Monoclonal antibody for resisting shiga toxin type II as well as related product and application thereof

Abstract

The invention discloses an anti-type II Shiga toxin monoclonal antibody and a related product and application thereof, 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 a mouse infected with enterohemorrhagic escherichia coli and significantly improve the survival rate of the mouse, and the monoclonal antibody can be used for preparing a monoclonal antibody for resisting type II Shiga toxin. The invention provides a new strategy for early diagnosis of enterohemorrhagic Escherichia coli infection related diseases and development of drugs for prevention or treatment of enterohemorrhagic Escherichia coli infection related diseases, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a monoclonal antibody against type II Shiga toxin and related products and applications thereof. Background Art

[0002] Enterohemorrhagic Escherichia coli (EHEC) infection is a highly threatening zoonosis, presenting a global epidemic. Characterized by explosive spread, high pathogenicity, and lethality, EHEC infection has become a severe global public health challenge and a major threat to human health. The O157:H7 serotype is the primary cause of severe human infection. EHEC infection can cause diarrhea, hemorrhagic colitis, and other serious complications, including hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS, in particular, can cause irreversible kidney damage and carries a high mortality rate.

[0003] The primary virulence factor of EHEC is phage-encoded Shiga toxin (Stx), which exists in two antigenic forms: type I Shiga toxin (Stx1) and type II Shiga toxin (Stx2). Both toxins consist of a 1A5B structure consisting of one A subunit and five B subunits. The A subunit is intracellularly toxic, interacting with 28S rRNA to halt protein synthesis and underlying the clinical manifestations of E. coli O157:H7. The B subunit possesses cell-binding properties, binding to cells with specific receptors (Gb3), thereby directing the action of the A subunit. Compared with Stx1, Stx2 is more associated with severe systemic disease, and the incidence of renal injury and neurological sequelae is significantly higher in cases of Stx2-mediated EHEC infection. Furthermore, Stx2 is the most common cause of hemolytic-uremic syndrome. Currently, treatment for EHEC infection primarily relies on antibiotics, but this approach carries significant side effects. Therefore, the development of a neutralizing monoclonal antibody against Shiga toxin type II has important clinical application value and urgent practical needs for blocking the pathogenic pathway of the toxin and improving the prognosis of severe illness. Summary of the Invention

[0004] In light of this, the present invention aims to provide a monoclonal antibody against type II Shiga toxin, as well as related products and applications. This invention successfully screened and obtained a novel, fully human, neutralizing antibody (YG12-1) against type II Shiga toxin of EHEC. This antibody can be used to detect Stx2, Stx2a, Stx2b, and EHEC. Furthermore, this antibody can be used to prevent and / or treat diseases caused by Stx2, Stx2a, Stx2b, and EHEC. This provides new insights and strategies for the research and development of related detection reagents, detection products, and therapeutic drugs, and has promising application prospects.

[0005] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0006] The first aspect of the present invention provides a neutralizing monoclonal antibody against type II Shiga toxin, wherein the monoclonal antibody comprises 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 NOs: 1-3, respectively;

[0008] The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NOs: 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 of the present 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 the CDR1, CDR2, and CDR3 in the heavy chain variable region and light chain variable region of the present invention as described above using any CDR numbering scheme are all within the scope of protection of the present invention.

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

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

[0014] The second aspect of the present invention provides a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the present invention.

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

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

[0017] In some embodiments, the nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.).

[0018] The 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 viral-derived vector, and the viral-derived vector includes but is not limited to a lentiviral vector, a retroviral vector, an adeno-associated viral vector, an adenoviral vector, a poxvirus vector, or a herpesvirus vector.

[0020] In the present invention, the recombinant vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The recombinant vector includes a vector that is a self-replicating nucleic acid structure and a vector that is incorporated into the genome of a host cell into which it has been introduced.

[0021] The fourth aspect of the present invention provides a recombinant host cell, which comprises 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 modification on 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, the nucleic acid can be transiently or stably transfected into the desired host cells according to any suitable method known to those skilled in the art.

[0024] A fifth aspect of the present 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 detection kit comprising the monoclonal antibody or the detection reagent described in the first aspect of the present invention;

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

[0028] (4) A biological preparation comprising the monoclonal antibody or the pharmaceutical composition according to the first aspect of the present invention.

[0029] In some embodiments, the pharmaceutical composition or formulation may further comprise a variety of pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, but are not limited to, solvents, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances may also be used, such as pH adjusters, buffers, tonicity adjusters, stabilizers, wetting agents, and the like. Non-limiting exemplary carriers include saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0030] In some embodiments, the pharmaceutical composition can be formulated into a liquid, solid, semisolid or gaseous form; the dosage form of the biologic includes but is not limited to: solution, tablet, capsule, powder, granule, suppository, enema, inhalant and aerosol.

[0031] In some embodiments, the monoclonal antibodies of the present invention as described above can be formulated into dosage forms for injection, including subcutaneous administration, by dissolving, suspending or emulsifying the monoclonal antibodies of the present invention as described above in an aqueous or non-aqueous solvent such as vegetable oil or other oils, 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 biologics of the present invention as described above can be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

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

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

[0035] (1) A method for preparing the monoclonal antibody according to the first aspect of the present invention, 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 a sample to be tested with the monoclonal antibody described in the first aspect of the present invention, the detection reagent or the detection kit described in the fifth aspect of the present invention, and detecting the formation of an antigen-antibody immune complex;

[0037] (3) A method for preparing the recombinant host cell 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 a host cell to obtain the recombinant host cell according to the fourth aspect of the present invention.

[0038] In the present invention, Stx2a is the A subunit of type II Shiga toxin (Stx2), the primary pathogenic active molecule of Stx2. The A subunit consists of two fragments, A1 and A2. The A1 fragment is the toxic active fragment, with the toxicity center located at tyrosine 77. The A2 fragment inserts into the pore formed by the five B subunits, exerting its toxic effects through binding of the B subunits to cellular receptors. Studies have shown that enterohemorrhagic Escherichia coli carrying Stx2a are more likely to cause hemolytic uremic syndrome (HUS), and compared with Stx2, Stx2a-induced renal damage progresses more rapidly, with a higher incidence of persistent proteinuria and glomerulosclerosis in patients.

[0039] In the present invention, Stx2b is the B subunit of type II Shiga toxin (Stx2), which can specifically bind to the cell membrane receptors globulin Gb3 or globulin Gb4. Gb3 is present on the surface of eukaryotic cells and is abundant on 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 internalized into the cell by endosomes. Stx2b has a stronger targeting effect on macrophages in the intestinal mucosal lamina propria, and can trigger 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 HUS, they are more likely to cause hemorrhagic colitis.

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

[0041] (1) Use of the monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the recombinant vector of the third aspect of the present invention, and / or the recombinant host cell of 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) 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) Use of the monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the recombinant vector of the third aspect of the present invention, the recombinant host cell of the fourth aspect of the present invention, and the detection reagent and / or detection kit of the fifth aspect of the present invention for non-diagnostic and non-therapeutic detection of type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli;

[0044] (4) 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 preparation of diagnostic products for diagnosing or assisting in the diagnosis of diseases caused by type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection;

[0045] (5) 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 preventing and / or treating diseases caused by infection with type II Shiga toxin Stx2, Stx2a, Stx2b, and / or enterohemorrhagic Escherichia coli.

[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 preparation for preventing and / or treating type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection diseases.

[0047] In some embodiments, Shiga toxin type II (Stx2) and its subtypes (Stx2a and Stx2b) are produced by bacteria such as enterohemorrhagic Escherichia coli (EHEC) and can cause infectious diseases characterized by localized intestinal damage and systemic multi-organ involvement. EHEC infection is often caused by ingestion of contaminated food or water. Typical symptoms include hemorrhagic colitis (manifested by abdominal pain and bloody stools) and hemolytic uremic syndrome (HUS). HUS, caused by Stx2 damaging glomerular endothelial cells, leads to microthrombosis and acute kidney injury. Severe cases can present with oliguria, hematuria, and irreversible renal damage. Stx2a, due to its stronger binding affinity for host cell receptors, is more likely to cause HUS and neurological sequelae (such as seizures and coma). Stx2b, on the other hand, is more targeted to intestinal mucosal macrophages, often causing hemorrhagic colitis and potentially associated with chronic renal fibrosis.

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

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

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

[0051] For purposes of the present invention, an effective amount refers to an amount of the monoclonal antibodies, pharmaceutical compositions, and / or biologics (i.e., active ingredients) described herein, sufficient to produce the desired activity when administered to a subject in need thereof. When a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would 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 drug type used, the route of administration, and other factors.

[0052] In the present invention, the 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 a condition. In addition, the prevention and / or treatment also refers to stopping, delaying the onset (i.e., the period before the clinical manifestation of the disease) and / or reducing the risk of developing or worsening the disease. In addition, the prevention and / or treatment may also include: (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the state, condition or illness in a subject who may have or is 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) inhibiting 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 thereof; or (3) ameliorating the disease, i.e., causing the regression of the state, condition or illness or at least one of its clinical or subclinical symptoms. In the present invention, the prevention refers to preventing the spread of infection in a subject exposed to a toxin, such as preventing the toxin from entering the subject's body.

[0053] In some embodiments, the monoclonal antibodies, pharmaceutical compositions and / or biologics described herein can be administered in vivo by various routes, including but not limited to: intravenous, subcutaneous, oral, intraarterial, parenteral, intranasal, intramuscular, intracardial, rectal, intraperitoneal, inhalation, intradermal, transdermal and intrathecal, or in other ways, such as by implantation.

[0054] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0055] The present invention discloses a novel fully human neutralizing antibody YG12-1 against enterohemorrhagic Escherichia coli type II Shiga toxin. The antibody YG12-1 can specifically bind to Stx2, Stx2a, and Stx2b and has high binding activity. In addition, the antibody YG12-1 can significantly prolong the survival time of mice infected with enterohemorrhagic Escherichia coli and significantly improve their survival rate. The present invention provides a new strategy for the early diagnosis of diseases related to enterohemorrhagic Escherichia coli infection and the development of preventive or therapeutic drugs for diseases related to enterohemorrhagic Escherichia coli infection, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Figure 1 is the result of monoclonal identification;

[0057] Figure 2 The electrophoresis results of the monoclonal antibody YG12-1 are shown in Figure 1, where A is the reduced electrophoresis and B is the non-reduced electrophoresis.

[0058] Figure 3 This is the result of identifying the binding activity of monoclonal antibody YG12-1 with Stx2;

[0059] Figure 4 This is the result of identifying the binding activity of monoclonal antibody YG12-1 with Stx2a;

[0060] Figure 5 This is the result of identifying the binding activity of monoclonal antibody YG12-1 with Stx2b;

[0061] Figure 6 This is the result graph corresponding to the neutralizing activity of monoclonal antibody YG12-1 tested in animal models. DETAILED DESCRIPTION

[0062] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental consumables, reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art. Unless otherwise specified, they can be obtained commercially. The experimental methods for which specific conditions are not specified in the present invention are typically tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

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

[0064] 1. Experimental Materials

[0065] PBS: Seville, G4202, skim milk powder: BD, 232100, immunotube: Thermo, 470319, PBST: Lamblide, P7207B, TBST: Lamblide, P7209B, 2YT: Sangon Biotechnology, A507019, agar powder: A100637, ampicilin: Beyotime, ST008, Kanamycin ST102, protein A: Full-Form Gold, DP301-01, restriction endonuclease NdeI / SalI: Takara, 1621 / 1636, colored prestained protein marker: Jinpulai, P06M01, HRP-conjugated goat anti-human IgG: Jackson, 109-036-003, TSB: OXOID, CM0129, 8-week-old C57BL / 6J female mice: Weitonglihua.

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

[0067] A. Stx2 group

[0068] (1) First round of affinity selection

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

[0070] ② Take the immunotube, 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 immune tube that has completed step ②, add sterile PBS buffer to wash 3 times, and then add the phage antibody library that has completed step ③ (the phage input amount is about 1.2×10 12 ), let it stand at room temperature for 1 h.

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

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

[0075] ⑦ Combine 500 μL of the eluate from step ⑤ with 10 mL of the culture medium from step ⑥, incubate at 37°C for 30 min (for infection), then centrifuge at 4000 rpm for 15 min, collect the cells, spread them evenly on a 2YTAG plate, and culture 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. Use PEG / NaCl precipitation to obtain phage. This phage is the phage obtained after the first round of panning.

[0077] (2) Second round of affinity selection

[0078] The phage antibody library in step (1) ③ is replaced with the phage obtained in step (1) ⑧, and the other steps remain unchanged to obtain a phage. This phage is the phage obtained after the second round of panning.

[0079] (3) The third round of affinity selection

[0080] The phage antibody library in step (1) is replaced by the phage obtained in step (2), and the other steps remain unchanged to obtain a phage. This phage is the phage obtained after three rounds of panning.

[0081] B. Control group

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

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

[0084] Table 1 Number of phages in each round of affinity panning

[0085] 3. Screening of anti-Stx2 monoclonal antibody positive clones

[0086] (1) After completing the above steps, 192 clones obtained after three rounds of selection in the Stx2 group were taken and inoculated into 1 mL of 2YTAG medium. The culture was shaken at 37°C and 220 rpm overnight to obtain a culture solution.

[0087] (2) Take 30 μL of culture solution and inoculate it into 900 μL of 2YTAG medium. Shake and culture at 37°C and 220 rpm until the OD 600 nm The value reaches 0.6-0.8, then add 5×10 10 The helper phage M13KO7 was incubated at 37°C for 30 min.

[0088] (3) After completing step (2), centrifuge at 4°C and 4000 rpm for 15 min, collect the precipitate and resuspend it in 1 mL of 2YTAK, and incubate it at 28°C and 220 rpm overnight.

[0089] (4) After completing step (3), positive clones were identified by phage-ELISA and sequenced.

[0090] The experimental results are shown in Figure 1 . Figure 1 1 in the middle 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 <OD 450 nm<1.0, N=4, 4 corresponds to OD 450 nm <0.5, N=184. The results showed a 4.17% positive clone rate. Eight clones with high ELISA signal values ​​were selected for sequencing, resulting in an antibody sequence named YG12-1. The sequence information for the YG12-1 antibody is shown in Table 2.

[0091] Table 2 Sequence information of YG12-1 antibody

[0092] Example 2 Functional identification of anti-Stx2 monoclonal antibody YG12-1

[0093] 1. Preparation of YG12-1 Antibody

[0094] The vector pCDNA3.1 was a product of Invitrogen.

[0095] (1) Construction of recombinant plasmid

[0096] The heavy chain expression vector was obtained by replacing the small fragment between the restriction endonuclease NdeI and SalI recognition sequences of the vector pCDNA3.1 with the corresponding DNA molecule. The light chain expression vector was obtained by replacing the small fragment between the restriction endonuclease NdeI and SalI recognition sequences of the vector pCDNA3.1 with the corresponding DNA molecule.

[0097] (2) Antibody preparation

[0098] The light chain and heavy chain variable region genes of the YG12-1 antibody were cloned into the pCDNA3.1 vector containing the light chain and heavy chain constant region genes, and transfected into 293T cells for secretory expression of the whole antibody. After protein A purification and ultrafiltration tube concentration, the whole antibody protein was obtained after changing the medium. The experimental results are shown in Figure 2 (A is a reduction electrophoresis, and B is a non-reduction electrophoresis.) The results showed that the anti-Stx2 monoclonal antibody YG12-1 was successfully prepared.

[0099] 2. ELISA detection of the binding activity of YG12-1 antibody to Stx2

[0100] (1) Take an ELISA plate and coat the antigen (Stx2 protein) with coating solution at a concentration of 10 μg / mL, 100 μL / well, and coat overnight at 4°C.

[0101] (2) After completing step (1), take the ELISA plate and add PBST buffer to wash three times.

[0102] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 hour.

[0103] (4) Using PBS buffer, the YG12-1 antibody was diluted three-fold starting from 10 μg / mL (a total of 10 dilution gradients) to obtain YG12-1 antibody solutions of different concentrations.

[0104] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or YG12-1 antibody solution of different concentrations obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0105] (6) After completing step (5), take the ELISA plate and add PBST buffer to wash three times (250 μL per well).

[0106] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution (HRP-labeled goat anti-human IgG secondary antibody is diluted 1:40,000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0107] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and develop the color at room temperature for 5 minutes. The colorimetric reagent is a component of the TMB colorimetric kit.

[0108] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0109] 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 effective concentration (EC50) value was 6.50 ng / mL.

[0110] 3. ELISA detection of the binding activity of YG12-1 antibody to Stx2a

[0111] (1) Take an ELISA plate and coat the antigen (Stx2a protein) with coating solution at a concentration of 10 μg / mL, 100 μL / well, and coat overnight at 4°C.

[0112] (2) After completing step (1), take the ELISA plate and add PBST buffer to wash three times.

[0113] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 hour.

[0114] (4) Using PBS buffer, the YG12-1 antibody was diluted three-fold starting from 10 μg / mL (a total of 10 dilution gradients) to obtain YG12-1 antibody solutions of different concentrations.

[0115] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or YG12-1 antibody solution of different concentrations obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0116] (6) After completing step (5), take the ELISA plate and add PBST buffer to wash three times (250 μL per well).

[0117] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution (HRP-labeled goat anti-human IgG secondary antibody is diluted 1:40,000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0118] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and develop the color at room temperature for 5 minutes. The colorimetric reagent is a component of the TMB colorimetric kit.

[0119] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0120] 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 effective concentration EC50 value was 99.56 ng / mL.

[0121] 4. ELISA detection of the binding activity of YG12-1 antibody to Stx2b

[0122] (1) Take an ELISA plate and coat the antigen (Stx2b protein) with coating solution at a concentration of 10 μg / mL, 100 μL / well, and coat overnight at 4°C.

[0123] (2) After completing step (1), take the ELISA plate and add PBST buffer to wash three times.

[0124] (3) After completing step (2), take the ELISA plate, add 5% (v / v) Milk-PBST buffer, and block at 37°C for 1 hour.

[0125] (4) Using PBS buffer, the YG12-1 antibody was diluted three-fold starting from 10 μg / mL (a total of 10 dilution gradients) to obtain YG12-1 antibody solutions of different concentrations.

[0126] (5) After completing steps (3) and (4), take the ELISA plate, add PBS buffer (as a control) or YG12-1 antibody solution of different concentrations obtained in step (4) (100 μL per well), and incubate at 37°C for 1 h.

[0127] (6) After completing step (5), take the ELISA plate and add PBST buffer to wash three times (250 μL per well).

[0128] (7) After completing step (6), take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution (HRP-labeled goat anti-human IgG secondary antibody is diluted 1:40,000 in 5% (v / v) Milk-PBST buffer), and incubate at 37°C for 30 min.

[0129] (8) After completing step (7), take the ELISA plate, add the colorimetric reagent (100 μL per well), and develop the color at room temperature for 5 minutes. The colorimetric reagent is a component of the TMB colorimetric kit.

[0130] (9) After completing step (8), take the ELISA plate, add 10% (v / v) H2SO4 aqueous solution to stop color development (50 μL per well), and then detect the OD value at a wavelength of 450 nm.

[0131] 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 effective concentration (EC50) value was 76.93 ng / mL.

[0132] 5. Detection of YG12-1 Neutralizing Activity in Animal Models

[0133] (1) Pick Escherichia coli O157:H7 and inoculate it into fresh TSB medium. Culture it at 37℃ and 220 rpm overnight.

[0134] (2) Centrifuge at 4000 rpm for 5 min, discard the supernatant, wash twice with PBS, and adjust the OD 600 nm =0.5.

[0135] (3) Eight-week-old C57BL / 6J female mice were randomly divided into two groups, with 8 mice in each group, and intraperitoneally injected with 200 μL PBS, cIgG1 (200 μg), YG12-1 (100 μg), and YG12-1 (200 μg), respectively.

[0136] (4) One hour later, inject 200 μL of the bacterial solution from step (1) into the abdominal cavity again.

[0137] (5) Observe and record the survival status of mice.

[0138] 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 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 NOs: 1-3, respectively; The amino acid sequences of LCDR1-3 in the light chain variable region are shown in SEQ ID NOs: 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 light chain variable region is shown in SEQ ID NO:

12.

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

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

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

7. Any of the following products, characterized in that: The products include: (1) A detection reagent comprising the monoclonal antibody according to any one of claims 1 to 3; (2) A detection kit comprising the monoclonal antibody or the detection reagent according to any one of claims 1 to 3; (3) A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 3; (4) A biological preparation comprising the monoclonal antibody or the pharmaceutical composition according to any one of claims 1 to 3.

8. Any of the following methods, characterized in that The method comprises: (1) A method for preparing the monoclonal antibody according to any one of claims 1 to 3, the method comprising: culturing the recombinant host cell according to claim 6, and isolating the monoclonal antibody according to any one of claims 1 to 3 from the culture; (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 a sample to be tested with the monoclonal antibody according to any one of claims 1 to 3, the detection reagent or the detection kit according to claim 7, and detecting the formation of an antigen-antibody immune complex; (3) A method for preparing the recombinant host cell according to claim 6, the method comprising: introducing the recombinant vector according to claim 5 into a host cell to obtain the recombinant host cell according to claim 6.

9. Any of the following applications, characterized in that: The applications include: (1) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, and / or the recombinant host cell according to claim 6 in the preparation of a detection reagent for detecting type II Shiga toxin Stx2, Stx2a, Stx2b, and / or enterohemorrhagic Escherichia coli; (2) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, and / or the detection reagent according to claim 7 in the preparation of a detection kit for detecting type II Shiga toxin Stx2, Stx2a, Stx2b, and / or enterohemorrhagic Escherichia coli; (3) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the detection reagent and / or the detection kit according to claim 7 for non-diagnostic and non-therapeutic detection of type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli; (4) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6, the detection reagent and / or the detection kit according to claim 7 in the preparation of a diagnostic product for diagnosing or assisting in the diagnosis of Shiga toxin type II Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli infection; (5) Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5 and / or the recombinant host cell according to claim 6 in the preparation of a pharmaceutical composition for preventing and / or treating infections caused by type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli.

10. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant host cell according to claim 6 and / or the pharmaceutical composition according to claim 7 in the preparation of a biological agent for preventing and / or treating infections caused by type II Shiga toxin Stx2, Stx2a, Stx2b and / or enterohemorrhagic Escherichia coli.

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