A single-domain antibody against ε-toxin of Clostridium perfringens and its application

Abstract

This invention belongs to the field of biological products and relates to a single-domain antibody against ε-toxin from *Clostridium perfringens* and its application. Specifically, this invention discloses a single-domain antibody against ε-toxin from *Clostridium perfringens* with the amino acid sequence shown in SEQ ID NO. 18. This single-domain antibody can neutralize ε-toxin in vivo and can treat animals poisoned by ε-toxin, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biological products and relates to a single-domain antibody against Clostridium perfringens ε toxin and its application. Background Technology

[0002] *Clostridium perfringens*, formerly known as *Clostridium wiltii* or *Clostridium perfringens*, is an important zoonotic pathogen widely distributed in nature, capable of causing various diseases under specific conditions. This bacterium primarily causes disease through more than 20 exotoxins it secretes. Based on the combination of four main lethal toxins (α, β, ε, and ι), it is classified into five toxin types, A to E; subsequent studies have added type F (primarily producing α toxin and enterotoxin CPE) and type G (primarily producing α toxin and NetB toxin). Among these, ε toxin is one of the most potent exotoxins, mainly secreted by type B and D strains, and can cause lethal enterotoxemia in animals such as sheep, goats, and calves. ε toxin is extremely toxic, with a very low median lethal dose (LD50); even minute exposure can lead to rapid death in livestock, causing significant economic losses to the livestock industry. Due to its high toxicity, it has even been classified as a Category B biological agent by the U.S. Centers for Disease Control and Prevention (CDC), posing a potential risk as a biological warfare agent.

[0003] Etatoxin is 296 amino acids in length with a molecular weight of approximately 32.9 kDa, and is secreted extracellularly in precursor form. This precursor is cleaved by host trypsin, chymotrypsin, or bacterial gamma proteases, removing amino acids 11-13 from the N-terminus and 22-29 from the C-terminus, transforming into a cytotoxic mature toxin. Etatoxin exhibits high selectivity for specific cell types, particularly kidney-derived cells, with MDCK cells being the most sensitive. Its pathogenic mechanism involves binding to cell membrane receptors, oligomerization, and the formation of transmembrane pores, ultimately leading to permeabilizing lysis of the cell.

[0004] Currently, Clostridium perfringens infection is mainly treated with antibiotics, but these drugs only inhibit bacterial proliferation and are ineffective against the ε-toxin already released into the bloodstream. While specific antitoxin therapy shows promise, its clinical application is limited by issues such as large batch-to-batch variations, allergy risks, high production costs, and short in vivo half-life of traditional antisera. Furthermore, antibiotic overuse and the emergence of drug-resistant strains make the treatment of this infection increasingly difficult. Therefore, the development of novel biological agents that can effectively neutralize ε-toxin is particularly urgent.

[0005] Nanobodies are a class of naturally occurring heavy chain antibodies derived from camel-like animals (such as alpacas and camels) and cartilaginous fish, lacking the light chain. Their variable region (VHH) possesses independent antigen-binding capability and is also known as a single-domain antibody. This antibody has a molecular weight of only about 15 kDa, approximately one-tenth that of traditional IgG. It not only retains complete antigen recognition capability but also possesses advantages such as small molecular size, strong tissue penetration, high stability, and ease of engineering modification. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies, such as the lack of specific treatments that can efficiently neutralize highly toxic ε-toxin and the high production costs and insufficient stability of traditional antibodies. It provides a nanobody with significant comprehensive advantages. This nanobody exhibits both high sensitivity and high specificity affinity for ε-toxin, demonstrating significant neutralizing activity and protective effects in both in vitro cell experiments and in vivo animal models. Due to its small molecular weight, this antibody possesses strong tissue penetration capabilities, enabling rapid distribution to the target site and exerting its function. Furthermore, the nanobody has a stable structure and can be efficiently recombinantly expressed in prokaryotic expression systems (such as *E. coli*), showing potential for large-scale preparation and economical production. The nanobody provided by this invention can be used to prepare drugs for the prevention and / or treatment of diseases caused by *Clostridium perfringens* ε-toxin and has the potential for development into an ε-toxin detection and diagnostic kit.

[0007] In this invention, a nanobody library targeting ε-toxin was constructed by immunizing alpacas and combining it with phage display technology. After multiple rounds of bioscreening, three specific nanobody sequences were obtained and successfully expressed efficiently in the *E. coli* system. Identification results showed that all three antibodies exhibited high affinity and highly sensitive binding activity to ε-toxin; two of them also showed significant toxin neutralization capabilities, demonstrating potential value as biological agents for the prevention or treatment of Clostridium perfringens ε-toxin-related diseases.

[0008] The first aspect of the present invention provides a single-domain antibody that maintains specific binding activity to a virulent peptide of Clostridium perfringens ε-toxin or a fusion protein containing a virulent peptide of Clostridium perfringens ε-toxin.

[0009] The single-domain antibodies include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4;

[0010] The amino acid composition of CDR1 is shown in positions 25-34 of SEQ ID NO.18;

[0011] The amino acid composition of CDR2 is shown in positions 50-57 of SEQ ID NO.18;

[0012] The amino acid composition of CDR3 is shown in positions 97-108 of SEQ ID NO.18.

[0013] In some embodiments, the single-domain antibody further includes a protein tag for separating and purifying the protein, and one, two, or three of a tag peptide and a signal peptide for protein-protein recognition.

[0014] In some embodiments, the amino acid sequence of the ε-toxin is as shown in SEQ ID NO.2 or SEQ ID NO.4;

[0015] The amino acid sequence of the fusion protein containing the virulent peptide of Clostridium perfringens ε-toxin is shown in SEQ ID NO.2 or SEQ ID NO.4; or

[0016] The amino acid sequence of the toxic active peptide is shown in positions 14-274 of SEQ ID NO.2 or positions 14-274 of SEQ ID NO.4.

[0017] In some embodiments, the single-domain antibody is an alpaca-derived monoclonal antibody.

[0018] In some embodiments, the amino acid sequence of the combination of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 is as shown in SEQ ID NO.18.

[0019] A second aspect of the present invention provides a method for detecting ε-toxin in Clostridium perfringens for non-diagnostic purposes, the method comprising the following steps:

[0020] S1: The sample to be tested is fixed on a solid surface to obtain a fixed surface;

[0021] S2: Add the single-domain antibody described in the first aspect of the present invention to the immobilized surface and incubate it to obtain the immobilized surface after the first incubation;

[0022] The single-domain antibody includes a tag peptide;

[0023] S3: Add a monoclonal antibody to the tag peptide linked to a chemiluminescent enzyme to the immobilized surface after the first incubation and incubate to obtain an immobilized surface for the second incubation.

[0024] S4: Add the substrate of the chemiluminescent enzyme to the immobilized surface after the second incubation, and determine whether Clostridium perfringens ε-toxin exists in the sample to be tested based on the enzymatic reaction results.

[0025] In some embodiments, the sample to be tested is selected from Clostridium perfringens ε-toxin, a fusion protein containing Clostridium perfringens ε-toxin peptides, a solution containing Clostridium perfringens ε-toxin, Clostridium perfringens cells that produce ε-toxin, secretions of Clostridium perfringens that produce ε-toxin, lysates of Clostridium perfringens that produce ε-toxin, and the sample to be tested that does not contain Clostridium perfringens ε-toxin.

[0026] The third aspect of the present invention provides the use of the single-domain antibody described in the first aspect of the present invention in the preparation of formulations for improving health conditions or reducing health risks;

[0027] The improvements in health status or reductions in health risks include:

[0028] Neutralizes Clostridium perfringens ε-toxin in vitro on animals;

[0029] Neutralizes the ε-toxin of Clostridium perfringens in animals;

[0030] Treatment, prevention, or mitigation of diseases or sub-health conditions caused by Clostridium perfringens ε-toxin or Clostridium perfringens containing ε-toxin.

[0031] A fourth aspect of the present invention provides a kit containing the single-domain antibody described in the first aspect of the present invention. Attached Figure Description

[0032] Figure 1 This is a statistical chart showing the titer of specific antibodies in alpaca serum.

[0033] Figure 2 Electrophoresis image of antibody gene amplification.

[0034] Figure 3 This is a photograph of a primary library plate containing bacterial colonies.

[0035] Figure 4 Gel electrophoresis image of a bacterial culture used for PCR identification of VHH fragment insertion.

[0036] Figure 5 The statistical results of the identification of phage-positive clones are shown.

[0037] Figure 6 Electrophoretic images of the purification process of three nanobodies for SDS-PAGE analysis are shown. A: Nb E1 purification process; B: Nb E2 purification process; C: Nb E3 purification process.

[0038] The loading order for the three antibody purifications was as follows: lane 1 was for protein molecular weight standard (marker), lane 2 was for uninduced control, lane 3 was for total protein after induction, lane 4 was for lysis supernatant, lane 5 was for gradient imidazole washing, and lanes 6 to 13 were for gradient imidazole elution.

[0039] Figure 7 Images showing the results of Western blot detection of the binding of nanobodies to ε-toxin protein. A: Results of specific binding of Nb E1 to precursor ε-toxin and activated ε-toxin; B: Results of specific binding of Nb E2 to precursor ε-toxin and activated ε-toxin; C: Results of specific binding of Nb E3 to precursor ε-toxin and activated ε-toxin; M: Protein Marker; 1: Precursor ε-toxin; 2 and 4 are irrelevant control proteins; 3: Activated ε-toxin.

[0040] Figure 8 Photographs of cells and experimental results, where the scale bar represents 300 nm. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0042] Materials and instruments not described in this invention are conventional materials and instruments in the art. Operational details not described in this invention are conventional operations in the art. The software used in this invention is operated by conventional methods in accordance with the software provider's instructions. The reagent kits used in this invention are operated by conventional methods in accordance with the reagent kit's instruction manual.

[0043] Applications of non-diagnostic detection of Clostridium perfringens ε-toxin (not the process of identifying, studying and determining the cause or lesion state in living humans or animals) include, but are not limited to, the following: (1) exploring the biodegradation patterns of Clostridium perfringens ε-toxin; (2) providing environmental assessments for developing environmental disinfection strategies for Clostridium perfringens.

[0044] Example 1: Prokaryotic expression of ε-toxin prototype (ETX) and ε-toxin attenuated mutant (ETX-Y71A)

[0045] 1. Prokaryotic expression and purification of recombinant ε-toxin prototype (ETX):

[0046] This invention uses the Clostridium perfringens ε toxin Etx gene, and the nucleotide sequence after codon optimization is shown in SEQ ID NO.1.

[0047] The ETX protein sequence encoded by the aforementioned Etx gene is shown in SEQ ID NO.2.

[0048] The Etx gene was cloned into the pET-22b vector (purchased from Merck, Germany), transformed into E. coli BL21(DE3) (purchased from Shanghai Qincheng Biotechnology Co., Ltd.), and exogenous ETX protein was induced to express. The purified recombinant ETX protein was then purified by nickel column affinity chromatography using the His-tagged protein carried on the expression vector. Mature ETX toxin was obtained by cleaving the N-terminal 13 amino acids and C-terminal 22 amino acids from ETX using trypsin. This invention will subsequently use both pre- and post-activated ETX protein forms for the screening and evaluation of nanobodies.

[0049] 2. Prokaryotic expression and purification of recombinant Etx-Y71A protein:

[0050] This invention uses the attenuated mutant of Clostridium perfringens ε toxin ETX-Y71A. The nucleotide sequence of the attenuated ε toxin protein (ETX-Y71A) after codon optimization is shown in SEQ ID NO.3.

[0051] The amino acid sequence of the attenuated ε protein (ETX-Y71A) encoded by the aforementioned Etx-Y71A gene is shown in SEQ ID NO.4.

[0052] The gene containing the above mutant Etx-Y71A was cloned into the pET-22a vector, transformed into E. coli (E. coli BL21), and the exogenous protein ETX-Y71A mutant ε toxin protein was induced to express. The His tag protein was introduced by primer design, and the obtained protein was then purified by the His purification system to obtain the purified ETX-Y71A recombinant protein, which was used for subsequent alpaca immunization.

[0053] Example 2: Preparation of ε-toxin nanobodies

[0054] I. Evaluation of Alpaca Immunity and Serum Titer

[0055] (1) Immunization

[0056] The attenuated ε-toxin protein ETX-Y71A prepared in step one was diluted to a 400 μg / mL ETX-Y71A antigen solution using sterile PBS (0.01 mol / L, pH 7.4, the same below). The antigen solution was then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified to obtain composition 1. The antigen solution was then mixed with an equal volume of Freund's incomplete adjuvant and thoroughly emulsified to obtain composition 2.

[0057] Healthy one-year-old male alpacas were selected and immunized using the attenuated ε-toxin protein (ETX-Y71A) prepared in step one. The immunization procedure was as follows: the first immunization was performed by subcutaneous injection of composition 1, with an injection dose of 2 mL. Thereafter, booster immunizations were performed every 14 days by subcutaneous injection of composition 2, with an injection dose of 2 mL. A total of three booster immunizations were completed.

[0058] (2) Serum collection

[0059] Blood samples were collected before immunization (day 0) and on days 7, 21, 35, and 49 after immunization, and serum was separated.

[0060] (3) Valence determination

[0061] Serum specific antibody titers were assessed using an indirect ELISA method. The steps were as follows: The prokaryotically expressed attenuated Clostridium perfringens ε-toxin protein prototype (ETX) prepared in Example 1 was diluted to 2 μg / mL with carbonate coating buffer, and 100 μL / well was added to an ELISA plate and incubated overnight at 4°C; after washing with PBST, 200 μL / well of 5% skim milk powder (solvent: sterile PBS) was added, and the plate was blocked at 37°C for 2 hours; after washing with PBST, 10 μL / well of sterile PBS was added to... 2 Up to 10 7 100 μL / well of serially diluted ε-toxin protein-positive serum samples were added to the coated plate and incubated at 37°C for 1 hour. After washing with PBST, HRP-labeled goat anti-alpaca IgG (purchased from Jackson ImmunoResearch Laboratories Inc.) diluted 10000 times with PBS was added as a secondary antibody and incubated at 37°C for 1 hour. After washing with PBST, 50 µL of TMB substrate chromogenic solution was added to each well and the reaction was carried out at 37°C in the dark for 10 minutes. Finally, the reaction was terminated by adding 50 µL of 2 M H2SO4 to each well, and the absorbance (OD) was measured at 450 nm. 450 The positive cutoff value was determined by using 2.1 times the pre-immunization serum OD value.

[0062] See results Figure 1 Therefore, it can be seen that after four immunizations, the specific antibody titer in alpaca serum exceeded 10. 6 This indicates a good immune response, which meets the requirements for subsequent library construction.

[0063] II. VHH gene amplification

[0064] 200 mL of peripheral blood was collected from alpacas after the fourth immunization and diluted with PBS buffer at a 1:1 volume ratio. Peripheral blood lymphocytes were then separated using density gradient centrifugation: 15 mL of diluted blood was carefully added above an equal volume of lymphocyte separation medium, and the mixture was centrifuged horizontally at 1000 ×g for 20 minutes at room temperature. After centrifugation, the white membrane layer (i.e., the lymphocyte layer) at the boundary between plasma and separation medium was aspirated, resuspended in cell washing buffer, and centrifuged at 300 ×g for 10 minutes. This washing process was repeated three times. Lymphocytes were collected, counted, and total RNA was extracted using the Trizol method.

[0065] The extracted total RNA was used to synthesize cDNA using the Hifair® AdvanceFast 1st Strand cDNA Synthesis Kit (No Dye) (purchased from Yisheng Biotechnology Co., Ltd.) according to the kit instructions. This cDNA was then used as a template for nested PCR to amplify the VHH gene fragment. The first round of PCR used CALL001 (SEQ ID NO. 5) and CALL002 (SEQ ID NO. 6) as primers. Agarose gel electrophoresis analysis of the amplification products revealed two specific bands of approximately 900 bp and 700 bp (see [link to kit]). Figure 2 A). The 700 bp band corresponds to the variable region (VHH) gene of the alpaca heavy chain antibody. The target band was recovered by gel excision and used as a template, along with VHH-FR1-F (VHH-FR1-F1).<SEQ ID NO.7> With VHH-FR1-F2<SEQ ID NO.8> (equimolar mixture) and VHH-FR4-R (VHH-FR4-R1)<SEQ ID NO.9> Using an equimolar mixture of VHH-FR4-R2 (SEQ ID NO.10), VHH-FR4-R3 (SEQ ID NO.11), and VHH-FR4-R4 (SEQ ID NO.12) as primers, a second round of nested PCR was successfully performed, yielding an amplified fragment of approximately 500 bp (see [link to primer]). Figure 2 B). The above results indicate that the alpaca VHH gene sequence has been successfully amplified.

[0066] III. Construction and Identification of Immune Libraries

[0067] (1) Construction of the library

[0068] The obtained VHH gene amplification fragment and pCOMB3 phage vector (purchased from Beijing Baokewei Food Safety Biotechnology Co., Ltd.) were digested with Sfi I restriction enzyme at 50℃ for 1 hour. After verifying complete digestion by agarose gel electrophoresis, the digested products were purified using a DNA purification kit (purchased from TransGen Biotech Co., Ltd.). Subsequently, the purified VHH fragment and the digested vector were mixed at a molar ratio of approximately 1:3, and T4 DNA ligase was added, and ligation was performed overnight at 16℃. After the ligation product was recovered using a DNA purification kit, 20 μL of the ligation product was gently mixed with 180 μL of pre-prepared TG1 competent E. coli cells and transferred to a pre-chilled electroporation cuvette for electroporation transformation at 1800 V. Immediately after electroporation, 1 mL of SOC medium was added, and the cells were incubated at 37℃ with shaking for 1 hour.

[0069] After resuscitation, the bacterial culture was centrifuged at 5000 ×g to collect the bacterial cells, resuspended in 5 mL of SOC medium, and evenly spread on 2×YT AG solid plates containing ampicillin. The plates were then incubated upside down overnight at 30°C. Once a dense bacterial growth had formed on the plate surface, the bacterial cells were scraped off, resuspended in 10 mL of 2×YT liquid medium, and a suitable amount of sterile glycerol was added and mixed thoroughly to obtain the primary library of ε-toxin nanobody.

[0070] (2) Authentication of the document library

[0071] Take 100 μL of the primary library bacterial culture and perform 10-fold serial dilutions using 2×YT liquid medium. Spread 100 μL of each dilution onto 2×YT solid plates containing ampicillin and incubate overnight at 37°C. Library capacity is assessed by colony counting. Results show that within 10... -4 Single colonies with clear boundaries grew on the dilution plate. Figure 3 Based on this, the primary library capacity is calculated to be 3.5 × 10⁻⁶. 7 CFU.

[0072] To assess antibody gene insertion efficiency, 53 single clones were randomly selected and inoculated into 5 mL of 2×YTAG liquid medium, and cultured overnight at 37°C with shaking. Colony PCR was performed using 1 μL of bacterial culture as a template, employing primers VHH-FR1-F and VHH-FR4-R as described above. An empty vector-free control was used. Results showed that all 53 clones amplified bands of the expected size (see [link to relevant documentation]). Figure 4 The insertion rate is 100%.

[0073] Further evaluation of library diversity was conducted by randomly selecting 29 PCR-positive clones and performing Sanger sequencing analysis on the inserted fragments, successfully obtaining 29 VHH gene sequences. After translation into amino acid sequences, 25 different sequences were identified, representing a diversity of 86.2%. Nanobody structural analysis revealed four backbone regions (FR1-FR4) and three complementarity-determining regions (CDR1-CDR3). Sequence alignment showed high conservation in the backbone regions across all clones, while significant sequence variation was observed in the CDR3 region, indicating that the constructed immune library possesses good diversity and is suitable for subsequent antibody screening studies.

[0074] IV. Screening and Identification of Specific Nanobodies

[0075] (1) Affinity screening of phage display libraries

[0076] After activating 0.5 mL of the primary library bacterial suspension, it was co-infected with M13KO7 helper phage at 37°C for 1 hour. Subsequently, the bacterial cells were collected by centrifugation at 3000 ×g for 10 minutes and resuspended in 2×YTAK medium (2×YT medium supplemented with ampicillin and kanamycin), and cultured overnight at 30°C with shaking to amplify phage particles. The culture supernatant was collected by centrifugation at 5000 ×g, and precipitated with 20 mL of PEG / NaCl solution. After incubating on ice for 1 hour, the phage precipitate was collected by centrifugation at 4°C and 12000 ×g to obtain the amplified phage library.

[0077] Immunotherapy tubes were coated with 2 mL of 20 μg / mL ε-toxin (ETX) prepared according to this invention (in PBS solvent) and incubated overnight at 4°C to achieve antigen fixation. The coating solution was discarded, and the tubes were washed three times with PBST, followed by blocking with MPBS (PBS solution with 3% skim milk added; skim milk powder was purchased from BD Biosciences) at room temperature for 1 hour. The collected phage pellet was resuspended in PBS and added to the immunotherapy tubes, incubated at room temperature for 2 hours, and then vigorously washed 20 times with PBST to remove non-specific or low-affinity bound phages. Finally, elution was performed with 2 mL of 100 mM triethylamine solution, incubated at room temperature for 15 minutes, and the eluent was immediately neutralized with an equal volume of 1 M Tris-HCl (pH 7.4).

[0078] The neutralized eluent was mixed with TG1 bacterial culture in the logarithmic growth phase (OD). 600 Mix equal volumes of approximately 0.5 g of the bacterial culture and infect at 37°C for 1 hour. Take a portion of the infected bacterial culture for phage titer determination, and concentrate the remaining bacterial cells by centrifugation before spreading them onto 2×YTAG plates and incubating overnight at 30°C.

[0079] Through the above process, a phage-display nanobody library targeting ε-toxin was successfully constructed. Subsequently, using immobilized ε-toxin (prepared in Example 1) as the target, the library was specifically enriched through a three-round biopanning process of "binding-washing-elution-amplification".

[0080] First screening results: Phage input amount (I) was 7.92 × 10⁻⁶. 12 The phage elution volume (O) was 1.76 × 10⁻⁶. 6 The recovery rate (O / I) was 1.76 × 10⁻⁶. -7 .

[0081] Second screening results: Phage input amount (I) was 7.68 × 10⁻⁶ 11 The phage elution volume (O) was 6.48 × 10⁻⁶. 7 The recovery rate (O / I) was 8.44 × 10⁻⁶. -5 The enrichment level is 479.55.

[0082] The third screening result showed that the phage input amount (I) was 1.48 × 10⁻⁶. 11 The phage elution volume (O) was 1.55 × 10⁻⁶. 8 The recovery rate (O / I) was 1.05 × 10⁻⁶. -3 The enrichment level is 12.44.

[0083] This shows that, under the condition that the amount of phage input in each round is basically the same, the titer of the eluted phage increases significantly with each round of panning, indicating that nanobodies with high affinity for ε-toxin are effectively enriched.

[0084] (2) Identification of positive clones

[0085] Ninety-eight monoclonal antibodies were randomly selected from the culture plates after the third round of bioscreening and amplified by phage. Their specific binding ability to ε-toxin protein was then identified using phage-ELISA. The specific steps were as follows: Different ELISA plates were coated with 2 μg / mL ε-toxin protein (Etx prepared in Example 1 of this invention) and 2 μg / mL BSA, respectively, 100 μL / well, and incubated overnight at 4°C. For each ELISA plate, 98 monoclonal phage supernatants were added as primary antibody, 100 μL / well. After washing with PBST, HRP-labeled mouse anti-M13 antibody IgG (purchased from Sinocare Biotechnology Co., Ltd.) diluted 1:5000 with PBS was used as secondary antibody, and the plates were incubated at 37°C for 1 hour. After washing with PBST, 50 µl of TMB substrate chromogenic solution was added to each well, and the reaction was carried out at 37°C in the dark for 10 minutes. Finally, the reaction was terminated by adding 50 µl of 2 M H2SO4 to each well, and the absorbance (OD) was measured at 450 nm. 450 ). (with "OD")450 The criterion for a positive result was "value ≥ twice the OD value of the negative control". Results showed ( Figure 5 As the number of screening rounds increased, the proportion of positive clones rose significantly, reaching 100% by the third round.

[0086] The M13 antibody is an antibody that specifically recognizes the capsid proteins of M13 phage (mainly pVIII and pIII proteins). It does not directly recognize nanobodies, but rather the phage itself. The helper phage M13KO7 provides all the proteins needed to produce a complete phage for bacteria containing only "incomplete" phage particles. Simultaneously, it "rescues" phage DNA (preferably packaging phage DNA containing nanobody genes into new phage particles), ultimately producing phage particles with functional nanobodies on their surface and possessing infectivity, ready for further screening or monoclonal ELISA detection.

[0087] Furthermore, using BSA as a negative control serves the core purpose of establishing a baseline of background noise. By comparing the signal from the target protein wells with this baseline, highly specific positive nanobody clones can be accurately and reliably identified, while non-specifically binding "false positive" clones, including "anti-BSA antibody" or "sticky" clones, can be eliminated. In our experiments, using BSA as a blocking solution also resulted in false positives against "anti-BSA" or "sticky" clones; therefore, using BSA as a negative control to remove background noise is essential.

[0088] Subsequently, 25 strongly positive clones were randomly selected and sequenced using the Sanger method against the inserted sequence. After aligning the obtained sequences with the vector sequence, the coding region nucleotide sequences of the ε-toxin-specific nanobodies were obtained. Through amino acid sequence translation and alignment analysis, three representative full-length nanobodies with significant sequence differences in the CDR3 region were selected and named NbE1, NbE2, and NbE3, respectively. The corresponding full-length single-chain antibody nucleotide coding sequences and amino acid sequence information are detailed below.

[0089] The nucleotide sequence of Nb E1 is shown in SEQ ID NO.13.

[0090] Among them, FR1 is located at positions 1-72, CDR1 is located at positions 73-105, FR2 is located at positions 106-156, CDR2 is located at positions 157-186, FR3 is located at positions 187-285, CDR3 is located at positions 286-312, and FR4 is located at positions 313-339.

[0091] The amino acid sequence of Nb E1 is shown in SEQ ID NO. 14.

[0092] Among them, FR1 is located at positions 1-24, CDR1 is located at positions 25-35, FR2 is located at positions 36-52, CDR2 is located at positions 53-62, FR3 is located at positions 63-95, CDR3 is located at positions 96-104, and FR4 is located at positions 105-113.

[0093] The nucleotide sequence of Nb E2 is shown in SEQ ID NO.15.

[0094] Among them, FR1 is located at positions 1-72, CDR1 is located at positions 73-102, FR2 is located at positions 103-147, CDR2 is located at positions 148-171, FR3 is located at positions 172-288, CDR3 is located at positions 289-318, and FR4 is located at positions 319-351.

[0095] The amino acid sequence of Nb E2 is shown in SEQ ID NO.16.

[0096] Among them, FR1 is located at positions 1-24, CDR1 is located at positions 25-34, FR2 is located at positions 35-49, CDR2 is located at positions 50-57, FR3 is located at positions 58-96, CDR3 is located at positions 97-106, and FR4 is located at positions 107-117.

[0097] The nucleotide sequence of Nb E3 is shown in SEQ ID NO.17.

[0098] Among them, FR1 is located at positions 1-72, CDR1 is located at positions 73-102, FR2 is located at positions 103-147, CDR2 is located at positions 148-171, FR3 is located at positions 172-288, CDR3 is located at positions 289-324, and FR4 is located at positions 325-357.

[0099] The amino acid sequence of Nb E3 is shown in SEQ ID NO.18.

[0100] Among them, FR1 is located at positions 1-24, CDR1 is located at positions 25-34, FR2 is located at positions 35-49, CDR2 is located at positions 50-57, FR3 is located at positions 58-96, CDR3 is located at positions 97-108, and FR4 is located at positions 109-119.

[0101] V. Expression and purification of ε-toxin nanobodies

[0102] (I) Construction of recombinant expression vectors

[0103] For the three strongly positive clones corresponding to antibodies Nb E1, Nb E2 and Nb E3, the nanobody gene fragment inserted in the pComb3 vector was used as a template for PCR amplification using universal primers F1 (SEQ ID NO.19) and R1 (SEQ ID NO.20) to obtain the target fragment of about 400 bp, which was separated by agarose gel electrophoresis and then excised and recovered.

[0104] The recovered nanobody gene fragment and the pCold-SUMO-his empty vector (pCold vector purchased from Thermo Fisher Scientific, later modified by our experiment to add SUMO and his tags) were double-digested with BamHI and HindIII, respectively. After purification, they were ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α competent cells, and after verification by colony PCR and Sanger sequencing, the recombinant plasmids were extracted and transformed into the expression host bacterium E. coli BL21(DE3) for subsequent protein expression.

[0105] (II) Induction, Expression and Purification of Nanobodies

[0106] E. coli BL21(DE3) containing the recombinant plasmid was inoculated into LB medium containing ampicillin and cultured at 30°C with shaking at 220 rpm until OD200. 600 The concentration was 0.6-0.8, and 0.1 mM IPTG was added. Expression was induced overnight at 16℃ and 220 rpm. Cells were collected by centrifugation, sonicated, and the supernatant was used for subsequent purification. The supernatant was filtered through a 0.22 μm filter membrane and Ni was equilibrated using Binding Buffer. 2+ Affinity chromatography column was used. After loading the filtrate, non-specifically adsorbed proteins were removed by washing with Binding Buffer containing 40 mM imidazole, followed by elution with Binding Buffer containing 250 mM imidazole to collect the target protein. Samples at each stage of purification were analyzed by SDS-PAGE (90 V for 20 min, then 120 V for 90 min) to verify the elution efficiency. The results showed that a single, clear band was visible at approximately 35 kDa in the eluted fraction. Figure 6 The molecular weight is consistent with the theoretical molecular weight of the SUMO-nanobody fusion protein. The purified protein was concentrated by ultrafiltration and replaced with PBS buffer. After sterilization by filtration, it was aliquoted and stored at -80°C.

[0107] Example 3. Identification and Application of Nanobodies

[0108] I. Affinity Identification of Nanobodies and E-toxin

[0109] (1) Indirect ELISA detection of nanobody affinity

[0110] To evaluate the affinity of nanobodies for ε-toxin, an indirect ELISA method was used for detection. Pre-activation and post-activation ε-toxin (diluted to 5 μg / mL with carbonate coating buffer) were coated onto ELISA plates, 100 μL / well, and incubated overnight at 4°C. Uncoated wells served as blank controls. Pre-activation ε-toxin refers to ETX prepared in Example 1, and post-activation ε-toxin refers to ETX prepared in Example 1 treated with 1% trypsin at 37°C for 30 min, the same applies below. After washing three times with PBST, the plates were blocked with 100 μL / well of 5% skim milk at 37°C for 1 hour. After washing three times with PBST, the three nanobodies (Nb E1, Nb E2, Nb E3) prepared in Example 2 of this invention, serially diluted with PBS, and positive serum from ε-toxin-immunized mice were added as primary antibodies, and incubated at 37°C for 1 hour. After washing three times with PBST, mouse anti-SUMO monoclonal antibody IgG (purchased from Genscript Biotech Inc.) diluted 1:5000 with PBS and HPR-labeled rabbit anti-mouse IgG secondary antibody were added sequentially. The mixtures were incubated at 37°C for 1 hour. After thorough washing with PBST, TMB substrate was added for color development. The reaction was allowed to proceed at room temperature for 15 minutes, and the reaction was terminated with 2 M H₂SO₄. The OD was then measured. 450 value.

[0111] The results showed that all three nanobodies exhibited high affinity binding to ε-toxin. For unactivated ε-toxin, the detection sensitivities of NbE1, NbE2, and NbE3 were 204 ng / mL, 71.3 ng / mL, and 16.1 ng / mL, respectively; while for activated ε-toxin, the sensitivities were 25.5 ng / mL, 35.7 ng / mL, and 32.3 ng / mL, respectively. These data indicate that the obtained nanobodies possess high affinity and recognition ability for ε-toxin.

[0112] (2) Western blot detection of nanobody affinity

[0113] To further verify the binding specificity of nanobodies to ε-toxin, purified ε-toxin (prepared in Example 1) and its activated form were used as detection targets and analyzed by Western blotting. ε-toxin and activated ε-toxin (0.3 mg / mL, PBS solvent) were mixed with 5×SDS loading buffer at a ratio of 1:4 (v / v) and boiled at 100°C for 10 minutes. SDS-PAGE was performed using a 12% separating gel and a 5% stacking gel, with 20 μL loaded into each well. The same concentration of pCOLD-SUMO-his vector tag protein was included as a control. Electrophoresis conditions were: concentration at 80 V for 30 minutes, followed by separation at 120 V for 60 minutes.

[0114] After electrophoresis, the proteins were transferred to a 0.22 μm PVDF membrane and blocked at room temperature for 3 hours with 5% skim milk (prepared with PBST). The membranes were then incubated overnight at 4°C with 1 μg / mL nanobodies (Nb E1, Nb E2, and Nb E3). After washing with PBST, mouse anti-SUMO-tagged monoclonal antibody IgG (diluted 1:5000 in PBS) and HRP-labeled anti-mouse IgG secondary antibody (diluted 1:1000 in PBS) were added sequentially, and incubated at room temperature for 1 hour. After thorough washing with PBST, the membranes were developed using chemiluminescent substrates, and signals were acquired using a chemiluminescent imaging system (eBlot).

[0115] See results Figure 7 Therefore, it can be seen that Nb E1, Nb E2 and Nb E3 can specifically recognize ε toxin and its activated form, and no non-specific binding was observed in the irrelevant protein control group, further confirming that these three nanobodies have good binding specificity.

[0116] II. Identification of Neutralizing Activity of Nanobodies

[0117] Neutralization activity is a key indicator for evaluating the function of antitoxin nanobodies. This invention evaluates the neutralization ability of nanobodies against ε-toxin using cytotoxicity neutralization experiments.

[0118] (1) Evaluation of neutralization activity in cells

[0119] 50 μL of nanobodies of different concentrations (NbE1, NbE2, and NbE3) were respectively mixed with an equal volume containing 10 CT 50 The activated Clostridium perfringens ε-toxin solution (prepared in Example 1, using 1% trypsin, treated at 37°C for 30 min) was pre-incubated at 37°C for 30 min, and then added to the MDCK cell culture system. The experiment included a blank cell control (MDCK cells without antibody and ε-toxin), an ε-toxin control (using an equal amount of ε-toxin without nanobodies), and a Trizol control (as a cell lysis control to demonstrate the cytotoxic effect of ε-toxin, serving as a positive control for cell lysis by toxin) to verify the reliability of the experimental system. Cell morphology changes were observed, and cell viability was detected using the CCK-8 assay: 10 μL of CCK-8 reagent was added to each well, with three replicates per group, and the OD was measured. 450 The average value is the final result. OD is compared with that of blank cells. 450 Based on the value (1.018), the OD of the sample group 450 A mean value ≥ 1.018 is considered completely neutralized.

[0120] As a result, for Nb E1, the antibody dilution factor was increased from 2...0 to 2 7 The obtained CCK-8 OD 450nm The values ​​(outside parentheses) and the calculated concentrations (inside parentheses) are 0.526 (102 ng / μL), 0.191 (51 ng / μL), 0.098 (25.5 ng / μL), 0.122 (12.75 ng / μL), 0.124 (6.375 ng / μL), 0.289 (3.1875 ng / μL), 0.078 (1.59375 ng / μL), and 0.246 (0.796875 ng / μL), respectively.

[0121] For Nb E2, the antibody dilution factor is from 2. 0 to 2 7 The obtained CCK-8 OD 450nm The values ​​(outside parentheses) and the calculated concentrations (inside parentheses) are 1.222 (571 ng / μL), 1.061 (285.5 ng / μL), 1.372 (142.75 ng / μL), 1.178 (71.375 ng / μL), 1.047 (35.6875 ng / μL), 1.09 (17.84375 ng / μL), 0.811 (8.921875 ng / μL), and 0.492 (4.4609375 ng / μL), respectively.

[0122] For Nb E3, the antibody dilution factor is from 2. 0 to 2 7 The obtained CCK-8 OD 450nm The values ​​(outside parentheses) and the calculated concentrations (inside parentheses) are 1.167 (258 ng / μL), 1.372 (129 ng / μL), 1.222 (64.5 ng / μL), 1.15 (32.25 ng / μL), 1.097 (16.125 ng / μL), 0.672 (8.0625 ng / μL), 0.378 (4.03125 ng / μL), and 0.314 (2.015625 ng / μL), respectively.

[0123] CCK-8 OD obtained from blank cell control 450nm The value was 1.018, the OD value of CCK-8 obtained from the ε-toxin control. 450nm The value was 0.225, the CCK-8 OD obtained by Trizol control. 450nm The value is 0.183.

[0124] See photos of cell and experimental results. Figure 8 .

[0125] Therefore, 892 ng Nb E2 or 806 ng Nb E3 can completely neutralize 10 CT. 50 The ε-toxin significantly inhibited the cytopathic effect induced by the toxin; however, even with 5.1 μg Nb E1, MDCK cells still showed significant lysis and death pathological changes similar to the toxin control group, indicating that it did not possess neutralizing ability. In summary, Nb E2 and Nb E3 exhibited significant neutralizing activity at the cellular level.

[0126] (2) Evaluation of neutralizing activity in mice

[0127] To evaluate the neutralizing efficacy of nanobodies in vivo, 21 healthy female 6-week-old BALB / c mice were randomly divided into the following three groups:

[0128] Experimental Group 1: 10 μg of Nb E1, Nb E2 and Nb E3 were mixed with 1 MLD (minimum lethal dose for adult mice, the same below) of activated ε-toxin (ETX prepared in Example 1, treated with 1% trypsin at 37°C for 30 min), and the mixture was brought to a final volume of 200 μL. After pre-incubation at 37°C for 30 min, the mixture was injected intraperitoneally into mice (3 mice for each antibody, 9 mice in total).

[0129] Second experimental group: The treatment method is the same as the first group, but the antibody dose is 1 μg (9 animals in total);

[0130] Blank control group: 1 MLD ε toxin was mixed with an equal volume of PBS, incubated in the same manner, and then injected into 3 mice.

[0131] The survival and physiological status of mice were observed for 7 consecutive days. Results: All mice treated with Nb E1 (10 μg and 1 μg groups) and the blank control group died; however, all mice in the 10 μg and 1 μg Nb E2 or Nb E3 treatment groups survived, and their mental state, activity, and feeding behavior were normal. These results indicate that only 1 μg of Nb E2 or Nb E3 can completely neutralize a 1 MLD ε-toxin challenge, further confirming that both have significant neutralizing activity in animals.

[0132] III. Evaluation of Nanobody Therapy Efficacy

[0133] To evaluate the therapeutic effect of neutralizing nanobodies, nine 6-week-old healthy female BALB / c mice were randomly divided into three groups (n=3 per group). All mice were challenged intraperitoneally with 1 MLD of ε-toxin (ETX prepared in Example 1). Ten minutes later, the first group was injected with 1 μg of Nb E2, the second group with 1 μg of Nb E3, and the third group with an equal volume of PBS as a control. The mice were then observed for 7 consecutive days, and their survival status was recorded.

[0134] The results showed that all mice in the Nb E2 and Nb E3 treatment groups survived, while all mice in the PBS control group died within 24 hours after challenge. These results indicate that administration of 1 μg of Nb E2 or Nb E3 after a lethal dose of ε-toxin challenge effectively protects mouse survival, demonstrating that both have significant in vivo therapeutic effects.

[0135] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A single-domain antibody, which maintains a virulence active peptide segment of epsilon toxin of Clostridium perfringens or specific binding activity of epsilon toxin of Clostridium perfringens; The single-domain antibody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; The amino acid of the CDR1 is shown in SEQ ID NO. 18, 25-34; The amino acid of the CDR2 is shown in SEQ ID NO. 18, 50-57; The amino acid of the CDR3 is shown in SEQ ID NO. 18, 97-108.

2. The single domain antibody of claim 1, wherein, The single-domain antibody further comprises one of a protein tag for isolating and purifying the protein, a tag peptide for protein-protein mutual recognition and a signal peptide, two or three.

3. The single domain antibody of claim 1 or 2, wherein The amino acid sequence of the epsilon toxin is shown in SEQ ID NO. 2 or SEQ ID NO. 4; or The amino acid sequence of the virulence active peptide segment is shown in SEQ ID NO. 2, 14-274 or SEQ ID NO. 4, 14-274.

4. The single-domain antibody of claim 1 or 2, wherein The single-domain antibody is a llama-derived single-domain antibody.

5. The single-domain antibody of claim 1 or 2, wherein The amino acid sequence of the combination of the FR1, the CDR1, the FR2, the CDR2, the FR3, the CDR3 and the FR4 is shown in SEQ ID NO.

18. 6.A method for detecting epsilon toxin of Clostridium perfringens for non-diagnostic purposes, the method comprising the following steps: S1:fixing a sample to be detected on a solid surface to obtain a fixed surface; S2:adding the single-domain antibody of any one of claims 2-5 to the fixed surface for incubation to obtain a first-incubated fixed surface; The single-domain antibody comprises a tag peptide; S3:adding a monoclonal antibody of the tag peptide connected with a chemiluminescent enzyme to the first-incubated fixed surface for incubation to obtain a second-incubated fixed surface; S4:adding a substrate of the chemiluminescent enzyme to the second-incubated fixed surface to determine whether the epsilon toxin of Clostridium perfringens exists in the sample to be detected according to the result of the enzymatic reaction. The sample to be detected is selected from the group consisting of epsilon toxin of Clostridium perfringens, a virulence active peptide segment of epsilon toxin of Clostridium perfringens, a solution containing epsilon toxin of Clostridium perfringens, Clostridium perfringens cells producing epsilon toxin, exudate of Clostridium perfringens producing epsilon toxin, lysate of Clostridium perfringens producing epsilon toxin and a sample to be detected without epsilon toxin of Clostridium perfringens.

7. The method of claim 6, wherein, 8.Use of the single-domain antibody of any one of claims 1-5 in the preparation of a preparation for improving health status or reducing health risk; The improvement of health status or reduction of health risk is selected from: neutralizing epsilon toxin of Clostridium perfringens in vitro in animals; neutralizing epsilon toxin of Clostridium perfringens in vivo in animals. 9.A kit comprising the single-domain antibody of any one of claims 1-5. ​

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