Multi-antigen epitope peptide recombinant protein of three lethal toxins of clostridium perfringens as well as design method and application of multi-antigen epitope peptide recombinant protein

Abstract

The invention is applicable to the technical field of immunology, and provides a multi-antigen epitope peptide recombinant protein of three lethal toxins of clostridium perfringens as well as a design method and application thereof. Composition fragments of the multi-antigen epitope peptide recombinant protein comprise B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes and helper T lymphocyte antigen epitopes of three lethal toxin proteins, and the antigen epitopes are combined through KK, GPGPG and AAY joints; the three types of lethal toxin proteins are Alpha, Beta1 and Beta2; the amino acid sequence of the multi-antigen epitope peptide recombinant protein is shown as SEQ ID NO. 1 in a sequence table. According to the present invention, three toxin proteins are adopted as target spots, peptide fragments with poor antigenicity, potential toxicity and sensitization are removed, and a plurality of antigen epitope peptides are integrated into a recombinant protein to be adopted as an immunogen so as to prepare the efficient, safe and broad-spectrum vaccine, and the vaccine has excellent biological safety in animal bodies, and can be widely used in animal bodies. The clostridium perfringens toxin vaccine has huge potential to replace the traditional clostridium perfringens toxin vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and in particular relates to a multi-epitope peptide recombinant protein of three lethal toxins of Clostridium perfringens, a design method and an application thereof. Background Art

[0002] Clostridium perfringens, a serious zoonotic pathogen, can cause fatal diseases such as enterotoxemia, necrotizing enterocolitis, and gas gangrene. Its virulence is due to multiple lethal exotoxins, including Alpha, Beta1, and Beta2. Vaccination of susceptible animals with toxinoids is the primary prevention and control measure. Outbreaks of C. perfringens can cause severe economic losses in the agricultural sector. Mortality rates in unvaccinated herds can exceed 50%, and for some infected young animals, mortality rates can reach 100%. Type C C. perfringens alone causes approximately $6 billion in production losses and control costs to the global poultry industry annually.

[0003] The production methods of pathogenic Clostridium toxin vaccines, including Clostridium perfringens, are similar. Different strains of Clostridium perfringens virulent strains are cultured under anaerobic conditions in a complex culture medium containing animal-derived raw materials, glucose or other fermentable carbohydrates, within the optimal pH range, and the culture containing the exotoxins secreted by the bacteria produced by fermentation is harvested. The culture is then chemically inactivated with formaldehyde at a final concentration of 0.2%, or the toxin is purified by chromatography and then inactivated to obtain a toxin that has lost its biological activity as a vaccine. However, due to the particularity of Clostridium perfringens bacteria itself, there are many problems with the vaccine itself and the production process. The shortcomings and problems of the current Clostridium perfringens toxin vaccine mainly include the following:

[0004] (1) The biosafety of the production process is worrying and the seeds used for production are prone to degeneration: the production strain of Clostridium perfringens toxin 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 a large plasmid in the bacteria, plasmid deletions often occur during the culture process, causing the degeneration of the seeds used for production;

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

[0006] (3) The secretion conditions of Clostridium perfringens exotoxin are very harsh, and factors such as culture medium composition 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 toxoid vaccine inactivation process. The "Regulations for the Inspection of Veterinary Biological Products (2010 Edition)" clearly stipulates that the formaldehyde residue in products containing Clostridium perfringens should not exceed 0.2%, while other biological products are required to contain no more than 0.08%.

[0008] (5) During the production process of Clostridium perfringens toxin-like vaccines, there is still a risk of residual toxicity after a long period of detoxification, which can bring disaster to the immunized animals.

[0009] (6) The culture of Clostridium perfringens contains not only the required immunogenic toxin, but also other toxins and proteins originally present in the culture medium, which may cause the vaccine to contain multiple unknown allergens.

[0010] Currently, many studies have shown that single or multivalent vaccines of recombinant Alpha, Beta1, and Epsilon toxins have good immunogenicity. Studies have shown that non-mutated recombinant toxins may still have the activity of natural toxins and may not be suitable for direct vaccination. Therefore, a large number of studies and strategies at home and abroad have focused on site-directed mutagenesis, expressing only the N-terminal or C-terminal protein domains, or using fusion genes to form chimeric toxins, etc., in an attempt to obtain non-toxic and immunogenic recombinant toxin proteins for vaccination. The shortcomings and problems of the current genetically engineered recombinant protein vaccines of Clostridium perfringens toxins mainly include the following:

[0011] (1) The genetically engineered recombinant protein vaccine of Clostridium perfringens toxin is produced by changing the essential toxic amino acid residues of the toxin protein to produce a non-toxic and immunogenic recombinant protein. The toxicity and immune protection of the recombinant proteins generated by site-directed mutagenesis need to be tested one by one, which is a lot of work.

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

[0013] (3) The polyvalent genetically engineered recombinant protein vaccine of Clostridium perfringens toxin is a recombinant chimeric protein of two or more antigens expressed heterologously. However, polyvalent toxin recombinant protein may result in an excessively large molecular weight of the expressed fusion protein and may also have inappropriate conformations, masking, or altering protective epitopes. Summary of the Invention

[0014] The purpose of the present invention is to provide a multi-epitope peptide recombinant protein of three lethal toxins of Clostridium perfringens, aiming to solve the problems raised in the background technology.

[0015] In response to the above problems, the present invention is achieved as follows: a multi-epitope peptide recombinant protein of three lethal toxins of Clostridium perfringens, wherein the constituent fragments of the multi-epitope peptide recombinant protein include B lymphocyte (B cell) antigen epitopes, cytotoxic T lymphocyte (Tc cell) antigen epitopes and helper T lymphocyte (Th cell) antigen epitopes of the three lethal toxin proteins, which are combined through KK, GPGPG and AAY linkers; the three lethal toxin proteins are Alpha, Beta1, and Beta2; the amino acid sequence of the multi-epitope peptide 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 is 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-mentioned encoding gene.

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

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

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

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

[0022] Another object of the present invention is to provide a vaccine for preventing and treating Clostridium perfringens infection, comprising an adjuvant and the above-mentioned multi-antigen epitope peptide recombinant protein.

[0023] Another object of the present invention is to provide a method for designing the above-mentioned multi-antigen epitope peptide recombinant protein, which comprises the following steps:

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

[0025] The B lymphocyte antigen epitopes were compared with the predicted peptides obtained by eight prediction methods using DNAMAN to select the common repeat sequences;

[0026] The amino acid sequences of the screened B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes, and helper T lymphocyte antigen epitopes were connected to construct a multi-antigen epitope peptide recombinant protein. At the same time, in order to avoid the formation of new epitopes by connection 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 connected through KK, GPGPG, and AAY linkers, respectively. This connection 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 connection is carried out in the order of Alpha-Beta1-Beta2 toxin (named AB12), which is convenient for replacing and adding other toxins or functional protein sequences.

[0028] Preferably, the biochemical properties of the multi-epitope peptide recombinant protein are evaluated by confirming antigenicity using the ANTIGENpro server; checking allergenicity and toxicity using the AllerTOPv2.0 and ToxinPred servers, respectively; using the Protein-Sol server to predict the solubility of the constructed candidate vaccine, and using the ProtParam tool to detect and obtain physicochemical properties;

[0029] Preferably, the secondary structure of the multi-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, which models the structure and function of multi-domain proteins through a progressive approach, with high accuracy in domain modeling and interdomain assembly. The best-scoring protein model is imported into the SAVES server, the PROCHECK tool generates a Ramachandran plot for model quality analysis, and ProSAweb analyzes the Z value to assess errors in the protein structure.

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

[0031] The present invention also provides a method for preparing the above-mentioned multi-antigen epitope peptide recombinant protein, comprising the following steps:

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

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

[0034] The present invention designs a multi-epitope peptide recombinant protein of three lethal toxins of Clostridium perfringens, Alpha, Beta1, and Beta2, through immunoinformatics and structural vaccinology technologies; specifically, 12 B cell epitopes, 10 Tc cell epitopes, and 4 Th cell epitopes of the Alpha, Beta1, and Beta2 toxin proteins of Clostridium perfringens are screened through immunoinformatics; structural vaccinology technology is used to detect that the multi-epitope peptide protein constructed based on the above epitopes is non-toxic, non-sensitizing, and has high antigenicity, and the protein structure quality is good. It can be effectively recognized by TLR-4 in molecular docking simulation, thereby completing the design of the multi-epitope peptide vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins; and the inclusion body-type multi-epitope peptide recombinant protein (AB12yh) that can be specifically recognized by monoclonal antibodies to Alpha, Beta1, and Beta2 toxins is successfully expressed and purified, and can be used for mouse immunization. The multi-epitope peptide recombinant protein (AB12yh) obtained by the present invention can effectively induce humoral immune response and Th1 and Th2 cellular immune response in mice, and successfully protect more than half of the 1×LD 100 The serum produced by mice immunized with the crude extract of the toxin of the standard strain of Clostridium perfringens type C, C59-44, was equal in volume to neutralize 1×LD 100 The protection rate of the crude toxin extract was 100%, which showed a good protection effect.

[0035] This invention directly targets the three main lethal toxins of Clostridium perfringens, Alpha, Beta1, and Beta2 proteins, for the first time. By using the ideas and techniques of structural vaccinology, immunoinformatics, and mature online tools and databases, it screens the B cell, Tc cell, and Th cell antigen epitopes to construct multi-antigen epitope recombinant proteins. Moreover, this invention breaks through the limitation that most current epitope peptide vaccine research remains at the bioinformatics level, and implements the theoretical feasibility of immunoinformatics vaccine design to the actual reliability of mouse immunization experiments. The bioinformatics vaccine design is verified and supported by the expression of epitope peptide recombinant proteins and a series of mouse immune effect evaluation experiments, thereby truly providing information resources and candidate vaccine strains for the further development of efficient, safe, and economical Clostridium perfringens vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1This is a schematic diagram of the structure 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 This is the predicted secondary structure of the recombinant protein of multiple antigenic epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins;

[0038] Figure 3 The tertiary structure prediction and structural quality analysis diagram of the multi-epitope peptide recombinant protein of Clostridium perfringens Alpha, Beta1, and Beta2 toxins;

[0039] Figure 4 This is a visualization analysis diagram of the interaction residues between the multi-epitope peptide recombinant proteins of Clostridium perfringens Alpha, Beta1, and Beta2 toxins and the TLR-4 complex;

[0040] Figure 5 Diagram showing 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 is the standard DNA molecular weight, 1 is the original plasmid, 2 is EcoRI single enzyme digestion, and 3 is EcoRI / NotI double enzyme digestion);

[0041] Figure 6 Figure 1 shows the induction expression and identification of recombinant proteins of multi-epitope peptides of Clostridium perfringens Alpha, Beta1, and Beta2 toxins (A is an SDS-PAGE image, B is a Western Blot image identified by Beta1 monoclonal antibody, C is a Western Blot image identified by Beta2 monoclonal antibody, and D is a Western Blot image identified by Alpha monoclonal antibody, where M is the molecular weight of the standard protein, 1 is pET28 empty vector, 2 is before AB12 induction, 3 is after AB12 induction, 4 is before AB12yh induction, and 5 is after AB12yh induction);

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

[0043] Figure 8 is the survival rate of mice challenged with poison (n=10);

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

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

[0046] Figure 11 Specific sIgA levels of 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 of immunized mice; A: IL-4, B: IFN-γ (n=3). DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present 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 only used to explain the present invention and are not intended to limit the present invention.

[0050] In the following examples, the experimental methods without specific experimental conditions are generally carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents, raw materials, and equipment used in the present invention can be obtained from commercial sources.

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

[0052] 1. Acquisition of Alpha, Beta1, and Beta2 toxin protein sequences: The three lethal exotoxin protein sequences of the main virulence factors of Clostridium perfringens, 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 antigen epitope peptides: The Alpha, Beta1, and Beta2 protein sequences of Clostridium perfringens were imported into the Immune Epitope Database (IEDB) for screening of B lymphocyte antigen epitope peptides. In order to circumvent the limitations of different antigen prediction design methods and improve the prediction accuracy, the B cell epitope prediction tools 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 Hydrophilicity were used respectively. Seven Prediction tools and the ABCpred database were used to predict the B cell antigen epitopes of the three toxins Alpha, Beta1, and Beta2, and the top ten peptides from each method were retained. DNAMAN was used to perform sequence alignment on the predicted peptides obtained by the eight prediction methods, and the common repeat sequences were selected as candidate B lymphocyte epitope peptides (LBL) for the next step of screening.

[0055] 3. Prediction of T lymphocyte antigen epitope peptides: The Alpha, Beta1, and Beta2 protein sequences of Clostridium perfringens were imported into the Immune Epitope Database (IEDB). The major histocompatibility complex MHC-I class molecule binding recognition server in the T cell epitope prediction tool was used to select the consensus prediction method. Twelve peptides were predicted based on the mouse 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 the next step of screening. The major histocompatibility complex MHC-II class molecule binding recognition server in the T cell epitope prediction tool was used to select the NetMHCII-2.3 prediction method. The mouse H-2-IAb, H-2-IAd, H-2-IA k, H-2-IA, H-2-IAu, H-2-IEd, and H-2-IEk alleles were predicted to produce fifteen peptides, and the peptides with the top 5% scores were retained as candidate helper T cell (HTL) epitope peptides for the next screening step.

[0056] 4. Evaluation and screening of candidate antigenic epitope peptides for sensitization, antigenicity, and toxicity: The selected antigenic epitope peptides were sequentially predicted using the VaxiJen2.0 server to predict the antigenicity of the selected epitopes, and epitopes with an antigenicity of ≥0.4 were further submitted to the AllerTOP2.0 and ToxinPred servers for checking allergenicity and toxicity, respectively. The AllerTOP2.0 server used the k-nearest neighbor algorithm (kNN, k=1) to predict the allergenicity of the epitope, and the ToxinPred server selected the protein toxicity region prediction tool, with the screening threshold set to 0.6, and the SVM (Swiss-Prot) + Motifbased method for segmented detection. Based on the above prediction results, epitope peptides with good antigenicity, non-toxicity, and non-sensitization were selected. Finally, the B lymphocyte epitope screening obtained 3 Alpha toxin epitopes, 5 Beta1 toxin epitopes, and 4 Beta2 toxin epitopes (the results are shown in Table 1). The cytotoxic T lymphocyte epitope screening obtained 2 Alpha toxin epitopes, 4 Beta1 toxin epitopes, and 4 Beta2 toxin epitopes (the results are shown in Table 2). For helper T cell epitope peptides, the IFNepitope server was used to predict the IFN-γ induction ability of candidate epitopes, and positive epitopes were selected as candidate epitope peptides. The helper T lymphocyte epitope screening obtained 2 Alpha toxin epitopes, 1 Beta1 toxin epitope, and 1 Beta2 toxin epitope (the results are shown in Table 3).

[0057] Table 1 LBL epitopes used for the construction of multi-epitope vaccines

[0058]

[0059] Table 2 CTL epitopes used for the construction of multi-epitope vaccines

[0060]

[0061] Table 3 HTL epitopes used for the construction of multi-epitope vaccines

[0062]

[0063] Example 2: This example provides a method for designing a multi-epitope peptide recombinant protein vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, which specifically includes the following steps:

[0064] 1. Construction of 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 connected according to the toxin as the structural unit, and the connection method was based on the Alpha-Beta1-Beta2 toxin sequence (named AB12); this connection method was designed based on the idea of ​​adding or replacing other toxin antigen peptides or other functional protein domains. To construct a multi-antigen epitope peptide recombinant protein vaccine, in order to avoid the formation of new epitopes by connection and ensure the independent immunogenicity of each epitope, appropriate linker proteins were added between the epitopes. All epitopes of LBL, HTL and CTL were connected through KK, GPGPG and AAY linkers, respectively. Dilysine (KK) maintained its independent immunogenic activity. AAY and GPGPG linkers were used to enhance the recognition ability of vaccine subunits. The multi-antigen epitope peptide recombinant protein finally constructed included 410 amino acids, such as Figure 1 shown.

[0065] 2. Evaluation of multi-epitope peptide recombinant protein vaccine: The Alpha, Beta1, and Beta2 protein epitope peptide recombinant proteins were predicted using the Vaxijenv2.0 server, with scores of 0.899, respectively, indicating that they can be used as antigens. The results of the ToxinPred and AllerTOP v2.0 servers showed that the multi-epitope peptide recombinant protein was non-toxic and non-allergenic. ProtParam server analysis showed that the multi-epitope 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-epitope peptide recombinant protein was approximately 33.88, indicating that its properties were stable. The aliphatic index of the multi-epitope peptide recombinant protein was 50.07, indicating good thermal stability. The multi-epitope peptide recombinant protein was predicted to have a half-life of more than 10 hours in Escherichia coli and a half-life of more than 20 hours in yeast cells in vivo. The results are shown in Table 4.

[0066] Table 4 Physicochemical properties and antigenicity, allergenicity and toxicity prediction of multi-epitope peptide recombinant proteins

[0067]

[0068] 3. Prediction and verification of secondary and tertiary structures of multi-antigen epitope peptide recombinant protein vaccines: The secondary structure of the multi-antigen epitope peptide recombinant protein was studied using the SOPMA server. The results showed that the multi-antigen epitope peptide recombinant protein was composed of 73 amino acids (18%) forming an α helix, 109 amino acids (27%) forming an extended β fold and 228 amino acids (55%) forming a random coil, such as Figure 2 shown.

[0069] For the tertiary structure identification of the multi-epitope peptide recombinant protein, the I-TASSER tool was used to generate the model, and the protein model with the best score was selected to generate a Ramachandran plot using the PROCHECK tool in the SAVES server for model quality analysis. The Ramachandran plot estimated that 64.2% of the residues in the multi-epitope peptide recombinant protein were located in the favorable region, 30.7% were located in the allowed region, and 2.3% were located in the unallowed region. The final model structure was scored and verified using the server ProSA Web, and the structural precision analysis showed a z value of -1.6, which is within the acceptable range for natural proteins. The results are as follows Figure 3 As shown, it shows that the AB12 multi-epitope peptide recombinant protein has a reliable and stable tertiary structure.

[0070] 4. Docking analysis of multi-epitope peptide recombinant protein vaccine and TLR-4 molecule: Protein-protein interactions are crucial for the function of biological molecules. Toll-like receptor 4 (TLR4) is a type I transmembrane protein expressed in a variety of cells, including monocytes and macrophages, and plays a key role in activating innate immunity by directly recognizing pathogen-associated molecular patterns (PAMPs). To evaluate the binding affinity between the multi-epitope peptide recombinant protein construct and TLR4, the server HADDOCK was used to dock the multi-epitope vaccine protein with the TLR-4 receptor protein. 24 models were generated in this process, of which model 24 (AB12) ranked first among all predicted models. The PDBsum server displays the specific docking sites to further evaluate their binding affinity. The AB12 multi-epitope peptide recombinant protein-TLR4 complex formed a total of 2 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, and 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, it shows that the constructed multi-antigen epitope peptide recombinant protein has a strong interaction with TLR-4 and can effectively bind to it.

[0071] Example 3: This example provides a method for preparing a multi-epitope peptide recombinant protein vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, which specifically includes the following steps:

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

[0074] 3. Induction expression of multi-antigen epitope peptide recombinant protein: Take the BL21 (DE3) pET28-AB12yh recombinant strain that has been correctly identified and frozen in a -80°C ultra-low temperature refrigerator, inoculate it on an LB solid culture medium plate containing 40 μg / mL kanamycin and place it in a 37°C constant temperature incubator for inversion culture for 16 hours; pick a single colony of the resuscitated culture strain, inoculate it into 10 mL LB liquid culture medium containing 40 μg / mL kanamycin, and culture it in a shaking incubator at 37°C and 180 rpm for 16 hours. After shaking, inoculate it into a 200 mL large bottle of LB culture medium at 1% and culture it in a shaking incubator at 37°C and 180 rpm for 3 hours; add the inducer IPTG to the culture flask to make the final concentration reach 1 mmol / L, and culture it in a shaking incubator at 37°C and 180 rpm for 3 hours. Prepare protein samples at each stage, and observe the molecular weight and expression level of the protein by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. The blot technique was used to observe the binding of recombinant protein to monoclonal antibodies of Alpha, Beta1 and Beta2 toxins. Figure 6 As shown, the multi-epitope peptide recombinant protein AB12yh was successfully expressed after codon optimization and was able to specifically bind to Alpha, Beta1, and Beta2 toxin monoclonal antibodies.

[0075] 4. Purification of multi-antigen epitope peptide recombinant protein: Select the transformed recombinant strain with good expression for large-scale culture. After induction expression for 3 hours, collect the bacterial sludge and centrifuge by adding lysozyme and ultrasonic disruption to obtain the multi-antigen epitope peptide recombinant protein in the form of inclusion bodies in the precipitate. In order to remove the impurities in the precipitate, select 0.1-2M urea gradient denaturation and washing to finally obtain the purified inclusion body multi-antigen epitope peptide recombinant protein. The results are as follows: Figure 7 shown.

[0076] Example 4: This example provides a method for evaluating the immune protection of a recombinant protein vaccine containing multiple antigenic epitopes of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as follows:

[0077] 1. Mouse Immunization Protocol: Balb / c mice were randomly divided into two groups, 10 mice each. The AB12yh experimental group was immunized with the epitope peptide recombinant protein prepared by the aforementioned method at a concentration of 1 μg / μL, mixed with 2% aluminum hydroxide gel in a 1:1 ratio, and immunized via multiple subcutaneous injections, with a dose of 200 μL per mouse. A saline group served as a negative control group (Control group), receiving 200 μL of saline per mouse. A secondary immunization was performed 14 days after the primary immunization, with a dose of half the primary dose. Following the primary immunization, five mice were randomly selected every three days for tail-chopping and bleeding, and serum was isolated.

[0078] 2. Mouse LD of crude extract of toxin of Chinese standard strain of Clostridium perfringens type C 100 Determination: In the clean bench, the standard strain of Clostridium perfringens type C, C59-44, was inoculated into the enrichment medium and cultured anaerobically at 37°C for 8 hours. After repeated inoculation for two generations, it was inoculated into the toxin production medium and cultured at 37°C overnight. The culture solution was centrifuged at 6000rpm for 30 minutes, and the supernatant was filtered with a 0.22μm 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 to 200μL with sterile saline at different doses, so that the final crude toxin extract content in each group was 20, 40, 60, 70, and 80μL. It was intraperitoneally injected into BALB / c mice, with 4 mice in each group. The death of mice in each group was observed after 48 hours to determine the absolute lethal dose (LD) of BALB / c mice. 100 ) was 70 μL (see Table 5 for the results).

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

[0080]

[0081] 3. Protection test of immune mice: 28 days after the first immunization, LD 100The challenge experiment was carried out with a crude extract of fresh Clostridium perfringens C59-44 toxin, and the survival rate of mice was observed and counted for 48 hours, and a survival curve was prepared. The experimental results are shown in the figure. Figure 8 As shown, after challenge, two mice in the control group died within 12 hours, most died by 24 hours, and all died by 36 hours. Mice in the immunized group began to die by 24 hours, and four mice in the AB12yh group died 48 hours later. Ultimately, the protection rate in 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 effect of a multi-epitope recombinant protein vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as follows:

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

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

[0085] (2) The next day, after discarding the coating solution, wash the cells three times with PBST, 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 three times with PBST, 3 min each time, and pat dry;

[0087] (4) 100 μL of serum from each group of mice at different stages (500-fold dilution) was added to each well, and PBS was added as a blank control. The cells were incubated at 37°C for 2 h, washed 5 times with PBST, and the liquid was patted dry.

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

[0089] (6) Add 100 μL of OPD colorimetric solution to each well, develop at 37°C for 15 minutes, then add 50 μL of stop solution and read the OD value on a microplate reader. 490 Numeric value.

[0090] The results of serum antibody changes are as follows Figure 9 As shown in the figure, mice produced specific serum antibodies to the Alpha, Beta1, and Beta2 toxin proteins after vaccination. The antibody levels reached their highest level one week after the second vaccination, and then showed a slow decline. Among them, the Alpha protein produced high antibody levels one week after the first vaccination, while the Beta2 toxin protein showed high antibody levels only after the second vaccination.

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

[0092] 2. Detection of antigen-specific sIgA in the jejunal mucosa of immunized mice: 28 days after the initial immunization, 6 eyeballs were randomly collected from each of the 3 groups for blood sampling and then immunized. The intestinal contents of the mice were removed after bleeding, the jejunum was peeled off, and the ileum mucus was washed and scraped. The sIgA ELISA detection kit was used to determine the antigen-specific sIgA content in the jejunal mucosa of mice in different groups. The specific experimental steps were referred to the product manual. The detection of all samples was repeated three times. The results are as follows: Figure 11 As shown in the figure, the sIgA content in the AB12yh group was significantly higher than that in the Control group (P < 0.0001), which played a protective role in the intestine of mice after challenge.

[0093] Example 6: This example provides a method for evaluating the cellular immune effect of a multi-epitope recombinant protein vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as follows:

[0094] 1. Spleen lymphocyte proliferation assay in immunized mice: To investigate whether the designed multi-epitope peptide recombinant protein has the ability to stimulate cellular immune responses, a spleen lymphocyte proliferation assay was performed to detect the stimulation index of spleen lymphocytes in each group of mice after stimulation with Alpha, Beta a1, and Beta 2 recombinant proteins.

[0095] (1) Mice were killed by cervical dislocation 28 days after primary immunization, and then immersed in 75% ethanol. 4 mL of mouse lymphocyte separation solution equilibrated at room temperature was placed in a culture dish.

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

[0097] (3) Transfer the spleen cell suspension to a 15 mL centrifuge tube, slowly add 500 μL of RPMI1640 medium along the tube wall to make the liquid surface boundary clear, and centrifuge at 800 g for 30 min at room temperature using a swing-out rotor centrifuge;

[0098] (4) Aspirate the middle lymphocyte layer, add 5 mL of RPMI1640 medium, wash by inversion, and centrifuge at 250 g for 10 min at room temperature;

[0099] (5) Remove the supernatant, resuspend the cells in RPMI1640 complete medium, and count them to make 2×10 6 cells / mL cell suspension;

[0100] (6) Set up a blank control group and add 100 μL RPMI1640 complete medium to each well;

[0101] (7) Add 100 μL of cell suspension to each well;

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

[0103] (9) Set up a negative control group and add 10 μL RPMI1640 complete 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 10 μg / mL);

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

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

[0107] (13) The above groups were placed in a cell culture incubator at 37°C for 44 h, 10 μL cck-8 was added to each well, and the culture was continued for 4 h;

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

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

[0110] The experimental results are as follows Figure 12As shown in the figure, the spleen lymphocyte stimulation index of the immunized mice was significantly higher than that of the control group (P < 0.01; P < 0.001; P < 0.0001), indicating that the multi-epitope peptide recombinant protein can induce cellular immune response.

[0111] 2. Detection of cytokine levels in the supernatant of spleen lymphocytes of immunized mice: 14 days after the second immunization, the supernatant of stimulated spleen lymphocytes was prepared and the changes in IL-4 and IFN-γ cytokine levels were detected by ELISA kit to analyze the ability of the multi-epitope peptide recombinant protein to induce Th1 and Th2 immune responses. Figure 13 As shown, among Th2 cytokines, the IL-4 content in the supernatant of spleen cells stimulated by Alpha protein was 11.64±2.78pg / mL (Control group) and 27.54±5.81pg / mL (AB12yh group), the IL-4 content in the supernatant of spleen cells stimulated by Beta1 protein was 22.54±8.86pg / mL (Control group) and 39.59±3.48pg / mL (AB12yh group), and the IL-4 content in the supernatant of spleen cells stimulated by Beta2 protein was 8.34±0.52pg / mL (Control group) and 16.75±2.90pg / mL (AB12yh group). The levels of IFN-γ 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 levels of IFN-γ 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 levels of IFN-γ 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, which were significantly higher in the immune groups than in the Control group (P<0.001). The above results indicate that the multi-epitope peptide recombinant protein AB12yh 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 multi-epitope recombinant protein vaccine of Clostridium perfringens Alpha, Beta1, and Beta2 toxins, as follows:

[0113] The ability of serum obtained from immunized mice to neutralize the toxicity of crude toxin in culture filtrate was determined by in vivo neutralization assay.100 One unit of crude toxin was mixed with an equal volume of mouse serum (mouse serum was diluted 1:1 and 1:2) and diluted to a total volume of 0.8 mL with normal saline. The mixture was incubated at 37°C for 2 hours. Subsequently, 0.2 mL of the incubated mixture was intraperitoneally injected into healthy, unimmunized mice (4 per group). The animals were closely observed for 48 hours, and survival was recorded.

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

[0115] Table 6 Neutralization protection of immunized mice serum

[0116]

[0117] In summary, the method provided by the embodiments of the present invention for expressing three lethal toxins of Clostridium perfringens - Alpha, Beta1, and Beta2 antigen epitope peptides in a prokaryotic expression system has the advantages of being safe, inexpensive, having stable expression conditions, and not requiring the cultivation of strong strains or the use of formaldehyde for inactivation. At the same time, in view of the problems of large screening workload and small amount of antigenic epitopes in the single or multivalent versions of recombinant Alpha, Beta1 and other toxins for screening common non-toxic mutations, the present invention screens the B, Tc and Th cell antigenic epitopes of the three major lethal toxins of type C Clostridium perfringens Alpha, Beta1 and Beta2 through immunoinformatics and structural vaccinology methods. The selected epitope peptides are connected through protein linkers to form recombinant proteins, and then combined with online tools such as Phyre2.0 and SWISS-M ODEL to complete the secondary and tertiary structure modeling of the recombinant protein. Finally, the Clus Pro2.0 protein-protein docking and PatchDock online tools can be used to predict the preferred binding mode of the antigen epitope recombinant protein after docking with TLR4. Escherichia coli BL21 (DE3) is used as the expression host to express the antigen epitope peptide recombinant protein. After identification and purification, mice are immunized, and their immune effects are comprehensively evaluated through three aspects: protection from toxicity, humoral immunity and cellular immunity. The results show that the Clostridium perfringens Al The multi-epitope peptide recombinant proteins of the three main lethal toxins, pha, Beta1, and Beta2, 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 above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A recombinant protein of multiple antigenic epitope peptides of three lethal toxins of Clostridium perfringens, characterized in that: 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 linkers; the three lethal toxin proteins are Alpha, Beta1 and Beta2; the amino acid sequence of the multi-antigen epitope peptide recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding a multi-epitope peptide recombinant protein according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. A recombinant vector, including an empty vector, characterized in that: Also included is the encoding gene according to claim 2.

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

5. A recombinant strain comprising a host bacterium, characterized in that: It also comprises the coding gene according to claim 2 or the recombinant vector according to any one of claims 3-4.

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

7. Use of the multi-antigen epitope peptide recombinant protein according to claim 1, the encoding gene according to claim 2, the recombinant vector according to any one of claims 3 to 4, or the recombinant strain according to any one of claims 5 to 6 in the preparation of a vaccine for preventing and treating Clostridium perfringens infection.

8. A vaccine for preventing and treating Clostridium perfringens infection, comprising an adjuvant, characterized in that: Also included is the multi-antigen epitope peptide recombinant protein according to claim 1.

9. A method for designing a multi-epitope peptide recombinant protein according to claim 1, characterized in that: The following steps are involved: The immune epitope database and ABCpred database were used to predict B lymphocyte epitopes, cytotoxic T lymphocyte epitopes, and helper T lymphocyte epitopes of Clostridium perfringens. VaxiJen2.0, AllerTOP2.0, and ToxinPred servers were used to select non-toxic, non-allergenic, and highly antigenic dominant epitopes. The B lymphocyte antigen epitopes are compared with the predicted peptides obtained by various prediction methods by DNAMAN, and the common repeated sequences are selected; The amino acid sequences of the screened B lymphocyte antigen epitopes, cytotoxic T lymphocyte antigen epitopes, and helper T lymphocyte antigen epitopes were connected to construct a multi-antigen epitope peptide recombinant protein. At the same time, in order to avoid the formation of new epitopes by connection 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 connected through KK, GPGPG, and AAY linkers, respectively. This connection method was designed based on the idea of ​​adding or replacing other toxins or other functional protein domains produced by Clostridium perfringens.

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