A method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen

Abstract

The invention discloses a method for preparing a Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen, comprising constructing a soluble E toxin mutant and a soluble Ferritin protein respectively and purifying them, and then self-assembling the soluble E toxin mutant and the soluble Ferritin protein to form a nanoparticle antigen. The epsilon-ferritin nanoparticles prepared by the invention significantly promote the production of IgG in antigen-immunized mice and successfully induce a cellular immune response.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticle antigen preparation, and in particular to a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen. Background Art

[0002] Clostridium perfringens (C. perfringens) is a Gram-positive anaerobic bacterium that produces endospores. It can survive normal cooking temperatures. If not consumed or refrigerated immediately, the surviving spores may germinate and produce toxins in cooked food. C. perfringens is widespread in nature, causing not only severe losses to the livestock industry but also systemic and gastrointestinal diseases in humans. For example, in humans, C. perfringens can cause gas gangrene, food poisoning, gastrointestinal disorders, and liver and kidney damage. In animals, particularly pigs and poultry, C. perfringens is the primary pathogen of gangrenous dermatitis, enterotoxemia, and necrotic enteritis.

[0003] Currently available vaccines are primarily natural toxin-based vaccines prepared from inactivated Clostridium culture supernatants. These traditional vaccines, based on inactivated whole-cell bacteria or heat-inactivated culture supernatants, have played an important role in preventing C. perfringens disease. However, these vaccines suffer from low production yields and high costs. Furthermore, vaccinated animals can experience local inflammation, nonspecific and uncontrolled immune responses, and short-lived immunity. The primary pathogenicity of C. perfringens is its exotoxins, which secrete at least 18 types of toxins, primarily α, β, E, and I, that can cause disease in humans and animals. Epsilon toxin (Etx, E), secreted by types B and D of C. perfringens, can cause enterotoxemia in sheep, goats, and cattle. The toxin protein consists of 311 amino acids. In its inactive form, it is known as the protoxin and exhibits low toxicity. However, activation by proteases, which removes certain amino acid fragments at the C- and N-termini, significantly enhances its toxicity. This toxin induces potassium ion efflux from cells, leading to apoptosis and sudden death in animals. Once absorbed, the activated Epsilon species enters multiple internal organs, including the brain. In the brain, the toxin acts on blood vessels, crosses the blood-brain barrier, produces perivascular edema, and affects several types of brain cells, including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, edema also occurs in the blood vessel walls of affected animals. This edema can separate astrocyte end feet from the affected blood vessels, causing hypoxia in nervous system tissue.

[0004] Given the limitations of toxoid vaccines, many laboratories have attempted to develop subunit protein vaccines using expressed proteins. However, current technologies suffer from two major drawbacks: first, the E. coli-based expression products are poorly soluble and prone to inclusion body formation; second, the small molecular weight and poor immunogenicity of individual proteins make it difficult to meet vaccine production requirements. Currently, most technologies are still in the laboratory development stage and have yet to enter commercial application. Therefore, a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigens is needed. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems of the prior art and to provide a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention comprises the steps of constructing a soluble E toxin mutant and a soluble Ferritin protein respectively, purifying the soluble E toxin mutant and the soluble Ferritin protein, and then self-assembling the soluble E toxin mutant and the soluble Ferritin protein to form a nanoparticle antigen.

[0008] Furthermore, the method for constructing the soluble E toxin mutant includes mutating the histidine at position 106 of the E toxin of Clostridium perfringens to proline, adding a Spy docking tag and a flexible linker to the carboxyl terminus of the polypeptide chain of the E toxin of Clostridium perfringens, connecting the fusion sequence to the cold expression vector pCold II, constructing the pCold II-E plasmid, and transforming it into the Escherichia coli expression strain BL21 to obtain a soluble E toxin mutant. The encoding nucleotide sequence of the soluble E toxin mutant is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The soluble E toxin mutant was deposited by the General Microbiology Center of the China Culture Collection Administration on May 13, 2024, and was classified and named Escherichia coli, with a deposit number of CGMCC No. 30558.

[0009] Furthermore, the construction method of the soluble Ferritin protein includes fusing Ferritin with the Catcher tag gene and the 6His tag gene, cloning them into the pET28a vector after codon optimization, constructing the pET28a-Ferritin plasmid, and transforming the plasmid into the Escherichia coli expression strain BL21 to obtain the Ferritin-Catcher fusion gene. The encoding nucleotide sequence of the Ferritin-Catcher fusion gene is SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The soluble Ferritin protein was deposited by the General Microbiology Center of the China Culture Collection Administration on May 13, 2024, and was classified and named Escherichia coli, with a deposit number of CGMCC No. 30559.

[0010] Furthermore, when the soluble E toxin mutant was purified, the pColdII-E protein was expressed in large quantities using 2×YT medium and purified using a Ni-NTA column.

[0011] Furthermore, when self-assembling the nanoparticle antigen, the soluble E toxin mutant and the soluble Ferritin protein were pipetted and mixed at a mass ratio of 1:1, and allowed to stand at 37° C. for 2 hours.

[0012] The preparation method of the Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen is used in the following aspects:

[0013] (1) Application in the preparation of anti-Clostridium perfringens specific antibodies;

[0014] (2) Application in the preparation of drugs for preventing diseases caused by Clostridium perfringens.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention docked soluble epsilon toxin and ferritin protein with the help of SpyCatcher technology to prepare epsilon-ferritin nanoparticles. Antigen immunization of mice significantly promoted the production of IgG and successfully induced cellular immune responses. The immune response to the epsilon-ferritin nanoparticle antigen was stronger than that of a single protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a pCold-E plasmid map for the preparation method of Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0018] Figure 2This is an SDS-PAGE image of the pCold II-E protein induced expression in the preparation method of Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0019] Wherein: Marker: protein molecular weight standard, 1 is the precipitate after pCold II-E induction, 2 is the supernatant after pCold II-E induction, 3 is the precipitate after pCold II-E induction, 4 is the supernatant after pCold II-E induction, 5 is the whole pCold II empty cell;

[0020] Figure 3 This is a pCold II-E protein purification diagram for a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0021] Wherein: Marker: protein molecular weight standard, 1 is the supernatant of pCold II-E before purification, 2 is the flow-through, 3 is the wash solution with 20 mM imidazole, 4 is the wash solution with 40 mM imidazole, 5-13 are the eluates with 250 mM imidazole;

[0022] Figure 4 A pET28a-Ferritin protein plasmid map for a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0023] Figure 5 This is a pET28a-Ferritin protein-induced SDS-PAGE image of a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0024] Among them: 1 is the pET28a empty supernatant, 2 is the pET28a empty precipitate, 3 is the pET28a-Ferritin uninduced supernatant, 4 is the pET28a-Ferritin uninduced precipitate, 5 is the pET28a-Ferritin induced supernatant, and 6 is the pET28a-Ferritin induced precipitate;

[0025] Figure 6 This is a pET28a-Ferritin protein purification diagram for a method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0026] Wherein: 1 is the supernatant of pET28a-Ferritin before purification, 2 is the flow-through, 3 is the wash solution with 20 mM imidazole, 4 is the wash solution with 40 mM imidazole, and 5-11 are the eluates with 250 mM imidazole;

[0027] Figure 7This is the SDS-PAGE electrophoresis diagram of the E-Ferritin fusion protein of the preparation method of Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0028] Among them: 1 is Ferritin protein, 2 is E protein, 3 is fusion protein

[0029] Figure 8 Transmission electron microscopy image of E-Ferritin fusion protein of the preparation method of Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention

[0030] Figure 9 This is a diagram showing the IgG detection results of the method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0031] Figure 10 A schematic diagram of the serum IgG1 / IgG2a ratio for the method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0032] Figure 11 This is a schematic diagram of the level of IFN-γ produced by spleen cells stimulated by E toxin in the method for preparing Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention;

[0033] Figure 12 This is an in vitro neutralization test diagram of the preparation method of Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen proposed in the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the embodiments and accompanying drawings. However, it should be understood that the embodiments and drawings are only used to illustrate the present invention and do not constitute any limitation on the scope of protection of the present invention.

[0035] The present invention is further illustrated below with reference to specific embodiments:

[0036] Example 1 Expression and purification of soluble E toxin mutants

[0037] 1.1 Vector construction and expression identification

[0038] like Figure 1 and 2As shown, the histidine at position 106 of Clostridium perfringens E toxin was mutated to proline, a Spy docking tag and a flexible linker were added to the C-terminus, and the fusion sequence was ligated into the cold expression vector pCold II to construct the pCold II-E plasmid. The plasmid was transformed into the Escherichia coli expression strain BL21(DE3). The encoding nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. A clone was obtained and shaken overnight in 2×YT medium. The next day, the clone was transferred to 10 mL of 2×YT at 1% and rapidly shaken at 37°C at 220 rpm to an OD600nm of 0.6-0.8. The culture was cooled to 16°C, and 20 μL of IPTG was added for induction at 16°C at 180 rpm for 20 h. An empty control was also performed.

[0039] After induction, centrifuge the bacterial suspension at 8000 rpm for 5 minutes, collect the precipitate, resuspend in 1 mL of PBS, sonicate, and centrifuge at 12000 × g for 15 minutes at 4°C. Collect the supernatant and perform SDS-PAGE electrophoresis.

[0040] The soluble E toxin mutant Epsilon-Spy was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024, with the deposit number: CGMCC No.30558, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification number: Escherichia coli;

[0041] Results: The non-toxic and soluble E toxin mutant was successfully expressed.

[0042] 1.2 Large-scale expression and purification

[0043] like Figure 3 As shown, pCold II-E protein was then expressed in large quantities using 500 mL of 2×YT medium and purified using a Ni-NTA column. The purification results were verified by SDS-PAGE and the purified protein was collected.

[0044] Example 2 Expression and purification of soluble Ferritin protein

[0045] 2.1 Vector construction and initial expression

[0046] like Figure 4 and 5As shown, Ferritin was fused with the Catcher tag gene and the 6His tag gene, and after codon optimization, cloned into the pET28a vector. The constructed plasmid was named pET28a-Ferritin. This plasmid was transformed into the E. coli expression strain BL21 (DE3). The encoding nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The clone was picked and shaken overnight in 2×YT medium. The next day, it was transferred to 10 mL of 2×YT at 1% and rapidly shaken at 37°C and 220 rpm to an OD600 of 0.6-0.8. 20 μL IPTG was added and induced at 28°C and 180 rpm for 6 h. An empty control was also performed.

[0047] After induction, centrifuge the bacterial suspension at 8000 rpm for 5 minutes, collect the precipitate, resuspend in 1 mL of PBS, sonicate, and centrifuge at 12000 × g for 15 minutes at 4°C. Collect the supernatant and perform SDS-PAGE electrophoresis.

[0048] The soluble Ferritin protein was deposited in the General Microbiology Center of the China Culture Collection Administration on May 13, 2024, with the deposit number: CGMCC No. 30559, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, classification number: Escherichia coli.

[0049] 2.2 Large-scale expression and purification

[0050] like Figure 6 As shown, 500 mL of 2×YT medium was then used to express pET28a-Ferritin protein in large quantities and purified using a Ni-NTA column. After purification, SDS-PAGE was used to verify the purification results and the purified protein was collected.

[0051] Example 3 Docking of ferritin and toxin protein

[0052] 3.1 BCA assay to determine the concentrations of E protein and Ferritin protein

[0053] The purified E protein and Ferritin protein were detected using the BCA kit of Thermo Company, and BSA was used as the standard protein to draw a standard curve. The concentrations of E protein and Ferritin were 1.476 mg / mL and 1.064 mg / mL, respectively.

[0054] Docking of 3.2E protein and ferritin protein

[0055] like Figure 7As shown, E protein and ferritin were mixed by pipetting at a 1:1 mass ratio and incubated at 37°C for 2 hours. After incubation, SDS-PAGE electrophoresis revealed a distinct band at approximately 70 kDa, confirming successful docking of E protein and ferritin.

[0056] 3.3 Electron microscopy images of docked proteins

[0057] like Figure 8 As shown, the recombinant fusion protein was analyzed by electron transmission microscopy, which showed a self-assembled nanoparticle structure with a diameter of 20-22 nm, which was larger than the diameter of Ferritin protein (20 nm), indicating that E protein was successfully assembled with Ferritin protein to form Ferritin protein nanoparticles.

[0058] Example 4 Immunological Evaluation of Particle Antigens in Mice

[0059] 4.1 Immunization groups and immunization procedures

[0060] Subunit vaccines were prepared by mixing recombinant E and E-ferritin proteins with equal volumes of aqueous solution and aqueous adjuvant, respectively. The E antigen content in the vaccine was 10 μg / mL, ensuring equal E protein doses in each group. Six-week-old female Balb / c mice were randomly divided into five groups (n=5): a PBS control group, an E aqueous solution group, an E-ferritin aqueous solution group, an E aqueous adjuvant group, and an E-ferritin aqueous adjuvant group. Each mouse in each group was immunized subcutaneously with 100 μL of the solution. A booster immunization was administered four weeks after immunization. Blood was collected from the sinus vein of the mice before and every seven days after immunization, and serum was separated and stored at -20°C.

[0061] 4.2 Detection of specific antibody levels against recombinant proteins in sera of immunized mice

[0062] 4.2.1 Detection of recombinant protein IgG levels in immunized mouse serum

[0063] like Figure 9 To evaluate the changes in E protein antibody levels after immunization, an indirect ELISA assay was used to measure IgG antibody levels in immunized mice. The results showed that three weeks after the first immunization, the IgG levels produced by the E-ferritin-water adjuvant group were significantly higher than those in the E-water adjuvant group; and the IgG levels produced by the E-ferritin-water solution group were significantly higher than those in the E-water solution group, indicating that ferritin has a good antigen presentation function.

[0064] The antibody level of the E-Ferritin docked recombinant protein water adjuvant group was significantly higher than that of the E single protein water adjuvant group, and the antibody level of the E-Ferritin docked recombinant protein without adjuvant group was also significantly higher than that of the E single protein without adjuvant group, and the antibody level of the E-Ferritin docked recombinant protein without adjuvant group was almost reaching the antibody level of E protein water adjuvant, confirming that ferritin has the function of antigen presentation.

[0065] 4.2.2 Detection of IgG subtypes of recombinant protein in sera of immunized mice

[0066] like Figure 10 As shown, in mice, a Th1 immune response produces IgG2a antibodies, while a Th2 immune response produces IgG1 antibodies. The indirect ELISA method was used to measure the expression of IgG subtypes, such as IgG1 and IgG2a, in the serum of immunized mice. As shown in the figure, 21 days after primary immunization, the immune levels of both IgG1 and IgG2a subtypes in the adjuvanted and unadjuvanted fusion protein groups were higher than those in the adjuvanted and unadjuvanted single protein groups. Furthermore, the IgG2a / IgG1 ratio in the fusion protein group was higher than that in the single protein group, indicating that the fusion protein triggered a stronger Th1 immune response.

[0067] 4.2.3 Detection of IFN-γ

[0068] like Figure 11 As shown, spleen cells were harvested from mice and stimulated with 5 μg of E protein for 48 hours. The supernatant was diluted fourfold and then assayed for IFN-γ immune levels. The results showed that the E-Ferritin aqueous adjuvant group produced significantly higher levels of IFN-γ cytokines than the E-water adjuvant group (p<0.001), indicating that the E-Ferritin aqueous adjuvant group induced a cellular immune response.

[0069] 4.2.4 Determination of serum neutralizing antibody titer

[0070] 4.2.4.1 Minimum lethal dose (LD50) concentration of CV-81 natural toxin

[0071] like Figure 12 As shown in the figure, the concentration of CV-81 natural toxin was determined to be 2.474 mg / mL by BCA method. The toxin was diluted 2-fold continuously and acted on MDCK cells to observe the number of cytopathic effects. The experiment showed that the minimum cell half-lethal dose of CV-81 natural toxin was 0.015 μg / mL.

[0072] 4.2.4.2 Determination of in vitro neutralization test

[0073] Serum was serially diluted two-fold and mixed with the minimum lethal dose (LD50) of CV-81 natural toxin at a 1:1 ratio by volume. The mixture was incubated at 37°C for 1 hour. 100 μL of the mixture was added to 96-well plates seeded with MDCK cells, and cytopathic effects were observed over 24 hours. Results showed that 21 days after the second immunization, the E-water solution group diluted 512-fold, the E-ferritin solution group diluted 2048-fold, the E-water adjuvant group diluted 2048-fold, and the E-ferritin aqueous adjuvant group diluted 4096-fold, neutralized the minimum lethal dose (LD50) of CV-81 natural toxin. In vitro neutralization studies demonstrate that ferritin has a strong immune presentation function, providing new avenues for the development of Clostridium perfringens vaccines.

[0074] The present invention successfully constructed highly soluble epsilon toxin and ferritin protein, and docked the soluble epsilon toxin and ferritin protein with the help of SpyCatcher technology to prepare epsilon-ferritin nanoparticles. Antigen immunization of mice significantly promoted the production of IgG and successfully induced a cellular immune response. The antigen immune response of epsilon-ferritin nanoparticles was stronger than that of a single protein.

[0075] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen, characterized in that: After constructing a soluble ε toxin mutant and a soluble Ferritin protein and purifying them respectively, the soluble ε toxin mutant and the soluble Ferritin protein are self-assembled to form a nanoparticle antigen. The construction method of the soluble ε toxin mutant comprises mutating the 106th histidine of the ε toxin of Clostridium perfringens to proline, adding a Spy docking tag and a flexible linker to the carboxyl end of the polypeptide chain of the ε toxin of Clostridium perfringens, connecting the fusion sequence to the cold expression vector pCold II, constructing a pCold II-ε plasmid, and transforming it into an Escherichia coli expression strain BL21 to obtain a soluble ε toxin mutant. The amino acid sequence of the soluble ε toxin mutant is SEQ ID NO: 2, the construction method of the soluble Ferritin protein includes fusing Ferritin with a Catcher tag gene and a 6His tag gene, cloning into a pET28a vector after codon optimization, constructing a pET28a-Ferritin plasmid, and transforming the plasmid into an Escherichia coli expression strain BL21 to obtain a Ferritin-Catcher fusion gene. The encoding nucleotide sequence of the Ferritin-Catcher fusion gene is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO:

4.

2. The Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen according to claim 1, characterized in that: When the soluble ε toxin mutant is purified, 2×YT medium is used to express pCold II-ε protein in large quantities, and Ni-NTA column is used for purification.

3. The Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen according to claim 1, characterized in that: When self-assembling nanoparticle antigens, the soluble ε toxin mutant and the soluble Ferritin protein were mixed by pipetting at a mass ratio of 1:1 and allowed to stand at 37° C. for 2 hours.

4. A subunit vaccine, characterized in that: The method comprises the Clostridium perfringens epsilon toxin-ferritin nanoparticle antigen as described in any one of claims 1 to 3 and a water adjuvant.

5. The use of the subunit vaccine as claimed in claim 4 in the following aspects: (1) Application in the preparation of anti-Clostridium perfringens specific antibodies; (2) Use in the preparation of drugs for preventing diseases caused by Clostridium perfringens.

Images