CN107875377B - Preparation method of clostridium perfringens type E toxoid vaccine - Google Patents
Preparation method of clostridium perfringens type E toxoid vaccine Download PDFInfo
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- A61K39/02—Bacterial antigens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
Abstract
The invention discloses a preparation method of an E-type clostridium perfringens toxoid vaccine, which comprises the following steps: (1) inoculating the clostridium perfringens strain E on a sterile blood plate culture medium, carrying out anaerobic culture, and recovering the strain; (2) inoculating the recovered Clostridium perfringens type E strain into a liquid thioglycollate fluid culture medium, and performing strain increasing under an anaerobic condition to prepare a seed solution; (3) inoculating the seed solution into a toxin-producing culture medium, and carrying out anaerobic culture to prepare an exotoxin solution; (4) and inactivating and emulsifying the prepared exotoxin solution, and adding thimerosal to prepare the E-type clostridium perfringens toxoid vaccine. The invention develops and prepares the E-type clostridium perfringens toxoid vaccine with strong immunity and no toxic or side effect. The vaccine can effectively prevent diseases such as dysentery of calves and lambs caused by the bacteria.
Description
Technical Field
The invention relates to the technical field of veterinary biological products, in particular to a preparation method of an E-type clostridium perfringens toxoid vaccine.
Background
Clostridium perfringens, old known as clostridium welchii, is classified into five toxin types a, B, C, D, E according to the 4 major exotoxins (α, β, E, iota) it produces. Bohenel states: the pathogenic factors of the clostridium welchii are that the clostridium welchii forms secreted toxin and metabolite, and no clinical symptoms can be generated when the toxin is generated. Clostridium perfringens type E, one of the members of this bacterium, secretes mainly alpha and iota exotoxins. Although there are few typical symptoms associated with clostridium perfringens type E disease in animal production, there are reports that clostridium perfringens type E accounts for 5% of all clostridium perfringens isolates, while clostridium perfringens type E5% can be associated with bovine lethal hemolytic enteritis of 50%. And the clostridium perfringens type E mainly attacks ruminants such as lambs, calves and the like. In the uk, the occurrence of iota toxin enterotoxemia was reported in calves and lambs at the beginning of the 20 th century, at which time a certain number of bovine haemolytics, necrotic enteritis, i.e. dysentery, had been reported, and type E strains and iota toxins were also detected from the diseased sheep and cattle. Clostridium perfringens type E is a relatively large proportion of the isolated strains in brazil, and iota toxin is carried in nearly 30% of samples in healthy and diarrheal cattle, of which 9% of cattle undergo sudden death. Meanwhile, the type E bacteria can also infect a plurality of species, and the type E clostridium perfringens strain is also separated from goats, deer and wild pigs with clinical symptoms of enterotoxemia. Therefore, in order to prevent the potential harm of the clostridium perfringens type E to calves and lambs, research on vaccines of the clostridium perfringens type E is necessary.
The main exotoxin formed by clostridium perfringens is a protein which has better immunogenicity. Therefore, according to the pathogenesis of clostridium perfringens, different types of clostridium perfringens toxins are prepared, and are inactivated to be prepared into toxoid vaccines for livestock and poultry prevention and treatment, so that reliable immunization and treatment effects can be achieved. There are currently reports on the preparation of clostridium perfringens type a-D toxoid vaccines, but there has been no report on the preparation of clostridium perfringens type E toxoid vaccines.
Disclosure of Invention
In view of the prior art, the invention aims to provide a preparation method of a clostridium perfringens type E toxoid vaccine.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a clostridium perfringens type E toxoid vaccine comprises the following steps:
(1) inoculating the clostridium perfringens strain E on a sterile blood plate culture medium, carrying out anaerobic culture, and recovering the strain;
(2) inoculating the recovered Clostridium perfringens type E strain into a liquid thioglycollate fluid culture medium, and performing strain increasing under an anaerobic condition to prepare a seed solution;
(3) inoculating the seed solution into a toxin-producing culture medium, carrying out anaerobic culture, and carrying out filtration sterilization to prepare an exotoxin solution;
(4) and inactivating and emulsifying the prepared exotoxin solution, and adding thimerosal to prepare the E-type clostridium perfringens toxoid vaccine.
Preferably, in step (1), the clostridium perfringens Type E strain is a standard strain, Type E NCTC8084(National Collection of Type Cultures, british culture Collection). The strain can be directly obtained through conventional channels in the prior art. After a plurality of tests, the strain has the best poison producing effect compared with other clostridium perfringens type E strains.
Preferably, in step (1), the sterile blood plate culture medium is prepared by the following method:
dissolving 3.8g blood agar base and 1g glucose in 100mL deionized water, autoclaving at 121 deg.C for 15min, cooling to 50 deg.C, adding 5mL defibered sheep blood, shaking, and pouring into sterilized plate.
Preferably, in step (1), the temperature of anaerobic culture is 37 ℃, and the time of anaerobic culture is 36 h.
Preferably, in the step (2), the strain is increased by anaerobic culture at 41 ℃ for 18 h. The conditions for the strain increasing culture of different types of clostridium perfringens are obviously different, and aiming at the clostridium perfringens type E, the temperature and time conditions for the strain increasing culture are optimized and investigated, and the result shows that the growth of the clostridium perfringens type E is not facilitated due to overhigh temperature or overlow temperature, and the strain increasing effect is not obvious due to too short culture time; the long culture time can affect the exotoxin production in the later period of the strain. When the culture temperature is 41 ℃ and the culture time is 18h, the bacterium increasing effect is optimal.
Preferably, in step (3), the toxin-producing medium is prepared by the following method:
dissolving 2g of peptone, 1g of dextrin, 2g of yeast extract and 1.2g L-arginine in 100mL of PBS buffer solution, adjusting the pH value to 7.5 by using concentrated hydrochloric acid, and carrying out autoclaving to obtain the peptone.
Preferably, in step (3), the anaerobic culture conditions are as follows: anaerobic shaking culture at 43 deg.C for 6 h.
Further preferably, in the anaerobic culture, the gas composition under anaerobic conditions is, in terms of volume fraction: 88% N2、5％CO2、7％H2。
The preparation of exotoxin solution by anaerobic culture in a toxin-producing culture medium is a key step for preparing the clostridium perfringens type E toxoid vaccine, because under the same adjuvant condition, the immune effect of the vaccine is always in direct proportion to the toxin content, and the higher the toxin content is, the better the vaccine effect is.
As the conditions of the clostridium perfringens secreted toxins of different types are different and difficult to refer to each other, aiming at the preparation of the clostridium perfringens exotoxin solution of type E, the invention explores the nutrient components, the pH condition, the temperature and the time of anaerobic culture, the gas composition of the anaerobic condition and the like of a toxin production culture medium, and finds that the toxin production culture medium particularly meets the requirements of the clostridium perfringens secreted toxins of type E on the nutrient components. The pH conditions are also critical to affect toxin production, with highest toxin production at pH 7.5 and lower or higher pH than 7.5 reducing toxin production. More importantly, the temperature and time conditions of anaerobic culture are that although the culture temperature is increased in the range of suitable temperature for bacterial growth to be beneficial to the growth of thalli, the growth of thalli and the toxin production of bacteria belong to different stages, the culture temperature most beneficial to the growth of thalli can not necessarily obtain the highest toxin yield, and multiple experiments show that the highest toxin yield can be obtained by anaerobic oscillation culture at 43 ℃ for 6 hours. In addition, the gas composition of the anaerobic environment can influence the growth and toxin secretion of the clostridium perfringens type E, and if the gas components are changed, such as the proportion of the mixed gas components is changed, the clostridium perfringens type E can not grow, and simultaneously can not secrete toxin.
Preferably, in the step (4), the inactivation is performed by the following method: adding formaldehyde into exotoxin solution to make its final concentration be 0.3%, placing at 37 deg.C, fully oscillating, rotating speed is 140rmp, inactivating for 24 h.
The exotoxin secreted by clostridium perfringens of different types has different treatment conditions for inactivating the exotoxin solution, the effect of inactivating the exotoxin solution is mainly related to the concentration, temperature, inactivation time and the like of formaldehyde, and the high-concentration formaldehyde and high-temperature inactivation are adopted, so that although the inactivation time can be reduced, the loss of antigens is larger; the inactivation time is too short, so that the inactivation is insufficient, the inactivation time is too long, on one hand, the cost is increased, and on the other hand, the antigen is lost. The inactivation condition is optimized by comprehensively considering the inactivation efficiency and the influence on the antigenicity, and the result shows that the optimal inactivation effect can be obtained by using 0.3% of formaldehyde and inactivating at 37 ℃ for 24 hours.
Preferably, in the step (4), white oil adjuvant is added into the inactivated exotoxin solution for emulsification, and the volume ratio of the exotoxin solution to the white oil adjuvant is 1: 2.
Preferably, the white oil adjuvant consists of: 12 parts of white oil and 1 part of Span-80 are mixed, and then aluminum stearate accounting for 1.5 percent of the total mass of the mixed liquid of the white oil and the Span-80 is added; the white oil contains 10-20% by weight of alkanes with normal carbon number of C16-C20. Through a plurality of experiments, the vaccine prepared by the white oil adjuvant with the structure has the highest antibody titer and the minimum side effect.
The invention has the beneficial effects that:
the invention develops and prepares the E-type clostridium perfringens toxoid vaccine with strong immunity and no toxic or side effect for the first time. The vaccine can effectively prevent diseases such as dysentery of calves and lambs caused by the bacteria.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. Among them, blood agar base, thioglycollate fluid medium (FT), yeast extract, etc. are all conventional commercially available products.
Example 1: preparation of E-type clostridium perfringens toxoid vaccine for calf and lamb dysentery
The method comprises the following specific steps:
(1) preparation of exotoxin: taking a preservation solution of the clostridium perfringens strain NCTC8084(National Collection of Type Cultures8084) of the E Type by a pipette, coating the preservation solution on a sterile blood plate culture medium (the culture medium of the sterile blood plate is 3.8g of blood agar base, 1g of glucose is dissolved in 100mL of deionized water, sterilizing at 121 ℃ for 15min under high pressure, cooling to about 50 ℃, adding 5mL of defibered sheep blood, shaking uniformly, pouring into a sterilized plate), and carrying out anaerobic culture at 37 ℃ for 36 h. Inoculating 5-10 colonies with sterile inoculating loop in sterile fluid culture medium (FT), and anaerobically culturing at 41 deg.C for 18h to increase bacteria. Taking E8084 seed liquid after culture according to the ratio of 7: 100 volume ratio of the cells were inoculated into a self-made sterile toxigenic medium (toxigenic medium: 2g peptone, 1g dextrin, 2g yeast extract, 1.2g L-arginine dissolved in 100mL PBS buffer, pH adjusted to 7.5 with concentrated hydrochloric acid, autoclaving), anaerobic at 43 deg.C (gas composition: 88% N)2，5％CO2，7％H2) Shaking and culturing for 6 h. Centrifuging the culture solution at 4 deg.C and 10000rpm for 30min, collecting supernatant, and filtering with bacteria filter to remove bacteria to obtain exotoxin.
(2) Inactivation of exotoxin: adding formaldehyde into the prepared exotoxin to make the final concentration be 0.3%, adjusting pH to 7.5, placing at 37 deg.C, fully oscillating at 140rpm, and inactivating for 24 hr to obtain completely inactivated exotoxin.
(3) Preparing a toxoid vaccine: emulsifying the inactivated toxoid and the white oil adjuvant in a volume ratio of 1:2, adding thimerosal to enable the final concentration to be 0.01%, and fully emulsifying to obtain the toxoid vaccine.
Example 2:
(1) security detection
About 3 rabbits (1.5 kg) were injected subcutaneously with 5mL of the toxoid vaccine (prepared in example 1) and observed continuously for 10 days, showing that the injection site was free from swelling, granuloma, and the like.
(2) Vaccine efficacy test
6 Kunming mice with the weight of about 22g are divided into two groups, 0.2mL of toxoid vaccine (prepared in example 1) and 0.2mL of normal saline are respectively injected into the two groups, after 28 days, 0.3mL of minimum lethal dose E type clostridium perfringens toxin is injected into the two groups, the mice in a control group die completely after observation for 5 days; 3 mice in the immune group are not killed, and are kept to be observed for 5 days, so that the mice are healthy and survive without disease symptoms such as dysentery and the like. The vaccine of the invention is proved to have 100 percent of protection rate on experimental animals.
Comparative example 1:
taking a preservation solution of the clostridium perfringens strain NCTC8084(National Collection of Type Cultures8084) of the E Type by a pipette, coating the preservation solution on a sterile blood plate culture medium (the culture medium of the sterile blood plate is 3.8g of blood agar base, 1g of glucose is dissolved in 100mL of deionized water, sterilizing at 121 ℃ for 15min under high pressure, cooling to about 50 ℃, adding 5mL of defibered sheep blood, shaking uniformly, pouring into a sterilized plate), and carrying out anaerobic culture at 37 ℃ for 36 h. Inoculating 5-10 colonies with sterile inoculating loop in sterile fluid culture medium (FT), and anaerobically culturing at 37 deg.C for 12 hr for enrichment. Taking E8084 seed liquid after culture according to the ratio of 7: 100 volume ratio of the cells were inoculated into a self-made sterile toxigenic medium (toxigenic medium: 2g peptone, 1g dextrin, 2g yeast extract, 1.2g L-arginine dissolved in 100mL PBS buffer, pH adjusted to 7.4 with concentrated hydrochloric acid, autoclaving), anaerobic at 40 deg.C (gas composition: 88% N)2，5％CO2，7％H2) Shaking and culturing for 5 h. Centrifuging the culture solution at 4 deg.C and 10000rpm for 30min, collecting supernatant, and filtering with bacteria filter to remove bacteria to obtain exotoxin.
Comparative example 2:
taking a preservation solution of the clostridium perfringens strain NCTC8084(National Collection of Type Cultures8084) of the E Type by a pipette, coating the preservation solution on a sterile blood plate culture medium (the culture medium of the sterile blood plate is 3.8g of blood agar base, 1g of glucose is dissolved in 100mL of deionized water, sterilizing at 121 ℃ for 15min under high pressure, cooling to about 50 ℃, adding 5mL of defibered sheep blood, shaking uniformly, pouring into a sterilized plate), and carrying out anaerobic culture at 37 ℃ for 46 h. Inoculating 5-10 colonies with sterile inoculating loop in sterile fluid culture medium (FT), and anaerobically culturing at 38 deg.C for 14 hr for enrichment. Collecting cultured E8084 speciesThe seed liquid is prepared according to the following steps of 7: 100 volume ratio of the cells were inoculated into a self-made sterile toxigenic medium (toxigenic medium: 2g peptone, 1g dextrin, 2g yeast extract, 1.2g L-arginine dissolved in 100mL PBS buffer, pH adjusted to 7.4 with concentrated hydrochloric acid, autoclaving), anaerobic at 38 deg.C (gas composition: 85% N)2，9％CO2，6％H2) Shaking and culturing for 5 h. Centrifuging the culture solution at 4 deg.C and 10000rpm for 30min, collecting supernatant, and filtering with bacteria filter to remove bacteria to obtain exotoxin.
Comparative example 3:
taking a preservation solution of the clostridium perfringens strain NCTC8084(National Collection of Type Cultures8084) of the E Type by a pipette, coating the preservation solution on a sterile blood plate culture medium (the culture medium of the sterile blood plate is 3.8g of blood agar base, 1g of glucose is dissolved in 100mL of deionized water, sterilizing at 121 ℃ for 15min under high pressure, cooling to about 50 ℃, adding 5mL of defibered sheep blood, shaking uniformly, pouring into a sterilized plate), and carrying out anaerobic culture at 37 ℃ for 36 h. Inoculating 5-10 colonies with sterile inoculating loop in sterile fluid culture medium (FT), and anaerobically culturing at 39 deg.C for 12 hr for enrichment. Taking E8084 seed liquid after culture according to the ratio of 7: 100 volume ratio of the cells were inoculated into a self-made sterile toxigenic medium (toxigenic medium: 2g peptone, 1g dextrin, 2g yeast extract, 1.2g L-arginine dissolved in 100mL PBS buffer, pH adjusted to 7.4 with concentrated hydrochloric acid, autoclaving), anaerobic at 45 deg.C (gas composition: 88% N)2，5％CO2，7％H2) Shaking and culturing for 4 h. Centrifuging the culture solution at 4 deg.C and 10000rpm for 30min, collecting supernatant, and filtering with bacteria filter to remove bacteria to obtain exotoxin.
The exotoxins prepared in example 1 and comparative examples 1 to 3 were each diluted and injected into tail vein of mice weighing 16 to 18g, 2 mice were injected per one drop, and 0.2ml of each mouse was injected, and the mice were observed for death within 72 hours after injection. The minimum amount of toxin that caused 2/2 death in the mice was recorded. As a result, it was found that the minimum toxin amounts of the exotoxins prepared in comparative examples 1 to 3 were 2.4 times, 5.6 times and 3.8 times as large as those of example 1, respectively. The difference of the toxin production amount of the clostridium perfringens type E is obvious under different culture conditions, and the toxin production amount of the clostridium perfringens type E is the highest when the clostridium perfringens type E is cultured by the method.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (5)
1. A preparation method of a clostridium perfringens type E toxoid vaccine is characterized by comprising the following steps:
(1) inoculating the clostridium perfringens strain E on a sterile blood plate culture medium, carrying out anaerobic culture, and recovering the strain;
(2) inoculating the recovered Clostridium perfringens type E strain into a liquid thioglycollate fluid culture medium, and performing strain increasing under an anaerobic condition to prepare a seed solution;
(3) inoculating the seed solution into a toxin-producing culture medium, carrying out anaerobic culture, and carrying out filtration sterilization to prepare an exotoxin solution;
(4) inactivating and emulsifying the prepared exotoxin solution, and adding thimerosal to prepare the E-type clostridium perfringens toxin vaccine;
in the step (1), the sterile blood plate culture medium is prepared by the following method:
dissolving 3.8g of blood agar base and 1g of glucose in 100mL of deionized water, sterilizing at 121 ℃ for 15min under high pressure, cooling to 50 ℃, adding 5mL of defibered sheep blood, uniformly shaking, and pouring into a sterilized plate to obtain the feed additive;
in the step (3), the toxin-producing culture medium is prepared by the following method:
dissolving 2g of peptone, 1g of dextrin, 2g of yeast extract and 1.2g L-arginine in 100mL of PBS buffer solution, adjusting the pH value to 7.5 by using concentrated hydrochloric acid, and performing autoclaving to obtain the extract;
during anaerobic culture, the gas composition under anaerobic conditions is as follows according to volume fraction: 88% N2、5％CO2、7％H2；
In the step (4), the inactivation adopts the following method: adding formaldehyde into exotoxin solution to make its final concentration be 0.3%, placing at 37 deg.C, fully oscillating, rotating speed is 140rmp, inactivating for 24 h.
2. The method according to claim 1, wherein the temperature of the anaerobic culture in the step (1) is 37 ℃ and the time of the anaerobic culture is 36 hours.
3. The method according to claim 1, wherein the step (2) comprises culturing the microorganism anaerobically at 41 ℃ for 18 hours.
4. The method according to claim 1, wherein in the step (3), the anaerobic culture conditions are: anaerobic shaking culture at 43 deg.C for 6 h.
5. The method according to claim 1, wherein in step (4), the inactivated exotoxin solution is emulsified by adding a white oil adjuvant, and the volume ratio of the exotoxin solution to the white oil adjuvant is 1: 2.
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| CN108892725A (en) * | 2018-04-18 | 2018-11-27 | 山东农业大学 | A kind of preparation method of E type C.perfringens antitoxic serum |
| CN110314227A (en) * | 2019-06-20 | 2019-10-11 | 青海省畜牧兽医科学院 | A kind of preparation method of the overworked macaque vaccine of sheep |
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| CN103160555A (en) * | 2013-03-19 | 2013-06-19 | 武汉中博生物股份有限公司 | Culture medium, culture method and application of high-yield exotoxin of clostridium perfringens |
| CN103432579B (en) * | 2013-09-12 | 2015-02-25 | 山东农业大学 | Preparation method of lamb-dysentery clostridium perfringens type B toxoid vaccine |
| CN103432578B (en) * | 2013-09-12 | 2015-03-25 | 山东农业大学 | Preparation method of C-type clostridium perfringens toxin vaccine of piglet red dysentery |
| CN104829712B (en) * | 2015-04-23 | 2018-03-27 | 山东农业大学 | C, D types C.perfringens antitoxic serum and preparation method thereof |
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