An ASP-ETX fusion protein and a Clostridium perfringens ε toxin nanoparticle vaccine and preparation method thereof

Abstract

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. The ASP-ETX fusion protein of the present invention comprises Clostridium perfringens epsilon toxin and an activation-related secretory protein; the amino acid sequence of the ASP-ETX fusion protein is shown in SEQ ID NO.1. The Clostridium perfringens epsilon toxin nanoparticle vaccine is obtained by co-incubating the ASP-ETX fusion protein with cell membrane-wrapped nanoparticles. 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 attack has great potential to replace traditional ETX vaccines.

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 serotypes B and D. 50 ) at 50ng / kg, can cause fatal intestinal diseases in sheep, lambs, cattle, and other animals, causing severe economic losses to the livestock industry. Its toxicity ranks third after botulinum neurotoxin and tetanus neurotoxin, making it the third most harmful toxin. Therefore, it has been classified as a Category B biological agent by the Centers for Disease Control and Prevention, a major US public health institute, and a potential threat to biosafety. Diseases caused by ETX toxins are characterized by rapid onset, a short course, and a high mortality rate. Once symptoms develop, death can occur quickly if treatment is not available within a short period of time. Therefore, preventive measures are primarily focused on ETX toxins.

[0003] At present, the traditional method for ETX toxin is to produce toxinoids through chemical detoxification. Although some toxinoid vaccines have been used clinically, there are still problems such as large batch-to-batch differences, complex preparation process and incomplete formaldehyde detoxification. With the rapid development of biotechnology, new methods include using genetic engineering technology to modify toxins to generate non-toxic subunits or attenuated toxin mutants while retaining immunogenic recombinant subunit vaccines. For example, in previous studies, our laboratory analyzed the key sites of ETX toxins and used site-directed mutagenesis technology to screen out an ETX mutant rETX Y196E -C (abbreviated as ETX in this article) 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, there is an urgent need 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, with nanoparticles demonstrating significant advantages in disease treatment, monitoring, and prevention. In vaccines, nanoparticles offer advantages such as protecting antigens from degradation, enhancing lymph node drainage, and improving immune efficacy. Modification of cell membranes imparts multiple functions not possessed by traditional vaccines, such as targeting, increasing antigenic epitopes, and prolonging circulation time, greatly enhancing 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 a nucleotide encoding the ASP-ETX fusion protein, and the nucleotide sequence is shown in SEQ ID NO.3.

[0014] The present invention also provides the use of the ASP-ETX fusion protein in preparing Clostridium perfringens epsilon 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 1-2 minutes to obtain ETX. Y196E -PLGA solution; ETX Y196E -PLGA solution is mixed with polyvinyl alcohol solution, 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, the precipitate is discarded, the supernatant is centrifuged at a centrifugal force of 21000-23000×g for 35-45 minutes, the supernatant is discarded, the precipitate is purified 2-3 times, and freeze-dried to obtain the loaded ETX Y196Eprotein nanoparticles;

[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 cell membrane-wrapped nanoparticles;

[0023] (3) The cell membrane-wrapped nanoparticles were mixed with ASP-ETX fusion protein and incubated for 50 to 70 minutes to obtain Clostridium perfringens epsilon 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 epsilon 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 with the ETX toxin. The constructed ASP-ETX fusion protein has the ability to enhance humoral immunity. The Clostridium perfringens epsilon toxin nanoparticle vaccine is obtained by co-incubating the ASP-ETX fusion protein with cell membrane-coated nanoparticles. The obtained Clostridium perfringens epsilon toxin nanoparticle vaccine has the efficacy of inducing strong humoral immunity. The vaccine exhibits excellent biosafety in both cells and animals. After vaccination in mice, it can induce 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. 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 Schematic diagram of the amino acid sequence of ASP-ETX fusion protein;

[0032] Figure 3 The figure shows the electrophoresis graph of low-dose induced expression of ASP-ETX fusion protein (lane M is a 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 cytotoxicity test results 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 ETX for load Y196E Encapsulation efficiency of protein nanoparticles (lane M is the protein molecular weight standard, lane 1 is the loaded ETX Y196E Protein nanoparticle encapsulation of ETX Y196E Lane 2 is ETX in the supernatant Y196E );

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

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

[0039] Figure 10 To evaluate the cell-based safety of Clostridium perfringens epsilon toxin nanoparticle vaccine;

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

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

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

[0043] Figure 14 The levels of specific IgG1 and IgG2a antibodies and the ratio of IgG2a to IgG1 induced by different vaccines are shown in Table 1 (a is the level of specific IgG1 antibodies, b is the level of specific IgG2a antibodies, and c is the ratio of IgG2a to IgG1);

[0044] Figure 15 The results 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] Figure 16 The neutralizing antibody titer of the serum of mice in the immunized group was determined by MTS method (n=3);

[0046] Figure 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: YNCPGGKLTALERKKIVGQNNKYRSDLINGKLKNRNGTYMPRGKNMLELTWDCKLES SAQRWANQCIFGHSPRQQREGVGENVYAYWSSVSVEGLKKTAGTDAGKSWWSKLPK LYENNPSNNMTWKVAGQGVLHFTQ.

[0050]

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

[0052] The experimental materials used in the examples of the present invention were as follows: poly(lactic-co-glycolic acid) (PLGA) was purchased from LACTEL Absorbable Polymers, USA; polyvinyl alcohol (PVA) was purchased from Sigma, USA; dichloromethane (DCM) was purchased from Sinopharm Chemical Reagent Co., Ltd., China; 200 nm polycarbonate membrane was purchased from Avanti Polar Lipids, USA; MDCK cells were obtained 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 Taicang Experimental Equipment Factory, Jiangsu, 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 examples 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 approximately 20 g, and housed in an SPF-grade animal laboratory approved by the Experimental Animal Center of the Academy of Military Medical Sciences. Experimental procedures were performed therein, and all animal experimental procedures strictly complied with the operating procedures for 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 solution A: 8.18g chloride Sodium chloride, 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] Purified 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 eluate is collected until A 280 The UV absorption peak completely returned to the baseline. The collected eluate was identified by SDS-PAGE. Figure 1 As shown, ETX Y196E The theoretical molecular weight of the protein is 32kDa, and a large amount of ETX was successfully obtained. Y196E Mutant protein was removed by ultrafiltration and the buffer system was replaced with PBS. ETX was determined by BCA method. Y196E Protein concentration, calculated the amount of protein obtained, the results showed that a total of 24mg ETX was obtained Y196E protein.

[0060] Amino acid sequence design of ASP-ETX fusion protein

[0061] An Ov-ASP-1 sequence fragment (shown in SEQ ID NO.2) with appropriate immunogenicity was selected for fusion expression with the ETX toxin. This fragment removed multiple B cell dominant epitopes to reduce autoimmunogenicity. By introducing three flexible linkers (GGGGS) between ASP-ETX, the fusion protein was able to fold correctly to ensure activity. A 6×His tag was introduced at the C-terminus for easy subsequent purification. Based on 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 ensure activity while increasing expression. The amino acid structure diagram of the ASP-ETX fusion protein is shown in the figure below. 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) connecting peptide, the ETX amino acid sequence, the 6×His tag, and the stop codon. The nucleotide sequence encoding ASP-ETX is shown in SEQ ID NO. 3.

[0062] Example 2

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

[0064] Appropriate restriction sites were selected for the pET-28a(+) vector, and the nucleotide sequence encoding the ASP-ETX recombinant protein shown in SEQ ID NO. 3 was submitted to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. After the recombinant vector was returned, a tube of BL21(DE3) competent cells was removed from a -80°C freezer and thawed on ice. 10 μL of the recombinant vector was added to the BL21(DE3) competent cells. After gentle mixing, the cells were incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, and incubated on ice for 2 minutes. 500 μL of antibiotic-free LB liquid medium was added and the cells were incubated in a constant temperature shaking incubator at 37°C, 180 rpm, for 1 hour. The cells were centrifuged at 1000 × g for 4 minutes, and the supernatant discarded. The pellet was gently aspirated and evenly spread onto LB solid medium containing 100 μg / mL kanamycin using a sterile L-shaped spreader. The cells were incubated inverted at 37°C for 16 hours. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin. Culture was shaken at 37°C and 180 rpm for 6 h to obtain recombinant bacterial suspension. The suspension was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0065] Pipette 100 μL of recombinant bacterial solution and inoculate into 10 mL of LB liquid medium containing 100 μg / mL kanamycin, and 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 liquid before and after induction, centrifuge at 4°C and 8000×g for 10 minutes, and collect the bacterial liquid. 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 an amplitude of 06 frequency of 75%, a program of supersonication for 3s / pause for 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 them 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 run SDS-PAGE gel electrophoresis for 45 minutes. After the electrophoresis is completed, 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. Based on 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 incubated overnight in His A solution containing 8M urea, 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 eluate is collected until A 280 The UV absorption peak completely returned to near the baseline. Take 24 μL of the eluate collected at each time period, mix it with 8 μL of 4× protein loading buffer, boil it 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 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] ASP-ETX inclusion body protein was renatured using concentration gradient dialysis. The purified ASP-ETX fusion protein was diluted with Solution A to a final concentration of 0.1 mg / mL, stirred, and then loaded into a pre-treated dialysis bag. The bag was clamped at both ends with the bag and placed in a beaker containing the renaturation solution for 48 hours. The replacement system was then placed in 8, 6, 4, and 2 M urea PBS buffer solutions, with the solution changed every 4 hours. The ASP-ETX fusion protein was observed to be stable in 2 M urea PBS buffer without aggregation. The BCA method was used to determine the concentration of ASP-ETX fusion protein. 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 at a volume ratio of 50:1 to prepare the BCA working solution. The solution was stored in the dark. 0, 1, 2, 4, 8, 16, and 20 μL of the standard protein dilution were pipetted and added to a 96-well plate in sequence. 2.5, 5, 10, and 20 μL of the renatured ASP-ETX fusion protein solution were pipetted and added to a 96-well plate in sequence. PBS solution was used to make up the volume of samples with a volume less than 20 μL to 20 μL. 200 μL of the mixed working solution was added to each well and incubated at 37°C for 45 minutes. 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 test whether the ASP-ETX fusion protein retains the cytotoxic effects of ETX after refolding, a cytotoxicity assay was performed on the ASP-ETX fusion protein. MDCK cells are known to be the most sensitive cells to ETX toxins and are commonly used in ETX toxicity studies. Therefore, the in vitro cytotoxicity of the ASP-ETX fusion protein was assessed using MDCK cells. Well-grown MDCK cells were harvested and the culture medium was discarded in a biosafety cabinet. 5 mL of pre-chilled PBS was added and gently mixed. The supernatant was discarded. This step was repeated twice. 5 mL of trypsin was added and the cells were incubated at 37°C in a CO2 incubator for 5 minutes. The digestion was terminated by adding 3 mL of DMEM medium supplemented with 10% fetal bovine serum. The adherent cells were gently aspirated using a 3 mL Pasteur pipette. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 × g for 5 minutes at 4°C. The supernatant was discarded and the cells were resuspended in 2 mL of DMEM medium. Gently mix 10 μL of cell suspension with 10 μL of 2% trypan blue and add it dropwise to a cell counting plate for cell counting. 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 at 37°C in a CO2 incubator for 24 hours.

[0073] Dilute the ASP-ETX fusion protein to a starting concentration of 100 μg / mL using DMEM medium. Then serially dilute the concentrations to 15 concentrations: 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.390, 0.196, 0.098, 0.049, 0.024, 0.012, and 0.006 μg / mL. Remove the 96-well plate and discard the medium in the wells needed for the experiment. Add 100 μL of pre-chilled PBS to each well, gently mix, and discard the supernatant. Repeat this step twice. Add 100 μL of the diluted protein solution from each gradient, setting up three replicates for each gradient. Add 100 μL of DMEM medium as a negative control, setting up three replicates. Incubate at 37°C in a CO2-controlled incubator for 1 hour. Discard the medium in the wells needed for the experiment and gently rinse three times with PBS. Mix DMEM medium and MTS colorimetric solution at a ratio of 100:20, add 120 μL of colorimetric solution to each well, and incubate in a 37°C, CO2 incubator for 3 hours. Measure the absorbance of each well. 492 Calculate cell survival rate and half 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, indicating 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 to 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 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 of 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 well-grown MDCK cells and discard the culture medium in a biosafety cabinet. Add 5 mL of pre-chilled PBS, mix gently, and discard the supernatant. Repeat this step twice. Add 5 mL of trypsin and digest in a 37°C, CO2 incubator for 5 min. Add 3 mL of DMEM medium containing 10% FBS to terminate the digestion. Gently pipette with a 3 mL Pasteur pipette, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 × g for 5 min at 4°C, discard the supernatant, resuspend the MDCK cells in 10 mL of ultrapure water, incubate on ice for 6 h, and centrifuge at 16,000 × g for 20 min at 4°C. Discard the supernatant. Repeat this step twice to obtain MDCK cell membranes. Resuspend the MDCK cell membranes in 1 mL of PBS and store at 4°C until use. Take the MDCK cell membranes resuspended in 1 mL of PBS and mix them with 10 mg of NPs. Extrude the mixture 11 times through a 200 nm polycarbonate membrane to prepare cell membrane-encapsulated nanoparticles (MNPs). 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 wraps 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 encapsulated amount of protein was determined by hydrolysis extraction and SDS-PAGE. Y196E Protein nanoparticle encapsulation of ETX Y196E The concentration of the nanoparticles was used to calculate the encapsulation efficiency (encapsulation efficiency). Accurately weigh 20 mg of NP, slowly add 1 mL of acetonitrile, vortex mix, wait until it is completely dissolved into a slightly white emulsion, centrifuge at 22000×g for 10 minutes, discard the supernatant, and dry the precipitate in a 37°C vacuum drying oven overnight. Add 1 mL of PBS to re-disperse the precipitate, blow and mix, centrifuge at 22000×g for 10 minutes, collect the supernatant, further dissolve the precipitate in 100 μL of 0.1 mol / L sodium hydroxide, vortex mix thoroughly, centrifuge at 22000×g for 10 minutes, 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 program.

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

[0084] Example 7

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

[0086] To test 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 hour 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 hour, and MTS color development was performed for 3 hours. The experimental results are shown in Figure 2. Figure 8 As shown in the figure, 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 of PBS group and MNP group was 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 capacity = 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, we can know that the specific process of preparing Clostridium perfringens ε toxin nanoparticle vaccine is as follows: Figure 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 a 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 the digestion. Use a 3mL Pasteur pipette to gently blow out 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 a cell counting plate, count the cells, and dilute the MDCK cell suspension to 1×10 5 / mL. 100 μL of cell suspension was added to each well of a 96-well plate. Incubated at 37°C in a CO2 constant temperature incubator for 24 h. Using 10 μg of total antigen carried by the core as a standard, MNP-ASP-ETX, MNP, and ETX were mixed in DMEM medium. Y196E Dilute to 125μg / mL respectively, and dilute 15 times downward by 2 times. 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 different gradient protein solutions, and set up 3 replicates for each gradient. Add 100μL of DMEM culture medium as a negative control, set up 3 replicates, and use the culture wells with a completely lethal dose of toxin 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 color development solution at a ratio of 100:20, and add 120μL of color development solution to each well. Incubate in a 37°C and CO2 incubator for 3 hours. Measure the absorbance A of each well. 492 Calculate cell survival rate and half 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 Figure 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 calculated based on the total ETX loaded in the nanocore). Y196E The amount of protein is the standard, and the antigen amount of MNP-ASP-ETX is the amount of ASP-ETX toxin adsorbed on the surface + 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. Figure 11 As shown, the mice grew well during the entire immunization period, without adverse reactions, and their weight showed a steady growth 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 and 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 it is injected at 2 points, with 100μL injected subcutaneously for each animal. Figure 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 as 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 + ETX loaded on the nanocore. Y196E =10 μg), the immunization method and dosage were kept consistent 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 minutes to obtain serum, which was then aliquoted and stored at -20°C.

[0101] The ETX-specific IgG antibody titer in serum was detected by ELISA. The experimental results are as follows Figure 13 As shown, after the second immunization, the ETX-specific IgG antibody titer in 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 was 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] To explore the type of immune response produced by the nanoparticle vaccine, the titers of ETX-specific IgG1 and IgG2a antibodies in mice were measured 7 days after the third immunization. The differentiation direction of helper T cells (Th cells) was explored by IgG2a / IgG1. A 1× coating solution containing 5 μg / mL His-ETX was prepared and added to ELISA plates, 100 μL per well, and incubated at 4°C overnight. The next day, the liquid in the enzyme-linked plate was quickly poured out, washed 3 times with PBST, and 350 μL of 5% BSA was added to each well. The plate was incubated at 37°C for 1 hour. The serum of the immunized mice was diluted to 10% with 5% BSA. 2 , 10 3 , 10 4 and 10 5 , add 100 μL to each well, add mouse serum from the PBS group as a negative control, and the 5% BSA group as a positive control, and repeat 2 times for each sample. Incubate in a 37°C constant temperature box for 1 hour. Take out the enzyme-linked plate, quickly pour out the liquid in the enzyme-linked 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 100 μL to each well, and add the enzyme-linked plate. Incubate in a 37°C constant temperature box for 1 hour. Take out the enzyme-linked plate, quickly pour out the liquid in the enzyme-linked plate, and wash 3 times with PBST. Prepare TMB colorimetric solution at a ratio of 1:1 between colorimetric solution A and solution B, and add 100 μL to each well. React at room temperature in the dark for 10 minutes. Add 50 μL 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. Figure 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 4IgG2a antibody titers were significantly higher in mice. The IgG2a / IgG1 ratios indicated 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 primarily Th2-type, with a Th1 bias. These results demonstrate that the MNP-ASP-ETX ETX nanoparticle vaccine group induced 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. Seven days after the third immunization, the mice were randomly divided into two groups, 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 were prepared with PBS 50 and 100×LD 50 Different doses of toxin were injected intraperitoneally into the mice in each immunization group, and the survival status of the mice was observed for 3 consecutive days. Figure 15 As shown, in 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 challenge 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 hours, with a survival rate of 60%, indicating that MNP-ASP-ETX and NP have good protection against 100×LD 50 The protection of the MNP group was lower than that of the other groups. 50 GST-ETX toxin could only protect 60% of mice from surviving the challenge.

[0106] Example 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 50The GST-ETX mixture was placed on a rotating mixer and incubated at 37°C and 120 r / min for 30 minutes. The 96-well plate was removed, the culture medium was discarded, 100 μL of pre-cooled PBS solution was added to each well, and the mixture was gently mixed. The supernatant was discarded, and the above steps were repeated twice. 100 μL of serum with different gradients was added to obtain serum-toxin mixtures with different gradients. Each gradient was repeated at least 3 times. 100 μL of DMEM culture medium was added as a negative control, and 3 replicates were set. The culture wells with a completely lethal dose of toxin were used as the positive control group. The cells were cultured in a 37°C, CO2 incubator for 1 hour. The 96-well plate was removed, the culture medium was discarded, and the cells were gently rinsed 3 times with PBS solution. The DMEM culture medium and MTS color development solution were mixed at a ratio of 100:20. 120 μL of color development solution was added to each well. The cells were cultured in a 37°C, CO2 incubator for 3 hours. The absorbance A of each well was measured. 492 Calculate cell survival rate and half lethal dose. Neutralization titer is expressed as the reciprocal of the highest serum dilution that shows 50% 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 Figure 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 serum of mice after the third immunization was diluted 2 times to different concentrations and mixed with 64, 32, 16, 8, 4, and 2×CT 50 GST-ETX toxin mixture was used to determine the toxicity of the serum-toxin mixture by MDCK cell toxicity assay, calculate the MDCK cell survival rate, and evaluate the ability of the immune group serum to neutralize the toxin in vitro. Figure 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 the mice challenged with GST-ETX toxin was 100%, indicating that the nanoparticle vaccine group showed good protection against 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 a secretory protein Ov-ASP-1 sequence fragment associated with the activity of inducing 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 shows 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 principles of the present invention. These improvements and modifications should also be regarded as within 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; 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.

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

3.

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

4. A recombinant vector, characterized in that The recombinant vector is a pET-28a vector comprising the nucleic acid molecule according to claim 2.

5. A recombinant bacterium, characterized in that The recombinant bacterium is Escherichia coli BL21 (DE3) comprising the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 4 .

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

7. A method for preparing the Clostridium perfringens epsilon toxin nanoparticle vaccine according to claim 6, characterized in that: The steps include: (1) ETX Y196E The protein was mixed with poly(lactic acid-co-glycolic acid) solution and ultrasonicated for 1-2 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, the precipitate is discarded, the supernatant is centrifuged at a centrifugal force of 21000-23000×g for 35-45 minutes, the supernatant is discarded, the precipitate is purified 2-3 times, and freeze-dried to obtain the loaded ETX Y196E protein nanoparticles; (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 cell membrane-wrapped nanoparticles; (3) The cell membrane-wrapped nanoparticles were mixed with ASP-ETX fusion protein and incubated for 50-70 min to obtain Clostridium perfringens ε toxin nanoparticle vaccine.

8. The preparation method according to claim 7, characterized in that The ETX Y196E The mass volume ratio of protein to poly(lactic-co-glycolic acid) solution was 10 mg: 23-25 ​​mL; The preparation method of the polylactic acid-glycolic acid copolymer solution is as follows: mixing the polylactic acid-glycolic acid copolymer with dichloromethane to obtain; The ETX Y196E -The volume ratio of the mixed PLGA solution and polyvinyl alcohol solution is 1:12~14.

9. The preparation method according to claim 8, characterized in that The load ETX Y196E The mass volume ratio of the protein nanoparticles mixed with the MDCK cell membrane is 10 mg: 0.8~1.2 mL; The pore size of the polycarbonate membrane is 180-220 nm.

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