the total RNA as a template.

[0077] 2. Construction of a human-specific anti-Staphylococcus aureus Fab antibody library:

[0078] The inverted cDNA was mixed and used as a gene template. Human light chain (VL+CL) Kappa / Lambda and heavy chain Fd fragment gene sequences were amplified by PCR using Ig primers. The PCR product of the VL+CL light chain gene was digested with SacI-HF and XbaI-HF enzymes and ligated into the pComb3XSS phage display vector. After successful construction of the VL+CL (Kappa) and VL+CL (Lambda) light chain libraries, the heavy chain Fd fragment was cloned into the pComb3XSS vector containing the light chain gene fragment using XhoI-HF and SpeI-HF restriction enzyme sites, respectively, to construct Fab phage particles. The ligation product was transformed into TG1 competent cells and cultured overnight at 37°C on ampicillin-containing plates. Cells were collected to calculate the library size, and 10 single colonies were selected for PCR identification using specific primers. The reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min. 5 μL of the PCR product was analyzed by 2% agarose gel electrophoresis.

[0079] 3. Positive rate identification:

[0080] Screening results showed that the positive clone rates of both the Fab (Kappa light chain) and Fab (Lambda light chain) libraries exceeded 80%, and the library sizes exceeded 10. 8 .

[0081] Example 4: Screening of Human-Specific Anti-Staphylococcus aureus Fab Antibody Library

[0082] 1. Bacterial purification and antibody phage preparation

[0083] The frozen bacterial culture was inoculated into LB-ampicillin medium at a ratio of 1:100 for resuscitation and grown to OD. 600 When the pH value is 0.5-0.6, remove the bacterial culture and add helper phage M13 at a ratio of 1:1000. Incubate at 37°C for 30 min, then continue shaking for another 30 min. Centrifuge to collect the bacterial cells and incubate overnight at 30°C in LB medium containing ampicillin and kanamycin on a shaker. Centrifuge the bacterial culture at 4°C at 3000 g for 10 min to collect the supernatant phage. Add PEG / NaCl solution and mix thoroughly. Incubate on ice for 30 min, then centrifuge at 4°C at 3000 g for 20 min. Remove the supernatant, add PEG / NaCl solution to the precipitate and mix thoroughly. Incubate on ice for 30 min, then centrifuge at 4°C at 12000 g for 2 min. Remove the supernatant, resuspend the precipitate in 1 mL PBS, add glycerol, and store at -80°C for later use.

[0084] 2. Phage library selection

[0085] Add antibody-phage to the antigen-coated ELISA plate and incubate at 37°C for 20 min. Wash 10 times with PBST and PBS solution to remove unbound phage. Digest the bound phage with trypsin and then add the plate to culture until OD200. 600 TG1 bacteria with a pH of 0.5–0.6 were infected for 30 min, and the bacterial cells were collected by centrifugation and plated onto LB agar plates containing ampicillin, and incubated overnight at 37°C. The next day, the titers of the panned phage sub-libraries were determined by serial dilution, and the bacterial cells were collected. The phage sub-libraries were then repackaged using the method described above as input phage libraries for the next round of panning. The panning process was repeated three times to maximize the enrichment of high-affinity antibodies.

[0086] 3. Phage library screening

[0087] Enriched phages were plated on LB agar plates containing ampicillin to generate bacterial single clones. Single clones were picked and cultured in 96-well plates for phage ELISA screening. Antibody-coated phages were added to antigen-coated ELISA plates and incubated at 37°C for 20 min. The plates were washed three times with PBST to remove unbound phages. Fluorescently labeled antibodies were added and incubated at 37°C for 20 min, followed by three washes with PBST. Specific antibodies were screened by detecting the ELISA fluorescence signal intensity. Corresponding positive well antibody-coated phages were amplified and sequenced.

[0088] Example 5 Cloning, expression, and purification of fully human antibody against IsdB-H6

[0089] 1. Experimental Methods

[0090] Using the positive clone display vector selected in Example 3 as a template, human Ig VH and VK / L were amplified by PCR. The PCR products were identified by agarose gel electrophoresis. Antibody genes that were identified as positive and whose light and heavy chains could be paired were purified using the Qiagen PCR product purification kit. The purified products were sequenced in both forward and reverse directions, and the antibody gene family, mutation rate, and CDR region were analyzed using the IMGT online server (https: / / www.imgt.org / IMGT_vquest / input).

[0091] Using the TA cloning method, the PCR products of antibody variable region genes that were positive by gel electrophoresis and whose heavy and light chains could be paired were ligated into the pcDNA3.4 vector containing either the heavy chain constant region or the light chain constant region to construct a fully human expression vector for anti-IsdB-H6 antibody. This expression vector was then transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated overnight at 37°C. The next day, 10 single colonies were picked and identified by PCR using specific primers. The reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 100 s, 28 cycles; 72°C extension for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis.

[0092] The PCR product identified as positive vector plasmid was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, it was incubated with PEI transfection reagent at 37℃ for 15-20 min and then transfected into HEK 293F cells. The cells were cultured in a 37℃, 5% CO2 incubator. After 5 days of cell culture, the cell supernatant was collected by centrifugation at 3000 g, 4℃ for 30 min. The supernatant was purified by protein A affinity chromatography, and the expression and purification status of the antibody were detected by SDS-PAGE gel electrophoresis.

[0093] 2. Experimental Results

[0094] This approach successfully constructed a series of antibody heavy and light chain expression vectors. One of the antibody clones was named the fully human IsdB monoclonal antibody IsdB-H6 (abbreviated as IsdB-H6 antibody or IsdB-H6 monoclonal antibody).

[0095] SDS-PAGE test results are as follows: Figure 2As shown, the relative molecular weight of the IsdB-H6 antibody in the transfected cells is approximately 150 kDa, with the heavy chain at approximately 50 kDa and the light chain at approximately 25 kDa, indicating that the transfected cells can successfully express the antibody. The IsdB-H6 antibody sequence is shown in Table 1.

[0096] Table 1 IsdB-H6 antibody sequence

[0097]

[0098]

[0099] Example 6: IsdB-H6 monoclonal antibody and Staphylococcus aureus iron-regulated surface determinant B (IsdB- N2 ) binding activity detection

[0100] 1. Experimental Methods

[0101] Recombinant expression of IsdB- N2 100 μL of 2 μg / mL protein was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed the next day, and then blocked at 37°C for 2 h with 3% BSA blocking buffer. The IsdB-H6 antibody transfected and expressed in Example 5 was diluted to 100 μg / mL and then serially diluted with 1×PBST. 100 μL of each antibody was added to each well of the ELISA plate, with three replicates for each concentration. The positive control was 100 μL of vaccine serum (1:2000 dilution) per well, and the negative control was 100 μL of negative serum (1:100 dilution) and negative control-irrelevant antibody IgG1 (0.5 μg / mL) per well. 100 μL of PBST was added to the blank control, and the plates were incubated at 37°C for 1 h.

[0102] Wash the plate three times with PBST buffer. Add 100 μL of Goat-Anti-Human-IgG-Fab-HRP (secondary antibody) diluted 1:5000 with PBST to each well and incubate at 37°C for 1 h. Wash the plate three times with PBST buffer. Add 100 μL of TMB chromogenic solution to each well and incubate at 37°C in the dark for 5–10 min. Immediately stop the reaction by adding 50 μL of 2 M H₂SO₄. Detect the OD value at dual wavelengths of 450 / 650 nm. Calculate the mean value of the negative control unrelated antibody IgG1, determine the threshold (3 times the mean value), and calculate the EC value. Antibody values ​​greater than the threshold are considered positive antibodies. 50 .

[0103] 2. Experimental Results

[0104] Test results as follows Figure 3 As shown, the results indicate that the fully human IsdB monoclonal antibody IsdB-H6 can bind to Staphylococcus aureus iron-regulated surface determinant B (IsdB-H6).N2 ) combined. IsdB- N2 EC 50 The calculated result is 0.27 μg / mL.

[0105] Example 7: Determination of Epitope Type of IsdB-H6 Monoclonal Antibody

[0106] 1. Experimental Methods

[0107] The antibody-antigen binding reaction was detected using Western blotting (WB). The IsdB-H6 antibody was subjected to SDS-PAGE (reduced) gel electrophoresis at 140 V for 1–2 hours. Electrophoresis was stopped when the bromophenol blue just appeared. After electrophoresis, the PVDF membrane was transferred using a semi-dry transfer apparatus at 23 V for 20 minutes. The transferred membrane was then completely covered with TBST solution containing 5% skim milk powder and blocked, then placed on a shaker at room temperature for 1 hour. The PVDF membrane was washed three times with TBST solution for 5 min each time. Then, a solution containing 1% skim milk powder was prepared with TBST, and anti-human IgG-AP was added at a ratio of 1:7500. The PVDF membrane was placed in the solution and incubated at room temperature on a horizontal shaker for 1 h. Then, it was washed three times with TBST solution for 5 min each time. The PVDF membrane was placed in a clean Petri dish, and about 1 mL of AP colorimetric solution was added to each membrane in the dark. The color development of the bands was observed. When the bands were clear, water was added to stop the reaction.

[0108] 2. Experimental Results

[0109] Test results as follows Figure 4 As shown, the results indicate that the IsdB-H6 antibody can bind to IsdB- N2 Antigen binding indicates that the epitope of the IsdB-H6 antibody is a linear epitope.

[0110] Example 8: Inhibitory effect of anti-IsdB antibody on the growth of recombinant Staphylococcus aureus in a low-iron environment.

[0111] 1. Experimental Methods

[0112] By using anti-IsdB antibody under low iron conditions, we observed its effect on the iron uptake pathway of recombinant Staphylococcus aureus and evaluated the potential effect of the antibody in inhibiting bacterial growth.

[0113] First, USA300 cells were cultured using TSB as a base. Then, the activated bacterial culture was cultured overnight in RPMI 1640 (containing 2,2′-bipyridine and casein hydrolysates). The next day, the cells were collected by centrifugation and washed twice with RPMI 1640 (containing 2,2′-bipyridine and casein hydrolysates). Finally, the cells were resuspended in RPMI 1640 (containing 2,2′-bipyridine and / or human hemoglobin). For the antibody group, 1 μM and 100 μM IsdB-H6 antibody were mixed with an equal volume of resuspended bacterial culture (resuspended in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin)) and added to 96-well cell culture plates. For the control group, an equal volume of resuspended bacterial culture (resuspended in RPMI 1640) was added to PBS buffer. The bacterial suspension was resuspended in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) and added to a 96-well cell culture plate. For the control group, an equal volume of the resuspended bacterial culture (resuspended in RPMI 1640 (containing 2,2′-bipyridine)) was added to a 96-well cell culture plate using PBS buffer. The 96-well cell culture plates were incubated at 37°C for 24 hours. The OD values ​​of each well were periodically measured using a microplate reader. 600 The values ​​were used to monitor bacterial growth, plot bacterial growth curves, and further calculate the inhibition rate of antibody on bacterial growth.

[0114] 2. Experimental Results

[0115] The results are as follows Figure 5 The absorbance (OD6) of Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) for 24 hours was shown to be... 00 The inhibition rate was 100%; Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) with 1 μM antibody for 24 hours showed an inhibition rate of 12%; Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) with 100 μM antibody for 24 hours showed an inhibition rate of 58%.

[0116] Example 9: IsdB-H6 monoclonal antibody opsonizes and kills Staphylococcus aureus.

[0117] 1. Experimental Methods

[0118] Antibodies can promote the phagocytosis and killing of Staphylococcus aureus by immune cells, but Staphylococcus aureus possesses multiple virulence factors that prevent phagocytosis. To evaluate the ability of the IsdB-H6 monoclonal antibody to promote the killing of Staphylococcus aureus by phagocytes, the IsdB-H6 monoclonal antibody was tested in an opsonization phagocytosis-killing assay (OPK) to assess the ability of the monoclonal antibody of this invention to promote the killing of Staphylococcus aureus USA300 by phagocytes in the presence of complement. The procedure involved serially diluting the IsdB-H6 monoclonal antibody, positive serum, negative serum, and control antibody seven times in a 96-well plate; culturing Staphylococcus aureus USA300 in TSB and then resuspending it in OBB buffer to a concentration of 1.0 × 10⁻⁶. 5 CFU / mL was added to microplates (10 μL / well), and incubated with shaking at room temperature for 1 hour. Leukocytes were then isolated from fresh rabbit whole blood, washed, and the concentration adjusted to 2.0 × 10⁻⁶ using buffer. 6 Cells / mL, after being mixed with complement in a certain ratio, were incubated in an inverted incubator at 37°C for half an hour. The cell-complement mixture was then added to a microplate (50 μL / well) and phagocytosed at 37°C with 5% CO2 for 60 min. After phagocytosis, the microplate was placed on ice for 20 min, and 10 μL of the mixture was seeded onto MHA for counting.

[0119] 2. Experimental Results

[0120] The results are as follows Figure 6 As shown, the control antibody did not promote cell killing of Staphylococcus aureus, while the IsdB-H6 monoclonal antibody did promote cell killing of Staphylococcus aureus, with a maximum bactericidal rate of 73%.

[0121] Example 10: The role of IsdB-H6 monoclonal antibody in a systemic MRSA infection model

[0122] 1. Experimental Methods

[0123] This invention evaluates the protective efficacy of the IsdB-H6 monoclonal antibody against Staphylococcus aureus USA300 in a systemic infection model. Staphylococcus aureus USA300 was cultured in TSB, passaged, and grown to mid-log phase (OD). 600 =1.0). The culture was then washed twice in physiological saline and measured with OD. 600The bacterial suspension was resuspended in physiological saline at an optical density of 1.6. Mice were injected with 100 μL of the bacterial suspension via the tail vein. One day before infection, mice in the experimental group were treated with 100 mg / kg of IsdB-H6 monoclonal antibody, mice in the control group were treated with 100 mg / kg of hIgG1 (PcrV-A039), and mice in the blank group were treated with physiological saline. After challenge with 100 μL of the bacteria via the tail vein, the mice's physical condition was monitored until 7 days post-infection, and the number of surviving mice was recorded every 12 hours to calculate the survival rate.

[0124] 2. Experimental Results

[0125] The protection rate of monoclonal antibody containing 100 mg / kg IsdB-H6 was 60%, while the protection rate of monoclonal antibody containing 100 mg / kg hIgG1 (PcrV-A039) was 10%. Figure 7 ).

[0126] Example 11: In vivo prophylactic effect of IsdB-H6 monoclonal antibody on an acute pneumonia model of MRSA

[0127] 1. Experimental Methods

[0128] This invention evaluates the protective efficacy of the IsdB-H6 monoclonal antibody against Staphylococcus aureus USA300 in a pneumonia model. Staphylococcus aureus USA300 was cultured in BHI broth and passaged to OD. 600 The value was 1.0. The culture was then washed twice in physiological saline and diluted with 2 × 10⁻⁶ solution. 10 The bacterial suspension was resuspended in physiological saline at a density of cfu / mL. Mice were challenged with 20 μL of the bacterial suspension via endotracheal intubation. One day before infection, mice in the experimental group were treated with monoclonal antibody containing 100 mg / kg IsdB-H6, mice in the control group were treated with hIgG1 (PcrV-A039) containing 100 mg / kg, and mice in the blank group were treated with physiological saline. After endotracheal intubation and challenge with 20 μL of the bacterial suspension, the survival status of the mice was monitored until 7 days post-infection. The number of surviving mice was recorded every 12 hours, and the survival rate was calculated.

[0129] 2. Experimental Results

[0130] The protection rate of monoclonal antibody containing 100 mg / kg IsdB-H6 was 70%, while the protection rate of monoclonal antibody containing 100 mg / kg hIgG1 (PcrV-A039) was 10%. Figure 8 ).

[0131] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody against Staphylococcus aureus IsdB, characterized in that, The antibody comprises a CDR region in the heavy chain variable region as shown in SEQ ID NO:8; and a CDR region in the light chain variable region as shown in SEQ ID NO:16; The CDR area can be determined using any one of the following schemes: IMGT, Chothia, Kabat, AbM, or Contact.

2. The antibody according to claim 1, characterized in that, The antibody has the heavy chain variable region sequence shown in SEQ ID NO: 8 and the light chain variable region sequence shown in SEQ ID NO:

16.

3. An antibody derivative, characterized in that, The antibody derivative is a complex obtained by modifying the antibody according to any one of claims 1-2; the modification includes conjugation modification using a detectable marker, conjugation modification using a therapeutic agent, or conjugation modification using an imaging agent.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-2.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6.

8. A product for detecting Staphylococcus aureus IsdB, characterized in that, The product comprises the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6.

9. The use of the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of products for diagnosing or treating diseases caused by Staphylococcus aureus infection.

10. The use of the antibody according to any one of claims 1-2 in the preparation of the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the pharmaceutical composition according to claim 7, or the product for detecting Staphylococcus aureus IsdB according to claim 8.

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