Clostridium septicum alpha toxin mutant as well as preparation method and application thereof

Abstract

The invention provides a clostridium septicum alpha toxin mutant as well as a preparation method and application thereof, and relates to the technical field of biology, leucine at the 403rd site of an amino acid sequence as shown in SEQ ID NO.1 is mutated into amino acid X, the amino acid X is any one of isoleucine, valine or methionine, and the amino acid X is any one of isoleucine, valine or methionine. Compared with the existing recombinant clostridium septicum alpha toxin, the clostridium septicum alpha toxin mutant has the advantages that the protein expression quantity is further improved, the storage life is longer, and the stability is better.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Clostridium putrefaciens alpha toxin mutant and a preparation method and application thereof. Background Art

[0002] Clostridium septium is a Gram-positive anaerobic bacillus that can cause malignant edema, muscle necrosis, gas gangrene, and necrotizing enteritis in animals and humans. It is also the pathogen that causes sheep blight. Alpha toxin, a member of the aerolysin family of perforating toxins, is the primary lethal virulence factor of Clostridium septium, causing hemolysis, lethality, and necrosis. Furthermore, this toxin exhibits excellent immunogenicity, and a toxoid prepared from it is effective against infection with Clostridium septium, serving as its primary pathogenic virulence factor and immunoprotective antigen.

[0003] Diseases caused by Clostridium putrefaciens, such as ephemeral blight, typically have a very short course, often resulting in death before treatment is available. Sheep, for example, are particularly susceptible to C. putrefaciens, and the nutritional status of infected sheep is often above average, posing a significant threat to the livestock and poultry industry. Currently, vaccination is one of the most effective ways to prevent the disease. However, existing recombinant C. putrefaciens α-toxin protein (48 kDa) degrades after one month of storage, hindering the long-term storage and efficacy of the vaccine.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a mutant of Clostridium putrefaciens α-toxin to solve the technical problems of the existing recombinant Clostridium putrefaciens α-toxin protein (48KDa) being short in storage time and easily degraded.

[0006] The second object of the present invention is to provide a gene encoding the Clostridium putrefaciens alpha toxin mutant.

[0007] The third object of the present invention is to provide a method for preparing the above-mentioned Clostridium putrefaciens α-toxin mutant.

[0008] A fourth object of the present invention is to provide the above-mentioned Clostridium putrefaciens α-toxin mutant, the above-mentioned gene, and the use of the Clostridium putrefaciens α-toxin mutant prepared by the above-mentioned preparation method.

[0009] A fifth object of the present invention is to provide a Clostridium putrefaciens vaccine.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: In a first aspect, the present invention provides a mutant of Clostridium putrefaciens alpha toxin, wherein the leucine at position 403 of the amino acid sequence shown in SEQ ID NO.1 is mutated to amino acid X, wherein amino acid X is any one of isoleucine, valine or methionine.

[0011] Furthermore, the amino acid X is any one of isoleucine and valine.

[0012] Furthermore, the amino acid X is valine.

[0013] Furthermore, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown in any one of SEQ ID NO.3 to SEQ ID NO.5.

[0014] Furthermore, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown in SEQ ID NO.3 or SEQ ID NO.5; Preferably, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown as SEQ ID NO.5.

[0015] In a second aspect, the present invention provides a gene encoding the Clostridium putrefaciens alpha toxin mutant, wherein the nucleotide sequence of the gene is shown in any one of SEQ ID NO.10 to SEQ ID NO.12; Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.10 or SEQ ID NO.12; more preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.12.

[0016] In a third aspect, the present invention provides a method for preparing the above-mentioned Clostridium putrefaciens α-toxin mutant, comprising constructing a recombinant expression plasmid based on the gene encoding the above-mentioned Clostridium putrefaciens α-toxin mutant, performing protein recombinant expression in a host cell, and obtaining the Clostridium putrefaciens α-toxin mutant.

[0017] Furthermore, the host cell is a prokaryotic cell; Preferably, the prokaryotic cell comprises Staphylococcus epidermidis or Staphylococcus aureus.

[0018] Furthermore, the recombinant expression of the protein in the host cell includes introducing the recombinant expression plasmid into the host cell, and collecting the protein expressed by the host cell to obtain the Clostridium putrefaciens alpha toxin mutant; Preferably, the collection further includes purification.

[0019] In a fourth aspect, the present invention provides the use of the aforementioned Clostridium putrefaciens α-toxin mutant, the aforementioned gene, and the Clostridium putrefaciens α-toxin mutant obtained by the aforementioned preparation method in the following B1 to B5: B1. Preparation of Clostridium putrefaciens vaccine; B2. Preparation of antibodies against alpha toxin of Clostridium putrefaciens; B3. Preparation of diagnostic antigens for alpha toxin of Clostridium putrefaciens; B4. preparing reagents and / or kits for detecting alpha toxin of Clostridium putrefaciens; B5. Prepare reagents and / or kits for detecting antibodies against alpha-toxin of Clostridium putrefaciens.

[0020] In a fifth aspect, the present invention provides a Clostridium putrefaciens vaccine comprising the aforementioned Clostridium putrefaciens α-toxin mutant or the aforementioned gene.

[0021] Furthermore, the vaccine also includes excipients, which include at least one of vaccine adjuvants, stabilizers or antibiotics.

[0022] The present invention provides a Clostridium putrefaciens alpha toxin mutant, which is obtained by mutating the 403rd amino acid to isoleucine, methionine or valine. The obtained Clostridium putrefaciens alpha toxin mutant has a shelf life of at least 1 month longer than that of the existing recombinant Clostridium putrefaciens alpha toxin protein, a higher protein expression level, better stability and good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The present invention provides the existing recombinant Clostridium putrefaciens alpha toxin protein ATX C86L N296A SDS-PAGE electrophoresis of degradation bands; Figure 2 This is the SDS-PAGE protein quantification graph provided in Example 3 of the present invention; wherein a: 500 μg / mL BSA standard; b: 250 μg / mL BSA standard; c: 125 μg / mL BSA standard; d: 62.5 μg / mL BSA standard; 1: rATX C86L-N296A Protein; 2: rATX C86L-N296A-L403V protein; Figure 3 The standard curve diagram drawn for the BSA standard in Example 3; Figure 4 The SDS-PAGE electrophoresis diagram of the α-toxin mutants of Clostridium putrefaciens at different storage times provided in Example 3 of the present invention; A is rATX stored for 1 to 6 months C86L-N296A-L403V Protein, B is rATX stored for 1 to 6 monthsC86L-N296A protein. DETAILED DESCRIPTION

[0024] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0025] Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, and protein and nucleic acid chemistry described herein, and the techniques thereof are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. The nomenclature used in connection with the medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof are those well known and commonly used in the art.

[0026] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] In one aspect, the present invention provides a mutant of Clostridium putrefaciens alpha toxin, wherein the leucine at position 403 of the amino acid sequence shown in SEQ ID NO.1 is mutated to amino acid X, wherein amino acid X is any one of isoleucine, valine or methionine.

[0028] In some specific embodiments, the amino acid X is either isoleucine or valine.

[0029] In some specific embodiments, the amino acid X is valine.

[0030] In some specific embodiments, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown in any one of SEQ ID NO.3 to SEQ ID NO.5.

[0031] In some specific embodiments, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown as SEQ ID NO.3 or SEQ ID NO.5.

[0032] In some specific embodiments, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown as SEQ ID NO.5.

[0033] According to another aspect of the present invention, a gene encoding the Clostridium putrefaciens alpha toxin mutant is also provided, and the nucleotide sequence of the gene is shown in any one of SEQ ID NO.10 to SEQ ID NO.12.

[0034] In some specific embodiments, the nucleotide sequence of the gene is shown as SEQ ID NO.10 or SEQ ID NO.12.

[0035] In some specific embodiments, the nucleotide sequence of the gene is shown as SEQ ID NO.12.

[0036] According to another aspect of the present invention, a method for preparing the above-mentioned Clostridium putrefaciens α-toxin mutant is also provided, comprising constructing a recombinant expression plasmid based on the gene encoding the above-mentioned Clostridium putrefaciens α-toxin mutant, performing protein recombinant expression in a host cell, and obtaining the Clostridium putrefaciens α-toxin mutant.

[0037] In some specific embodiments, the host cell is a prokaryotic cell.

[0038] In some specific embodiments, the prokaryotic cell comprises Staphylococcus epidermidis or Staphylococcus aureus.

[0039] According to another aspect of the present invention, there is also provided the use of the aforementioned Clostridium putrefaciens α-toxin mutant, the aforementioned gene, and the Clostridium putrefaciens α-toxin mutant obtained by the aforementioned preparation method in the following B1 to B5: B1. Preparation of Clostridium putrefaciens vaccine; B2. Preparation of antibodies against alpha toxin of Clostridium putrefaciens; B3. Preparation of diagnostic antigens for alpha toxin of Clostridium putrefaciens; B4. preparing reagents and / or kits for detecting alpha toxin of Clostridium putrefaciens; B5. Prepare reagents and / or kits for detecting antibodies against alpha-toxin of Clostridium putrefaciens.

[0040] According to another aspect of the present invention, a Clostridium putrefaciens vaccine is provided, comprising the aforementioned Clostridium putrefaciens α-toxin mutant or the aforementioned gene.

[0041] Furthermore, the vaccine also includes excipients, which include at least one of vaccine adjuvants, stabilizers or antibiotics.

[0042] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0043] Material: The plasmid vector pYL and RN4220 competent cells were frozen in the inventor's laboratory. The pYL vector construction method is disclosed in Chinese Patent Application No. 202010907945.7, specifically: the pYL plasmid vector is a recombinant pYL plasmid vector obtained by inserting the Xyl / tet fragment, ori region, and Erm resistance gene into the multiple cloning site and resistance region of the pRB373 plasmid; Strains RN4220 and Staphylococcus epidermidis (SE) were purchased from ATCC; E. coli DH5α competent cells were purchased from Takara Biotechnology (Dalian) Co., Ltd.; High-fidelity FastPfu DNA polymerase and dNTPs were purchased from Quanshijin Company; Restriction endonucleases (Asc Ⅰ; Pme Ⅰ), DL2000 DNA Marker, 10× Loading Buffer, 4× Protein SDS PAGE Loading Buffer, and PCR product recovery and purification kit were all purchased from Takara Biotechnology (Dalian) Co., Ltd. Plasmid mini-extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Clostridium putrefaciens C55-1 was purchased from the Culture Collection Center of the China Veterinary Drug Administration; Mice weighing 15–20 g and rabbits weighing 1.5–2 kg were purchased from Xi’an Yifeng Biotechnology Co., Ltd.

[0044] Example 1 Construction of a recombinant strain expressing a mutant protein of Clostridium putrefaciens alpha toxin 1. Gene synthesis Recombinant Clostridium putrefaciens alpha toxin ATX C86L N296A The amino acid sequence (SEQ ID NO.1) at position 403 leucine (Leu, L) was mutated to amino acid X to obtain rATX C86L-N296A-L403X .

[0045] As shown in Table 1, based on hydrophobicity matching, side chain volume, and secondary structure compatibility, the amino acid X was selected as isoleucine (I), methionine (M), valine (V), alanine (A), phenylalanine (F), aspartic acid (Asp, D), and proline (Pro, P). After codon optimization of the nucleotide sequences of these mutants, the sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0046] Table 1

[0047] PCR amplification was performed using the nucleotide sequence of a synthetic mutant of Clostridium putrefaciens α-toxin as a template using the primers listed in Table 2. The PCR amplification system is shown in Table 3. The PCR amplification protocol was as follows: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 1 minute, annealing at 53°C for 1 minute, and extension at 72°C for 90 seconds; and a final extension at 72°C for 10 minutes. The target DNA band was amplified and recovered, then digested with the vector pYL using double enzymes (Asc I / Pme I), and the digestion product was recovered.

[0048] Table 2 PCR primers

[0049] Table 3 PCR system

[0050] 2. Ligation and transformation of target gene fragment with pYL vector 10μL ligation system: Pipette 1μL of DNA Ligase Buffer, 4μL of target fragment recovery product, 2μL of expression vector recovery product, 1μL of T4 DNA Ligase, and 2μL of double-distilled water into a PCR tube. Gently tap the tube to mix the reaction mixture, centrifuge briefly, and incubate in a ligation apparatus at 16°C overnight. Transform the ligation product into E. coli DH5α competent cells, plate onto LB solid culture plates containing 300μg / mL Erm, and incubate in a 36-38°C incubator for 16-24 hours. Pick individual colonies, extract the recombinant expression plasmid, and perform PCR verification and sequencing. After the sequenced recombinant plasmid is transformed into RN4220, the plasmid extracted from RN4220 is transformed (by electroporation) into Staphylococcus epidermidis (SE), generating the recombinant strain SE / PYL ATX that inducibly expresses the mutant alpha toxin protein of Clostridium putrefaciens. C86L-N296A-L403X .

[0051] Example 2 Identification of Clostridium putrefaciens alpha toxin mutant protein The recombinant strain SE / PYL ATX prepared in Example 1 C86L-N296A-L403XThe cells were inoculated into TSB liquid medium (containing 5 μg / ml Erm) at a 1% inoculation ratio to induce expression of the target protein. The induction conditions were: a final concentration of 300 ng / mL of inducer ATC, an induction temperature of 36-37°C, an incubator speed of 200 rpm, and a culture time of 18-24 hours. After induction, the culture was centrifuged at 12,000 rpm, and the supernatant was collected. The target protein in the supernatant was then analyzed by SDS-PAGE and Western Blot. The results were consistent with expectations, and the resulting Clostridium putrefaciens α-toxin mutant was named rATX. C86L-N296A-L403X protein.

[0052] Example 3 Expression and stability study of recombinant Clostridium putrefaciens alpha toxin protein 1. Expression of recombinant Clostridium putrefaciens α-toxin protein According to the methods of Examples 1 and 2, the mutant proteins of Clostridium putrefaciens α-toxin were constructed and expressed, and the protein expression levels were determined. The specific methods were as follows: (1) BSA standards of different masses (500 μg, 250 μg, 125 μg, and 62.5 μg) and the samples to be tested were added with 6× Protein loading buffer, mixed, and heated at 100°C for 5 min to obtain SDS-PAGE electrophoresis samples, as shown in FIG. Figure 2 As shown; (2) After SDS-PAGE electrophoresis, the gel is stained and destained and the image is collected. The gray value of each target protein band area is automatically calculated according to the image analysis software; (3) The known concentration and corresponding gray value of each BSA standard are recorded, and the BSA standard concentration is used as the X-axis and its gray value as the Y-axis to draw the BSA standard curve, as shown Figure 3 As shown in Table 4, the R² (coefficient of determination) is 0.999; (4) The grayscale value of the target protein of the sample to be tested is substituted into the standard curve to obtain the concentration of the sample to be tested, i.e., the quantitative result. The protein expression level is shown in Table 4.

[0053] Table 4 Protein expression

[0054] The results showed that rATX C86L-N296A-L403I Protein, rATX C86L-N296A-L403M Protein and rATX C86L-N296A-L403V The protein expression levels at 18h and 24h were higher than those at rATX C86L-N296A The expression level of rATX C86L-N296A-L403V The highest expression level was 225 μg / mL, while the expression levels of the other mutant strains were lower than 100 μg / mL.

[0055] 2. Stability of recombinant Clostridium putrefaciens α-toxin protein The recombinant Clostridium putrefaciens α-toxin mutant protein prepared in step 1 was stored at 2-8°C for 6 months. During this period, protein degradation was detected by SDS-PAGE every month. The results are shown in Table 5.

[0056] Table 5 Stability results

[0057] Among them, “+” represents normal, “×” represents degradation, and “-” represents no test.

[0058] The results showed that rATX C86L-N296A-L403V Protein, rATX C86L-N296A-L403I Protein and rATX C86L-N296A-L403M Proteins were higher than rATX C86L-N296A The protein was increased, among which the mutant rATX C86L-N296A-L403V The protein has the best stability and can be stored for at least 6 months. Its SDS-PAGE electrophoresis diagram is as follows: Figure 4 As shown; followed by rATX C86L-N296A-L403I , can be stored for at least 5 months; rATX C86L-N296A-L403M , can be stored for 1 month.

[0059] Example 4 Study on the immunogenicity of recombinant Clostridium putrefaciens α-toxin protein 1. Vaccine preparation The mutant protein rATX of Clostridium putrefaciens α-toxin prepared in Example 3 was selected C86L-N296A 、rATX C86L-N296A-L403I 、rATX C86L-N296A-L403V and rATX C86L-N296A-L403M They were mixed with water-in-oil adjuvant (ISA35A) at a volume ratio of 4:1 so that the protein content in the vaccine was 100 μg / mL, namely Vaccine 1, Vaccine 2, Vaccine 3 and Vaccine 4.

[0060] 2. Safety inspection Twenty healthy rabbits weighing 1.5-2.0 kg were divided into five groups of four rabbits each and vaccinated with Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4, and saline, respectively. Each rabbit in the experimental group received an intramuscular injection of 4 mL of the vaccine, while the control group received the same dose of saline. The animals were observed for 14 days, and their body temperature, appetite, and mental state were monitored. All animals in both the experimental and control groups remained healthy and alive, with normal body temperature, appetite, and mental state.

[0061] 3. Effectiveness test Immunization grouping: 18 healthy rabbits weighing 1.5-2.0 kg were divided into 4 groups, with 4 rabbits in each group, and vaccinated with vaccine 1, vaccine 2, vaccine 3 and vaccine 4 respectively. Each rabbit was injected intramuscularly with 0.25 mL of vaccine. The other 2 rabbits served as the control group.

[0062] Neutralizing Antibody Detection: 21 days after immunization, rabbit blood was collected and serum separated. Equal amounts of serum were pooled from each rabbit. Then, 0.4 mL of the pooled serum was mixed with 0.8 mL of Clostridium putrefaciens alpha toxin (containing 12 mouse MLDs) and incubated at 36-37°C for 40 minutes. This mixture was then injected intravenously into mice (weighing 15-20 g) at a volume of 0.3 mL per mouse, with two mice per group. The control group received 0.2 mL of Clostridium putrefaciens toxin (containing 12 mouse MLDs). Three days later, the mice were observed for survival. All mice in the immunized group survived, while all mice in the control group died.

[0063] Immune challenge experiment: 21 days after immunization, 1.0 mL (1 rabbit MLD of Clostridium putrefaciens α-toxin) was injected into the marginal ear vein of the rabbits in the experimental group. Two rabbits in the control group were selected under the same conditions as the experimental group and were also injected with 1.0 mL (1 rabbit MLD of Clostridium putrefaciens α-toxin). The death of the rabbits was observed after the challenge.

[0064] The experimental results are shown in Table 6. It can be seen that the neutralization titer of serum in each vaccine immunization group against Clostridium putrefaciens toxin reached 3 (0.1 mL of immunized animal serum neutralized 3 MLD of toxin); after intravenous injection of 1 MLD of Clostridium putrefaciens toxin, 4 / 4 of the immunization groups were protected, while all the control groups died. It can be seen that the three mutant proteins rATX of the present invention C86L-N296A-L403I 、rATX C86L-N296A-L403V and rATX C86L-N296A-L403M with rATX C86L-N296A The immunogenicity of the proteins was consistent and met the vaccine efficacy requirements.

[0065] Table 6

[0066] Example 5 Study on the Shelf Life of Recombinant Clostridium putrefaciens Alpha Toxin Egg Mutant Subunit Vaccine Vaccines 1-4 in Example 4 were stored for 3 months, 6 months, 12 months, and 18 months, respectively, and the efficacy test method in Example 4 was repeated. The results are shown in Tables 7 to 10, showing that rATX C86L-N296A-L403I The shelf life of protein-based vaccines is 12 months. C86L-N296A-L403V The shelf life of the protein vaccine is 18 months, which is comparable to rATX C86L-N296A Compared with other vaccines, the quality has been improved.

[0067] Table 7 Vaccine efficacy test results after 3 months of storage

[0068] Table 8 Vaccine potency test results after 6 months of storage

[0069] Table 9 Vaccine potency test results stored for 12 months

[0070] Table 10 Vaccine efficacy test results stored for 18 months

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Clostridium putrefaciens alpha toxin mutant, characterized in that: The leucine at position 403 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to amino acid X, wherein amino acid X is any one of isoleucine, valine or methionine.

2. The alpha toxin mutant of Clostridium putrefaciens according to claim 1, characterized in that The amino acid X is any one of isoleucine and valine; Preferably, the amino acid X is valine.

3. The alpha toxin mutant of Clostridium putrefaciens according to claim 1, characterized in that The amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown in any one of SEQ ID NO.3 to SEQ ID NO.

5.

4. The alpha toxin mutant of Clostridium putrefaciens according to claim 2, characterized in that The amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown in SEQ ID NO.3 or SEQ ID NO.5; Preferably, the amino acid sequence of the Clostridium putrefaciens alpha toxin mutant is shown as SEQ ID NO.

5.

5. A gene encoding a mutant of Clostridium putrefaciens alpha toxin, characterized in that: The nucleotide sequence of the gene is shown in any one of SEQ ID NO.10 to SEQ ID NO.12; Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.10 or SEQ ID NO.12; more preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.

12.

6. A method for preparing the alpha toxin mutant of Clostridium putrefaciens according to any one of claims 1 to 4, characterized in that: The method comprises constructing a recombinant expression plasmid based on the gene encoding the alpha toxin mutant of Clostridium putrefaciens according to claim 5, and performing protein recombinant expression in a host cell to obtain the alpha toxin mutant of Clostridium putrefaciens.

7. The preparation method according to claim 6, characterized in that The host cell is a prokaryotic cell; Preferably, the prokaryotic cell comprises Staphylococcus epidermidis or Staphylococcus aureus.

8. Use of the Clostridium putrefaciens alpha toxin mutant according to any one of claims 1 to 4, the gene according to claim 5, and the Clostridium putrefaciens alpha toxin mutant obtained by the preparation method according to claim 6 or 7 in the following B1 to B5: B1. Preparation of Clostridium putrefaciens vaccine; B2. Preparation of antibodies against alpha toxin of Clostridium putrefaciens; B3. Preparation of diagnostic antigens for alpha toxin of Clostridium putrefaciens; B4. preparing reagents and / or kits for detecting alpha toxin of Clostridium putrefaciens; B5. Prepare reagents and / or kits for detecting antibodies against alpha-toxin of Clostridium putrefaciens.

9. A Clostridium putrefaciens vaccine, characterized in that: It includes the Clostridium putrefaciens alpha toxin mutant according to any one of claims 1 to 4 or the gene according to claim 5.

10. The Clostridium putrefaciens vaccine according to claim 9, characterized in that The vaccine further comprises an adjuvant, which comprises at least one of a vaccine adjuvant, a stabilizer or an antibiotic.

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