A monoclonal antibody against Shiga toxin type II, related products and applications

Abstract

This invention discloses a monoclonal antibody against type II Shiga toxin, its related products, and applications. The monoclonal antibody can specifically bind to Stx2, Stx2a, and Stx2b and has high binding activity. In addition, the monoclonal antibody can significantly prolong the survival time of mice infected with enterohemorrhagic Escherichia coli and significantly improve their survival rate. This invention provides a new strategy for the early diagnosis of enterohemorrhagic Escherichia coli-related diseases and the development of drugs for the prevention or treatment of enterohemorrhagic Escherichia coli-related diseases, and has good prospects for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against type II Shiga toxin and its related products and applications. Background Technology

[0002] Enterohemorrhagic Escherichia coli (EHEC) infection is a highly threatening zoonotic disease, prevalent globally. Its infection is characterized by explosive transmission, high pathogenicity, and lethality, posing a significant global public health challenge and a major threat to human health. The O157:H7 serotype is the main type causing severe infection in humans. EHEC infection can cause diarrhea, hemorrhagic colitis, and serious complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Hemolytic uremic syndrome, in particular, can cause irreversible kidney damage and has a high mortality rate.

[0003] The primary virulence factor of EHEC is phage-encoded Shiga toxin (Stx), which exists primarily in two antigenic forms: type I Shiga toxin (Stx1) and type II Shiga toxin (Stx2). Both toxins have a 1A5B structure, consisting of one A subunit and five B subunits. The A subunit is endotoxic, interacting with 28S rRNA to halt protein synthesis, which is the pathological basis for the clinical manifestations of E. coli O157:H7. The B subunits have cell-binding properties, binding to cells with a specific receptor (Gb3) to guide the A subunit's function. Compared to Stx1, Stx2 is more associated with severe systemic diseases, and the incidence of kidney damage and neurological sequelae is significantly higher in Stx2-mediated EHEC infections. Furthermore, Stx2 is the most common cause of hemolytic uremic syndrome in clinical practice. Currently, treatment for EHEC infection is mainly based on antibiotics, but this treatment has significant side effects. Therefore, developing a neutralizing monoclonal antibody against type II Shiga toxin is of great clinical value and an urgent practical need for blocking the pathogenic pathway of the toxin and improving the prognosis of severe cases. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a monoclonal antibody against Shiga toxin type II, related products, and applications. This invention has successfully screened and obtained a novel fully human neutralizing antibody (YG12-1) against Shiga toxin type II of enterohemorrhagic Escherichia coli. This antibody can be used for the detection of Stx2, Stx2a, Stx2b, and enterohemorrhagic Escherichia coli. Furthermore, this antibody can also be used for the prevention and / or treatment of diseases caused by Stx2, Stx2a, Stx2b, and enterohemorrhagic Escherichia coli. This provides a novel approach and strategy for the research and development of related diagnostic reagents, diagnostic products, and therapeutic drugs, and has promising application prospects.

[0005] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0006] A first aspect of the present invention provides a neutralizing monoclonal antibody against type II Shiga toxin, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The amino acid sequences of HCDR1-3 in the heavy chain variable region are shown in SEQ ID NO:1-3, respectively;

[0008] The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NO:9-11, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4.

[0010] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0011] In some embodiments, the HCDR1, HCDR2, and HCDR3 in the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 in the light chain variable region of the monoclonal antibody described in this invention are not limited to the amino acid sequences shown above. The amino acid sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable region and light chain variable region described above in this invention using any CDR numbering scheme are all within the protection scope of this invention.

[0012] In some implementations, the CDR numbering scheme includes, but is not limited to, any one or any combination of two or more of the following: Chothia numbering scheme, Kabat numbering scheme, Contact numbering scheme, IMGT numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, and Aho numbering scheme.

[0013] In some embodiments, antibody sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the corresponding amino acid sequences of the heavy chain variable region, the light chain variable region, the HCDR1, HCDR2, and HCDR3 sequences in the heavy chain variable region, or the LCDR1, LCDR2, and LCDR3 sequences in the light chain variable region of the monoclonal antibody provided by the present invention as described above are all within the scope of protection of the present invention.

[0014] A second aspect of the present invention provides a nucleic acid molecule that encodes the monoclonal antibody described in the first aspect of the present invention.

[0015] In some embodiments, the nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO:8 or a nucleotide sequence having at least 70% homology with SEQ ID NO:8.

[0016] In some embodiments, the nucleotide sequence of the variable region of the light chain of the monoclonal antibody is as shown in SEQ ID NO:16 or a nucleotide sequence having at least 70% homology with SEQ ID NO:16.

[0017] In some embodiments, the nucleic acid molecule may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases, as readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as non-electrolyte linkages (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.), charged linkages (e.g., thiophosphates, dithiophosphates, etc.), side-linked portions (e.g., peptides), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.).

[0018] A third aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0019] In some embodiments, the vector is a DNA vector, an RNA vector, or a virus-derived vector, including but not limited to: lentiviral vectors, retroviral vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, or herpesvirus vectors.

[0020] In this invention, the recombinant vector refers to a nucleic acid molecule capable of amplifying another nucleic acid linked thereto. The recombinant vector includes a vector serving as a self-replicating nucleic acid structure and a vector that binds to the genome of a host cell in which it has already been introduced.

[0021] A fourth aspect of the present invention provides a recombinant host cell comprising the recombinant vector described in the third aspect of the present invention.

[0022] In some embodiments, the host cells include, but are not limited to: COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; NSO cells; yeast, etc. Specific eukaryotic host cells are selected based on their ability to perform the desired post-translational modifications to the heavy and / or light chains of the monoclonal antibodies of the present invention as described above.

[0023] In some embodiments, the nucleic acid molecules of the present invention as described above can be introduced into the desired host cells by any method, including but not limited to: DEAE-dextran-mediated transfection, calcium phosphate transfection, cationic lipid-mediated transfection, electroporation, etc. In the present invention, nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method known to those skilled in the art.

[0024] The fifth aspect of the invention provides any of the following products:

[0025] (1) A detection reagent comprising the monoclonal antibody described in the first aspect of the present invention;

[0026] (2) A test kit comprising the monoclonal antibody or the test reagent described in the first aspect of the present invention;

[0027] (3) A pharmaceutical composition comprising the monoclonal antibody described in the first aspect of the present invention;

[0028] (4) A biological agent comprising the monoclonal antibody or the pharmaceutical composition described in the first aspect of the present invention.

[0029] In some embodiments, the pharmaceutical composition or pharmaceutical formulation may further comprise a variety of pharmaceutically acceptable carriers. These pharmaceutically acceptable carriers include, but are not limited to, solvents, adjuvants, and diluents. Furthermore, various pharmaceutically acceptable excipients may be used, such as pH adjusters, buffers, tonic modifiers, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include saline, glucose, water, glycerol, ethanol, and combinations thereof.

[0030] In some embodiments, the pharmaceutical composition may be formulated as a liquid, solid, semi-solid, or gaseous formulation; the dosage forms of the biological agent include, but are not limited to, solutions, tablets, capsules, powders, granules, suppositories, enemas, inhalers, and aerosols.

[0031] In some embodiments, the monoclonal antibody as described above can be formulated into an injection dosage form, including subcutaneous administration, by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent such as vegetable oil or other oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and, if necessary, adding conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0032] In some embodiments, the pharmaceutical compositions or biological agents of the present invention as described above can be formulated for inhalation, for example, using pressurized, acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, etc.

[0033] In some embodiments, the pharmaceutical composition or biological agent as described above may also be formulated into sustained-release microcapsules together with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. The sustained-release microcapsules can be prepared using certain methods well known to those skilled in the art for preparing such formulations.

[0034] The sixth aspect of the present invention provides any of the following methods:

[0035] (1) A method for preparing a monoclonal antibody according to the first aspect of the present invention, the method comprising: culturing the recombinant host cell according to the fourth aspect of the present invention, and isolating the monoclonal antibody according to the first aspect of the present invention from the culture;

[0036] (2) A method for detecting type II Shiga toxin Stx2, Stx2a and / or Stx2b proteins for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the sample to be tested with the monoclonal antibody described in the first aspect of the present invention, the detection reagent or detection kit described in the fifth aspect of the present invention, and detecting the formation of antigen-antibody immune complexes;

[0037] (3) A method for preparing recombinant host cells according to the fourth aspect of the present invention, the method comprising: introducing the recombinant vector according to the third aspect of the present invention into host cells to obtain the recombinant host cells according to the fourth aspect of the present invention.

[0038] In this invention, Stx2a is the A subunit of type II Shiga toxin (Stx2), and is the most important pathogenic active molecule of Stx2. The A subunit includes two fragments, A1 and A2. The A1 fragment is the toxic active fragment, with the virulence active site located at tyrosine residue 77. The A2 fragment is inserted into a pore formed by five B subunits, exerting its virulence through binding to cell receptors via the B subunits. Studies have shown that enterohemorrhagic Escherichia coli carrying Stx2a is more likely to induce hemolytic uremic syndrome (HUS), and compared with Stx2, Stx2a-induced kidney damage progresses more rapidly, with a higher proportion of patients developing persistent proteinuria and glomerulosclerosis.

[0039] In this invention, Stx2b is the B subunit of type II Shiga toxin (Stx2), which specifically binds to the cell membrane receptors globulinylsphingosine Gb3 or globulinylsphingosine Gb4. Gb3 is present on the surface of eukaryotic cells, and is abundant in the cell membranes of intestinal epithelial cells, renal endothelial cells, and central nervous system cells. After Stx2 binds to the receptor Gb3 via its B subunit, it is endocytosed into the cell by an internal protein vesicle. Stx2b has a stronger targeting effect on macrophages in the lamina propria of the intestinal mucosa, and can induce a severe intestinal inflammatory response by inducing macrophages to release IL-1β and TNF-α. Although some strains carrying Stx2b have a lower risk of causing hemorrhagic colitis (HUS), they are more likely to cause hemorrhagic colitis.

[0040] The seventh aspect of the present invention provides for any of the following applications:

[0041] (1) The use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention and / or the recombinant host cell described in the fourth aspect of the present invention in the preparation of a detection reagent for detecting type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli;

[0042] (2) The use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, and / or the detection reagent described in the fifth aspect of the present invention in the preparation of a detection kit for detecting type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli;

[0043] (3) The use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, and the detection reagent and / or detection kit described in the fifth aspect of the present invention in the detection of type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli for non-diagnostic and non-therapeutic purposes;

[0044] (4) The use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, the detection reagent and / or detection kit described in the fifth aspect of the present invention in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection;

[0045] (5) The use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, and / or the recombinant host cell described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition for the prevention and / or treatment of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection.

[0046] The eighth aspect of the present invention provides the use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, the recombinant host cell described in the fourth aspect of the present invention, and / or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a biological agent for the prevention and / or treatment of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection.

[0047] In some implementations, Shiga toxin type II (Stx2) and its subtypes (Stx2a and Stx2b) are produced by bacteria such as enterohemorrhagic Escherichia coli (EHEC), which can cause infectious diseases characterized by local intestinal damage and systemic multi-organ involvement. EHEC infection is often caused by ingestion of contaminated food or water, with typical symptoms including hemorrhagic colitis (manifested as abdominal pain and bloody stools) and hemolytic uremic syndrome (HUS). HUS occurs because Stx2 damages glomerular endothelial cells, leading to microthrombus formation and acute kidney injury; severe cases can present with oliguria, hematuria, and irreversible kidney damage. Stx2a, due to its stronger affinity for host cell receptors, is more likely to induce HUS and neurological sequelae (such as epilepsy and coma), while Stx2b has a prominent targeting effect on intestinal mucosal macrophages, often leading to hemorrhagic colitis and has a potential association with chronic renal fibrosis.

[0048] The present invention also provides a method for diagnosing or assisting in the diagnosis of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection, the method comprising: contacting a test sample from a subject with the monoclonal antibody described in the first aspect of the present invention, the detection reagent and / or detection kit described in the fifth aspect of the present invention, detecting the formation of antigen-antibody immune complexes, and based on the result diagnosing or assisting in the diagnosis of whether the subject has Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection or the risk of having Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection.

[0049] The present invention also provides a method for preventing and / or treating Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the monoclonal antibody described in the first aspect of the present invention, the pharmaceutical composition described in the fifth aspect of the present invention and / or the biological agent.

[0050] In this invention, the subject refers to a human, veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.), veterinary poultry, and experimental animal models of diseases (e.g., mice, rats, ferrets, monkeys, etc.). In one specific embodiment, the subject of this invention is a human.

[0051] In this invention, the effective amount refers to the amount of the monoclonal antibody, pharmaceutical composition, and / or biological agent (i.e., the active ingredient) as described above, sufficient to produce the desired activity when administered to a subject in need. When a combination of active ingredients is administered, the effective amount of the combination may or may not include the amounts of each ingredient that might be effective when administered alone. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the severity of the condition being treated, the specific type of drug used, the method of administration, etc.

[0052] In this invention, prevention and / or treatment refers to alleviating or reducing at least one symptom associated with a specified condition, or slowing or reversing the progression of such condition. Furthermore, prevention and / or treatment also refer to stopping, delaying onset (i.e., the period prior to clinical manifestations of the disease), and / or reducing the risk of developing or worsening the disease. Additionally, prevention and / or treatment may include: (1) preventing or delaying the onset of at least one clinical or subclinical symptom of the state, condition, or illness in a subject who may have or be susceptible to the state, condition, or illness but has not yet experienced or displayed clinical or subclinical symptoms of the state, condition, or illness; or (2) suppressing the state, condition, or illness, i.e., preventing, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom; or (3) alleviating the disease, i.e., causing the resolution of at least one of the state, condition, or illness or its clinical or subclinical symptoms. In this invention, prevention refers to preventing the spread of infection in a subject exposed to a toxin, for example, preventing the toxin from entering the subject's body.

[0053] In some embodiments, the monoclonal antibodies, pharmaceutical compositions and / or biological agents of the present invention as described above can be administered in vivo via various routes, including but not limited to: intravenous, subcutaneous, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, rectal, intraperitoneal, inhalation, intradermal, percutaneous and intrathecal, or otherwise, such as by implantation.

[0054] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0055] This invention discloses a novel fully human neutralizing antibody, YG12-1, against Shiga toxin of enterohemorrhagic Escherichia coli type II. The antibody YG12-1 specifically binds to Stx2, Stx2a, and Stx2b with high binding activity. Furthermore, the antibody YG12-1 significantly prolongs the survival time and improves the survival rate of mice infected with enterohemorrhagic Escherichia coli. This invention provides a new strategy for the early diagnosis of enterohemorrhagic Escherichia coli-related diseases and the development of drugs for the prevention or treatment of these diseases, showing promising clinical application prospects. Attached Figure Description

[0056] Figure 1 This is a graph showing the results of monoclonal antibody identification.

[0057] Figure 2 The image shows the electrophoresis results of the monoclonal antibody YG12-1, where A represents reducing electrophoresis and B represents non-reducing electrophoresis.

[0058] Figure 3 The image shows the results of the binding activity assay of monoclonal antibody YG12-1 with Stx2.

[0059] Figure 4 The image shows the results of the binding activity assay of monoclonal antibody YG12-1 with Stx2a.

[0060] Figure 5 The image shows the results of the binding activity assay of monoclonal antibody YG12-1 with Stx2b.

[0061] Figure 6 The figure shows the results of detecting the neutralizing activity of the monoclonal antibody YG12-1 in an animal model. Detailed Implementation

[0062] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0063] Example 1: Screening of anti-Stx2 monoclonal antibody YG12-1

[0064] 1. Experimental Materials

[0065] PBS: Sewell, G4202; Skim milk powder: BD, 232100; Immunotherapy tubes: Thermo, 470319; PBST: Lamborghini, P7207B; TBST: Lamborghini, P7209B; 2YT: Sangon Biotech, A507019; Agar powder: A100637; Ampicilin: Beyotime, ST008; Kanamycin ST102; Protein A: TransGold, DP301-01; Restriction endonuclease NdeI / SalI: Takara, 1621 / 1636; Color pre-stained protein marker: Jinpulai, P06M01; HRP-labeled goat anti-human IgG: Jackson, 109-036-003; TSB: OXOID, CM0129; 8-week-old C57BL / 6J female mice: Vital River.

[0066] 2. Biopanning of anti-Stx2 monoclonal antibodies

[0067] Group A, Stx2

[0068] (1) First round of affinity screening

[0069] ① Dilute Stx2 protein to 10 μg / mL with 500 μL PBS and coat the immunotubes overnight at 4°C.

[0070] ② Take the immunotherapy tube, add 5% (v / v) Milk-PBST buffer, and block at room temperature for 1 h.

[0071] ③ Take the phage antibody library, add 5% (v / v) Milk-PBST buffer, and block at room temperature for 1 h.

[0072] ④ Take the immunotherapy tube from step ②, wash it three times with sterile PBS buffer, and then add the phage antibody library from step ③ (the amount of phage added is approximately 1.2 × 10⁻⁶). 12 Let it stand at room temperature for 1 hour.

[0073] ⑤ After completing step ④, take the immunotherapy tube, first add an appropriate amount of sterile PBS buffer to wash (the purpose is to wash away unbound phages), then add 500 μL of pH 2.2, 0.1 M HCl-Glycine to elute phage-Abs, collect the eluent and add 1.5 M Tris-HCl (pH 8.8) to adjust the pH to 7.4.

[0074] ⑥ Inoculate a single TG1 clone of Escherichia coli into LB liquid medium and culture at 37°C with shaking at 200 rpm until the logarithmic growth phase to obtain the culture solution.

[0075] ⑦ Mix 500 μL of elution buffer from step ⑤ with 10 mL of the culture medium obtained in step ⑥, incubate at 37°C for 30 min (for infection), then centrifuge at 4000 rpm for 15 min, collect the cells and spread them evenly on 2YTAG plates, and incubate at 37°C overnight.

[0076] ⑧ After completing step ⑦, scrape the colonies from the 2YTAG plate and inoculate them into 2YTAG medium for phage display. Precipitate with PEG / NaCl to obtain the phage. This phage is the one obtained after a round of panning.

[0077] (2) Second round of affinity selection

[0078] Replace the “phage antibody library” in step (1) ③ with the phage obtained in step (1) ⑧, while keeping all other steps unchanged, to obtain the phage. This phage is the one obtained after two rounds of screening.

[0079] (3) Third round of affinity selection

[0080] Replace the “phage antibody library” in step (1) ③ with the phage obtained in step (2), while keeping all other steps unchanged, to obtain the phage. This phage is the one obtained after three rounds of screening.

[0081] B. Control Group

[0082] Following the three-round affinity panning procedure for the Stx2 group, the 10 μg / mL Stx2 protein solution was replaced with PBS buffer, while all other steps remained unchanged, to obtain the corresponding phages (as a control).

[0083] The number of phages obtained in each round of affinity panning is shown in Table 1. The results indicate that after three rounds of panning, the number of recombinant phages obtained in the Stx2 group showed a significant increasing trend, while the number of phages in the control group remained relatively unchanged. This suggests that phages binding to Stx2 were significantly enriched.

[0084] Table 1. Number of phages selected in each round of affinity screening

[0085]

[0086] 3. Screening of anti-Stx2 monoclonal antibody-positive clones

[0087] (1) After completing the above steps, take 192 clones obtained after three rounds of panning in Stx2 group, inoculate them into 1 mL 2YTAG medium, and culture overnight at 37℃ and 220 rpm to obtain the culture solution.

[0088] (2) Take 30 μL of the culture medium and inoculate it into 900 μL of 2YTAG medium. Incubate at 37℃ and 220 rpm with shaking until OD. 600 nm Once the value reaches 0.6-0.8, then add 5 × 10. 10 The helper phage M13KO7 was incubated at 37°C for 30 min.

[0089] (3) After completing step (2), centrifuge at 4℃ and 4000 rpm for 15 min, collect the precipitate and resuspend it with 1 mL of 2YTAK, and incubate overnight at 28℃ and 220 rpm.

[0090] (4) After completing step (3), phage-ELISA is used to identify positive clones and the positive clones are sequenced.

[0091] The experimental results are shown in Figure 1 . Figure 1 1 corresponds to OD 450 nm >1.5, N=3, 2 corresponds to 1.0 <OD 450 nm <1.5, N=1, 3 corresponds to 0.5 <OD450 nm <1.0, N=4, 4 corresponds to OD 450 nm <0.5, N=184. The results showed that the positive rate among clones was 4.17%. Eight clones with high ELISA signal values ​​were selected for sequencing, and one antibody sequence was obtained, named YG12-1 antibody. The sequence information of the YG12-1 antibody is shown in Table 2.

[0092] Table 2 Sequence information of YG12-1 antibody

[0093]

[0094] Example 2: Functional identification of the anti-Stx2 monoclonal antibody YG12-1

[0095] 1. Preparation of YG12-1 antibody

[0096] The vector pCDNA3.1 is a product of Invitrogen.

[0097] (1) Construction of recombinant plasmids

[0098] The small fragment between the restriction endonuclease NdeI and SalI recognition sequences of vector pCDNA3.1 was replaced with the corresponding DNA molecules to obtain the heavy chain expression vector. The small fragment between the restriction endonuclease NdeI and SalI recognition sequences of vector pCDNA3.1 was replaced with the corresponding DNA molecules to obtain the light chain expression vector.

[0099] (2) Antibody preparation

[0100] The light and heavy chain variable region genes of the YG12-1 antibody were cloned into the pCDNA3.1 vector containing the light and heavy chain constant region genes, transfected into 293T cells, and expressed as a complete antibody secretion. After purification with protein A, the complete antibody protein was obtained by ultrafiltration and medium replacement. The experimental results are shown in [Figure number missing]. Figure 2 (A is reducing electrophoresis, B is non-reducing electrophoresis). The results show that the monoclonal antibody YG12-1 against Stx2 was successfully prepared.

[0101] 2. ELISA detection of the binding activity of YG12-1 antibody to Stx2.

[0102] (1) Take an ELISA plate and coat the antigen (Stx2 protein) with coating solution. The antigen concentration is 10 μg / mL, 100 μL / well, and the coating is carried out overnight at 4℃.

[0103] (2) After completing step (1), take the ELISA plate and wash it three times with PBST buffer.

[0104] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 h.

[0105] (4) YG12-1 antibody was serially diluted three times starting from 10 μg / mL using PBS buffer (a total of 10 dilution gradients were set) to obtain YG12-1 antibody solutions of different concentrations.

[0106] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or different concentrations of YG12-1 antibody solution obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0107] (6) After completing step (5), take the ELISA plate and wash it three times with PBST buffer (250 μL per well).

[0108] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution solution (obtained by diluting HRP-labeled goat anti-human IgG secondary antibody at 1:40000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0109] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and incubate at room temperature for 5 min. The colorimetric reagent is a component of the TMB colorimetric kit.

[0110] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop the color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0111] The experimental results are shown in Figure 3 The results showed that the YG12-1 antibody could bind to the Stx2 protein with high binding activity, and its half-maximal effective concentration (EC50) was 6.50 ng / mL.

[0112] 3. ELISA detection of the binding activity between YG12-1 antibody and Stx2a.

[0113] (1) Take an ELISA plate and coat the antigen (Stx2a protein) with coating solution. The antigen concentration is 10 μg / mL, 100 μL / well, and the coating is carried out overnight at 4℃.

[0114] (2) After completing step (1), take the ELISA plate and wash it three times with PBST buffer.

[0115] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 h.

[0116] (4) YG12-1 antibody was serially diluted three times starting from 10 μg / mL using PBS buffer (a total of 10 dilution gradients were set) to obtain YG12-1 antibody solutions of different concentrations.

[0117] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or different concentrations of YG12-1 antibody solution obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0118] (6) After completing step (5), take the ELISA plate and wash it three times with PBST buffer (250 μL per well).

[0119] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution solution (obtained by diluting HRP-labeled goat anti-human IgG secondary antibody at 1:40000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0120] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and incubate at room temperature for 5 min. The colorimetric reagent is a component of the TMB colorimetric kit.

[0121] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop the color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0122] The experimental results are shown in Figure 4 The results showed that the YG12-1 antibody could bind to the Stx2a protein with high binding activity, and its half-maximal effective concentration (EC50) was 99.56 ng / mL.

[0123] 4. ELISA detection of the binding activity between YG12-1 antibody and Stx2b.

[0124] (1) Take an ELISA plate and coat the antigen (Stx2b protein) with coating solution. The antigen concentration is 10 μg / mL, 100 μL / well, and the coating is carried out overnight at 4℃.

[0125] (2) After completing step (1), take the ELISA plate and wash it three times with PBST buffer.

[0126] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 h.

[0127] (4) YG12-1 antibody was serially diluted three times starting from 10 μg / mL using PBS buffer (a total of 10 dilution gradients were set) to obtain YG12-1 antibody solutions of different concentrations.

[0128] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or different concentrations of YG12-1 antibody solution obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0129] (6) After completing step (5), take the ELISA plate and wash it three times with PBST buffer (250 μL per well).

[0130] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution solution (obtained by diluting HRP-labeled goat anti-human IgG secondary antibody at 1:40000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0131] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and incubate at room temperature for 5 min. The colorimetric reagent is a component of the TMB colorimetric kit.

[0132] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop the color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0133] The experimental results are shown in Figure 5 The results showed that the YG12-1 antibody could bind to the Stx2b protein with high binding activity, and its half-maximal effective concentration (EC50) was 76.93 ng / mL.

[0134] 5. Animal model detection of YG12-1 neutralizing activity

[0135] (1) Pick Escherichia coli O157:H7 and inoculate it into fresh TSB medium and incubate overnight at 37°C and 220 rpm.

[0136] (2) Centrifuge at 4000 rpm for 5 min, discard the supernatant, wash twice with PBS, and adjust OD. 600 nm =0.5.

[0137] (3) Eight-week-old female C57BL / 6J mice were randomly divided into groups of eight and injected intraperitoneally with 200 μL PBS, cIgG1 (200 μg), YG12-1 (100 μg), and YG12-1 (200 μg), respectively.

[0138] (4) One hour later, inject 200 μL of bacterial solution from step (1) into the peritoneum again.

[0139] (5) Observe and record the survival status of the mice.

[0140] The experimental results are shown in Figure 6 The results showed that YG12-1 could significantly prolong the survival time of mice infected with Escherichia coli, and YG12-1 could significantly improve the survival rate.

Claims

1. A neutralizing monoclonal antibody against type II Shiga toxin, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of HCDR1-3 in the heavy chain variable region are shown in SEQ ID NO:1-3, respectively; The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NO:9-11, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

12.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody as described in any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 4.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant vector of claim 5.

7. A detection reagent, characterized in that, The detection reagent comprises the monoclonal antibody as described in any one of claims 1-3.

8. A test kit, characterized in that, The test kit comprises the monoclonal antibody of any one of claims 1-3 or the test reagent of claim 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody according to any one of claims 1-3.

10. A biological agent, characterized in that, The biological agent comprises the monoclonal antibody of any one of claims 1-3 or the pharmaceutical composition of claim 9.

11. A method for preparing a monoclonal antibody according to any one of claims 1-3, characterized in that, The method includes: culturing the recombinant host cell of claim 6, and isolating the monoclonal antibody of any one of claims 1-3 from the culture.

12. A method for non-diagnostic and non-therapeutic detection of type II Shiga toxin Stx2, Stx2a, and / or Stx2b proteins, characterized in that, The method includes: contacting the sample to be tested with the monoclonal antibody of any one of claims 1-3, the detection reagent of claim 7, or the detection kit of claim 8 to detect the formation of antigen-antibody immune complexes.

13. A method for preparing recombinant host cells according to claim 6, characterized in that, The method includes: introducing the recombinant vector of claim 5 into a host cell to obtain the recombinant host cell of claim 6.

14. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, and / or the recombinant host cell of claim 6 in the preparation of a detection reagent for detecting type II Shiga toxins Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli.

15. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant host cell of claim 6, and / or the detection reagent of claim 7 in the preparation of a detection kit for detecting type II Shiga toxins Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli.

16. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant host cell of claim 6, the detection reagent of claim 7, and / or the detection kit of claim 8 in the non-diagnostic and non-therapeutic target detection of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli.

17. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant host cell of claim 6, the detection reagent of claim 7, and / or the detection kit of claim 8 in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection.

18. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, and / or the recombinant host cell of claim 6 in the preparation of a pharmaceutical composition for the prevention and / or treatment of Shiga toxin type II Stx2, Stx2a, Stx2b, and / or enterohemorrhagic Escherichia coli infection.

19. The use of the monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, the recombinant host cell of claim 6, and / or the pharmaceutical composition of claim 9 in the preparation of a biological agent for the prevention and / or treatment of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection.

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