ASP-ETX fusion protein, clostridium perfringens epsilon toxin nanoparticle vaccine and preparation method thereof

Abstract

The invention relates to the technical field of vaccine preparation, in particular to ASP-ETX fusion protein, a clostridium perfringens epsilon toxin nano-particle vaccine and a preparation method of the clostridium perfringens epsilon toxin nano-particle vaccine. The ASP-ETX fusion protein disclosed by the invention comprises epsilon toxin of clostridium perfringens and secretory protein related to activation; the amino acid sequence of the ASP-ETX fusion protein is as shown in SEQ ID NO. 1. The clostridium perfringens epsilon toxin nano-particle vaccine is obtained by co-incubation of ASP-ETX fusion protein and nano-particles wrapped by cell membranes, the vaccine shows excellent biological safety in cells and animal bodies, ETX specific antibodies as high as 105 are generated through induction in mouse bodies, the neutralizing antibody titer is as high as 64, mice can be protected against 100 * LD50GST-ETX toxin attack, and the vaccine can be used as a vaccine for treating the epsilon toxin of the clostridium perfringens. The vaccine has a huge potential of replacing a conventional ETX vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to an ASP-ETX fusion protein and a Clostridium perfringens epsilon toxin nanoparticle vaccine and a preparation method thereof. Background Art

[0002] Clostridium perfringens ε toxin (ETX) is a major pathogenic factor produced by Clostridium perfringens type B and D. The median lethal dose (LD50) in mice is 50 ) is 50ng / kg, which can cause fatal intestinal diseases in sheep, lambs, cattle and other animals, causing serious economic losses to the animal husbandry industry. Its toxicity is second only to botulinum neurotoxin and tetanus neurotoxin, and is listed as the third most harmful toxin. Therefore, it is listed as a Class B biological agent by the main public health institutes and the Centers for Disease Control and Prevention in the United States, and it poses a potential threat to biosafety. Since the diseases caused by ETX toxins are characterized by rapid onset, short course, and high mortality, once the disease occurs, the patient will die quickly if there is no time for treatment. Therefore, for ETX toxins, preventive measures are mainly taken.

[0003] At present, the traditional method for ETX toxin is to make toxinoids by chemical detoxification. Although some toxinoid vaccines have been used clinically, there are still problems such as large batch differences, complex preparation process and incomplete formaldehyde detoxification. With the rapid development of biotechnology, new methods include the use of genetic engineering technology to transform toxins to generate non-toxic subunits or attenuated toxin mutants, while retaining immunogenic recombinant subunit vaccines. For example, in the previous study, our laboratory analyzed the key sites of ETX toxin and screened out an ETX mutant rETX by using site-directed mutagenesis technology. Y196E -C (hereinafter referred to as ETX Y196E)(Yao W, Kang J, Kang L, et al, Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice[J]. Scientific reports, 2016, 6: 24162). This mutant mutates the tyrosine at position 196 to glutamic acid and connects it to the C-terminus of the toxin. While maintaining a good conformation, it achieves large-scale soluble expression in the Escherichia coli expression system and has good immunogenicity. However, this recombinant subunit vaccine only targets specific surface antigens and lacks other additional antigenic epitopes. In addition, a single protein or polysaccharide antigen cannot induce a lasting immune response by itself and often needs to be used in combination with an adjuvant system. Therefore, it is urgent to develop a new ETX vaccine with good biosafety and the ability to induce strong immunity.

[0004] In recent years, the application of nanotechnology in the biomedical field has become increasingly widespread, and nanoparticles have shown great advantages in disease treatment, monitoring and prevention. In terms of vaccines, nanoparticles have the advantages of protecting antigens from degradation, enhancing lymph node drainage and improving immune efficiency. The modification of cell membranes gives traditional vaccines multiple functions such as targeting, increasing antigen epitopes and prolonging circulation time in the body, which greatly enhances the flexibility of nanoparticles.

[0005] Activation-associated secreted protein-1 (ASP-1) is a non-toxin protein secreted by Onchocerca volvulus that can effectively induce humoral and cellular immune responses, enhance the immunogenicity of various forms of antigens, and induce strong humoral immune responses.

[0006] The prior art has not found a solution for using ASP-1 to construct a fusion protein to prepare a nano vaccine.

[0007] The present invention is proposed based on this. Summary of the invention

[0008] The purpose of the present invention is to develop a novel ETX nanoparticle vaccine that induces strong humoral immunity, and in particular to an ASP-ETX fusion protein and a Clostridium perfringens epsilon toxin nanoparticle vaccine and a preparation method thereof.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an ASP-ETX fusion protein, wherein the ASP-ETX fusion protein comprises Clostridium perfringens ε toxin and an activation-related secretory protein;

[0011] The amino acid sequence of the ASP-ETX fusion protein is shown in SEQ ID NO.1.

[0012] Preferably, the activation-related secretory protein is Ov-ASP-1 protein, and the amino acid sequence of the Ov-ASP-1 protein is shown in SEQ ID NO.2.

[0013] The present invention also provides nucleotides encoding the ASP-ETX fusion protein, and the nucleotide sequence is shown as SEQ ID NO.3.

[0014] The present invention also provides the use of the ASP-ETX fusion protein in preparing Clostridium perfringens ε toxin nanoparticle vaccine.

[0015] The present invention also provides a recombinant vector, comprising the nucleotide and an empty vector;

[0016] The empty vector is a pET-28a vector.

[0017] The present invention also provides a recombinant bacterium, comprising the nucleotide or the recombinant vector and a host bacterium;

[0018] The host bacteria is Escherichia coli BL21 (DE3).

[0019] The present invention also provides a Clostridium perfringens ε toxin nanoparticle vaccine, comprising the ASP-ETX fusion protein, the cell membrane of MDCK cells and the loaded ETX Y196E Protein nanoparticles.

[0020] The present invention also provides a method for preparing the Clostridium perfringens ε toxin nanoparticle vaccine, comprising the following steps:

[0021] (1) ETX Y196E The protein was mixed with poly(lactic acid-co-glycolic acid) solution and ultrasonicated for 1 to 2 minutes to obtain ETX. Y196E -PLGA solution; ETX Y196E -PLGA solution is mixed with polyvinyl alcohol solution, and ultrasonicated for 25-35 minutes to obtain an intermediate product; the intermediate product is centrifuged at a centrifugal force of 6500-7500×g for 12-18 minutes, and the precipitate is discarded; the supernatant is centrifuged at a centrifugal force of 21000-23000×g for 35-45 minutes, and the supernatant is discarded, and the precipitate is taken, the precipitate is purified 2-3 times, and freeze-dried to obtain the loaded ETX Y196ENanoparticles of proteins;

[0022] (2) Load ETX Y196E The protein nanoparticles are mixed with the cell membrane of MDCK cells to obtain a mixture, and the mixture is extruded 10 to 12 times using a polycarbonate membrane to obtain the nanoparticles wrapped by the cell membrane;

[0023] (3) The cell membrane-wrapped nanoparticles are mixed with the ASP-ETX fusion protein and incubated for 50 to 70 minutes to obtain a Clostridium perfringens ε toxin nanoparticle vaccine.

[0024] Preferably, the ETX Y196E The mass volume ratio of the protein and the poly(lactic-co-glycolic acid) solution is 10 mg: 23-25 ​​mL;

[0025] The preparation method of the polylactic acid-glycolic acid copolymer solution is as follows: mixing the polylactic acid-glycolic acid copolymer with dichloromethane;

[0026] The ETX Y196E -The volume ratio of the PLGA solution to the polyvinyl alcohol solution is 1:12-14.

[0027] Preferably, the load ETX Y196E The mass volume ratio of the protein nanoparticles mixed with the cell membrane of MDCK cells is 10 mg: 0.8-1.2 mL;

[0028] The pore size of the polycarbonate membrane is 180-220 nm.

[0029] The present invention provides an ASP-ETX fusion protein and a Clostridium perfringens ε toxin nanoparticle vaccine and a preparation method thereof. The ASP-ETX fusion protein of the present invention is obtained by fusion expression of an immunogenic Ov-ASP-1 sequence fragment and an ETX toxin. The constructed ASP-ETX fusion protein has the ability to enhance humoral immunity. The Clostridium perfringens ε toxin nanoparticle vaccine is obtained by co-incubating the ASP-ETX fusion protein with nanoparticles wrapped in cell membranes. The obtained Clostridium perfringens ε toxin nanoparticle vaccine has the effect of inducing strong humoral immunity. The vaccine exhibits excellent biosafety in both cells and animals. After inoculation of mice, it can induce up to 10 5 ETX-specific antibodies with a neutralizing antibody titer of up to 64 can protect mice against 100×LD 50 GST-ETX toxin challenge can neutralize 16×CT in vitro 50 GST-ETX and 2×LD 50 GST-ETX protects cells and blank mice from damage and has great potential to replace traditional ETX vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 ETX after purification Y196E SDS-PAGE image (lane M is the protein molecular weight standard, lanes 1 to 8 are the purified protein solutions);

[0031] Figure 2 It is a schematic diagram of the structure of the amino acid sequence of ASP-ETX fusion protein;

[0032] Figure 3 It is the electrophoresis diagram of low-dose induced expression of ASP-ETX fusion protein (lane M is the protein molecular weight standard, lane 1 is the supernatant of the recombinant bacterial solution after induction at 37°C, lane 2 is the precipitate of the recombinant bacterial solution after induction at 37°C, lane 3 is the supernatant of the recombinant bacterial solution after induction at 25°C, lane 4 is the precipitate of the recombinant bacterial solution after induction at 25°C, lane 5 is the supernatant of the recombinant bacterial solution after induction at 16°C, and lane 6 is the precipitate of the recombinant bacterial solution after induction at 16°C);

[0033] Figure 4 The electrophoresis diagram of the purified ASP-ETX fusion protein (lane M is the protein molecular weight standard, and lanes 1 to 6 are the purified protein solutions);

[0034] Figure 5 The results of cytotoxicity test of ASP-ETX fusion protein;

[0035] Figure 6 The structures of NP and MNP were observed under transmission electron microscopy (a is NP, b is MNP);

[0036] Figure 7 For load ETX Y196E Encapsulation efficiency of protein nanoparticles (lane M is the protein molecular weight standard, lane 1 is the ETX loaded Y196E Protein Nanoparticle Encapsulation of ETX Y196E Lane 2 is ETX in the supernatant Y196E );

[0037] Figure 8 The adsorption of MNP on ASP-ETX was determined by MTS method;

[0038] Fig. 9 The figure is a flow chart of the preparation of Clostridium perfringens ε toxin nanoparticle vaccine;

[0039] Fig.10 To evaluate the cell-based safety of Clostridium perfringens ε toxin nanoparticle vaccine;

[0040] Fig.11This is an in vivo animal safety evaluation of Clostridium perfringens epsilon toxin nanoparticle vaccine (a represents the change in the proportion of the body weight of mice to the original body weight, and b represents the survival rate of mice (n=10));

[0041] Fig.12 Plan for the immunization experiment at different time points (n=10);

[0042] Fig.13 The levels of specific IgG antibodies induced by different vaccines (n=5) (the horizontal axis from left to right is the first immunization result, the second immunization result and the third immunization result);

[0043] Fig.14 The levels of specific IgG1 and IgG2a antibodies and IgG2a / IgG1 induced by different vaccines (a is the level of specific IgG1 antibodies, b is the level of specific IgG2a antibodies, and c is the ratio of IgG2a / IgG1);

[0044] Fig.15 is the result of the challenge test (a is 10×LD 50 GST-ETX toxin challenge results, b is 100×LD 50 GST-ETX toxin challenge results, (n=5));

[0045] Fig.16 The neutralizing antibody titer of the serum of mice in the immunized group was determined by MTS method (n=3);

[0046] Fig.17 In vitro neutralization test (a is in vitro neutralization cytotoxicity test (n=3), b is in vitro neutralization animal toxicity test (n=5)). DETAILED DESCRIPTION

[0047] In the present invention, the amino acid sequence of the ASP-ETX fusion protein is shown in SEQ ID NO.1; SEQ ID NO.1: MGYNCPGGKLTALERKKIVGQNNKYRSDLINGKLKNRNGTYMPRGKNMLELTWDCKLESSAQRWANQCIFGHSPRQQREGVGENVYAYWSSVSVEGLKKTAGTDAGKSWWSKLPKLYENNPSNNMTWKVAGQGVLHFTQGGGGSGGGGSGGGGSKASYDNVDTLIEKGRYNTKYNYLKRMEKYYPNAMAYFDKVTINPQGNDFYINNP KVELDGEPSMNYLEDVYVGKALLTNDTQQEQKLKSQSFTCKNTDTVTATTTHTVGTSIQATAKFTVPFNETGVSLTTSYSFANTNTNTNSKEITHNVPSQDILVPANT TVEVIAYLKKVNVKGNVKLVGQVSGSEWGEIPSYLAFPRDGYKFSLSDTVNKSDLNEDGTININGKGNYSAVMGDELIVKVRNLNTNNVQEYVIPVDKKHHHHHHLE.

[0048] The structure of the ASP-ETX fusion protein includes NcoⅠ (CCATGG) restriction site, XhoⅠ (CTCGAG) restriction site, Ov-ASP-1 protein sequence fragment, Linker, EXT, and His-Tag. Figure 2 shown.

[0049] The amino acid sequence of the Ov-ASP-1 protein is shown in SEQ ID NO. 2: YNCPGGKLTALERKKIVGQNNKYRSDLINGKLKNRNGTYMPRGKNMLELTWDCKLESSAQRWANQCIFGHSPRQQREGVGENVYAYWSSVSVEGLKKTAGTDAGKSWWSKLPK LYENNPSNNMTWKVAGQGVLHFTQ.

[0050]

[0051] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0052] Experimental materials used in the examples of the present invention: poly(lactic-co-glycolic acid), PLGA was purchased from LACTEL Absorbable Polymers, USA; polyvinylalcohol (PVA) was purchased from Sigma, USA; dichloromethane (DCM) was purchased from Sinopharm Chemical Reagent Co., Ltd., China; 200nm polycarbonate membrane was purchased from Avanti Polar Lipids, USA; MDCK cells were from the Unknown Pathogen Analysis Laboratory, Institute of Microbiology and Epidemiology, Military Medical College, Academy of Military Sciences, China; pET-His-rETX Y196E -C gene vector (stored in the Unknown Pathogen Analysis Laboratory, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Academy of Military Sciences, China); pET-28a(+) vector was purchased from GenScript Biotech Co., Ltd., Nanjing, China; DMEM medium was purchased from Gibco, USA; MTS colorimetric solution was purchased from Abcam (Shanghai) Trading Co., Ltd., China.

[0053] The instruments used in the embodiments of the present invention are as follows: Class II biological safety cabinet purchased from NUAIR, USA; constant temperature shaking incubator purchased from Jiangsu Taicang Experimental Equipment Factory, China; electric constant temperature water bath purchased from Beijing Liuyi Instrument Factory, China; cell counter purchased from ThermoFisher, USA; ultrasonic disruptor purchased from Qsonica LLC, USA; protein electrophoresis instrument purchased from Bio-Rad, USA; desktop high-speed centrifuge purchased from SAKURA, Japan; magnetic stirrer purchased from Jintan Instrument Factory, China; AKTA protein purifier purchased from GE Healthcare, USA; gel image analysis system purchased from Biometra, USA; electronic analytical balance purchased from Sartorius, Germany.

[0054] The experimental animals used in the embodiments of the present invention are BALB / c mice obtained from Spef (Beijing) Biotechnology Co., Ltd. They are all 6-8 weeks old SPF-grade female BALB / c mice, weighing about 20 g, and kept in an SPF-grade animal laboratory approved by the Experimental Animal Center of the Academy of Military Medical Sciences. Experimental operations were performed therein, and all animal experimental operations strictly complied with the operating procedures of experimental animals.

[0055] The configuration of the reagents used in the embodiments of the present invention is:

[0056] HisA solution: 5.54g disodium hydrogen phosphate, 0.7g sodium dihydrogen phosphate, 29.22g sodium chloride, 1.36g imidazole, add purified water, dilute to 1L, pH7.4; HisB solution: 5.54g disodium hydrogen phosphate, 0.7g sodium dihydrogen phosphate, 29.22g sodium chloride, 34.04g imidazole, add purified water, dilute to 1L, pH7.4; GST-tagged protein purification A solution: 8.18g chloride Sodium hydroxide, 0.2g potassium chloride, 3.58g disodium hydrogen phosphate, 0.25g potassium dihydrogen phosphate, 0.6g dithiothreitol, add purified water, dilute to 1L, pH 7.3; refolding solution: 120g urea, 0.61g reduced glutathione, 2.4g oxidized glutathione, 34.84g arginine, 0.58g ethylenediaminetetraacetic acid, 100mL tris(hydroxymethyl)aminomethane hydrochloride (1M), dilute to 1L.

[0057] Example 1

[0058] Purification of ETX Y196E protein

[0059] According to ETX Y196E Ni 2+ The 6×His tag at the end can specifically bind to nickel ions. 2+ Affinity chromatography for ETX Y196E When the target protein reaches 18% in HisB solution, A 280 The UV absorption peak rises significantly, and the eluent is collected until A 280 The UV absorption peak completely dropped back to near the baseline. The collected eluate was identified by SDS-PAGE. Figure 1 As shown, ETX Y196E The theoretical molecular weight of the protein is 32 kDa, and a large amount of ETX was successfully obtained. Y196E Mutant protein, remove impurities by ultrafiltration and replace the buffer system with PBS, and determine ETX by BCA method Y196E Protein concentration, calculated the amount of protein obtained, the result showed that a total of 24 mg ETX was obtained Y196E protein.

[0060] Design of amino acid sequence of ASP-ETX fusion protein

[0061] An Ov-ASP-1 sequence fragment with appropriate immunogenicity (shown in SEQ ID NO.2) was selected for fusion expression with ETX toxin. This fragment removed multiple B cell dominant epitopes and reduced autoimmunogenicity. Three flexible linkers (GGGGS) were introduced between ASP-ETX to enable the fusion protein to fold correctly and ensure activity. A 6×His tag was introduced at the C-terminus for subsequent purification. According to the restriction sites of pET-28a, NcoⅠ (CCATGG) and XhoⅠ (CTCGAG) restriction sites were introduced at both ends of the sequence to construct the pET-28a-ASP-ETX expression vector. At the same time, the sequence was codon-optimized according to the host codon preference to increase the expression level while ensuring activity. The amino acid structure diagram of the ASP-ETX fusion protein is shown in the figure. Figure 2 The amino acid sequence is shown in SEQ ID NO.1, starting from the start codon, followed by the Ov-ASP-1 sequence fragment (17-152aa), the flexible linker (G4S) peptide, the ETX amino acid sequence, the 6×His tag and the stop codon. The coding nucleotide sequence of ASP-ETX is shown in SEQ ID NO.3.

[0062] Example 2

[0063] Low-dose inducible expression of ASP-ETX fusion protein

[0064] According to the pET-28a(+) vector, select the appropriate restriction site, and submit the nucleotide sequence encoding the ASP-ETX recombinant protein shown in SEQ ID NO.3 to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. After the recombinant vector is returned, take out a BL21(DE3) competent cell from the -80℃ refrigerator, put it on ice to melt, take 10μL of the recombinant vector and add it to the BL21(DE3) competent cell, mix it gently, incubate it on ice for 30min, heat shock it at 42℃ for 45s, incubate it on ice for 2min, add 500μL of antibiotic-free LB liquid culture medium, incubate it in a constant temperature shaking incubator at 37℃, 180rpm, incubate it for 1h, centrifuge it at 1000×g for 4min, and discard the supernatant. Gently blow and aspirate the precipitate, and use an L-shaped sterile coating rod to evenly coat it on the LB solid culture medium containing 100μg / mL of kanamycin. Invert and culture it at 37℃ for 16h. Pick a single clone colony and inoculate it into 5 mL of LB liquid medium containing 100 μg / mL kanamycin. Incubate at 37°C and 180 rpm for 6 h to obtain a recombinant bacterial solution. The bacterial solution was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0065] Pipette 100 μL of the recombinant bacterial solution and inoculate it into 10 mL of LB liquid medium containing 100 μg / mL kanamycin. Incubate the culture in a constant temperature shaking incubator at 37°C and 180 rpm for 6 h until the absorbance reaches A600 Reach 0.25. Add IPTG to a final concentration of 1mM. Place in a constant temperature shaking incubator at 37, 25, and 16°C at 180rpm and shake for 16 hours. Collect the bacterial solution before and after induction, centrifuge at 4°C and 8000×g for 10 minutes, and collect the bacterial solution. Add 3mL of pre-cooled PBS to resuspend the bacteria, centrifuge at 4°C and 8000×g for 10 minutes, and discard the supernatant. Add 3mL of pre-cooled PBS to resuspend the bacteria, use an ultrasonic disruptor with a frequency of 75% at 06 horn, supersonic 3s / stop 2s, and sonicate on ice for 5 minutes. Centrifuge at 4°C and 8000×g for 10 minutes, collect the supernatant, add 3mL of pre-cooled PBS to resuspend the precipitate, take 24μL of the supernatant and precipitate under each induction condition, mix with 8μL of 4× protein loading buffer, and boil for 10 minutes. Take 20 μL and load it onto a 4-20% protein precast gel, set the voltage to 180V, and perform SDS-PAGE gel electrophoresis for 45 minutes. After the electrophoresis, observe the molecular weight and expression level of the protein under different temperature conditions. The results are as follows: Figure 3 As shown, the theoretical molecular weight of the ASP-ETX target protein is 46 kDa. After induction at 16°C, the ASP-ETX protein is expressed in the form of inclusion bodies, and the expression level is significantly increased. This induction condition will be used to purify the ASP-ETX protein in the future.

[0066] Example 3

[0067] Purification and refolding of ASP-ETX fusion protein

[0068] According to the characteristics of ASP-ETX fusion protein with 6×His tag at the C-terminus, Ni 2+ Purification of ASP-ETX fusion protein by affinity chromatography. According to the results of low-dose induction expression in Example 2, ASP-ETX fusion protein was expressed in the form of inclusion bodies. The collected cells were placed in His A solution containing 8M urea overnight, the cells were disrupted, and then purified using His A and B solutions containing 8M urea. The experimental results are shown in Figure 2. Figure 4 As shown, most of the ASP-ETX inclusion body proteins can bind to Ni 2+ Column binding, elution with 8M urea HisB solution, from A 280 When the UV absorption peak starts to rise, the eluent is collected until A 280 The UV absorption peak completely dropped back to near the baseline. Take 24 μL of the eluate collected in each time period, mix with 8 μL 4× protein loading buffer, boil for 10 minutes, take 20 μL and load it on 4-20% protein precast gel, set the voltage to 180V, and perform SDS-PAGE gel electrophoresis for 45 minutes. After the electrophoresis, observe the molecular weight and expression level of the protein under different temperature conditions. The experimental results are as follows: Figure 4As shown, the theoretical molecular weight of the ASP-ETX fusion protein is 46 kDa, 90% of the protein in the eluate is the ASP-ETX fusion protein, and a large amount of high-purity ASP-ETX inclusion body protein is successfully obtained.

[0069] The ASP-ETX inclusion body protein was renatured using the concentration gradient dialysis method. The purified ASP-ETX fusion protein was diluted with solution A to a final concentration of 0.1 mg / mL, stirred and mixed, and then placed in a pre-treated dialysis bag. Both ends were clamped with dialysis bags, and the dialysis bag was placed in a beaker containing the renaturation solution for 48 hours. Then, the replacement system was placed in 8, 6, 4, and 2M urea PBS buffers, and the solution was changed every 4 hours. It was observed that the ASP-ETX fusion protein can be stably present in 2M urea PBS buffer without aggregation. The concentration of ASP-ETX fusion protein was determined by BCA method. The standard protein in the BCA kit was diluted to a final concentration of 0.5 mg / mL using PBS solution. Solution A and Solution B were mixed evenly at a volume ratio of 50:1 to prepare BCA working solution, which was stored in the dark. 0, 1, 2, 4, 8, 16, and 20 μL of the standard protein dilution were respectively added to a 96-well plate. 2.5, 5, 10, and 20 μL of the renatured ASP-ETX fusion protein solution were respectively added to a 96-well plate. The sample volume less than 20 μL was supplemented to 20 μL using PBS solution. 200 μL of the mixed working solution was added to each well, and the mixture was incubated at 37°C for 45 min. A was determined by microplate reader 562 The absorbance was measured and the concentration of ASP-ETX fusion protein was calculated. A total of 10 mg of ASP-ETX fusion protein was obtained.

[0070] Example 4

[0071] In vitro toxicity analysis of ASP-ETX fusion protein

[0072] To detect whether the ASP-ETX fusion protein retains the toxic effect of ETX after renaturation, a cytotoxicity experiment was performed on the ASP-ETX fusion protein. MDCK cells are known to be the most sensitive cells to ETX toxins and are often used in ETX toxicity studies. Therefore, MDCK cells were used to evaluate the in vitro cytotoxicity of ASP-ETX fusion proteins. Take MDCK cells with good growth status, discard the culture medium in a biosafety cabinet, add 5mL of pre-cooled PBS solution, mix gently, discard the supernatant, repeat the above steps twice, add 5mL of trypsin, stand in a 37℃, CO2 incubator for 5min, and add 3mL of DMEM medium containing 10% fetal bovine serum to terminate digestion. Use a 3mL Pasteur pipette to gently blow and aspirate the digested adherent cells, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 4℃, 1000×g for 5min, discard the supernatant, and add 2mL of DMEM medium to resuspend the cells. Gently mix 10 μL of cell suspension with 10 μL of 2% trypan blue, add it to the cell counting plate, count the cells, and dilute the MDCK cell suspension to 1×10 5 / mL. Add 100 μL of cell suspension to each well of a 96-well plate and culture in a 37°C, CO2 constant temperature incubator for 24 hours.

[0073] Use DMEM medium to dilute the starting concentration of ASP-ETX fusion protein to 100 μg / mL, and then dilute it downward to 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.390, 0.196, 0.098, 0.049, 0.024, 0.012, 0.006 μg / mL 15 gradients, take out the 96-well plate, and discard the medium in the wells required for the experiment. Add 100 μL of pre-cooled PBS solution to each well, mix gently, and discard the supernatant. Repeat the above steps twice. Add 100 μL of diluted different gradient protein solutions, and set 3 replicates for each gradient. Add 100 μL of DMEM medium as a negative control, and set 3 replicates. Incubate at 37°C, CO2 constant temperature incubator for 1 hour. Discard the medium in the wells required for the experiment and gently rinse 3 times with PBS solution. Mix DMEM medium and MTS colorimetric solution at a ratio of 100:20, add 120 μL of colorimetric solution to each well, incubate in a 37°C, CO2 incubator for 3 hours, and measure the absorbance A of each well. 492 , calculate the cell survival rate and the median lethal dose. (Cell survival rate (SurvivalPercent) = (experimental group A 492 - Positive control group A 492 ) / (negative control group A 492 - Positive control group A 492 )×100%). The experimental results are as follows Figure 5As shown in the figure, ASP-ETX fusion protein has toxic effects on MDCK cells, and the cell survival rate gradually decreases with the increase of toxin concentration. The complete lethal concentration of ASP-ETX fusion protein to MDCK cells is 25 μg / mL, and the concentration that causes 50% MDCK cell death (CT 50 ) was 1.491 μg / mL, and the results showed that the recombinantly expressed ASP-ETX fusion protein retained the toxic effect on ETX.

[0074] Example 5

[0075] Cell membrane-wrapped nanoparticles

[0076] 1.2 g of polyvinyl alcohol (PVA) solid was slowly added into 320 mL of ultrapure water and stirred continuously in a fume hood until completely dissolved to obtain a polyvinyl alcohol solution (PVA solution).

[0077] 0.24 g of poly(lactic acid-co-glycolic acid) (PLGA) was dissolved in 24 mL of dichloromethane (DCM) to obtain a poly(lactic acid-co-glycolic acid) solution (PLGA solution).

[0078] Take 10 mg of ETX purified in Example 1 Y196E The protein was slowly added to 24 mL of PLGA solution and ultrasonicated in an ice bath in a fume hood for 1 min until it was completely emulsified to obtain ETX. Y196E -PLGA solution. 24 mL ETX Y196E -PLGA solution was slowly added dropwise to 320mL PVA solution, and stirring was continued on a magnetic stirrer until the mixture was evenly mixed. Ultrasonication was performed in an ice bath for 30 minutes until the solution turned milky white, and stirring was continued on a magnetic stirrer for 12 hours to fully evaporate the DCM. Centrifuge at 4°C and 7000×g for 15 minutes, discard the precipitate, centrifuge at 22000×g for 40 minutes, and discard the supernatant. Add 45mL ultrapure water to resuspend the precipitate, centrifuge at 4°C and 22000×g for 40 minutes, discard the supernatant, and repeat the above steps twice. Finally, take 10mL of ultrapure water to resuspend the precipitate, wait until the precipitate is completely dissolved, divide it into portions, freeze it at -80°C for 12 hours, and freeze-dry it in a freeze dryer for 24 hours to make loaded ETX. Y196E Protein nanoparticles (NPs).

[0079] Take a bottle of MDCK cells in good growth condition and discard the culture medium in a biosafety cabinet. Add 5 mL of pre-cooled PBS solution, gently mix and discard the supernatant, repeat the above steps twice, add 5 mL of trypsin, digest for 5 minutes in a 37°C, CO2 incubator, add 3 mL of DMEM culture medium containing 10% FBS to terminate digestion, use a 3 mL Pasteur pipette to gently blow and aspirate, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 4°C, 1000×g for 5 minutes, discard the supernatant, resuspend the MDCK cells in 10 mL of ultrapure water, ice bath for 6 hours, centrifuge at 4°C, 16000×g for 20 minutes, discard the supernatant, repeat the above steps twice to obtain MDCK cell membrane. Add 1 mL of PBS solution to resuspend the MDCK cell membrane and store it at 4°C for use. Take the MDCK cell membrane resuspended in 1 mL of PBS solution and mix it with 10 mg of NP, squeeze the mixture 11 times on a 200nm polycarbonate membrane to prepare cell membrane-encapsulated nanoparticles (MNP). The structures of NPs and MNPs were observed using a transmission electron microscope (TEM) at 40,000 times magnification. Figure 6 As shown, both NP and MNP are clearly spherical, and MNP exhibits a clear core-shell structure formed after the cell membrane is wrapped around the surface of the nanocore.

[0080] Example 6

[0081] Determination of ETX in NPs by hydrolysis extraction Y196E Protein concentration

[0082] To verify the load ETX Y196E Protein nanoparticles for ETX Y196E The amount of protein encapsulated was determined by hydrolysis extraction and SDS-PAGE. Y196E Protein Nanoparticle Encapsulation of ETX Y196E The concentration of nanoparticles was calculated to calculate the encapsulation efficiency. Accurately weigh 20 mg of NP, slowly add 1 mL of acetonitrile, vortex to mix, wait until it is completely dissolved into a slightly white emulsion, centrifuge at 22000×g for 10 min, discard the supernatant, and dry the precipitate in a 37°C vacuum oven overnight. Add 1 mL of PBS to re-disperse the precipitate, blow and mix, centrifuge at 22000×g for 10 min, collect the supernatant, further dissolve the precipitate in 100 μL of 0.1 mol / L sodium hydroxide, vortex to mix thoroughly, centrifuge at 22000×g for 10 min, collect the supernatant, combine the supernatants, add 100 μL of 0.1 mol / L hydrochloric acid to balance the pH, and quantify the protein using the BCA protein quantification kit. The experimental results are as follows: Figure 7 As shown, the load ETX Y196EThe encapsulation efficiency of protein nanoparticles was 37.5% per mg of ETX loaded Y196E Protein nanoparticles can encapsulate 50 μg of ETX Y196E Protein, the nanoparticles prepared at one time are enough to complete the entire immunization procedure.

[0083] Encapsulation efficiency (%) = (ETX encapsulated in nanoparticles Y196E Protein / Total ETX Y196E Protein amount) × 100%.

[0084] Example 7

[0085] Adsorption rate of ASP-ETX by cell membrane-wrapped nanoparticles (MNPs)

[0086] To detect the adsorption effect of MNP on ASP-ETX, 100, 50, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390, and 0.195 μg / mL of ASP-ETX were incubated with 2 mg of MNP and 100 μL of PBS for 1 h to obtain MNP-toxin mixtures (MNP-ASP-ETX) and PBS-toxin mixtures (PBS-ASP-ETX). MNP-ASP-ETX and PBS-ASP-ETX were incubated with MDCK cells for 1 h, and MTS color development was performed for 3 h. The experimental results are shown in Figure 2. Figure 8 As shown in Figure 2, with the increase of toxin concentration, the survival rate of MDCK cells gradually decreased. The cell survival rate of MNP-ASP-ETX was higher than that of PBS-ASP-ETX, indicating that MNP can indeed effectively bind to ASP-ETX toxin. The CT values ​​of PBS group and MNP group were calculated. 50 The difference was used to obtain the adsorption capacity of MNPs. 2 mg of MNPs could adsorb 187 ng of ASP-ETX.

[0087] MNP adsorption amount = MNP-ASP-ETX CT 50 Corresponding toxin concentration-PBA-ASP-ETX CT 50 The corresponding toxin concentration.

[0088] It can be seen that when preparing Clostridium perfringens ε toxin nanoparticle vaccine, the optimal mass ratio of cell membrane-wrapped nanoparticles to ASP-ETX fusion protein is 2000000:187.

[0089] From the above, it can be seen that the specific process for preparing Clostridium perfringens ε toxin nanoparticle vaccine is as follows Fig. 9 shown.

[0090] Example 8

[0091] Safety evaluation

[0092] The safety of the vaccine is one of the primary prerequisites for its application. The in vitro safety of the new ETX nanoparticle vaccine was evaluated by in vitro cytotoxicity experiments. Take MDCK cells in good growth condition, discard the culture medium in the biosafety cabinet, add 5mL of pre-cooled PBS solution, mix gently, discard the supernatant, repeat the above steps twice, add 5mL of trypsin, let it stand in a 37℃, CO2 incubator for 5min, and add 3mL of DMEM culture medium containing 10% fetal bovine serum to terminate digestion. Use a 3mL Pasteur pipette to gently blow and aspirate the digested adherent cells, transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 4℃, 1000×g for 5min, discard the supernatant, and add 2mL of DMEM culture medium to resuspend the cells. Gently mix 10μL of cell suspension with 10μL of 2% trypan blue, add it dropwise to the cell counting plate, count the cells, and dilute the MDCK cell suspension to 1×10 5 / mL. Add 100 μL of cell suspension to each well of a 96-well plate. Incubate in a 37°C, CO2 incubator for 24 h. Using 10 μg of total antigen carried by the core as the standard, use DMEM medium to culture MNP-ASP-ETX, MNP and ETX Y196E Dilute to 125μg / mL respectively, and dilute down 2-fold for 15 gradient ratios. Take out the 96-well plate and discard the culture medium in the wells required for the experiment. Add 100μL of pre-cooled PBS solution to each well, mix gently, and discard the supernatant. Repeat the above steps twice. Add 100μL of diluted protein solutions of different gradients, and set up 3 replicates for each gradient. Add 100μL of DMEM culture medium as a negative control, set up 3 replicates, and the culture wells with a completely lethal dose of toxin are used as the positive control group. Incubate in a constant temperature incubator at 37°C and CO2 for 1 hour. Discard the culture medium in the wells required for the experiment and gently rinse 3 times with PBS solution. Mix DMEM culture medium and MTS colorimetric solution at a ratio of 100:20, and add 120μL of colorimetric solution to each well. Incubate in a 37°C, CO2 incubator for 3 hours. Measure the absorbance A of each well. 492 , calculate the cell survival rate and the median lethal dose. (Cell survival rate (Survival Percent) = (experimental group A 492 - Positive control group A 492 ) / (negative control group A 492 - Positive control group A 492 )×100%). The experimental results are as follows Fig.10 As shown, at a concentration of up to 125 μg / mL, the survival rate of MDCK cells was 100%, indicating that the Clostridium perfringens ε toxin nanoparticle vaccine did not cause damage to MDCK cells and had good cell safety.

[0093] The in vivo safety of Clostridium perfringens epsilon toxin nanoparticle vaccine was evaluated by detecting the weight change and survival rate of mice in each immunization group during the entire immunization period. The immunization was divided into 4 groups, MNP-ASP-ETX group, MNP group, NP group and PBS negative control group. The vaccination dose was 10 μg (the antigen amount in the MNP and NP groups was based on the total ETX loaded in the nanocore). Y196E The amount of protein is used as the standard, and the amount of antigen in MNP-ASP-ETX is the amount of ASP-ETX toxin adsorbed on the surface + the amount of ETX loaded on the nanocore. Y196E =10 μg). After the first immunization, the survival of the mice was observed and the weight of the mice was monitored every 3 days. Fig.11 As shown, the mice grew well during the entire immunization period, without adverse reactions, and their body weight showed a steady increase trend, indicating that the nanoparticle vaccine has good in vivo safety.

[0094] Example 9

[0095] Immunization groups and immunization procedures

[0096] The immune groups are shown in Table 1, which are divided into 4 groups.

[0097] Table 1 Immunization groups

[0098]

[0099]

[0100] The immunization method is subcutaneous immunization, the immunization dose is 10μg, the injection volume is 200μL, and the injection is divided into 2 points, with 100μL injected subcutaneously for each. Fig.12 As shown, immunization was performed on day 0, day 14, and day 28 (the amount of antigen in the MNP and NP groups was calculated based on the total ETX loaded in the nanocore). Y196E The amount of protein was used as the standard, and the amount of antigen in the MNP-ASP-ETX group was the amount of ASP-ETX toxin adsorbed on the surface + the amount of ETX loaded on the nanocore Y196E =10 μg), the immunization method and dosage were kept the same each time, and blood was collected from the tail vein 7 days after immunization. The blood was allowed to stand at room temperature for two hours, and then centrifuged at 4000×g for 10 min to obtain serum, which was then packaged and stored at -20°C.

[0101] The ETX-specific IgG antibody titer in serum was detected by ELISA. The experimental results are as follows Fig.13 As shown, after the second immunization, the ETX-specific IgG antibody titer of the NP group reached 10 5 , the IgG antibody titer of MNP-ASP-ETX increased to 10 4 , while the ETX-specific IgG antibody titer of MNP reached 10 3After the third immunization, the ETX-specific IgG antibody titers of both MNP-ASP-ETX and MNP increased to 10 4 , NP group: 10 5 , indicating that the MNP-ASP-ETX group can induce mice to produce high levels of ETX-specific IgG antibodies and has good immunogenicity.

[0102] In order to explore the type of immune response produced by the nanoparticle vaccine, the titers of ETX-specific IgG1 and IgG2a antibodies in mice were detected 7 days after the third immunization, and the differentiation direction of helper T cells (Th cells) was explored through IgG2a / IgG1. A 1× coating solution containing 5μg / mL His-ETX was prepared, and 100μL was added to each well of the ELISA special strips. The plate was incubated at 4°C overnight. The next day, the liquid in the enzyme-linked plate was quickly poured out, and PBST was washed 3 times. 350μL 5% BSA was added to each well, and the plate was incubated at 37°C for 1h. The serum of the immunized mice was diluted to 10 with 5% BSA. 2 , 10 3 , 10 4 and 10 5 , add 100μL to each well, add mouse serum from the PBS group as a negative control, and the 5% BSA group as a positive control, and do 2 replicates for each sample. Incubate in a 37°C constant temperature box for 1 hour. Take out the ELISA plate, quickly pour out the liquid in the ELISA plate, and wash 3 times with PBST. Dilute the horseradish peroxidase-labeled goat anti-mouse IgG, IgG 1, and IgG 2a secondary antibodies with 5% BSA at a ratio of 1:5000, add 100μL to each well, and add to the ELISA plate. Incubate in a 37°C constant temperature box for 1 hour. Take out the ELISA plate, quickly pour out the liquid in the ELISA plate, and wash 3 times with PBST. Prepare TMB colorimetric solution in a ratio of 1:1 between colorimetric solution A and solution B, and add 100μL to each well. React at room temperature in the dark for 10 minutes. Add 50μL of stop solution to each well to terminate the reaction. Absorbance A 450 Read the absorbance value. Subtract the 5% BSA blank control. If it is greater than 2.1 times the serum value of the PBS immunized mice, it is considered positive. Fig.14 As shown in the figure, after three immunizations, MNP-ASP-ETX, MNP and NP could simultaneously induce mice to produce up to 10 5 IgG1 antibody titers, and up to 10 4The IgG2a / IgG1 ratio showed that MNP-ASP-ETX, MNP, and NP were able to produce high levels of IgG2a / IgG1, indicating that the immune response induced by the ETX nanoparticle vaccine was mainly Th2-type and produced a Th1-type bias. The results showed that the ETX nanoparticle vaccine MNP-ASP-ETX group could induce high levels of ETX-specific IgG, IgG1, and IgG2a antibodies in mice.

[0103] Example 10

[0104] Challenge test

[0105] The protective effect of the vaccine was evaluated by the challenge test. Mice were randomly divided into two groups 7 days after the third immunization, with 5 mice in each group. The median lethal dose (LD50) of the ETX toxin with a soluble GST tag (GST-ETX) was used to evaluate the protective effect of the vaccine. 50 ), 10×LD 50 and 100×LD 50 Different doses of toxin were injected intraperitoneally into mice in each immunization group, and the survival status of the mice was observed for 3 consecutive days. Fig.15 As shown, at 10×LD 50 Under the GST-ETX toxin challenge, all mice in the PBS negative control group died within 6 hours, while all mice in the other immunization groups survived. 50 Under the attack of GST-ETX toxin, all mice in the MNP-ASP-ETX and NP groups survived, while two mice in the MNP immunization group died within 12 h, with a survival rate of 60%, indicating that MNP-ASP-ETX and NP had good protection against 100×LD 50 GST-ETX toxin challenge, while the protection of the MNP group was lower than that of the other groups. 50 GST-ETX toxin challenge could only protect 60% of mice from surviving.

[0106] Embodiment 11

[0107] Neutralization test

[0108] Neutralizing antibodies are one of the important aspects of evaluating vaccine quality. In order to evaluate the protective effect of neutralizing antibodies, the serum of mice after the third immunization was diluted to 2 6 , 2 7 , 2 8 , 2 9 , 2 10 , 2 11 and 2 12 Multiple gradients, with 10× CT 50GST-ETX mixture, placed in a rotating mixer, incubated at 37°C, 120r / min for 30min, removed the 96-well plate, discarded the culture medium, added 100μL of pre-cooled PBS solution to each well, gently mixed, discarded the supernatant, repeated the above steps twice, added 100μL of serum with different gradients to obtain serum-toxin mixtures with different gradients, and set at least 3 replicates for each gradient; added 100μL of DMEM culture medium as a negative control, set 3 replicates, and used the culture wells with a completely lethal dose of toxin as the positive control group, and cultured in a 37°C, CO2 incubator for 1h, removed the 96-well plate, discarded the culture medium, gently rinsed 3 times with PBS solution, mixed the DMEM culture medium and MTS color development solution at a ratio of 100:20, added 120μL of color development solution to each well, and cultured in a 37°C, CO2 incubator for 3h, and measured the absorbance A of each well. 492 The cell survival rate and the median lethal dose were calculated. The neutralization titer was expressed as the reciprocal of the highest serum dilution that showed 50% cell survival rate. (Cell survival rate (Survival Percent) = (experimental group A 492 - Positive control group A 492 ) / (negative control group A 492 - Positive control group A 492 )×100%). The experimental results are as follows Fig.16 As shown, MNP-ASP-ETX, MNP and NP can all induce mice to produce neutralizing antibody titers as high as 64, indicating that nanoparticle vaccines can produce high levels of neutralizing antibodies in mice.

[0109] The in vitro neutralization ability of the nanoparticle vaccine group was verified by neutralization test. The first was in vitro neutralization cytotoxicity test. The mouse serum after the third immunization was diluted 2-fold to different concentrations and mixed with 64, 32, 16, 8, 4, and 2×CT 50 GST-ETX toxin mixture, the toxicity of serum-toxin mixture was determined by MDCK cell toxicity test, the survival rate of MDCK cells was calculated, and the ability of immunized group serum to neutralize toxin in vitro was evaluated. Fig.17 As shown, the serum of MNP-ASP-ETX, MNP and NP groups can completely protect 16×CT 50 , MNP-ASP-ETX and NP at 32×CT 50 GST-ETX toxin protected more than 50% of cells from surviving. The results of in vitro neutralization animal toxicity experiments showed that the nanoparticle vaccine could protect mice against 2×LD 50 The survival rate of GST-ETX toxin challenge was 100%, indicating that the nanoparticle vaccine group showed good protection for cells and animals in vitro.

[0110] As can be seen from the above examples, the present invention provides an ASP-ETX fusion protein and a Clostridium perfringens ε toxin nanoparticle vaccine and a preparation method thereof. The ASP-ETX fusion protein of the present invention is obtained by fusion expression of an activity-related secretory protein Ov-ASP-1 sequence fragment that can induce a strong humoral immune response with the ETX toxin. The constructed ASP-ETX fusion protein has the ability to enhance humoral immunity. The Clostridium perfringens ε toxin nanoparticle vaccine is obtained by co-incubating the ASP-ETX fusion protein with cell membrane-wrapped nanoparticles. The obtained Clostridium perfringens ε toxin nanoparticle vaccine has good biosafety and the efficacy of inducing strong humoral immunity, and can produce high levels of neutralizing antibodies in mice. The vaccine exhibits excellent biosafety in both cells and animals, and induces the production of up to 10 5 ETX-specific antibodies with a neutralizing antibody titer of up to 64 can protect mice against 100×LD 50 GST-ETX toxin challenge can neutralize 16×CT in vitro 50 GST-ETX and 2×LD 50 GST-ETX protects cells and blank mice from damage and has great potential to replace traditional ETX vaccines.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ASP-ETX fusion protein, characterized in that: The ASP-ETX fusion protein includes Clostridium perfringens ε toxin and activation-related secretory protein; The amino acid sequence of the ASP-ETX fusion protein is shown in SEQ ID NO.

1.

2. The ASP-ETX fusion protein according to claim 1, characterized in that: The activation-related secretory protein is Ov-ASP-1 protein, and the amino acid sequence of the Ov-ASP-1 protein is shown in SEQ ID NO.

2.

3. The nucleotide encoding the ASP-ETX fusion protein according to claim 1 or 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

3.

4. Use of the ASP-ETX fusion protein according to claim 1 or 2 in the preparation of Clostridium perfringens epsilon toxin nanoparticle vaccine.

5. A recombinant vector, characterized in that: Comprising the nucleotide according to claim 3 and an empty vector; The empty vector is a pET-28a vector.

6. A recombinant bacterium, characterized in that: Comprising the nucleotide according to claim 3 or the recombinant vector according to claim 5 and a host bacterium; The host bacteria is Escherichia coli BL21 (DE3).

7. A Clostridium perfringens epsilon toxin nanoparticle vaccine, characterized in that: The method comprises the ASP-ETX fusion protein according to claim 1 or 2, a cell membrane of MDCK cells and a loaded ETX Y196E Protein nanoparticles.

8. A method for preparing the Clostridium perfringens epsilon toxin nanoparticle vaccine according to claim 7, characterized in that: The steps include: (1) ETX Y196E The protein was mixed with poly(lactic acid-co-glycolic acid) solution and ultrasonicated for 1 to 2 minutes to obtain ETX. Y196E -PLGA solution; ETX Y196E -PLGA solution is mixed with polyvinyl alcohol solution, and ultrasonicated for 25-35 minutes to obtain an intermediate product; the intermediate product is centrifuged at a centrifugal force of 6500-7500×g for 12-18 minutes, and the precipitate is discarded; the supernatant is centrifuged at a centrifugal force of 21000-23000×g for 35-45 minutes, and the supernatant is discarded, and the precipitate is taken, the precipitate is purified 2-3 times, and freeze-dried to obtain the loaded ETX Y196E Nanoparticles of proteins; (2) Load ETX Y196E The protein nanoparticles are mixed with the cell membrane of MDCK cells to obtain a mixture, and the mixture is extruded 10 to 12 times using a polycarbonate membrane to obtain the nanoparticles wrapped by the cell membrane; (3) The cell membrane-wrapped nanoparticles are mixed with the ASP-ETX fusion protein and incubated for 50 to 70 minutes to obtain a Clostridium perfringens ε toxin nanoparticle vaccine.

9. The preparation method according to claim 8, characterized in that: The ETX Y196E The mass volume ratio of the protein and the poly(lactic-co-glycolic acid) solution is 10 mg: 23-25 ​​mL; The preparation method of the polylactic acid-glycolic acid copolymer solution is as follows: the polylactic acid-glycolic acid copolymer is mixed with dichloromethane to obtain; The ETX Y196E -The volume ratio of the PLGA solution to the polyvinyl alcohol solution is 1:12-14.

10. The preparation method according to claim 9, characterized in that: The load ETX Y196E The mass volume ratio of the protein nanoparticles mixed with the cell membrane of MDCK cells is 10 mg: 0.8-1.2 mL; The pore size of the polycarbonate membrane is 180-220 nm.

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