Multi-antigenic epitope peptide recombinant proteins of three lethal toxins of clostridium perfringens and design method and application

Abstract

The application belongs to the technical field of immunology, and provides a multi-antigen epitope peptide recombinant protein of three lethal toxins of Clostridium perfringens, a design method and application, wherein the constituent fragments of the multi-antigen epitope peptide recombinant protein include B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes and helper T lymphocyte antigen epitopes of three lethal toxin proteins, which are combined through KK, GPGPG and AAY connectors; the three lethal toxin proteins are Alpha, Beta1 and Beta2; and the amino acid sequence of the multi-antigen epitope peptide recombinant protein is shown in the sequence table SEQ ID NO. 1. The application uses three toxin proteins as target points, integrates multiple antigen epitope peptides into a recombinant protein as an immunogen by eliminating antigenic poor, potentially toxic and sensitizing peptide segments, thereby preparing a high-efficiency, safe and broad-spectrum vaccine, and the vaccine shows excellent biosafety in an animal body and has great potential to replace traditional Clostridium perfringens toxoid vaccines.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, and particularly relates to a recombinant protein with multiple antigenic epitope peptides of Clostridium perfringens, three lethal toxins, and its design method and application. Background Technology

[0002] Clostridium perfringens, a highly dangerous zoonotic pathogen, can cause fatal diseases such as enterotoxemia, necrotizing enteritis, and gas gangrene. Its causative agents are multiple lethal exotoxins, including Alpha, Beta1, and Beta2, three of which are produced by the bacterium. Vaccination of susceptible animals with toxoids is the primary control measure. Outbreaks of Clostridium perfringens can cause extremely severe economic losses in agriculture. In unvaccinated herds, mortality rates can exceed 50%, and in some infected young animals, the mortality rate can even reach 100%. Clostridium perfringens type C alone causes approximately $6 billion in production losses and control costs annually for the global poultry industry.

[0003] The production method for pathogenic Clostridium perfringens toxoid vaccines is similar. It involves culturing virulent strains of Clostridium perfringens under anaerobic conditions within an optimal pH range in a complex culture medium containing animal-derived raw materials, glucose, or other fermentable carbohydrates. The resulting fermentation culture containing the exotoxin secreted by the bacteria is harvested. The culture is then chemically inactivated using formaldehyde at a final concentration of 0.2%, or the toxin is purified by chromatography before inactivation to obtain a biologically inactive toxoid for use as a vaccine. However, due to the unique characteristics of Clostridium perfringens, both the vaccine itself and the production process present numerous problems. The current shortcomings and problems of Clostridium perfringens toxoid vaccines mainly include the following:

[0004] (1) The biosafety of the production process is worrying and the production seeds are prone to degeneration: The production strain of Clostridium perfringens toxoid vaccine is a highly virulent strain that can secrete a large amount of exotoxin. Since multiple lethal exotoxin genes of this bacterium are located on the large plasmid of the bacterial cell, plasmid deletion often occurs during the culture process, resulting in the degeneration of the production seeds.

[0005] (2) Different strains secrete different immunogenic toxins, and the ratio after fermentation will also cause different vaccine efficacy.

[0006] (3) The secretion conditions of Clostridium perfringens exotoxin are very harsh, and factors such as the composition of the culture medium can also affect the preparation of toxoid vaccines.

[0007] (4) Clostridium perfringens has the characteristic of producing spores, and high doses of formaldehyde are required for inactivation during the inactivation process of toxoid vaccines. The "Inspection Regulations for Veterinary Biological Products (2010 Edition)" clearly stipulates that the formaldehyde residue in products containing Clostridium perfringens should not exceed 0.2%, while the formaldehyde residue in other biological products should not exceed 0.08%.

[0008] (5) Clostridium perfringens toxoid vaccine still carries the risk of residual toxicity after a long detoxification process, which could bring disaster to immunized animals.

[0009] (6) Clostridium perfringens cultures do not contain only the required immunogenic toxins, but also other toxins and proteins originally present in the culture medium, which may result in vaccines containing multiple unknown allergens.

[0010] Numerous studies have demonstrated that single or multivalent vaccines using recombinant Alpha, Beta1, and Epsilon toxins exhibit good immunogenicity. Research indicates that non-mutated recombinant toxins may retain the activity of natural toxins and may not be suitable for direct use as vaccines. Therefore, much research and strategies both domestically and internationally focus on site-directed mutagenesis, expression of only N-terminal or C-terminal protein domains, or the use of fusion genes to form chimeric toxins, attempting to obtain non-toxic and immunogenic recombinant toxin proteins for vaccination. The current drawbacks and problems of Clostridium perfringens toxin-engineered recombinant protein vaccines mainly include the following:

[0011] (1) Clostridium perfringens toxin genetically engineered recombinant protein vaccines are produced by altering the essential amino acid residues of the toxin protein to produce non-toxic and immunogenic recombinant proteins. This requires testing the toxicity and immunoprotective properties of each site-directed mutagenized recombinant protein, which is a large workload.

[0012] (2) Although the toxin protein loses its toxicity after site-directed mutagenesis and has a certain degree of immunogenicity, as a complete toxin protein, it contains a part that is not related to the production of protective immune response (neutralizing antibody).

[0013] (3) Clostridium perfringens toxin multivalent genetically engineered recombinant protein vaccines are recombinant chimeric proteins of two or more antigens expressed heterologously. However, multivalent toxin recombinant proteins may result in excessively large molecular weights of the expressed fusion proteins, and may also have inappropriate conformations, masking, or alterations to protective epitopes. Summary of the Invention

[0014] The purpose of this invention is to provide a recombinant protein with multiple antigenic epitope peptides from three lethal toxins of Clostridium perfringens, in order to solve the problems mentioned in the background art.

[0015] To address the aforementioned problems, this invention provides a recombinant protein containing multiple antigenic epitope peptides from three lethal toxins of Clostridium perfringens. The recombinant protein comprises B lymphocyte (B cell) antigenic epitopes, cytotoxic T lymphocyte (Tc cell) antigenic epitopes, and helper T lymphocyte (Th cell) antigenic epitopes of the three lethal toxin proteins, which bind via KK, GPGPG, and AAY linkers. The three lethal toxin proteins are Alpha, Beta1, and Beta2. The amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1.

[0016] Another object of the present invention is to provide a gene encoding the above-mentioned multi-antigen epitope peptide recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO.2, and codon preference optimization was performed using Escherichia coli BL21(DE3) as the host (named AB12yh).

[0017] Another object of the present invention is to provide a recombinant vector, comprising an empty vector and the above-described coding gene.

[0018] Preferably, the empty carrier is a pET-28b carrier.

[0019] Another object of the present invention is to provide a recombinant strain comprising a host bacterium and the above-described coding gene or the above-described recombinant vector.

[0020] Preferably, the host bacterium is Escherichia coli; more preferably, the host bacterium is Escherichia coli BL21(DE 3).

[0021] Another object of the present invention is to provide the application of the above-mentioned multi-antigen epitope peptide recombinant protein, encoding gene, recombinant vector or recombinant strain in the preparation of a vaccine for the prevention and treatment of Clostridium perfringens infection.

[0022] Another object of the present invention is to provide a vaccine for preventing Clostridium perfringens infection, comprising an adjuvant and the above-described recombinant protein with multiple antigenic epitope peptides.

[0023] Another object of the present invention is to provide a method for designing the above-mentioned recombinant protein with multiple antigenic epitope peptides, which includes the following steps:

[0024] The immune epitope database and ABCpred database were used to predict B lymphocyte antigenic epitopes, cytotoxic T lymphocyte antigenic epitopes, and helper T lymphocyte antigenic epitopes of Clostridium perfringens. The dominant epitopes that are non-toxic, non-allergenic, and highly antigenic were selected using VaxiJen2.0, AllerTOP2.0, and ToxinPred servers.

[0025] The B lymphocyte antigen epitopes were compared with the predicted peptides obtained by eight prediction methods using DNAMAN, and common repetitive sequences were selected.

[0026] The amino acid sequences of the screened B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes, and helper T lymphocyte antigen epitopes were linked to construct a multi-antigen epitope peptide recombinant protein. To avoid the formation of new epitopes and ensure the independent immunogenicity of each epitope, linker proteins were added between the epitopes. The B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes, and helper T lymphocyte antigen epitopes were linked via KK, GGPPG, and AAY linkers, respectively. This linking method was designed based on the idea of ​​adding or replacing other toxins or other functional protein domains produced by Clostridium perfringens.

[0027] Preferably, the linkage is performed in the Alpha-Beta1-Beta2 toxin sequence (named AB12), which facilitates the replacement and addition of other toxin or functional protein sequences.

[0028] Preferably, the biochemical properties of the multi-antigenic epitope peptide recombinant protein are evaluated by confirming antigenicity using the ANTIGENpro server; allergenicity and toxicity are checked using AllerTOPv2.0 and ToxinPred servers, respectively; the solubility of the constructed candidate vaccine is predicted using the Protein-Sol server; and the physicochemical properties are detected and obtained using the ProtParam tool.

[0029] Preferably, the secondary structure of the multi-antigen epitope peptide recombinant protein is predicted by the SOPMA server, including α-helices, β-sheets, and random coil structures. The tertiary structure is generated using the I-TASSER tool. I-TASSER models the structure and function of multi-domain proteins using a progressive approach, exhibiting high accuracy in domain modeling and inter-domain assembly. The protein model with the best score is imported into the SAVES server. The PROCHECK tool generates a Laplace plot for analyzing model quality, and ProSAweb analyzes the Z-score to assess errors in the protein structure.

[0030] Preferably, the recombinant multi-epitope peptide protein is docked with the TLR-4 receptor protein using the HADDOCK server. The optimal docking model is refined, and the PDBsum server is used to analyze the interacting residues between the vaccine and TLR-4, thereby obtaining an immunogenic recombinant multi-epitope peptide protein that can recognize three toxins.

[0031] The present invention also provides a method for preparing the above-mentioned recombinant protein with multiple antigenic epitope peptides, comprising the following steps:

[0032] The nucleotide sequence of the gene encoding the above-mentioned recombinant multi-antigen epitope peptide was inserted into the pET-28b empty vector to obtain the recombinant vector; the recombinant vector was then purified by the Escherichia coli BL21(DE3) expression system to obtain the recombinant multi-antigen epitope peptide.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] This invention designs a recombinant multi-antigenic epitope peptide protein for three lethal toxins of Clostridium perfringens: Alpha, Beta1, and Beta2, using immunoinformatics and structural vaccinology techniques. Specifically, 12 B-cell epitopes, 10 Tc-cell epitopes, and 4 Th-cell epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxin proteins were screened using immunoinformatics. Structural vaccinology testing showed that the multi-antigenic epitope peptide protein constructed based on these epitopes was non-toxic, non-allergenic, and highly antigenic, with good protein structure quality. It was effectively recognized by TLR-4 in molecular docking simulations, thus completing the design of a multi-antigenic epitope peptide vaccine for Clostridium perfringens Alpha, Beta1, and Beta2 toxins. Furthermore, an inclusion body-type recombinant multi-antigenic epitope peptide protein (AB12yh) that can be specifically recognized by monoclonal antibodies against Alpha, Beta1, and Beta2 toxins was successfully expressed and purified, and can be used for mouse immunization. The recombinant protein (AB12yh) with multiple antigenic epitope peptides obtained in this invention can simultaneously and effectively induce humoral immune responses and Th1 and Th2 cellular immune responses in mice, and successfully protects more than half of 1×LD mice. 100 Mice containing crude toxin extract from the standard strain C59-44 of Clostridium perfringens type C were used to neutralize 1×LD by serum produced from immunized mice. 100 The crude toxin extract has a 100% protection rate, demonstrating excellent protective effects.

[0035] This invention is the first to directly target the three major lethal toxins of Clostridium perfringens—Alpha, Beta1, and Beta2 proteins—and, through structural vaccinology, immunoinformatics, and mature online tools and databases, screen for B-cell, Tc-cell, and Th-cell antigenic epitopes to construct multi-antigenic epitope recombinant proteins. Furthermore, this invention overcomes the limitation of most current epitope peptide vaccine research remaining at the bioinformatics level, translating the theoretical feasibility of immunoinformatics vaccine design into the practical reliability of mouse immunization experiments. The expression of the epitope peptide recombinant protein and a series of mouse immunization efficacy evaluation experiments validate and support bioinformatics-based vaccine design, thereby truly providing information resources and candidate vaccine strains for the further development of efficient, safe, and economical Clostridium perfringens vaccines. Attached Figure Description

[0036] Figure 1This is a schematic diagram of the amino acid sequence of the recombinant protein of multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins.

[0037] Figure 2 Predicted secondary structures of recombinant proteins containing multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins.

[0038] Figure 3 Schematic diagram of predicted tertiary structure and structural quality analysis of recombinant proteins with multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins.

[0039] Figure 4 A visualization analysis of the interaction residues of the multi-antigenic epitope peptide recombinant protein and the TLR-4 complex of Clostridium perfringens Alpha, Beta1, and Beta2 toxins.

[0040] Figure 5 The construction of recombinant protein expression vectors for multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins and the identification of plasmid enzyme digestion (A is pET28b-AB12, B is pET28b-AB12yh, where M: standard DNA molecular weight, 1: original plasmid, 2: EcoRI single enzyme digestion, 3: EcoRI / NotI double enzyme digestion);

[0041] Figure 6 The images show the induction and identification of recombinant proteins containing multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins (A is an SDS-PAGE image, B is a Western Blot image for Beta1 monoclonal antibody identification, C is a Western Blot image for Beta2 monoclonal antibody identification, and D is a Western Blot image for Alpha monoclonal antibody identification, where M: standard protein molecular weight, 1: pET28 empty vector, 2: before AB12 induction, 3: after AB12 induction, 4: before AB12yh induction, and 5: after AB12yh induction).

[0042] Figure 7 The purification diagram of recombinant proteins containing multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins (M: standard protein molecular weight, 1-7: urea gradient denaturation washing, 8: before induction, 9: after induction).

[0043] Figure 8 Survival rate of mice challenged with the virus (n=10);

[0044] Figure 9 The fluctuation pattern of specific antibodies in immunized mice; A: Alpha, B: Beta1, C: Beta2 (n=5);

[0045] Figure 10 The specific IgG levels in immunized mice; A: Alpha, B: Beta1, C: Beta2 (n=10);

[0046] Figure 11 The specific sIgA levels in immunized mice; A: Alpha, B: Beta1, C: Beta2 (n=10);

[0047] Figure 12 The proliferation stimulation index of spleen lymphocytes in immunized mice; A: Alpha, B: Beta1, C: Beta2 (n=3);

[0048] Figure 13 The levels of IL-4 and IFN-γ in the supernatant of spleen lymphocytes from immunized mice; A: IL-4, B: IFN-γ (n=3). Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] Experimental methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or as recommended by the manufacturer. Unless otherwise stated, the reagents, raw materials, and equipment used in this invention are all commercially available.

[0051] Example 1: This example provides a method for predicting and screening the antigenic epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxins:

[0052] 1. Acquisition of Alpha, Beta1, and Beta2 toxin protein sequences: The main virulence factors of Clostridium perfringens, namely Alpha, Beta1, and Beta2, were selected from the NCBI database.

[0053] The Alpha protein sequence is 398 amino acids long (GenBank:AAA99195.1); the Beta1 protein sequence is 336 amino acids long (GenBank:CAA58246.1); and the Beta2 protein sequence is 265 amino acids long (GenBank:AAC27654.1).

[0054] 2. Prediction of B lymphocyte antigenic epitope peptides: The Alpha, Beta1, and Beta2 protein sequences of *Clostridium perfringens* were imported into the Immunological Epitope Database (IEDB) for screening of B lymphocyte antigenic epitope peptides. To avoid the limitations of different antigen prediction design methods and improve prediction accuracy, the following B cell epitope prediction tools were used: Bepipred Linear Epitope Prediction, Bepipred Linear Epitope Prediction 2.0, Chou & Fasman Beta-Turn Prediction, Emini Surface Accessibility Prediction, Karplus & Schulz Flexibility Prediction, Kolaskar & Tongaonkar Antigenicity, and Parker Hydrophobicity. Seven Prediction tools and the ABCpred database were used to predict B-cell antigenic epitopes for Alpha, Beta1, and Beta2 toxins, retaining the top ten peptides from each method. DNAMAN was used to perform sequence alignment of the predicted peptides obtained from the eight prediction methods, and common repetitive sequences were selected as candidate B lymphocyte epitope peptides (LBLs) for further screening.

[0055] 3. Prediction of T lymphocyte antigenic epitope peptides: The Alpha, Beta1, and Beta2 protein sequences of *Clostridium perfringens* were imported into the Immunoeptope Database (IEDB). Using the Major Histocompatibility Complex (MHC) class I molecule binding recognition server in the T cell epitope prediction tool, the consensus prediction method was selected. Dodecapeptides were predicted using mouse-derived H-2-Db, H-2-Dd, H-2-Kb, H-2-Kd, H-2-Kk, and H-2-Ld alleles. The top 1% of peptides were retained as candidate cytotoxic T cell (CTL) epitope peptides for further screening. Using the Major Histocompatibility Complex (MHC) class II molecule binding recognition server in the T cell epitope prediction tool, the NetMHCII-2.3 prediction method was selected. Dodecapeptides were predicted using mouse-derived H-2-IAb, H-2-IAd, and H-2-IA... The alleles k, H-2-IA, H-2-IAu, H-2-IEd, and H-2-IEk predict fifteen peptides, and the top 5% of peptides by score are selected as candidate helper T cell (HTL) epitope peptides for further screening.

[0056] 4. Evaluation and screening of the sensitization, antigenicity, and toxicity of candidate antigenic epitope peptides: The selected antigenic epitope peptides were sequentially evaluated using the VaxiJen2.0 server to predict the antigenicity of the selected epitopes. Epitopes with an antigenicity ≥0.4 were further submitted to the AllerTOP2.0 and ToxinPred servers for sensitization and toxicity testing, respectively. The AllerTOP2.0 server used the k-nearest neighbor algorithm (kNN, k=1) to predict the allergenicity of the epitopes. The ToxinPred server used a protein toxicity region prediction tool with a screening threshold set to 0.6. Segmentation detection was performed using the SVM (Swiss-Prot) + Motif-based method. Based on the combined prediction results, epitope peptides with no toxicity, no sensitization, and good antigenicity were selected. Finally, B lymphocyte epitope screening yielded 3 Alpha toxin epitopes, 5 Beta1 toxin epitopes, and 4 Beta2 toxin epitopes (results shown in Table 1). Cytotoxic T lymphocyte epitope screening yielded 2 Alpha toxin epitopes, 4 Beta1 toxin epitopes, and 4 Beta2 toxin epitopes (results shown in Table 2). For helper T cell epitope peptides, the induction ability of candidate epitopes to IFN-γ was predicted using the IFNepitope server, and positive epitopes were selected as candidate epitope peptides. Helper T lymphocyte epitope screening yielded 2 Alpha toxin epitopes, 1 Beta1 toxin epitope, and 1 Beta2 toxin epitope (results shown in Table 3).

[0057] Table 1. LBL epitopes used for multiepitope vaccine construction.

[0058]

[0059] Table 2. CTL epitopes used for multi-epitope vaccine construction.

[0060]

[0061] Table 3 HTL epitopes used for multi-epitope vaccine construction

[0062]

[0063] Example 2: This example provides a method for designing a recombinant protein vaccine containing multiple antigenic epitope peptides from Clostridium perfringens Alpha, Beta1, and Beta2 toxins, specifically including the following steps:

[0064] 1. Construction of a multi-antigen epitope peptide recombinant protein vaccine: The B, Tc, and Th cell epitope amino acid sequences of the screened Clostridium perfringens Alpha, Beta1, and Beta2 toxins were linked according to the toxin as structural unit, in the Alpha-Beta1-Beta2 toxin sequence (named AB12). This linkage method was designed based on the idea of ​​adding or replacing other toxin antigenic peptides or other functional protein domains. To construct the multi-antigen epitope peptide recombinant protein vaccine, and to avoid the formation of new epitopes and ensure the independent immunogenicity of each epitope, appropriate linker proteins were added between the epitopes. All epitopes of LBL, HTL, and CTL were linked via KK, GGPPG, and AAY linkers, respectively. The dilysine (KK) maintained their independent immunogenic activity, and the AAY and GGPPG linkers enhanced the recognition ability of the vaccine subunits. The final constructed multi-antigen epitope peptide recombinant protein consisted of 410 amino acids, such as... Figure 1 As shown.

[0065] 2. Evaluation of the Multi-Epidermal Peptide Recombinant Protein Vaccine: The Alpha, Beta1, and Beta2 protein epitope peptide recombinant proteins were predicted using the Vaxijenv 2.0 server, with scores of 0.899, indicating their suitability as antigens. Results from the ToxinPred and AllerTOP v2.0 servers showed that the multi-epidermal peptide recombinant protein was non-toxic and non-allergenic. ProtParam server analysis indicated that the multi-epidermal peptide recombinant protein consisted of 410 amino acids, with a molecular weight of 45.74 kDa and a theoretical isoelectric point of 9.65. The instability index of the multi-epidermal peptide recombinant protein was approximately 33.88, indicating its stability. The aliphatic index of the multi-epidermal peptide recombinant protein was 50.07, indicating good thermal stability. The predicted half-life of this multi-epidermal peptide recombinant protein was over 10 hours in *E. coli* and over 20 hours in vivo in yeast cells, as shown in Table 4.

[0066] Table 4. Physicochemical properties and predicted antigenicity, allergenicity, and toxicity of recombinant proteins with multiple antigenic epitope peptides.

[0067]

[0068] 3. Prediction and Validation of Secondary and Tertiary Structures of Multi-Episode Peptide Recombinant Protein Vaccine: The secondary structure of the multi-episode peptide recombinant protein was studied using the SOPMA server. The results showed that the multi-episode peptide recombinant protein consists of 73 amino acids (18%) forming an α-helix, 109 amino acids (27%) forming an extended β-sheet, and 228 amino acids (55%) forming a random coil. Figure 2 As shown.

[0069] For the tertiary structure identification of recombinant proteins with multiple antigenic epitope peptides, the I-TASSER tool was used for modeling. The protein model with the best score was selected, and Raachandran plots were generated using the PROCHECK tool in the SAVES server for model quality analysis. The Raachandran plot estimated that 64.2% of the residues in the recombinant proteins with multiple antigenic epitope peptides were located in favorable regions, 30.7% in permissible regions, and 2.3% in disallowed regions. The final model structure was scored and validated using the ProSA Web server. Structural precision analysis showed a z-value of -1.6, which is within the acceptable range for native proteins. Results are as follows... Figure 3 As shown, the AB12 multi-antigen epitope peptide recombinant protein has a reliable and stable tertiary structure.

[0070] 4. Docking Analysis of Multi-Epitope Recombinant Protein Vaccine with TLR-4: Protein-protein interactions are crucial for the functioning of biomolecules. Toll-like receptor 4 (TLR4) is a type I transmembrane protein expressed in various cell types, including monocytes and macrophages, and plays a key role in activating innate immunity by directly recognizing pathogen-associated molecular patterns (PAMPs). To assess the binding affinity between the multi-episotope recombinant protein construct and TLR4, the multi-episotope vaccine protein was docked with the TLR-4 receptor protein using the HADDOCK server. This process generated 24 models, with model 24 (AB12) ranking first among all predicted models. The PDBsum server displayed the specific docking sites to further evaluate their binding affinity. The AB12 multi-antigen epitope peptide recombinant protein-TLR4 complex formed two salt bridges, 19 hydrogen bonds, and 210 non-contact binding sites. The vaccine amino acid residues involved in salt bridge formation were Asp72 and Asp31, while the residues involved in hydrogen bond formation were Ser263, Leu265, Arg286, Arg286, Arg264, Phe285, Ser201, Ser201, Asn208, Asp210, Ser252, Ser252, Glu254, Ile165, Ala189, Lys94, Asp72, Asn76, and Asp31. The results are as follows... Figure 4 As shown, the constructed multi-antigen epitope peptide recombinant protein interacts strongly with TLR-4 and can bind effectively.

[0071] Example 3: This example provides a method for preparing a recombinant protein vaccine containing multiple antigenic epitope peptides from Clostridium perfringens Alpha, Beta1, and Beta2 toxins, specifically including the following steps:

[0072] 1. Codon optimization and expression vector construction of recombinant multi-antigen epitope peptide protein sequence: Without altering the amino acid sequence, the amino acid sequence of the recombinant multi-antigen epitope peptide protein AB12 (as shown in SEQ ID NO.1) was optimized for codon preference using *E. coli* BL21(DE3) as the host. The GC content was optimized from 33.3% to 50.2% (ideal range 40%-60%); the codon fitness index was increased from 0.47 to 0.93. The determined coding gene was sent to Shanghai Sangon Biotech Co., Ltd. (www.sangon.com) for synthesis. The gene before and after codon optimization was cloned into the EcoRI and NotRI sites of the pET28b plasmid vector, respectively, to obtain the recombinant vector, named pET28-AB12yh. The optimized nucleotide sequence is shown in SEQ ID NO.2, and the expression vector of the recombinant multi-antigen epitope peptide protein is shown in [image missing]. Figure 5 As shown.

[0073] 2. Transformation of recombinant vector: The recombinant vector pET28-AB12yh was transformed into Escherichia coli BL21(DE3)plys competent cells using the heat stress method to obtain the recombinant strain.

[0074] 3. Induction of recombinant protein expression with multiple antigenic epitope peptides: The correctly identified BL21(DE3)pET28-AB12yh recombinant strain, frozen at -80℃, was inoculated onto LB solid medium plates containing 40 μg / mL kanamycin and incubated upside down at 37℃ for 16 h. Single colonies of the revived strain were picked and inoculated into 10 mL of LB liquid medium containing 40 μg / mL kanamycin, and incubated at 37℃ and 180 rpm for 16 h. After incubation, the inoculum was diluted to 1% and added to a 200 mL LB medium bottle, and incubated at 37℃ and 180 rpm for 3 h. IPTG was added to the culture bottle to a final concentration of 1 mmol / L, and the culture was incubated at 37℃ and 180 rpm for 3 h. Protein samples were collected at each stage. After SDS-PAGE gel electrophoresis, the molecular weight and expression level of the proteins were observed by Coomassie Brilliant Blue staining, and Western spectroscopy was performed. Blot analysis was used to observe the binding of recombinant proteins to monoclonal antibodies against Alpha, Beta1, and Beta2 toxins. The results are as follows: Figure 6 As shown, the recombinant protein AB12yh, a multi-antigen epitope peptide, was successfully expressed after codon optimization and can specifically bind to Alpha, Beta1, and Beta2 toxin monoclonal antibodies.

[0075] 4. Purification of recombinant multi-antigen epitope peptides: Transformed recombinant strains with good expression were selected and cultured in large quantities. After 3 hours of induction, bacterial sludge was collected. The precipitate was then centrifuged using lysozyme and ultrasonic disruption to obtain the recombinant multi-antigen epitope peptides in inclusion body form. To remove impurities from the precipitate, a 0.1-2M urea gradient was used to denature and wash the precipitate, ultimately obtaining the purified inclusion body-type recombinant multi-antigen epitope peptides. The results are as follows: Figure 7 As shown.

[0076] Example 4: This example provides a method for evaluating the immunoprotective efficacy of a Clostridium perfringens Alpha, Beta1, and Beta2 toxin multi-antigenic epitope recombinant protein vaccine, as detailed below:

[0077] 1. Mouse Immunization Protocol: Balb / c mice were randomly divided into two groups of 10 mice each. The AB12yh experimental group was immunized with recombinant epitope peptide protein prepared using the previously described method at a concentration of 1 μg / μL, mixed 1:1 with 2% aluminum hydroxide gel, and administered via multiple subcutaneous injections at a dose of 200 μL per mouse. A saline group served as the negative control group (Control group), with each mouse receiving 200 μL of saline. A second immunization was administered 14 days after the initial immunization, at half the dose of the initial immunization. Every three days after the initial immunization, 5 mice were randomly selected for tail amputation and blood collection, and serum was separated.

[0078] 2. Mouse LD50 of crude extract of Clostridium perfringens type C toxin (Chinese standard strain) 100 Assay: The standard strain C59-44 of *Clostridium perfringens* type C was inoculated into enrichment medium in a clean bench and anaerobically cultured at 37°C for 8 h. After two repeated inoculations, the culture was transferred to toxin-producing medium and incubated overnight at 37°C. The culture was centrifuged at 6000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was the crude extract of *Clostridium perfringens* type C toxin, which was stored at 4°C for later use. The crude toxin extract was diluted with sterile physiological saline to 200 μL at different doses, resulting in final crude toxin extract contents of 20, 40, 60, 70, and 80 μL for each group. BALB / c mice were intraperitoneally injected (n=4 per group). Mice mortality was observed 48 h later to determine the absolute lethal dose (LD50) of BALB / c mice. 100 The result was 70 μL (see Table 5 for the results).

[0079] Table 5 Clostridium perfringens C59-44 LD 100 Measurement

[0080]

[0081] 3. Protective test against challenge in immunized mice: 28 days after the initial immunization, mice were intraperitoneally injected with LD50. 100A challenge experiment was conducted on mice with a dose of fresh Clostridium perfringens C59-44 crude toxin extract. The survival rate of mice after 48 hours was observed and statistically analyzed, and a survival curve was constructed. The experimental results are as follows: Figure 8 As shown, after challenge, 2 mice in the Control group died within 12 hours, most died within 24 hours, and all died within 36 hours; mice in the immunized group began to die within 24 hours, and 4 mice in the AB12yh group died after 48 hours. Ultimately, the protection rate of the AB12yh group was 60%, while all mice in the Control group died.

[0082] Example 5: This example provides a method for evaluating the humoral immune efficacy of a recombinant protein vaccine containing multiple antigenic epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as detailed below:

[0083] 1. Analysis of serum antibody changes and specific IgG levels in immunized mice: From the initial immunization to 28 days later, blood was collected from the tail vein every 3 days, and serum was separated and stored at -20℃ for later use. Microplates were coated with recombinant proteins of Alpha, Beta1, and Beta2 toxins, respectively. Using the isolated serum from the three groups of mice as primary antibodies, the level of antigen-specific IgG in the serum was measured by indirect ELISA to detect the antibody production pattern of immunized mice against the three toxins in Clostridium perfringens type C. 14 days after the second immunization, blood was collected from the eyeballs of mice in each group, and serum was collected and the level of specific IgG in the serum was measured by indirect ELISA. The specific ELISA method is as follows:

[0084] (1) Dilute the Alpha, Beta1, and Beta2 toxin recombinant proteins with coating buffer to make the antigen concentration 20 μg / mL. Add 100 μL to each well of the ELISA plate and coat overnight at 4°C.

[0085] (2) On the second day, after discarding the coating solution, wash three times with PBST for 3 min each time, and pat dry.

[0086] (3) Add 200 μL of 5% skim milk to each well, block at 37°C for 1.5 h, wash 3 times with PBST for 3 min each time, and pat dry the liquid.

[0087] (4) Add 100 μL of mouse serum from different time periods (500-fold dilution) to each well, and add PBS as a blank control. Incubate at 37°C for 2 h, wash 5 times with PBST for 3 min each time, and pat dry.

[0088] (5) Add 100 μL of HRP-labeled secondary antibody diluted 1:3000 to each well, incubate at 37℃ for 1 h, wash 5 times with PBST for 3 min each time, and pat dry the liquid.

[0089] (6) Add 100 μL of OPD chromogenic solution to each well, incubate at 37℃ for 15 min, then add 50 μL of stop solution and read the OD value on the microplate reader. 490 Numerical value.

[0090] The results of serum antibody change patterns are as follows: Figure 9 As shown, mice developed specific serum antibodies against Alpha, Beta1, and Beta2 toxin proteins after vaccination. Antibody levels peaked on average one week after the second vaccination, and then gradually declined. Specifically, high antibody levels were observed for Alpha protein one week after the initial vaccination, while high antibody levels for Beta2 toxin protein were only observed after the second vaccination.

[0091] Specific IgG level results as follows Figure 10 As shown, the specific IgG content in the AB12yh group was significantly higher than that in the Control group (P < 0.0001), with the specific IgG levels being Alpha > Beta1 > Beta2.

[0092] 2. Detection of sIgA specific antigen in jejunal mucosa of immunized mice: 28 days after the initial immunization, blood was collected from the eyeballs of 6 mice in each of the 3 groups before immunization. The intestinal contents of the mice were removed, the jejunum was dissected, and ileal mucus was collected after rinsing and scraping. The content of sIgA specific antigen in the jejunal mucosa mucus of different groups of mice was measured using an sIgA ELISA kit. Specific experimental procedures were followed according to the product instructions. All samples were tested three times. Results are as follows: Figure 11 As shown, the sIgA content in the AB12yh group was significantly higher than that in the Control group (P < 0.0001), which has a protective effect on the intestines of mice after challenge.

[0093] Example 6: This example provides a method for evaluating the cellular immunogenicity of a recombinant protein vaccine containing multiple antigenic epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as detailed below:

[0094] 1. Splenic lymphocyte proliferation experiment in immunized mice: To investigate whether the designed multi-antigen epitope peptide recombinant protein has the ability to stimulate cellular immune response, the stimulation index of splenic lymphocytes in each group of mice after stimulation by Alpha, Beta1, and Beta2 recombinant proteins was detected through a splenic lymphocyte proliferation experiment.

[0095] (1) Mice that were immunized for the first time were euthanized by cervical dislocation and immersed in 75% ethanol. 4 mL of mouse lymphocyte separation solution that had been equilibrated at room temperature was placed in a culture dish.

[0096] (2) Remove the mouse spleen in a clean bench and place it in the separation solution (aseptic operation), and grind it in a 200-mesh cell sieve using a syringe plunger;

[0097] (3) Transfer the suspension containing spleen cells to a 15 mL centrifuge tube, slowly add 500 μL of RPMI 1640 medium along the tube wall to make the liquid surface distinct, and centrifuge at 800 g at room temperature for 30 min using a horizontal rotor centrifuge.

[0098] (4) Aspirate the middle lymphocyte layer, add 5 mL of RPMI 1640 medium, wash by inversion, and centrifuge at 250 g at room temperature for 10 min.

[0099] (5) Remove the supernatant, resuspend the cells in RPMI 1640 complete medium, count the cells, and prepare 2×10⁶ cells / cells. 6 Cells / mL cell suspension;

[0100] (6) Set up a blank control group and add 100 μL of RPMI 1640 complete culture medium to each well;

[0101] (7) Add 100 μL of cell suspension to each of the remaining wells;

[0102] (8) Set up a positive control group, add 10 μL ConA to each well (final concentration of 5 μg / mL);

[0103] (9) Set up a negative control group, adding 10 μL of RPMI 1640 complete culture medium to each well;

[0104] (10) Set up the Alpha protein experimental group and add 10 μL of Alpha protein to each well (final concentration of 10 μg / mL);

[0105] (11) Set up the Beta1 protein experimental group and add 10 μL Beta1 protein to each well (final concentration of 10 μg / mL);

[0106] (12) Set up the Beta2 protein experimental group and add 10 μL Beta2 protein to each well (final concentration of 10 μg / mL);

[0107] (13) All groups were placed in a cell culture incubator at 37°C for 44 hours. 10 μL of cck-8 was added to each well and cultured for another 4 hours.

[0108] (14) OD450 values ​​were measured and stimulation indices were calculated for each group. The stimulation index of the spleen lymphocyte proliferation experiment was used to reflect the cellular immune effect.

[0109] Stimulus Index (SI) = (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group)

[0110] Experimental results are as follows Figure 12As shown, the spleen lymphocyte stimulation index of the immunized group mice was significantly higher than that of the Control group (P<0.01; P<0.001; P<0.0001), indicating that the recombinant protein with multiple antigenic epitope peptides can induce a cellular immune response.

[0111] 2. Detection of cytokine levels in the supernatant of immunized mouse spleen lymphocytes: Fourteen days after the second immunization, the supernatant of stimulated spleen lymphocytes was prepared. Changes in IL-4 and IFN-γ cytokine levels were analyzed using an ELISA kit to assess the ability of recombinant multi-antigen epitope peptides to induce Th1 and Th2 immune responses. Results are as follows: Figure 13 As shown, among Th2 cytokines, the IL-4 levels in the supernatant of spleen cells stimulated by Alpha protein were 11.64±2.78 pg / mL (Control group) and 27.54±5.81 pg / mL (AB12yh group), respectively; the IL-4 levels in the supernatant of spleen cells stimulated by Beta1 protein were 22.54±8.86 pg / mL (Control group) and 39.59±3.48 pg / mL (AB12yh group), respectively; and the IL-4 levels in the supernatant of spleen cells stimulated by Beta2 protein were 8.34±0.52 pg / mL (Control group) and 16.75±2.90 pg / mL (AB12yh group), respectively. Except for Alpha... The levels of alpha protein in the CHLyh group and other immune groups were significantly higher than those in the Control group (P < 0.05; P < 0.01; P < 0.001). Among Th1 cytokines, the IFN-γ levels in the supernatant of spleen cells stimulated by alpha protein were 240.7 ± 104.1 pg / mL (Control group) and 9838.0 ± 195.2 pg / mL (AB12yh group), respectively; the IFN-γ levels in the supernatant of spleen cells stimulated by beta1 protein were 102.9 ± 61.9 pg / mL (Control group) and 9590.0 ± 235.4 pg / mL (AB12yh group), respectively; and the IFN-γ levels in the supernatant of spleen cells stimulated by beta2 protein were 20.22 ± 35.03 pg / mL (Control group) and 8450.0 ± 239.5 pg / mL (AB12yh group), respectively. These levels in the immune groups were significantly higher than those in the Control group (P < 0.001). The above results indicate that the recombinant protein AB12yh with multiple antigenic epitope peptides can induce Th1 and Th2 cellular immune responses.

[0112] Example 7: This example provides a method for evaluating the serum toxin neutralization effect of a recombinant protein vaccine containing multiple antigenic epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as detailed below:

[0113] The ability of serum obtained from immunized mice to neutralize the toxicity of crude toxins in culture filtrate was determined by an in vivo neutralization assay. (4×LD)100 One unit of crude toxin was mixed with an equal volume of mouse serum (diluted 1:1 or 1:2), and diluted with physiological saline to a total volume of 0.8 mL. The mixture was incubated at 37°C for 2 h, followed by intraperitoneal injection of 0.2 mL of the incubated mixture into unimmunized healthy mice (n=4 per group). The animals were closely monitored for 48 hours, and survival rates were recorded.

[0114] The experimental results are shown in Table 6. Serum prepared from mice immunized with the multi-antigen epitope peptide recombinant protein AB12yh neutralized 1×LD. 100 The crude toxin extract from the standard strain C59-44 of Clostridium perfringens type C showed a 100% protection rate. A 1:2 dilution of serum neutralized 1×LD50. 100 The protection rate of crude toxin extract from the standard strain C59-44 of Clostridium perfringens type C was 75%.

[0115] Table 6. Neutralization protection status of serum in immunized mice

[0116]

[0117] In summary, the method for expressing three lethal toxins of Clostridium perfringens—Alpha, Beta1, and Beta2—in a prokaryotic expression system provided by the embodiments of the present invention has the advantages of being safe, inexpensive, having stable expression conditions, and requiring no cultivation of virulent strains or the use of formaldehyde for inactivation. Meanwhile, addressing the common issues of large screening workload and limited antigenic epitope capacity in selecting single or multivalent versions of recombinant Alpha, Beta1, and other toxins with non-toxic mutations, this invention uses immunoinformatics and structural vaccinology methods to screen B, Tc, and Th cell antigenic epitopes of three major lethal toxins of Clostridium perfringens type C: Alpha, Beta1, and Beta2. After the selected epitope peptides are linked by protein linkers to form recombinant proteins, online tools such as Phyre 2.0 and SWISS-MODEL are used to model the secondary and tertiary structures of the recombinant proteins. Finally, online tools such as Clus Pro 2.0 protein-protein docking and PatchDock are used to predict the optimal binding mode of the antigenic epitope recombinant protein after docking with TLR4. The antigenic epitope peptide recombinant protein is expressed using Escherichia coli BL21(DE3) as the expression host. After identification and purification, it is used to immunize mice. The immunization effect is comprehensively evaluated through challenge protection, humoral immunity, and cellular immunity. The results show that the Clostridium perfringens Alpha, Beta1, and Beta2 antigenic epitope peptides designed in this embodiment of the invention are effective. The recombinant protein of the three main lethal toxins, pha, Beta1, and Beta2, with multiple antigenic epitope peptides can induce specific humoral and cellular immune responses in mice, providing information resources and laying an experimental foundation for the further development of efficient, safe, and economical Clostridium perfringens vaccines.

[0118] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A recombinant protein containing multiple antigenic epitope peptides from three lethal toxins of Clostridium perfringens, characterized in that, The recombinant multiantigen epitope peptide protein comprises B lymphocyte antigenic epitopes, cytotoxic T lymphocyte antigenic epitopes, and helper T lymphocyte antigenic epitopes of three lethal toxin proteins, which bind via KK, GGPPG, and AAY linkers; the three lethal toxin proteins are Alpha, Beta1, and Beta2; the amino acid sequence of the recombinant multiantigen epitope peptide protein is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant protein with multiple antigenic epitope peptides as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant vector, comprising an empty vector, characterized in that, It also includes the coding gene as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The empty carrier is the pET-28b carrier.

5. A recombinant bacterial strain, comprising a host bacterium, characterized in that, It also includes the coding gene as described in claim 2 or the recombinant vector as described in any one of claims 3-4.

6. The recombinant strain according to claim 5, characterized in that, The host bacterium is Escherichia coli.

7. The use of a recombinant protein with multiple antigenic epitope peptides as described in claim 1, the encoding gene as described in claim 2, the recombinant vector as described in any one of claims 3-4, or the recombinant strain as described in any one of claims 5-6 in the preparation of a vaccine for the prevention of Clostridium perfringens infection.

8. A vaccine for the prevention of Clostridium perfringens infection, comprising an adjuvant, characterized in that, It also includes the recombinant protein with multiple antigenic epitope peptides as described in claim 1.

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