A type D Clostridium perfringens 21-D-5, exotoxin, vaccine, preparation method and application thereof

Abstract

The present invention relates to the technical field of vaccine preparation, and specifically discloses a type D Clostridium perfringens 21-D-5, exotoxins, vaccines, preparation methods and applications thereof, wherein the type D Clostridium perfringens 21-D-5 is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on March 21, 2024, with a deposit number of CGMCC No. 30092. The exotoxins (CPA, ETX, CPE) produced by the type D Clostridium perfringens 21-D-5 separated and obtained by the present invention can be used to prepare vaccines, and have good immune effects.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to a type D Clostridium perfringens 21-D-5, exotoxin, vaccine, preparation method and application thereof. Background Art

[0002] In recent years, the large number of sheep deaths caused by Clostridium perfringens (C. perfringens) infection has brought huge economic losses to the sheep industry. Clostridium perfringens is an anaerobic bacterium formed by Gram-positive spores. It can colonize the normal gastrointestinal environment of humans and animals. By forming spores with strong resistance to the outside world, it can also be widely present in natural environments such as soil and rivers. As a conditional pathogen, Clostridium perfringens is difficult to cause disease in the body under normal conditions. When Clostridium perfringens is infected through trauma, a large number of Clostridium perfringens are ingested at one time, or the composition of the intestinal flora changes significantly, the bacteria can proliferate rapidly and secrete a large amount of protein toxins, thereby causing a variety of diseases. Clostridium perfringens can form a capsule and is one of the fastest growing microorganisms known to date. In addition to causing human infections such as food poisoning and infectious diarrhea, Clostridium perfringens can also cause a variety of animal diseases characterized by necrotic enteritis, hemorrhagic enteritis, and sudden death. It is an important zoonotic pathogen. Type A and D Clostridium perfringens are common in sheep and goats. Clostridium perfringens can produce more than 20 toxins in total, including some deadly toxins. Among them, α toxin can activate the inflammatory cascade in host cell metabolism, and both α toxin and PFO toxin can cause gas gangrene in the body; CPE toxin can destroy the tight junctions of intestinal epithelial cells and cause food poisoning and non-foodborne diarrhea; ETX toxin is currently considered to be the most toxic of all toxins produced by Clostridium perfringens and is associated with enterotoxemia in goats and sheep.

[0003] C. perfringens is classified into seven types (AG) based on the ability to produce six types of toxins, namely CPA, CPB, ETX, ITX, CPE and NetB, each of which is associated with different enteric diseases in animals. All strains carry CPA toxins, ETX toxins are mainly produced by types B and D of C. perfringens and can cause severe acute diseases in livestock populations, and CPE is the main virulence factor of type F strains, which can cause gastrointestinal diseases in humans and can lead to fatal enterotoxemia. It has been reported that type F C. perfringens has been isolated from diarrheal sheep samples, indicating its potential harm to sheep flocks. The toxins produced by the bacteria are absorbed into the circulation and can affect other organs. Vaccination is an effective means of preventing infectious diseases. Among the various types of vaccines that have been developed, toxoid vaccines have been shown to be effective against toxin-mediated bacterial infections. In general, it is almost impossible to eradicate diseases caused by C. perfringens toxins; therefore, vaccination is essential for controlling C. perfringens-related diseases. Vaccines can protect livestock from enteritis and enterotoxemia caused by the bacteria, but the current measures to prevent sheep Clostridium perfringens disease are mainly to vaccinate with single-valent and multivalent vaccines, which cannot effectively prevent diseases caused by Clostridium perfringens. Sheep enterotoxemia caused by type D Clostridium perfringens is rapid in onset and death, and can generally only be effectively prevented and controlled through vaccination. However, the commercial vaccines currently used have large doses and severe reactions in vaccinated livestock, and the immune effects of some vaccines are controversial.

[0004] Current commercial vaccines, such as the bacterial vaccine "Triple Four Protection", use vaccine strains of Clostridium perfringens type B (strain C58-1), Clostridium perfringens type D (strain C60-2) and Clostridium putrificum (C.putrificum) (strain C55-1). After the sheep are vaccinated with bacterial vaccines, the antibodies produced against toxins are low, resulting in poor vaccine immunity and frequent illness in the immunized sheep. Its quality also needs to be further improved. Summary of the invention

[0005] In order to develop a new vaccine that can effectively prevent sheep enterotoxemia, the present invention provides a type D Clostridium perfringens 21-D-5, exotoxins, vaccines, preparation methods and applications. The exotoxins (CPA, ETX, CPE) produced by the type D Clostridium perfringens 21-D-5 separated and obtained by the present invention can be used to prepare vaccines and have good immune effects.

[0006] The present invention provides a type D Clostridium perfringens 21-D-5, wherein the type D Clostridium perfringens 21-D-5 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on March 21, 2024, with a deposit number of CGMCC No. 30092.

[0007] Type D Clostridium perfringens 21-D-5 carries CPA, ETX and CPE toxins at the same time. The inactivated exotoxin is converted into toxoid, which is used as the antigen component to make a vaccine. After immunization of animals, it can effectively stimulate the body to produce antibodies. Vaccination with toxoid vaccine can solve the Clostridium perfringens disease caused by antibiotic resistance to a certain extent, and can also protect livestock against enteritis and enterotoxemia caused by the bacteria.

[0008] The present invention also provides an exotoxin produced by the type D Clostridium perfringens 21-D-5.

[0009] The present invention also provides a method for preparing the exotoxin, wherein the 21-D-5 strain is inoculated into a glycolate fluid culture medium for anaerobically culturing to obtain a FT bacterial culture solution, and then inoculated into Gordon soup for anaerobically culturing to obtain a Gordon soup bacterial solution, and the supernatant is collected after the first centrifugal filtration to obtain a 21-D-5 toxin solution;

[0010] Add saturated ammonium sulfate solution to the 21-D-5 toxin solution until the ammonium sulfate reaches 50% saturation, collect the precipitate by centrifugation and dissolve it in Trsi-HCL buffer, add saturated ammonium sulfate solution again until the ammonium sulfate reaches 40% saturation, collect the precipitate by centrifugation for the second time and dissolve it in Trsi-HCL buffer to obtain crude exotoxin.

[0011] Furthermore, the FT bacterial culture solution was inoculated into Gordon broth at an inoculation level of 3-5%.

[0012] Furthermore, the centrifugation conditions for the first centrifugation and the second centrifugation are both 4°C, 8000-10000 r / min, and 15-30 min.

[0013] The invention also provides a vaccine, which consists of the exotoxin and a vaccine adjuvant.

[0014] Furthermore, the exotoxin and the vaccine adjuvant are mixed in equal volume ratios.

[0015] Furthermore, the vaccine adjuvant is any one of aluminum adjuvant, oil emulsion adjuvant, propolis adjuvant, liposome adjuvant, traditional Chinese medicine adjuvant and small peptide adjuvant.

[0016] Furthermore, the aluminum adjuvant includes aluminum phosphate and aluminum hydroxide adjuvant.

[0017] The present invention also provides a method for preparing the vaccine, which specifically comprises: thoroughly mixing the inactivated crude exotoxin with an aluminum hydroxide adjuvant in a volume ratio of 1:1 to prepare an aluminum hydroxide adjuvant vaccine, namely, a type D Clostridium perfringens 21-D-5 toxoid vaccine.

[0018] The present invention also provides an application of the D-type Clostridium perfringens 21-D-5, the exotoxin or the vaccine in preparing a product for preventing diseases caused by Clostridium perfringens.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The type D Clostridium perfringens 21-D-5 isolated and obtained by the present invention carries CPA, ETX and CPE toxins at the same time. The inactivated exotoxins are converted into toxoids, which are used as antigen components to prepare vaccines. After being immunized in animals, the body can be effectively stimulated to produce antibodies. The vaccination of toxoid vaccines can solve the Clostridium perfringens diseases caused by antibiotic resistance to a certain extent, and can also protect livestock from enteritis and enterotoxemia caused by the bacteria.

[0021] 2. When reviving type D Clostridium perfringens, the present invention adjusts the culture temperature to 42°C to increase the growth rate of Clostridium; when culturing the colony in FTG, the temperature is appropriately lowered to 37°C and the bacterial growth time is extended to 15 hours to approach the optimal culture temperature of FTG.

[0022] 3. The present invention determines the inactivation time of type D Clostridium perfringens exotoxin and the concentration of formaldehyde. The exotoxin was inactivated with formaldehyde at a final concentration of 0.2%, but the effect was slow, and Kunming mice still died at 40 hours. The exotoxin was inactivated with formaldehyde at a final concentration of 0.3%. The results showed that 0.3% formaldehyde could not completely inactivate the exotoxin within 16 hours; and when the exotoxin was inactivated at 0.3% for 24 hours and 32 hours, the extracted exotoxin was not completely inactivated and still active; when the inactivation time was 40 hours and 48 hours, three Kunming mice survived completely and had no adverse reactions, indicating that the expected inactivation effect had been achieved. The exotoxin was inactivated with formaldehyde at a final concentration of 0.4%, and all Kunming mice survived at 32 hours, but their spirits were relatively depressed. Everything was normal in the blank control group. Considering that the inactivation time of 0.2% formaldehyde solution is longer, while the inactivation time of 0.4% formaldehyde solution is shorter but the Kunming mice are more depressed, in order to ensure the safety of animals and take into account the hazards of formaldehyde itself, for safety reasons, 37°C and 0.3% formaldehyde solution can be used for inactivation for 40 hours. This reduces the damage of the extracted D-type Clostridium perfringens exotoxin to livestock and reduces the time cost in industrial production.

[0023] 4. In the present invention, the vaccine was prepared for Kunming mice and injected at 0d and 14d. Before testing the immune serum, the optimization exploration of the antigen coating concentration and serum dilution of indirect ELISA was first carried out. After the experiment, it was finally determined that the optimal coating concentration of the antigen was 0.5μg / mL and the optimal dilution of the serum was 1:100. Under this condition, the P / N ratio was better.

[0024] 5. After the present invention immunizes Kunming mice by subcutaneous multi-point injection, the body temperature of Kunming mice rises on the first day, but slowly decreases and returns to normal on the second day, proving that the vaccine is less harmful to animals. Indirect ELISA was used to detect the change rules of immunization 0d, 7d, 14d, 21d, 28d, 35d, 42d and 49d. The Kunming mice in the immunized group all produced corresponding antibodies against the vaccine immunogen components. The serum antibody level continued to rise after the first immunization, and its antibody level reached the highest at 21d, and then slightly decreased, but still higher than the antibody level of the first immunization. The antibody titer of Kunming mice was 1:800 at 7d, and the antibody titer could reach 1:6400 at 21d of immunization, and then decreased to 1:3200, and then remained stable. The Kunming mice in each group were challenged with poison, and the results showed that the vaccine had a good protective effect on A, D and F types of Clostridium perfringens. The survival rate of Kunming mice after challenge was 100%, and the damage of bacteria to organs was greatly reduced.

[0025] 6. The present invention conducts immune tests on Guanzhong dairy goats in different dose groups (immune doses are 2, 1.5, and 1 mL / goat, respectively). The results show that after 1 day of vaccination, the body temperature of Guanzhong dairy goats rises by about 1.5 ° C, but decreases and gradually returns to normal at the 2nd day, proving that the vaccine is safe. Similarly, indirect ELISA is used to detect the antibody change rule after 0d, 7d, 14d, 21d, and 28d of immunity. The results show that the serum titer of Guanzhong dairy goats in the 2mL immune group is basically the same as that of the 1.5mL immune group. Therefore, in production, 1.5mL of immune dose can be selected for vaccination, which is lower than the immune dose in other studies. The positive antibody titer of Guanzhong dairy goats reaches 1:800 at 7d and 1:3200 at 21d, proving that the vaccine can produce a good immune effect and can stimulate the body to produce antibodies in a short time.

[0026] Information on the deposit of biological materials

[0027] 21-D-5, referred to as type D Clostridium perfringens 21-D-5 in this application, was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on March 21, 2024, with a deposit number of CGMCC No.30092. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and is classified and named Clostridium perfringens. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The colony morphology of strain 21-D-5 in Example 1 of the present invention on a TSC plate (A) and a blood plate (B) and the morphology after Gram staining (C) (optical microscope 10×100).

[0030] Figure 2 This is the gel electrophoresis diagram of the 16S rRNA gene PCR product of strain 21-D-5 in Example 1 of the present invention, where M is a marker and 1-2 are samples of strain 21-D-5.

[0031] Figure 3 This is the death curve of Kunming mice after being challenged with the strain 21-D-5 isolated in Example 1 of the present invention.

[0032] Figure 4 This is the SDS-PAGE and Western Blot of the crude exotoxin in Example 1 of the present invention. In Figure A, M is a marker, 1-5 are extracted samples, and 1-2 are samples.

[0033] Figure 5 This is a diagram of the autopsy results of Kunming mice after being challenged with the strain 21-D-5 in Example 1 of the present invention. In the figure, the autopsy results of Kunming mice in the challenge group (A, C, E) and the control group (B, D, F); wherein, A, B: Kunming mouse autopsy pictures; C, D: intestines; E, F: spleen; G, H: kidneys.

[0034] Figure 6 This is a diagram showing the results of HE staining of organs of Kunming mice after being challenged with the strain 21-D-5 in Example 1 of the present invention. In the figure, HE stained sections (10×40) of Kunming mice in the challenge group (a, c, e) and blank control Kunming mice (b, d, f); among them, a, b: intestine; c, d: spleen; e, f: kidney.

[0035] Figure 7 The figure shows the temperature changes of Kunming mice after being vaccinated with the vaccine prepared by the present invention. Numbers 1 to 9 are 9 randomly selected Kunming mice that were vaccinated.

[0036] Figure 8 The OD450nm values ​​of Kunming mice in different experimental groups of the present invention at different immunization times, wherein groups 1, 2 and 3 represent immunization groups.

[0037] Fig. 9 The antibody titers of Kunming mice in group 1 of the present invention at different immunization times.

[0038] Fig.10 These are the appearance pictures of Kunming mice in groups 1 and 4 after autopsy;

[0039] In the figure, A is the overall picture of the autopsy of one group of Kunming mice; B is the overall picture of the autopsy of four groups of Kunming mice; C is the picture of intestinal lesions at the autopsy of one group of Kunming mice; D is the picture of spleen lesions at the autopsy of one group of Kunming mice; E is the picture of kidney lesions at the autopsy of one group of Kunming mice; F is the picture of intestinal lesions at the autopsy of four groups of Kunming mice; G is the picture of spleen lesions at the autopsy of four groups of Kunming mice; H is the picture of kidney lesions at the autopsy of four groups of Kunming mice.

[0040] Fig.11 HE-stained sections (10×40) of Kunming mice in group 1 (a, c, e) and group 4 (b, d, f); a, b: intestine; c, d: spleen; e, f: kidney.

[0041] Fig.12 The appearance of Kunming mice after autopsy in group 2 (A) and group 5 (B);

[0042] In the figure, A is the overall picture of the autopsy of 2 groups of Kunming mice; B is the overall picture of the autopsy of 5 groups of Kunming mice; C is the picture of intestinal lesions at the autopsy of 2 groups of Kunming mice; D is the picture of spleen lesions at the autopsy of 2 groups of Kunming mice; E is the picture of kidney lesions at the autopsy of 2 groups of Kunming mice; F is the picture of intestinal lesions at the autopsy of 5 groups of Kunming mice; G is the picture of spleen lesions at the autopsy of 5 groups of Kunming mice; H is the picture of kidney lesions at the autopsy of 5 groups of Kunming mice.

[0043] Fig.13 HE-stained sections (10×40) of Kunming mice in two challenged groups (a, b, c) and five groups (d, e, f); in the figure, a, b: intestine; c, d: spleen; e, f: kidney.

[0044] Fig.14 The appearance of Kunming mice after autopsy in group 3 (A) and group 6 (B);

[0045] In the figure, A is the overall picture of the autopsy of 3 groups of Kunming mice; B is the overall picture of the autopsy of 6 groups of Kunming mice; C is the picture of intestinal lesions at the autopsy of 3 groups of Kunming mice; D is the picture of spleen lesions at the autopsy of 3 groups of Kunming mice; E is the picture of kidney lesions at the autopsy of 3 groups of Kunming mice; F is the picture of intestinal lesions at the autopsy of 6 groups of Kunming mice; G is the picture of spleen lesions at the autopsy of 6 groups of Kunming mice; H is the picture of kidney lesions at the autopsy of 6 groups of Kunming mice.

[0046] Fig.15 HE-stained sections (10×40) of organs of Kunming mice in three challenged groups (a, b, c) and six groups (d, e, f). In the figure, a, b: intestine; c, d: spleen; e, f: kidney.

[0047] Fig.16The external appearance of Kunming mice in group 4 (A) and group 8 (B) after autopsy;

[0048] In the figure, A is the overall picture of the autopsy of 4 groups of Kunming mice; B is the overall picture of the autopsy of 8 groups of Kunming mice; C is the picture of intestinal lesions at the autopsy of 4 groups of Kunming mice; D is the picture of spleen lesions at the autopsy of 4 groups of Kunming mice; E is the picture of kidney lesions at the autopsy of 4 groups of Kunming mice; F is the picture of intestinal lesions at the autopsy of 8 groups of Kunming mice; G is the picture of spleen lesions at the autopsy of 8 groups of Kunming mice; H is the picture of kidney lesions at the autopsy of 8 groups of Kunming mice.

[0049] Fig.17 HE-stained sections (10×40) of organs of Kunming mice in 4 challenged groups (a, b, c) and 8 groups (d, e, f). In the figure, a, b: intestine; c, d: spleen; e, f: kidney.

[0050] Fig.18 The figure shows the temperature changes of Guanzhong dairy goats after being vaccinated with the vaccine prepared by the present invention. No. 1-9 are Guanzhong dairy goats vaccinated with the vaccine.

[0051] Fig.19 Effect of different immunization doses and different immunization times on the OD of dairy goats during immunization 450 nm value.

[0052] Fig. 20 This is the ELISA result of the antibody titer of the 1.5 mL immunization group in Example 4 of the present invention. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Example 1: Isolation, identification and pathogenicity study of Clostridium perfringens type D.

[0055] 1. Experimental Materials and Methods

[0056] 1. Culture medium

[0057] Anaerobic liver broth medium was purchased from Haibo Biotechnology Co., Ltd.

[0058] Tryptone-cycloserine-sulfite (TSC) medium was purchased from Haibo Biotechnology Co., Ltd. Blood plates were purchased from Haibo Biotechnology Co., Ltd.

[0059] 2. Sampling

[0060] Anal swabs were collected from Guanzhong dairy goats with depression and diarrhea at a large-scale Guanzhong dairy goat farm in Fuping County, Shaanxi Province. After collection, the samples were placed in transport culture medium and stored in a 4°C sampling box and sent back to the Veterinary Infectious Diseases Laboratory of Northwest A&F University within 12 hours for bacterial isolation and identification.

[0061] 3. Isolation of Clostridium perfringens type D

[0062] Initial culture: Take 2g of the collected sample and mix it with 1mL of normal saline, then shake it at 37℃ and 150rpm for 30min to obtain a suspension. Take 50μL of the suspension and mix it with 5mL of anaerobic liver broth medium, then culture it in a 37℃ constant temperature incubator for 24h to perform bacterial amplification culture and obtain turbid bacterial liquid. Take 1mL of the turbid bacterial liquid in a 1.5mL EP tube, centrifuge it at 6000rpm for 3min, discard the supernatant, take the precipitate with an inoculation loop, inoculate it in a trypticase-cycloserine-sulfite (TSC) medium, and place it in an anaerobic incubator at 37℃ for 48h.

[0063] Purification culture: After the initial culture, a single colony with a black edge and translucent color was selected from the TSC medium, and streaked again on a new TSC medium, and cultured in an anaerobic incubator at 37°C for 48 hours. After the culture was completed, a single black colony on the TSC medium was picked, streaked on a blood plate, and cultured in an anaerobic incubator at 37°C for 24 hours. The isolated strain was recorded as strain 21-D-5.

[0064] 3. Morphological identification

[0065] Observe and record the morphology of the purified single colonies on the TSC plate and blood plate. Take a single colony with double hemolysis rings on the blood plate after separation and purification (strain 21-D-5) and place it on a slide with dd H 2 O and mix well, and bake with an alcohol lamp until the liquid evaporates completely. Gram staining microscopy and strain morphology observation were performed according to the staining process of crystal violet staining for 30 seconds, iodine solution for 60 seconds, alcohol for 15 seconds, and sand yellow for 30 seconds. The results are shown in Figure 1 .

[0066] like Figure 1 As shown in A, the isolated strain 21-D-5 appears black on the TSC plate with transparent edges, smooth surface and neat edges; B: It appears as a translucent and smooth small colony with double or single hemolytic rings on the blood plate; C: Gram staining microscopy shows that 21-D-5 is a Gram-positive medium-sized rod with straight edges and blunt ends.

[0067] 4. Molecular identification

[0068] A single colony (21-D-5 strain) with a double hemolytic ring on the blood plate was taken, mixed evenly with 1 mL of sterile saline, and treated at 100°C for 10 min as a DNA template for standby use. Universal primers 27F and 1495R were used as primers for amplification; the PCR reaction conditions were 94°C, 5 min; 94°C, 30 s; 55°C, 30 s; 72°C, 1 min, 35 cycles; 72°C, 5 min. The PCR product was identified by 1% agarose electrophoresis and sent to Xi'an Qingke Jersey Biotechnology Co., Ltd. for sequencing. The sequencing results were compared online using NCBIBlast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Results are shown in Figure 2 .

[0069] 27F: 5′-GAGAGTTTGATCCTGGCTCAG-3′;

[0070] 1495R: 5′-CTACGGCTACCTTGTTACGA-3′.

[0071] 5. Pathogenicity experiment of strain 21-D-5 to Kunming mice

[0072] The strain 21-D-5 was anaerobically revived at 42°C for 24 hours. A single colony was aseptically picked and inoculated into anaerobic meat liver soup to prepare seed liquid, which was inoculated into Gordon toxin-producing medium at a ratio of 1:20, and the toxin was sterilized by centrifugation to obtain the toxin. The toxin (0.5 mg / mL) was diluted in multiples and injected intraperitoneally into Kunming mice, 0.2 mL per mouse, for a total of 6 mice. The blank control group of 6 mice was injected with 0.2 mL of sterile saline. The survival status of the Kunming mice was observed for 12 consecutive hours to observe the pathogenicity of the 21-D-5 strain. The results are shown in Figure 3 .

[0073] The results are as follows Figure 3 As shown, strain 21-D-5 has a strong ability to produce toxins. Kunming mice began to die at 1 hour, and the mortality rate of the experimental Kunming mice reached 100% at 3 hours. Therefore, it can be used as a vaccine strain.

[0074] Example 2: Preparation method of sheep D-type Clostridium perfringens 21-D-5 vaccine.

[0075] 1. Experimental Methods

[0076] 1. Culture medium

[0077] Blood plates were purchased from Haibo Biotechnology Co., Ltd.;

[0078] Thioglycollate fluid medium (FTG) was purchased from Haibo Biotechnology Co., Ltd.;

[0079] Gordon soup: Accurately weigh 2 g of peptone, 1 g of dextrin, and 2 g of yeast extract, mix the above ingredients, dissolve and make up to 100 mL with PBS, adjust the pH to 7.4, and sterilize at 121°C for 15 min.

[0080] 2. Preparation of exotoxin of Clostridium perfringens 21-D-5

[0081] The 21-D-5 strain was inoculated into a blood plate and placed in an anaerobic chamber at 42°C (the anaerobic environment gas composition was (88% N 2 , 7% H 2 , 5% CO 2 ) for 36 hours; a single colony was selected and inoculated into thioglycollate fluid medium (FTG) at 37°C for anaerobic growth for 10 hours to obtain FT bacterial culture solution, 5% (v / v) FT bacterial culture solution was added to Gordon soup, and anaerobically cultured at 43°C for 6 hours to obtain Gordon soup bacterial solution. The Gordon soup bacterial solution was centrifuged at 4°C and 10,000 r / min for 15 minutes, and then filtered with a Cai filter with a pore size of 0.22 μm to obtain the supernatant to obtain the 21-D-5 toxin solution.

[0082] Saturated ammonium sulfate solution was slowly added to the 21-D-5 toxin solution until the ammonium sulfate in the mixed solution reached 50% saturation, and the mixture was allowed to stand overnight at 4°C. Then, the mixture was centrifuged at 4°C and 8000 r / min for 30 min, the supernatant was discarded, and the precipitate was dissolved with 1 mL of 0.005 M Tris-HCL buffer (pH 7.5), and saturated ammonium sulfate solution was added again until the ammonium sulfate in the mixed solution reached 40% saturation, and the mixture was allowed to stand overnight at 4°C. Then, the mixture was centrifuged at 4°C and 8000 r / min for 30 min, the supernatant was discarded, and the precipitate was dissolved in 5 mL of 0.005 M Tris-HCL buffer (pH 7.5) to obtain the crude exotoxin.

[0083] The crude exotoxin was concentrated. According to the amount of protein solution, the dialysis bag was cut into a suitable length. The dialysis bag was boiled with deionized water for 10 minutes each time, and the boiling was repeated 3 times. The dialysis bag containing the crude exotoxin was placed in a highly saturated sucrose solution and dialyzed with stirring to make the volume of the crude exotoxin about 2mL-3mL. The concentrated crude exotoxin was transferred to a 1.5mL centrifuge tube and stored at -20°C.

[0084] The crude exotoxin prepared was subjected to SDS-PAGE and Western Blot detection as follows:

[0085] (1) Polyacrylamide gel electrophoresis (SDS-PAGE) test

[0086] Preparation: Clean the equipment and dry it in a natural environment for later use.

[0087] Glue preparation: Assemble the gel plate. After the separation gel is prepared, immediately fill it with isopropanol for sealing. After the separation gel is completely solidified, pour out the isopropanol, use filter paper to absorb the remaining water, and prepare 5% concentrated gel.

[0088] Add samples: Assemble the electrophoresis apparatus, add 5× Running Buffer, make sure the electrophoresis solution covers the short plate of the inner layer of the gel plate, and the outer layer of the gel plate is 1 / 3, and covers the electrode line below the electrophoresis apparatus. Unplug the comb, mix the sample and buffer in a ratio of 4:1, boil for 10 minutes, and cool at 4℃ to room temperature. Add samples and record the order.

[0089] Gel running: Run the gel immediately after adding the sample. Run at 80V for 0.5h, then switch to 120V until the dye strip just runs out of the gel board.

[0090] Glue unloading: remove the glue plate, separate the glass plate, and cut off the concentrated glue.

[0091] Staining: Stain the separation gel for about 15 minutes.

[0092] Preservation results: Wash the separation gel with ultrapure water, place it on a white board and take a photo for preservation.

[0093] (2) Western Blot

[0094] After the crude exotoxin protein was concentrated and subjected to SDS-PAGE, it was transferred to PVDF membrane by wet transfer method, blocked with 5% skim milk powder, and incubated in CPA, ETX, and CPE positive serum at 4°C overnight. The dilution ratio of positive serum was 1:500.

[0095] After the primary antibody incubation, the membrane was washed three times and then incubated in horseradish peroxidase-labeled mouse anti-goat IgG antibody. The dilution ratio of the enzyme-labeled secondary antibody was 1:5000. The membrane was incubated at room temperature for 1 h and washed three times after incubation.

[0096] Color development and imaging, take photos and save.

[0097] The results are as follows Figure 4 As shown, Figure 4 A is the SDS-PAGE test result of crude exotoxin, where M is the protein molecular weight standard, 1-5 are crude 21-D-5 exotoxins, and it is known that the molecular weight of CPA exotoxin protein is 43kDa, the molecular weight of ETX exotoxin protein is 33kDa, and the molecular weight of CPE exotoxin protein is 35kDa. Figure 4 B is the result of Western Blot test of crude exotoxin, among which 1-2 is the crude 21-D-5 exotoxin. Therefore, it can be determined that the three exotoxins mainly produced in the 21-D-5 toxin-producing culture medium are CPA, ETX and CPE.

[0098] 3. Determination of minimum lethal dose (MLD)

[0099] Take 36 20g Kunming mice and divide them into 6 groups, 6 in each group, one of which is a blank control group, and the remaining five groups are experimental groups. Take the prepared crude exotoxin and dilute it 0, 25, 50, 100, and 200 times, and inject 0.5mL into the experimental group Kunming mice intraperitoneally. The blank control group is normal saline, and the number of deaths and mortality in each group are recorded. The results are shown in Table 1. The minimum lethal dose of crude exotoxin to Kunming mice is 50 times dilution of the toxin, that is, the dilution concentration is 0.0338mg / mL at 50 times, and the injection volume is 0.5mL, then the actual injection volume is 0.0169mg, Kunming mice weigh 20g, and the minimum lethal dose of crude exotoxin to Kunming mice is 0.845mg / kg.

[0100] Table 1 Determination of minimum lethal dose

[0101]

[0102] 4. Observation of pathological sections of Kunming mice challenged with exotoxin

[0103] The Kunming mice that died from the crude 21-D-5 exotoxin in the previous experiment were dissected, and their lungs, livers, spleens, kidneys, jejunums, colons and other organs were collected, their morphology was observed, photographed and recorded, and placed in a 4% paraformaldehyde solution. After 24 hours, the paraformaldehyde was replaced again for fixation. The water in the tissue block was removed with alcohol, placed in xylene and then immersed in wax and embedded. The paraffin block was cut into thin slices, ironed flat, spread on a slide, dried and dewaxed, stained with hematoxylin and eosin, dehydrated and transparent, and sealed to observe the pathological changes of the tissues of the experimental Kunming mice.

[0104] Observe the organ lesions of Kunming mice killed by exotoxins, collect the lungs, livers, spleens, kidneys, jejunum and colon, observe their morphology, take photos and record, put them in 4% paraformaldehyde solution, replace the paraformaldehyde again after 24 hours, and fix them. Use alcohol to remove the water in the tissue blocks, put them in xylene and then immerse them in wax, cut the paraffin blocks into thin slices, flatten them, spread them on slides, dry them and dewax them, stain them with hematoxylin and eosin, dehydrate them and make them transparent, and then seal them and observe the pathological changes of the tissues of the experimental Kunming mice.

[0105] Results Figure 5-6 , the dead Kunming mice had congested and swollen spleens and kidneys, thinned intestines, edema, bloating and bleeding. The intestines, spleens and kidneys of Kunming mice had varying degrees of lesions, among which the spleen bleeding was the most obvious, followed by the kidneys and intestines. Compared with the blank control group, the intestines had bleeding, cell shedding and vacuoles were seen at the tips of the intestinal villi, and the structural integrity was defective; the spleen had large-scale bleeding, a large number of lymphocytes infiltrated, and the number of parenchymal cells decreased; the kidneys had large-scale bleeding.

[0106] 5. Preparation of Clostridium perfringens type D 21-D-5 vaccine

[0107] (1) Effect of final formaldehyde concentration on the inactivation of crude exotoxins

[0108] The crude exotoxin was adjusted to pH 7.2 with 1M NaOH and divided into 3 groups. The total amount of formaldehyde was 0.2%, 0.3%, and 0.4%, respectively. The mixture was fully shaken and mixed, and then placed in a 37°C incubator for inactivation. The mixture was shaken every 6 hours. At 8h, 16h, 24h, 32h, 40h, and 48h, 1mL of inactivated crude exotoxin was taken and intraperitoneally injected into 3 Kunming mice to test the inactivation effect of crude exotoxin. At the same time, a blank control group was set up, and 3 Kunming mice were intraperitoneally injected with 1mL / mouse, and observed for 72h. The results are shown in Table 2. When the final concentration of formaldehyde was 0.2%, the Kunming mice still died at 48h. When the final concentration of formaldehyde was 0.3%, the three Kunming mice could survive completely at 40h without any discomfort, indicating that the expected inactivation effect had been achieved. When the final concentration of formaldehyde was 0.4%, all Kunming mice could survive at 32h, but their spirits were relatively depressed. Everything was normal in the blank control group. To sum up the above, considering the hazards of formaldehyde itself, for safety reasons, 0.3% formaldehyde and 40 hours can be selected as the inactivation condition during production.

[0109] Table 2 Results of inactivation of 21-D-5 exotoxin by different concentrations of formaldehyde at different times

[0110]

[0111] (2) Preparation of Clostridium perfringens Type D 21-D-5 Vaccine

[0112] The inactivated crude exotoxin was thoroughly mixed with aluminum hydroxide adjuvant in a volume ratio of 1:1 to prepare an aluminum hydroxide adjuvant vaccine, i.e., type D Clostridium perfringens 21-D-5 toxoid vaccine, and stored at 4°C.

[0113] (3) Basic testing of Clostridium perfringens 21-D-5 toxoid vaccine

[0114] ① Leave the D-type Clostridium perfringens 21-D-5 toxoid vaccine to stand for 14 days to observe whether its properties are stable. Refer to the "Chinese Veterinary Pharmacopoeia (2020 Edition)" Appendix 3306 Sterility (Pure) Test Method, take the prepared vaccine and inoculate it on the sheep blood plate, and culture it at 37℃ for 3 days; at the same time, take 0.5mL of the prepared vaccine and inoculate it into FTG, and culture it anaerobically at 37℃ for 7 days, and both should grow aseptically. Refer to the "Chinese Veterinary Pharmacopoeia (2020 Edition)" Appendix 3203 Formaldehyde Residue Determination Method for detection.

[0115] After 14 days of standing, the D-type Clostridium perfringens 21-D-5 toxoid vaccine was clearly stratified, with a relatively transparent upper layer and a white precipitate at the lower layer. The vaccine can be easily mixed to form a uniform suspension and needs to be fully mixed before use. The blood plates and FTG of the D-type Clostridium perfringens 21-D-5 toxoid vaccine were sterile and met the requirements of the "Veterinary Biological Products of the People's Republic of China". The formaldehyde residue was 0.125%, which was qualified and met the requirements of the "Veterinary Biological Products of the People's Republic of China".

[0116] ② Randomly select 10 healthy Kunming mice weighing 18-22g, and inoculate them subcutaneously with a large dose of 0.5mL of D-type Clostridium perfringens 21-D-5 toxoid vaccine. After 10 days, observe the health and survival of the Kunming mice. Then randomly select 4 healthy rabbits weighing 1.5-2kg, and inoculate them intramuscularly with a super large dose of 5mL of D-type Clostridium perfringens 21-D-5 toxoid vaccine. Observe for local or systemic abnormal reactions.

[0117] After 14 days of observation, none of the 10 Kunming mice injected with the D-type Clostridium perfringens 21-D-5 toxoid vaccine died; within 6 hours after injection, Kunming mice occasionally showed signs of depression, curling up and shivering, and reduced food and water intake, which returned to normal about 6 hours later; the injection site was initially swollen, and a hard nodule could be felt under the skin when touched, and then the hair at the site fell off, the skin became inflamed, and scabs formed. Therefore, in the subsequent immune effect evaluation test, subcutaneous multi-point injection (more than five points) was used, and the injection dose was strictly controlled.

[0118] After 10 days of observation, none of the four healthy rabbits injected with type D Clostridium perfringens 21-D-5 toxoid vaccine died and were in good mental condition. One rabbit developed bulges and hard nodules under the skin.

[0119] Example 3: Evaluation of the immune effect of Clostridium perfringens type D 21-D-5 toxoid vaccine on Kunming mice.

[0120] 1. Experimental Methods

[0121] 1. Experimental supplies

[0122] 18-22g Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0123] Negative serum: directly collect 18-22g of healthy Kunming mouse serum, which is negative serum.

[0124] Standard strains CVCC1125 (type A, carrying CPA toxin), CVCC81 (type D, carrying CPA and ETX toxins) and 1,2-tetracycline were purchased from China Veterinary Microbial Culture Collection Center. 12916 TM(Type F, carrying CPA and CPE) purchased from American Type Culture Collection.

[0125] 2. Kunming mouse immunization experiment

[0126] 80 healthy Kunming mice weighing 18-22g were prepared and randomly divided into 8 groups, each with 10 mice. Groups 1, 2 and 3 were immunized groups, groups 5, 6 and 7 were challenged groups, group 4 was an immunized control group, and group 8 was a blank control group. All mice were first immunized with type D Clostridium perfringens 21-D-5 toxoid vaccine by subcutaneous injection at the back, with an immunization dose of 0.2mL / mouse (actual protein concentration 0.338mg / mL), and the blank control group was injected with the same dose of normal saline.

[0127] Before the first immunization, blood was collected from the heart and serum was separated. After the first immunization, the temperature changes were observed for 7 consecutive days. On the 14th day, a booster immunization was given (secondary immunization). Blood was collected from the heart and serum was separated before the first immunization. During the immunization period, blood was collected from the heart and separated at 7d, 14d, 21d, 28d, 35d, 42d and 49d, and antibody titer was tested.

[0128] Fourteen days after the second immunization, six groups of Kunming mice, namely, groups 1, 2, 3, 5, 6 and 7, were challenged by intraperitoneal injection with the standard strains CVCC1125 (type A, carrying CPA toxin), CVCC81 (type D, carrying CPA and ETX toxins) and 12916 TM (Type F, carrying CPA and CPE), viable bacteria count 9×10 8 CFU / mL, and groups 4 and 8 were injected with sterile saline as blank control groups. Specific grouping and treatment are shown in Table 3.

[0129] Table 3: Groups in the challenge experiment

[0130]

[0131] Note: The vaccine is the aluminum hydroxide adjuvant vaccine obtained.

[0132] After Kunming mice were immunized with the prepared vaccine by subcutaneous multi-point injection, the trend of body temperature change within 7 days was shown in the figure. Figure 7 The body temperature of Kunming mice increased on the first day after vaccination, but slowly decreased and returned to normal on the second day ( Figure 7 ).

[0133] Kunming mouse sera were collected from the immunized group and the blank control group 7 days and 14 days after the first immunization and 7 days and 14 days after the second immunization, with 3 mice in each group. The collected and separated Kunming mouse sera were diluted and the antibody titer was detected by ELISA. The P / N value was determined by the chessboard titration method, and the optimal coating concentration of the antigen was 0.5 μg / mL, and the optimal serum dilution was 1:100 (Table 4).

[0134] Table 4 Determination of P / N value of samples

[0135]

[0136] The serum antibody level was determined by indirect ELISA. Three replicate wells were set for each sample, and the average value was taken as the result of each sample. The average value of three samples in each group was taken for plotting. The specific steps are as follows:

[0137] Coating: Crudely extracted CPA, ETX, and CPE toxins were diluted and used as antigens to coat the ELISA plate. The antigen amount was about 50 ng / well and coated overnight at 4°C. Then blocked with BSA.

[0138] Blocking: discard the coating solution and wash the ELISA plate with PBST, repeat three times, let it stand for 3 minutes each time, add 200 μL of 5% skim milk powder to each well as blocking solution, and block at 37°C for 2 hours.

[0139] Binding to primary antibody: discard blocking solution, wash three times, add diluted Kunming mouse serum to be tested, 100 μL / well, and incubate at 37°C for 1 hour.

[0140] Binding secondary antibody: discard the primary antibody, repeat washing three times, add 1:3000 diluted goat anti-mouse HRP-IgG, and culture in an incubator at 37°C for 1 hour.

[0141] Color development: discard the secondary antibody, wash three times, pat dry and add TMB color development solution, 100 μL / well, and stand in the dark for 10 minutes.

[0142] Stop: Add stop solution to stop the reaction, 50 μL / well, mix well, and place on the ELISA plate to detect the OD of each well. 450 value.

[0143] Result determination: value to be tested OD 450 nm(P) / negative value OD 450 nm(N)≥2.1 is positive, otherwise it is negative. P / N value = a / b, a and b are the absorbance values ​​of the test group and the blank control group at 450nm respectively.

[0144] The results are as follows Figure 8 As shown, the OD of the immune group from the first immunization 450Compared with the blank control group, the immunized Kunming mice produced corresponding antibodies against the immunogenic components of the vaccine. After the first immunization, the serum antibody levels of Kunming mice in groups 1, 2, and 3 had increased. OD 450 The value reached the highest, then dropped slightly, but it was still higher than the antibody level of the first immunization and remained stable.

[0145] The results are as follows Fig. 9 As shown, the immunization group 1 with better effect was selected for the experiment. Compared with the blank control group, the immunized Kunming mice in the immunized group produced corresponding antibodies against the immunogen components of the D-type Clostridium perfringens 21-D-5 toxoid vaccine. After the first immunization, the serum antibody level had increased, and the antibody level gradually increased after the first immunization, with an antibody titer of 1:800. The antibody level reached the highest 7 days after the second immunization, with a titer of 1:6400, and then stabilized at 1:3200.

[0146] 3. Effects of immune experiments on Kunming mouse organs

[0147] After the above-mentioned immune test, the liver, spleen, lung and kidney of each group of Kunming mice were collected and placed in 10% formalin. After 24 hours, the formalin was replaced again for fixation, and pathological sections were made in the same way as above. The pathological histological changes of each organ were observed, and the blank group Kunming mice were used as controls. The results showed that all Kunming mice in the immune group survived after the poison challenge, all Kunming mice in the poison challenge group died, and all the blank control group survived.

[0148] Organ lesions were observed. The Kunming mice in group 5 had intestinal edema, and congestion and edema of the spleen and kidneys, while the Kunming mice in group 1 only had slightly enlarged kidneys and no obvious intestinal lesions. The Kunming mice in group 6 had obvious intestinal bleeding, edema, and bloating, and congestion and edema of the spleen and kidneys, while the organ lesions in group 2 were relatively mild. The Kunming mice in group 7 had obvious intestinal edema and bleeding, and the congestion and edema of their spleen and kidneys were significantly more serious than those in group 3. The Kunming mice in group 3 had no intestinal bleeding, and their spleen and kidneys were close to those in groups 4 and 8. Fig.10 , Fig.12 , Fig.14 , Fig.16 .

[0149] Therefore, the type D Clostridium perfringens 21-D-5 toxoid vaccine prepared by the present invention has a better protective effect on the intestine of Kunming mice, and a slightly worse protective effect on the kidney and spleen than that on the intestine.

[0150] Pathological sections showed that the intestines of Kunming mice in group 5 showed obvious lesions, cell shedding, vacuoles, and defective integrity, while the situation of Kunming mice in group 1 was slightly better and the structure was relatively complete; the spleens of Kunming mice in group 5 had severe bleeding, while those in group 1 had almost no lesions and the overall structure was intact; the bleeding of Kunming mice in group 5 was severe, and the renal corpuscles were obviously edematous, while those in group 1 had milder lesions and the structure was intact. The intestinal structure of Kunming mice in group 6 was lost, accompanied by bleeding, while those in group 2 had almost no lesions; the spleens of Kunming mice in group 6 had severe bleeding, vacuoles, and unclear boundaries between the medulla and cortex, while those in group 2 had almost no lesions; the kidneys of Kunming mice in group 6 showed a large number of vacuolar degeneration, bleeding, and blurred renal corpuscle structure, while those in group 2 had milder kidney lesions and a small amount of bleeding. The intestinal structure of the 7th group of Kunming mice was blurred and there was bleeding, while the intestinal lesions of the 3rd group of Kunming mice were milder; the spleen of the 7th group of Kunming mice was severely bleeding and vacuoles appeared, while the intestinal cavitation of the 3rd group of Kunming mice was milder but the bleeding was milder; the kidney structure of the 7th group of Kunming mice was changed, bleeding, and vacuoles, while the intestinal lesions of the 3rd group of Kunming mice were milder and the structure was relatively intact. There were no obvious lesions in the 4th and 8th groups. Fig.11 , Fig.13 , Fig.15 , Fig.17 .

[0151] Example 4: Evaluation of the immune effect of Clostridium perfringens type D 21-D-5 toxoid vaccine on Guanzhong dairy goats.

[0152] 1. Experimental supplies

[0153] Guanzhong dairy goats: purchased from the Experimental Animal Center of Northwest Agriculture and Forestry University

[0154] Negative serum: Negative serum is serum collected directly from healthy Guanzhong dairy goats.

[0155] 2. Immunity experiment of Guanzhong dairy goats

[0156] Guanzhong dairy goats were divided into 4 groups, 3 in each group, 3 of which were injected with the D-type Clostridium perfringens 21-D-5 toxoid vaccine, with the immunization doses of 2, 1.5, and 1 mL / goat (actual protein concentrations of 3.38 mg / mL, 2.535 mg / mL, and 1.69 mg / mL), and the remaining group was injected with normal saline as a negative control, and a second immunization was performed at 14 days. Before the first immunization, venous blood was collected once, serum was separated, and the body temperature changes were observed for 7 consecutive days. During the immunization period, venous blood was collected and separated at 0d, 7d, 14d, 21d, and 28d.

[0157] Serum was collected from Guanzhong dairy goats in the immunization group and the blank control group at 0, 7, 14, 21, and 28 days after immunization, with 3 goats in each group. The collected serum was diluted with PBS and the antibody titer was detected by ELISA. The specific method was the same as in Example 3.

[0158] The temperature change trend of Guanzhong dairy goats after vaccination with the prepared vaccine is as follows: Fig.18 One day after vaccination, the body temperature of Guanzhong dairy goats increased by about 1.5℃, but it dropped and gradually returned to normal after 2 days ( Fig.18 ).

[0159] The results are as follows Fig.19 The results showed that the antibody level of Guanzhong dairy goats would increase with the increase of the vaccination dose of type D Clostridium perfringens 21-D-5 toxoid vaccine, but the titer of the serum of goats vaccinated with 2mL of type D Clostridium perfringens 21-D-5 toxoid vaccine was basically the same as that of the serum of goats vaccinated with 1.5mL of type D Clostridium perfringens 21-D-5 toxoid vaccine.

[0160] A group of Guanzhong dairy goat serum with the highest antibody titer was selected for antibody titer detection. The collected serum was diluted 100 times with PBS for sample incubation. First, 100 μL PBS was added to wells 2-7 of a 96-well plate, 200 μL diluted serum was added to well 1, and then 100 μL liquid in well 1 was drawn and added to well 2, and then diluted to well 7 in sequence. When it reached well 7, 100 μL serum was discarded, and the last column was a negative control. After adding the sample, incubate at 37°C for 1 hour. The remaining steps were the same as in Example 3.

[0161] 1.5 mL of immunized serum was selected for indirect ELISA to determine the antibody titer. The results showed that the positive antibody titer reached 1:800 on day 7 and 1:3200 on day 21, and then remained stable. Fig. 20 .

[0162] According to the results of the previous ELISA, a group of Guanzhong dairy goat serum with the best titer was selected for toxin neutralization test. The serum was diluted to 1:100, 1:200, 1:400, 1:600, and 1:800. Take 0.2mL of the Guanzhong dairy goat serum with the highest antibody titer and mix it with 2MLD of extracted toxin, make up the total volume to 1.0mL with sterile saline, and inject it intraperitoneally into 3 18-22g Kunming mice after 2h at 37℃, 0.5mL / mouse. Observe for 72h and count the number of deaths and survivals of Kunming mice. Neutralization titer means that 0.1mL of diluted serum can neutralize a minimum lethal dose of natural toxin. Even if all the injected Kunming mice survive, the maximum dilution multiple of the serum at this time is called the neutralization titer of the serum, and the unimmunized Guanzhong dairy goat serum is used as a negative control. According to the "Veterinary Dictionary of the People's Republic of China 2020 Edition", the vaccine can be judged as qualified if the serum neutralization titer against type D Clostridium perfringens toxin reaches 3 (0.1mL of immune animal serum neutralizes 3MLD toxin).

[0163] After the prepared D-type Clostridium perfringens 21-D-5 toxoid vaccine was used to immunize Guanzhong dairy goats, the antitoxin serum could be separated after blood collection. The neutralization titer of the antitoxin serum can more clearly reflect the protective power of the antitoxin serum. As shown in Table 5, the measured results show that the prepared antitoxin serum can neutralize 400 MLD.

[0164] Table 5 Neutralization titer of antitoxin serum

[0165] Antitoxin serum dilution multiple Number of Kunming rats (individuals) Number of deaths (nearly) mortality rate(%) 50 3 0 0 100 3 0 0 200 3 0 0 400 3 0 0 800 3 3 100

[0166] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0167] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A type D Clostridium perfringens ( Clostridium perfringens ) 21-D-5, characterized in that, The type D Clostridium perfringens 21-D-5 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on March 21, 2024, with the deposit number CGMCC No.30092.

2. A method for preparing exotoxin, characterized in that: The exotoxin is prepared from the type D Clostridium perfringens 21-D-5 of claim 1: the 21-D-5 strain is inoculated into a thioglycollate fluid culture medium for anaerobically culturing to obtain a FT bacterial culture solution, which is then inoculated into Gordon soup for anaerobically culturing to obtain a Gordon soup bacterial solution, and the supernatant is collected after the first centrifugal filtration to obtain a 21-D-5 toxin solution; Saturated ammonium sulfate solution was added to the 21-D-5 toxin solution until the ammonium sulfate reached 50% saturation, the precipitate was collected by centrifugation and dissolved in Trsi-HCL buffer, saturated ammonium sulfate solution was added again until the ammonium sulfate reached 40% saturation, the precipitate was collected by centrifugation for the second time and dissolved in Trsi-HCL buffer to obtain crude exotoxin.

3. The preparation method according to claim 2, characterized in that: The inoculation volume of FT bacterial culture into Gordon broth is 3-5%.

4. The preparation method according to claim 2, characterized in that: The centrifugation conditions for the first centrifugation and the second centrifugation are both 4°C, 8000-10000 r / min, 15-30 min.

5. Use of the D-type Clostridium perfringens 21-D-5 according to claim 1 in the preparation of products for preventing diseases caused by Clostridium perfringens.

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