CN116445373A - C clostridium perfringens toxigenic culture medium and preparation method and application thereof - Google Patents
C clostridium perfringens toxigenic culture medium and preparation method and application thereof Download PDFInfo
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- CN116445373A CN116445373A CN202310700911.4A CN202310700911A CN116445373A CN 116445373 A CN116445373 A CN 116445373A CN 202310700911 A CN202310700911 A CN 202310700911A CN 116445373 A CN116445373 A CN 116445373A
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- clostridium perfringens
- culture medium
- toxigenic
- toxin
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Links
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- 238000002360 preparation method Methods 0.000 title claims abstract description 54
- 230000001551 toxigenic effect Effects 0.000 title claims abstract description 46
- 231100000033 toxigenic Toxicity 0.000 title claims abstract description 44
- 239000003053 toxin Substances 0.000 claims abstract description 86
- 231100000765 toxin Toxicity 0.000 claims abstract description 86
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- 241001544469 Clostridium perfringens D Species 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of biological products, and particularly discloses a C clostridium perfringens toxigenic culture medium, and a preparation method and application thereof. The clostridium perfringens toxin-producing culture medium of the invention consists of water, monosaccharide, peptone, yeast extract powder, naCl and Na 2 HPO 4 ·12H 2 O、KH 2 PO 4 And a polysaccharide; the monosaccharide is glucose, and the polysaccharide is dextrin or crude dextrin. C-type pod produced by the inventionThe clostridium perfringens toxin medium has stable toxin producing effect, and the obtained clostridium perfringens toxin C has stable and stronger toxicity, and can be effectively used for preparing clostridium perfringens inactivated vaccines and testing the efficacy of related products.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a C clostridium perfringens toxigenic culture medium, a preparation method and application thereof.
Background
Clostridium disease of sheepClostridial Diseases of Sheep) Is prepared from clostridiumClostridium) A group of diseases caused by microorganisms. Comprises sheep epidemic diseaseBraxy, BradsotFrom clostridium putrefyingCl.septicumCause sheep intestinal toxemiaEnterotoxaemiaFrom Clostridium welchii Cl.perfringens，Also called as being caused by type D clostridium perfringens), and sudden sheep snipingStruckFrom clostridium welchiiCl.perfringensCause lamb dysenterylamb dysenteryFrom clostridium welchii type BCl.perfringensCause sheep black epidemic diseaseBlack diseaseFrom Clostridium novinarumClostridium novyiCaused) clostridium botulinum poisoningBotulismFrom clostridium botulinum type C, type DClostridium botulinumCaused), sudden death syndrome (caused by clostridium perfringens type a).
Clostridium is an anaerobic bacterial species, 60 species, more than 10 species of common pathogenic bacteria exist in soil, sewage and human and various animal excretions. Gram staining is positive and has flagella except for a few strains, and can move and form spores. Most strains can produce severe exotoxins, which are both the main factor of the pathogenesis and the main antigen, and after being converted into toxoids, can stimulate animals to produce antitoxin, and can be used for preventing corresponding clostridial diseases. The clostridium disease has rapid onset, short course of disease and high death rate. Livestock are usually found in cattle, sheep, pigs, birds, etc.
Clostridium perfringens C (Clostridium perfringens type C) is the main pathogen responsible for animal hemorrhagic, necrotic enteritis and enterotoxemia. Especially for young and young birds (especially newborn piglets), high pathogenicity and high mortality are easily caused. The main virulence factors are alpha, beta 1 and beta 2 toxins, which often cause sudden sheep sniping. Young sheep and adult sheep can both occur, but with most adult sheep, the sheep die within hours, showing the toxic blood symptoms of acute poisoning, and causing significant losses to the breeding industry.
When the forage grass is turned green, a large amount of concentrated feed is fed, daily ration is changed suddenly, trauma, management and adjustment, parasites or other abnormal conditions occur, an environment suitable for clostridium growth is created, and clostridium is propagated in a large amount and secretes strong exotoxins. Often clostridium infection has a poor prognosis, and often the animal dies when it is found. Since the success rate of treatment is low, the focus of the disease is on prevention, and extensive immunization can effectively reduce the loss caused by the disease.
At present, animal raw materials such as beef, fish meat, beef liver and the like are commonly used for preparing anaerobic meat liver and stomach enzyme digestion soup and fish liver and stomach enzyme digestion soup culture mediums, but the culture mediums are prepared by the method, the phenomenon of unstable toxigenic performance often occurs due to uneven quality of raw materials and large batch-to-batch differences, and particularly, the raising amount of dairy cows is rapidly increased along with the development of dairy industry, so that the quality of raw materials for preparing the vaccine culture mediums is reduced due to the fact that a large amount of obsolete dairy beef fills the market; meanwhile, the residual medicines in beef and beef liver can often inhibit bacterial growth, the quality of the vaccine is seriously affected by the above various conditions, and the increase of the epidemic of the clostridium in the beef and the sheep in recent years also reflects the decrease of the overall quality of the vaccine. In addition, the traditional culture medium has the disadvantages of complicated preparation process, long time consumption, more manpower, high price and low recovery rate, and also brings great waste and high cost to vaccine production, thus causing great trouble to vaccine production enterprises. The industry is in urgent need of a culture medium with convenient material taking, excellent quality, low cost and stable performance and a culture method which are suitable for the culture medium.
Disclosure of Invention
The invention aims to provide a C-type clostridium perfringens toxigenic culture medium with stable performance and low cost, and a preparation method and application thereof.
In order to achieve the object, the technical scheme of the invention is as follows:
a C clostridium perfringens toxin-producing culture medium is prepared from water, monosaccharide, peptone, yeast extract powder, naCl and Na 2 HPO 4 ·12H 2 O、KH 2 PO 4 And a polysaccharide; the monosaccharide is glucose, and the polysaccharide is dextrin or crude dextrin.
According to the invention, the research shows that when the clostridium perfringens C is cultured, the clostridium perfringens C produced by using specific monosaccharide and polysaccharide in combination with other proteins, buffer systems and trace element components has high toxicity, and the culture effect of each batch is stable, the cost is low, and the quality of the vaccine prepared by the clostridium perfringens C is high and stable.
The clostridium perfringens type B and C are two strains which are particularly similar in terms of growth and development, exotoxin generation and immunogenicity, and after the inventor groups obtain a new culture method of clostridium perfringens type B, further research shows that the addition of glucose can not improve the toxic effect, and finally the toxic level can be obviously reduced after the glucose is added into a culture medium of clostridium perfringens type B. However, surprisingly, when the culture of clostridium perfringens type C is carried out under conditions conventionally considered to be the same as clostridium perfringens type B, the addition of glucose to the culture medium can significantly enhance the toxic effect, which breaks the conventional knowledge of the culture of both bacteria (the same for the culture medium of both bacteria in the current national standard), and gives unexpected good results.
In addition, KH is added into the culture medium 2 PO 4 It is combined with Na 2 HPO 4 ·12H 2 O forms a strong phosphate buffer system, and can lead the basic bacteria number in the culture solution to reach a larger value and then lead the pH to drop (C clostridium perfringens has fast development and can produce gas and acid in the development process), thereby ensuring the realization of high toxin production level.
The clostridium perfringens type C toxigenic culture medium consists of a component A and a component B, wherein every 1000ml of the component A is prepared from the following raw materials in parts by weight: 15-25g of peptone; 2-4g of yeast extract powder; 2-4g of NaCl; na (Na) 2 HPO 4 ·12H 2 O 5-10g；KH 2 PO 4 5-10g; 5-10g of dextrin or crude dextrin; the balance being water; the component B is an aqueous solution of glucose, and the final concentration of the glucose in the clostridium perfringens type C toxigenic culture medium is 0.9-1.1% (preferably 0.98%).
Preferably, each 1000ml of the component A is prepared from the following raw materials in parts by weight: 20g of peptone; 3g of yeast extract powder; 3g of NaCl; na (Na) 2 HPO 4 ·12H 2 O 10g；KH 2 PO 4 10g; 10g of dextrin; the balance being water; the component B is an aqueous solution of glucose with the concentration of 50%.
In the invention, dextrin refers to biological reagent dextrin, and coarse dextrin refers to medicinal dextrin. The invention finds that when dextrin (biological reagent) is adopted as polysaccharide in the culture medium, the toxicity-producing culture effect is better.
The water used in the formula of the invention is water for injection.
The pH value of the clostridium perfringens toxin-producing culture medium is 7.8-8.2.
The invention also provides a method for preparing the clostridium perfringens type C virus-producing medium, which comprises the following steps: peptone, yeast extract, naCl, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 Mixing with water with the total volume of 80% of the formula, adjusting pH value, mixing with polysaccharide, adding the rest water to fix volume, and sterilizing to obtain a culture medium component A; preparing monosaccharide into aqueous solution, and sterilizing to obtain culture medium component B.
In the method, sodium hydroxide is adopted to adjust the pH value; and/or, the sterilization conditions of the culture medium component A are as follows: sterilizing with high pressure steam at 116 ℃ for 30 minutes; the sterilization conditions of the culture medium component B are as follows: and sterilizing with steam at 110deg.C for 30 min.
In the present invention, the pH adjustment may be performed using 2mol/L sodium hydroxide solution, and other suitable reagents may be used by those skilled in the art to adjust the pH of the above-mentioned culture medium, which are within the scope of the present invention.
As a specific embodiment, the invention provides a preparation method of the clostridium perfringens type C toxigenic medium, which comprises the following steps: the raw materials of peptone, yeast extract powder, naCl and Na for preparing the culture medium 2 HPO 4 ·12H 2 O、KH 2 PO 4 Adding into 80% of water for injection according to weight ratio to dissolve, regulating pH value to 7.8-8.2, adding dextrin or coarse dextrin according to weight ratio to fix volume, sterilizing under high pressure, preparing anhydrous glucose into 50% (V/V) water solution separately, sterilizing, and adding into required concentration before use to obtain the culture medium.
The clostridium perfringens toxigenic medium prepared by the method has the following beneficial effects: the culture medium preparation and sample adding sequence effectively ensures complete dissolution of nutrient substances in a visible state; the pH value is regulated after substances except dextrin or crude dextrin and anhydrous glucose in the raw materials are dissolved, the pH value is regulated, the constant volume does not change the pH value of the finally obtained culture medium, and the obtained solution has strong buffer capacity, so that the accuracy of the final proportioning volume can be ensured, the accurate regulation of the pH value is facilitated, and the relatively stable pH value in the use process of the culture medium is facilitated; meanwhile, as the dextrin or the crude dextrin belongs to starch reagents and has an adhesive effect on the pH meter, the pH is adjusted before the dextrin or the crude dextrin is added, so that the protection of the pH meter and the accurate adjustment of the pH value are facilitated. Meanwhile, glucose is sensitive to high temperature, carbonization can be caused by too high sterilization temperature, and the use efficiency of glucose is ensured by adopting independent preparation and sterilization.
The invention also provides a method for preparing the clostridium perfringens type C exotoxin, which comprises the step of fermenting clostridium perfringens type C by using the clostridium perfringens type C toxin-producing culture medium or the clostridium perfringens type C toxin-producing culture medium prepared by the method.
In the method of the invention, the inoculum size of the fermentation is 1-1.5% (v/v), provided that: culturing at 36-37deg.C for 12-14 hr.
The inventor unexpectedly discovers that if the fermentation culture time taught by the prior art is 10-16h, the finally obtained 10h fermentation culture time and the C clostridium perfringens toxin virulence under the condition of 16h fermentation culture time are not ideal in measurement, the 10h fermentation culture cannot meet the virulence standard regulated by 0.001-0.0025ml of C clostridium perfringens toxin in the three-combined inactivated vaccine for the epidemic disease, the sudden-death sniper and the enterotoxemia of sheep specified in the ' biological product code for livestock ' 2000 edition of China ' after 10h fermentation culture, and the 16h fermentation culture can reach the lower limit of the virulence standard of 0.0025ml, but the inactivation, purification and concentration losses are added, so that the immune effect is lost. Therefore, the inventor selects the fermentation culture time to be 12-14h through experiments, and the toxicity of the obtained clostridium perfringens C type toxin reaches or even exceeds the specified standard.
During fermentation, the primary seed liquid, the secondary seed liquid or the tertiary seed liquid of C clostridium perfringens is adopted as zymophyte; the preparation of the first-stage seed liquid adopts sugar-free anaerobic meat liver soup as a culture medium, the preparation of the second-stage seed liquid adopts anaerobic meat liver soup as a culture medium, and the preparation of the third-stage seed liquid adopts the C-type clostridium perfringens toxin-producing culture medium or the C-type clostridium perfringens toxin-producing culture medium prepared by the method as a culture medium;
and/or, the method further comprises the steps of centrifuging the obtained fermentation liquor after fermentation is completed, and filtering the centrifugated supernatant; preferably, the centrifugation is carried out at 3000-4000rpm for 20-30min and the filtration is carried out with a 0.22 μm filter.
As a specific embodiment, the present invention provides a method for preparing clostridium perfringens type C exotoxin, said method comprising the steps of:
seed liquid culture: inoculating clostridium perfringens type C into sugar-free anaerobic liver soup, and standing at 36-37deg.C for 10-16 hr to obtain clostridium perfringens type C seed solution;
fermentation culture: inoculating the seed solution into the clostridium perfringens type C toxin-producing culture medium for culture, collecting the culture, centrifuging, collecting supernatant and filtering, wherein the obtained filtrate is clostridium perfringens type C toxin.
In the preparation method of the clostridium perfringens type C exotoxin, the seed liquid of clostridium perfringens type C can be a primary seed liquid or a secondary seed liquid; the preparation method of the first-stage seed liquid comprises the following steps: inoculating clostridium perfringens C into sugar-free anaerobic liver soup, standing at 36-37deg.C for 10-16 hr, and performing pure detection to obtain first-stage seed solution; the preparation method of the secondary seed liquid comprises the following steps: inoculating the first-stage seed liquid into a 350ml/500ml bottle of anaerobic meat liver soup according to the inoculum size of 1-1.5% (v/v), standing at 36-37 ℃ for culturing for 10-16h, and obtaining a second-stage seed liquid after pure inspection; the second-level seed liquid can be inoculated into the clostridium perfringens C-type toxigenic culture medium component A, meanwhile, a sterile glucose solution (component B) with the concentration of 50% and accounting for 2% of the volume of the culture medium component A is added aseptically, the culture medium is placed at 37 ℃ for static culture for 12-16 hours, and then the third-level seed liquid is obtained after pure inspection. The seed liquid obtained by continuous expansion culture can also be used as seed liquid for producing C clostridium perfringens exotoxin by fermentation culture, and is also within the protection scope of the invention.
In the preparation method of the clostridium perfringens type C exotoxin, the inoculation amount of clostridium perfringens type C inoculation and the inoculation amount of the seed liquid inoculation are both 1-1.5% (v/v).
In the above-described method for producing clostridium perfringens type C exotoxin, the fermentation culture may be carried out in 500ml bottles or fermenters.
The invention further provides an application of the method in preparing a vaccine containing clostridium perfringens type C exotoxin.
It is still another object of the present invention to provide a clostridium perfringens type C exotoxin prepared according to the method for preparing clostridium perfringens type C exotoxin described above using the clostridium perfringens type C exotoxin culture medium described above. The method can be used for the triple inactivated vaccine of sheep fast epidemic, sudden sniper, lamb dysentery and enterotoxemia, the triple four-proofing inactivated vaccine of sheep fast epidemic, sudden sniper, lamb dysentery and enterotoxemia, the serum neutralization titer determination and animal toxicity attack protection test in the efficacy test of the multi-linked dry powder vaccine of sheep clostridial disease and the red diarrhea inactivated vaccine of piglets.
The use of the clostridium perfringens type C exotoxin described above for the preparation of a medicament or vaccine for the prevention of diseases associated with clostridium perfringens is also within the scope of the present invention. The application of toxoid prepared by inactivating and detoxication of the clostridium perfringens type C exotoxin in preparation of medicaments or vaccines for preventing related diseases caused by clostridium perfringens also belongs to the protection content of the invention. The related diseases are selected from any one or combination of sheep fast epidemic, sudden sniper, enterotoxemia, red diarrhea of piglets and necrotic enteritis of birds, and the vaccine is selected from at least one of sheep fast epidemic, sudden sniper, enterotoxemia triple inactivated vaccine and red diarrhea inactivated vaccine of piglets.
Still another object of the present invention is to provide an inactivated clostridium perfringens type C vaccine prepared by inactivating and detoxication of a culture obtained by the clostridium perfringens type C culture method.
The vaccine is prepared by uniformly mixing C clostridium perfringens culture solution after inactivation and detoxification with aluminum hydroxide gel according to the volume ratio of 5:1.
The vaccine is prepared by uniformly mixing an inactivated and detoxified culture (containing D clostridium perfringens inactivated and detoxified bacterial solution, C clostridium perfringens inactivated and detoxified bacterial solution and putrefying clostridium inactivated and detoxified bacterial solution according to the proportion of 1:1:1) and sterile aluminum hydroxide gel according to the volume ratio of 5:1. The exotoxin toxicity before the bacterial liquid inactivation and detoxification of the clostridium perfringens D is less than or equal to 1 MLD and less than or equal to 0.00025ml, the exotoxin toxicity before the bacterial liquid inactivation and detoxification of the clostridium perfringens C is less than or equal to 1 MLD and less than or equal to 0.0008ml, and the exotoxin toxicity before the bacterial liquid inactivation and detoxification of the clostridium putrefying is less than or equal to 1 MLD and less than or equal to 0.005ml.
By adopting the scheme, the C-type clostridium perfringens toxin production medium prepared by the method has stable toxin production performance, does not contain drug residues carried by traditional medium raw materials and the like, has no inhibition effect on the cultured clostridium perfringens bacteria, and meanwhile has the advantages of simple preparation method, convenient material obtaining, excellent quality and low cost, and compared with toxin virulence effect prepared by the traditional medium, the C-type clostridium perfringens toxin obtained by the method has more stable toxin virulence effect, and the vaccine prepared by the method has higher quality. The method has the following advantages:
(1) The invention uses commercial peptone, yeast extract powder and other finished products as raw materials to replace the original quality uncontrollable beef, beef liver and other raw materials, and the invention can be used for preparing veterinary C clostridium perfringens toxin by screening the formula of the culture medium and the optimal content of each component and optimizing the toxigenic culture condition, so that the toxigenic capacity of the culture medium reaches or even exceeds the original regulation standard and almost has no batch difference.
(2) The culture medium obtained by screening has stronger toxin producing capability no matter being cultured by a 500ml bottle or a fermentation tank, has stable toxin producing capability and controllable quality, is convenient to prepare and use and has low price.
(3) The effect of the C clostridium perfringens inactivated vaccine prepared by the invention is evaluated on rabbits and sheep. As a result, the neutralization titer of the serum of the triple inactivated vaccine for the fast epidemic, the sudden sniper and the enterotoxemia of sheep prepared by the invention reaches or even exceeds the corresponding standard of the regulations.
(4) The toxin-producing culture medium and the preparation method thereof have the advantages of convenient material acquisition, simple preparation, omitting the digestion processes of the anaerobic-meat-liver-stomach enzyme digestion soup and the fish-liver-stomach enzyme digestion soup, greatly shortening the preparation time and reducing the cost by 1/5 of the traditional anaerobic-meat-liver-stomach enzyme digestion soup. The highest toxin power of the clostridium perfringens C prepared by the method can be 4.165 times of the seedling preparation standard of China veterinary biological product code. In addition, the neutralizing titer of the inactivated vaccine prepared by the method in the corresponding serum of rabbits and sheep is also improved to 3-4 times of the rule standard. Meanwhile, the toxigenic culture medium does not contain animal raw material residues and drug residues, so that the pressure of developing and producing low-immune-dose toxoid inactivated vaccine in purification and concentration is reduced. The invention can be used for replacing the existing anaerobic-meat-liver-stomach enzyme digestion soup and fish-liver-meat-stomach enzyme digestion soup culture medium, is used for producing clostridium ovine multi-linked inactivated vaccine, and has wide prospect.
(5) After the clostridium perfringens exotoxin C prepared by the method or the clostridium perfringens exotoxin C prepared by the method is converted into toxoid after being inactivated, the centrifugate is concentrated by an ultrafiltration concentration system of 3-5KD, and the recovery rate of more than 90% can be achieved, so that the clostridium perfringens exotoxin C is used for preparing vaccines with low immune dose.
Detailed Description
Aiming at the defects of inconvenient material taking, complicated preparation process, time and labor consumption, high cost, low toxin producing capability, unstable toxin producing effect and the like of the culture medium raw materials such as anaerobic liver and stomach enzyme digestion soup and the like suitable for C clostridium perfringens in the prior art, the invention provides the toxin producing culture medium which is more suitable for the culture of the C clostridium perfringens, has the advantages of convenient material taking, simple and quick preparation method and low cost, has high toxin virulence and stable toxin producing effect of the C clostridium perfringens obtained by fermentation culture, and can be widely popularized and used.
Meanwhile, based on the C-type clostridium perfringens toxin-producing culture medium provided by the invention, the C-type clostridium perfringens exotoxin with higher virulence level is provided by the invention, and based on the exotoxin, the C-type clostridium perfringens inactivated vaccine is obtained after inactivation and detoxification, and the serum neutralization titer reaches or even exceeds the corresponding standard of the regulations.
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.
The clostridium perfringens C strain adopted in the invention is clostridium perfringens C59-2 strain for animals, and the strain numbers CVCC60102 and 2006.09.29 are purchased from the China center for type culture Collection of veterinary medicine inspection.
The dextrins used in the examples are biological agents, in particular biological agents dextrins from the company Beijing Oboc Biotechnology, inc., cat#01-034, which is white or off-white amorphous powder, has no odor and is slightly sweet. Technical standard: qualified water insoluble substances, ethanol solubility less than or equal to 1.0%, drying weight loss less than or equal to 10%, burning residue less than or equal to 0.5%, chloride (Cl) less than or equal to 0.002%, phosphate (SO) 4 ) Less than or equal to 0.002%, oxalate (C) 2 O 4 ) Less than or equal to 0.05 percent, ferric salt (Fe) less than or equal to 0.005 percent, heavy metal (calculated by Pb) less than or equal to 0.001 percent, reducing sugar less than or equal to 5.0 percent, and molecular formula (C) 6 H 10 O 5 )a·XH 2 O, execution standard QB/ABX 01-034-2005.
The crude dextrins used in the examples were medicinal dextrins, performing the standard: four parts of the 2015 edition of Chinese pharmacopoeia (specifically, medicinal dextrin from Shandong chat Anhua pharmaceutical Co., ltd.).
The anaerobic meat liver soup mentioned in the invention is prepared according to the composition and preparation method of China veterinary biological product regulations, and the specific composition is shown in the following table 1:
table 1 composition of anaerobic liver soup
The preparation method comprises the following steps:
1. taking beef to remove fat and fascia, mincing with a meat mincer, mixing with liver blocks cut into about 100g, adding distilled water, stirring thoroughly, and cold soaking for 20-24 hr.
2. Boiling for 20-60 min, supplementing water, filtering with white cloth, removing meat residue, and taking out liver block.
3. Adding peptone and sodium chloride into the filtrate, heating to melt, adjusting pH to 7.8-8.0 with sodium hydroxide solution, and boiling for 10-20 min.
4. Filtering with filter paper or flannelette, adding glucose, stirring, and thawing.
5. The filtered liver block is washed, cut into small blocks, fully washed by distilled water and then packaged in test tubes or neutral glass bottles, the amount of which is about 1/10 of the expected packaged meat liver soup amount.
6. Packaging the filtrate into neutral container (such as test tube, and optionally adding appropriate amount of liquid paraffin), and sterilizing with steam at 116 deg.C under high pressure for 30-40 min.
7. The application is as follows: for culturing and testing general avermectin. When used for strain preservation, glucose is not added.
The components and preparation methods of the diluents (peptone water) used in the examples are shown in the following Table 2, and are described in "China animal biological preparation protocol":
TABLE 2 peptone Water Components
The preparation method comprises the following steps:
1. mixing the above materials, heating for dissolving, and adjusting pH to 6.8-7.2 with sodium hydroxide solution.
2. Boiling, filtering, and packaging in neutral container.
And autoclaving at 3.116 ℃for 20 minutes.
4. The application is as follows: for sterile test dilutions (peptone 1 g) and bacterial count dilutions and sugar fermentation (peptone 10 g).
The anaerobic meat liver gastric enzyme digestion soup mentioned in the invention is prepared according to the composition and preparation method of China veterinary biological product regulations, and the specific composition is shown in the following table 3:
table 3 composition of decoction for digestion of liver and stomach by anaerobic fermentation
The preparation method comprises the following steps:
1. adding hydrochloric acid and minced beef and liver into 65deg.C warm water, stirring, adding pepsin, stirring, and mixing at 56-58 deg.C.
2. Digestion is carried out at 53-55 ℃ for 22-24 hours, and stirring is carried out fully every hour for the first 10 hours.
3. Extracting supernatant, heating to 80deg.C, adding peptone, boiling, and adjusting pH to 7.6-7.8. Boiling for 10 min, filtering or precipitating to obtain supernatant, adding dextrin, dissolving, and packaging.
4.116℃and steam sterilization for 40 minutes.
5. The application is as follows: for use in the manufacture of clostridium vaccines.
Comparative example C evaluation of the culture Effect of Clostridium perfringens in traditional anaerobic meat liver gastric enzyme digestion soup
1. Strain culture and toxin preparation:
opening an ampoule containing freeze-dried clostridium perfringens strains with good vacuum degree by a sterile burst method, extracting a proper amount of sterile nutrient broth to dissolve freeze-dried bacterial blocks, inoculating the freeze-dried bacterial blocks into a 6-branch tube of sugar-free anaerobic clostridium perfringens soup with the inoculum size of 1-1.5% (v/v), standing at 36-37 ℃ for 10-16h, and performing pure detection on the culture, wherein the pure culture is first-grade seed liquid.
Inoculating the first-stage seed solution into a 350ml/500ml bottle anaerobic meat liver gastric enzyme digestion soup culture medium according to the inoculum size of 1-1.5% (v/v), standing at 36-37 ℃ for culturing for 12-16h, collecting the culture, and centrifuging at 3000rpm for 30min; collecting supernatant, filtering with 0.22 μm filter membrane, sampling, sterilizing, and testing.
2. Toxin Effect assay
The inventor uses traditional anaerobic meat liver gastric enzyme digestion soup to prepare C clostridium perfringens toxin, and performs 28 batches of experiments, the toxin measuring method is described in example 3, and the toxin measuring effect is shown in the following table 4:
TABLE 4 toxicity test results of C Clostridium perfringens in traditional anaerobic liver and stomach enzyme digestion soups
As can be seen from the above Table 4, the effect of preparing C-type clostridium perfringens toxin by the conventional anaerobic-meat-liver-stomach-enzyme digestion broth culture is poor, and in the 28 batches of experiments, only 4 batches are qualified, and up to 24 batches are unqualified, so that the conventional anaerobic-meat-liver-stomach-enzyme digestion broth culture medium is unstable in toxin production effect and poor in effect. The inventor prepares 2 batches of sheep triple inactivated vaccine by adopting qualified batch inactivated detoxicated bacteria liquid. Detection result: wherein the neutralization titer of 1 batch of type C serum is 1 MLD, and the batch is judged to be in compliance with the regulations. The other 1 lot of type C serum had a neutralization titer < 1 MLD and was judged to be out of specification (see description of example 4 for vaccine formulation and detection methods). Therefore, the quality of the vaccine prepared from the traditional anaerobic meat liver and stomach enzyme digestion soup culture medium is not stable enough.
Example 1C Clostridium perfringens toxigenic Medium Screen
1. Preparation of C-type clostridium perfringens toxigenic culture medium
Formula 1: preparation of C clostridium perfringens toxigenic culture medium
1. 800ml of water for injection, 20g of peptone, 3g of yeast extract powder, 3g of sodium chloride and Na are added 2 HPO 4 ·12H 2 O 10g，KH 2 PO 4 10g, and dissolved.
2. The pH was adjusted to 7.8-8.2 with 2mol/L NaOH.
3. After adding 10g of crude dextrin, the volume of the water for injection was set to 1000ml. Sterilizing with high pressure steam at 116 ℃ for 30 minutes to obtain a culture medium component A for standby.
4. And (3) preparing a 50% glucose solution (V/V) by taking an appropriate amount of anhydrous glucose, and sterilizing the glucose solution by high-pressure steam at 110 ℃ for 30 minutes to obtain a culture medium component B for later use.
Formula 2: preparation of C clostridium perfringens toxigenic culture medium
1. 800ml of water for injection, 20g of peptone, 3g of yeast extract powder, 3g of sodium chloride and Na are added 2 HPO 4 ·12H 2 O 10g，KH 2 PO 4 10g, and dissolved.
2. The pH was adjusted to 7.8-8.2 with 2mol/L NaOH.
3. After adding 10g of dextrin (biological agent), the volume of the water for injection was set to 1000ml. Sterilizing with high pressure steam at 116 ℃ for 30 minutes to obtain a culture medium component A for standby.
4. And (3) preparing a 50% glucose solution (V/V) by taking an appropriate amount of anhydrous glucose, and sterilizing the glucose solution by high-pressure steam at 110 ℃ for 30 minutes to obtain a culture medium component B for later use.
2. Bacterial culture and toxin preparation
The ampoule containing freeze-dried clostridium perfringens strain with good vacuum degree is opened by a sterile burst method, a proper amount of sterile nutrient broth is extracted to dissolve freeze-dried bacteria blocks, inoculated into a 6-branch tube of sugar-free anaerobic liver soup, and placed at 37 ℃ for static culture for 16 hours. Meanwhile, the pure detection is carried out, and the pure seed liquid is the first-level seed liquid.
Inoculating the first-stage seed solution into C clostridium perfringens toxigenic medium component A in a 350ml/500ml bottle according to an inoculum size of 1% (v/v), aseptically adding 7.0ml of 50% concentration sterile glucose solution (component B), standing at 37 ℃ for 12 hours, collecting the culture, and centrifuging, wherein the centrifuging speed can be 3000rpm, and the time can be 30 minutes; collecting supernatant, filtering with 0.22 μm filter membrane, sampling, sterilizing, and testing.
3. The results of comparing the toxic effects of two clostridium perfringens type C toxic culture media are shown in table 5 below.
TABLE 5 comparison of the toxicity effects of two C-type Clostridium perfringens toxicity culture media
The data in Table 5 shows that the toxicity producing effect of the dextrin (biological reagent) of the formula 1 or the dextrin (biological reagent) of the formula 2 can reach the level of less than or equal to 0.0025ml of the mouse 1 MLD regulated by the veterinary biological product code by proportioning sterile glucose with the final concentration of 1 percent, compared with the result in a comparative example, the toxicity producing effect of the culture medium is more stable, the selection of the raw materials of the culture medium is widened, the selection of the raw materials of the culture medium is not limited to beef, beef and the like which are used in the traditional soup culture medium for the anaerobic meat liver and stomach enzyme digestion, the raw materials which are difficult to store and have high cost and inconvenient to obtain are not only easy to obtain the raw materials, but also the preparation process is simple and convenient, the time for preparing the culture medium is greatly saved, and the cost is reduced to 1/5 of the traditional soup for the anaerobic meat liver and stomach enzyme digestion. In addition, the formulation 1 using the crude dextrin has the same toxicity effect as the formulation 2 using the dextrin (biological agent), two times of the three experiments have the same toxicity effect according to the data of the above table 5, and one formulation 2 is better than the formulation 1, so that the formulation 2 has the relatively better effect of the dextrin (biological agent) group, the dextrin (biological agent) is adopted in the formulation as a preferred embodiment, and the subsequent expansion culture experiments all adopt the dextrin (biological agent) as the component of the culture medium to prepare the clostridium perfringens type C toxicity producing culture medium of the present invention.
EXAMPLE 2 preparation of C clostridium perfringens toxigenic Medium
C clostridium perfringens toxigenic medium was prepared and evaluated for toxigenic effect according to the following formulation shown in Table 6 using the same preparation method as formulation 2 in example 1.
Table 6C Clostridium perfringens toxin-producing medium formulation (1000 ml)
Note that: and (3) preparing a 50% glucose solution (V/V) by taking a proper amount of anhydrous glucose, and sterilizing for 30 minutes at 110 ℃ by high-pressure steam for later use.
In this example, the inventors prepared clostridium perfringens type C clostridium perfringens toxigenic medium for later use by the method described in example 1 according to the formulation listed in table 6 above, then opened a vacuum-friendly ampoule containing lyophilized clostridium perfringens type C strain in a sterile burst method, extracted an appropriate amount of sterile nutrient broth to dissolve the lyophilized pellet, inoculated in a sugarless anaerobic clostridium perfringens soup 6-branch tube, and placed in a stationary culture at 37 ℃ for 16 hours. Meanwhile, the pure detection is carried out, and the pure seed liquid is the first-level seed liquid.
Inoculating the first-level seed solution into C clostridium perfringens toxigenic medium component A prepared according to formulas 3, 4 and 5 in a triangular flask with the inoculum size of 1% (v/v) of 350ml/500ml respectively, simultaneously aseptically adding 7.0ml of 50% concentration sterile glucose solution (component B) respectively, standing at 37 ℃ for 12 hours, sampling, aseptically detecting to meet the regulations, and then evaluating the toxigenic effect. The results show that the toxicity effects of the culture media prepared by the culture media formulas 3, 4 and 5 listed in the above table 6 can reach similar toxicity effects as those of the culture media prepared by the culture media formula 2 in the above example 1, and the results of two parallel experiments are shown in table 7.
TABLE 7C results of the toxicity formulation of Clostridium perfringens toxicity production Medium (350 ml/500 ml)
Example 3 preparation of C clostridium perfringens exotoxin and evaluation of toxic Effect
1. Preparation of C clostridium perfringens secondary seed liquid exotoxin and evaluation of toxic Effect Using formulation 2 in example 1
Inoculating the first-stage seed solution into 350ml/500ml bottle of anaerobic meat liver soup according to the inoculum size of 1-1.5% (v/v), standing at 37deg.C for 16 hr, and taking the qualified product as the second-stage seed solution.
Inoculating qualified second-level seed solution into C clostridium perfringens toxigenic medium component A40000 ml according to inoculum size of 1.5% (v/v), adding 800ml sterile glucose solution (component B) with 50% concentration aseptically, standing at 37deg.C for 10-16h.
Sampling in 10h, 12h, 14h and 16h respectively, centrifuging sample bacterial liquid at 3000rpm/min for 30min, extracting supernatant, filtering with a 0.22 μm filter to obtain clostridium perfringens exotoxin C, diluting, testing toxin, and determining virulence of toxin in bacterial liquid in different time periods on mice to determine optimal toxin producing time.
The filtered toxin was taken and diluted with 1% peptone water as shown in the following Table 8, and 16-20g KM mice were injected into the tail vein, 2 mice were injected per titer, 0.2ml each, and the death of the mice was observed within 24 hours after the injection. The minimum toxin amount capable of enabling the mice to die by 2/2 is the MLD of C clostridium perfringens toxin to the mice. The results of the 1 st and 2 nd experiments (the 1 st and 2 nd are two parallel experiments for verifying the toxigenic stability) are shown in tables 9 and 10 below, wherein 0/2+ indicates that 2 mice were injected and 0 mice were dead; 1/2+ means that 2 mice were injected and 1 died. 2/2+ means that 2 mice were injected and 2 had died.
TABLE 8 toxin dilution route
TABLE 9 results of the 1 st experiment toxigenic (40000 ml Scale)
As can be seen from table 9 above: culturing for 10h to determine that the virulence 1MLD is less than or equal to 0.01ml; culturing for 12h, sampling and measuring virulence 1MLD less than or equal to 0.0006ml; culturing for 14h, sampling and measuring virulence 1MLD less than or equal to 0.001ml; culturing for 16h, sampling and measuring virulence 1MLD less than or equal to 0.002ml.
Table 10 results of the 2 nd experiment (40000 ml Scale)
As is clear from Table 10, the virulence 1MLD was measured to be not more than 0.01ml after 10 hours of cultivation; culturing for 12h, sampling and measuring virulence 1MLD less than or equal to 0.0006ml; culturing for 14h, sampling and measuring virulence 1MLD less than or equal to 0.002ml; culturing for 16h, sampling and measuring virulence 1MLD less than or equal to 0.0025ml.
2. Preparation of C-type clostridium perfringens three-level seed liquid exotoxin and toxin production effect evaluation
Inoculating 350ml/500ml bottle of anaerobic liver soup to the first-stage seed liquid according to the inoculation amount of 1%, standing at 37 ℃ for 16 hours, and taking the qualified product as the second-stage seed liquid after pure inspection.
Inoculating 8000ml/10000ml bottle C clostridium perfringens toxigenic medium component A with the second level seed liquid according to the inoculation amount of 1.5%, aseptically adding 160ml 50% concentration aseptic glucose solution (component B), standing at 37 ℃ for 12h, and obtaining the third level seed liquid after pure inspection.
Inoculating qualified three-stage seed solution into 400000ml of clostridium perfringens C toxigenic culture medium component A according to the inoculum size of 1.5%, adding sterile glucose solution (component B) with concentration of 50% of 800L in a sterile way, and standing at 37 ℃ for 10-16h.
Sampling after culturing for 10h, 12h, 14h and 16h, centrifuging for 30min at 3000rpm/min, collecting supernatant, filtering with 0.22 μm filter to obtain toxin, and diluting for testing. And determining the toxicity of toxin in bacterial liquid cultured for different time periods to mice so as to determine the optimal toxin production time.
The filtered toxin was taken and diluted with 1% peptone water as shown in Table 8 above, and 16-20gKM mice were injected into the tail vein, 2 mice per titer, 0.2ml each, and the death of the mice was observed within 24 hours after injection. The minimum toxin amount capable of enabling the mice to die by 2/2 is the MLD of C clostridium perfringens toxin to the mice. The results of the 3 rd experiment are shown in Table 11 below.
Table 11 results of the 3 rd experiment (400000 ml Scale)
As is clear from Table 11, the virulence 1MLD was measured to be not more than 0.01ml by culturing for 10 hours; culturing for 12h, sampling and measuring virulence 1MLD less than or equal to 0.0008ml; culturing for 14h, sampling and measuring virulence 1MLD less than or equal to 0.002ml; culturing for 16h, sampling and measuring virulence 1MLD less than or equal to 0.0025ml.
As shown by the results, the C clostridium perfringens toxin obtained by fermentation culture is 1MLD which is less than or equal to 0.0008ml when the fermentation culture time is 12h, and the toxicity of the C clostridium perfringens toxin obtained by fermentation culture exceeds the toxicity standard specified by the national veterinary biological product regulations of the people's republic of China (hereinafter referred to as the procedure) for 2000 edition, namely, the standard of 0.001-0.0025ml of the C clostridium perfringens toxin in the triple inactivated vaccine of sheep epidemic disease, sudden sniper and enterotoxemia. When the fermentation culture time is 14h and 16h, the toxicity of the C-type clostridium perfringens toxin obtained by fermentation culture is 1MLD less than or equal to 0.0025ml, and the toxicity standard of 0.001-0.0025ml of the C-type clostridium perfringens toxin in the triple inactivated vaccine for sheep fast epidemic, sudden sniping and enterotoxemia specified in the procedure 2000 edition is achieved. In combination with the subsequent toxin production and utilization conditions, the fermentation culture time is preferably 12-14h when the clostridium perfringens type C is fermented and cultured, so that the virulence standard specified in the code can be met or exceeded, the toxin production effect of the clostridium perfringens type C toxin production medium is stable, the obtained clostridium perfringens type C toxin is stable and strong, and the clostridium perfringens type C toxin can be effectively used for preparing clostridium perfringens type C inactivated detoxicated vaccines.
Example 4 preparation and efficacy evaluation of a triple inactivated vaccine against fast epidemic, sudden sniper and enterotoxemia in sheep
1. Preparation of a triple inactivated vaccine for sheep fast epidemic, sudden sniping and enterotoxemia:
cultures of various bacteria (clostridium perfringens type D, clostridium perfringens type C, clostridium putref) previously cultured (clostridium perfringens type C cultures were prepared in example 3), 0.8% formaldehyde solution (40% concentration) was added by volume, and the mixture was inactivated at 37 ℃ for 3-4 days (stirring 4 times/day). Inoculating the inactivated detoxified bacterial liquid into the anaerobic meat-liver soup, TSB and casein peptone agar slant, observing the aseptic growth for 5 days, and indicating complete inactivation; and meanwhile, the inactivated bacterial solutions are centrifuged at 3000rpm/min for 30min, bacterial precipitate is discarded, the supernatant is sucked, a 0.22 mu m filter membrane is used for filtering, 16-20 KM mice are injected into tail veins respectively, each of the mice is injected with 0.4ml, and the observation of the total health care time for 24 hours shows that the detoxification is complete.
And (3) taking all bacterial solutions with complete inactivation and detoxification, filtering the bacterial solutions in a sterilization container filled with 200-mesh copper yarns, and storing the bacterial solutions at 2-8 ℃ for later use.
Placing proper amount of aluminum hydroxide gel into glass bottle, adding proper amount of water for injection (sterilization evaporation amount), and sterilizing.
Taking a proper amount of inactivated and detoxified completely D-type clostridium perfringens bacterial liquid (D, 1 MLD is less than or equal to 0.00025 ml) stored at 2-8 ℃, C-type clostridium perfringens bacterial liquid (C, 1 MLD is less than or equal to 0.0008 ml) and putrefying clostridium bacterial liquid (S, 1 MLD is less than or equal to 0.005 ml), respectively placing the clostridium perfringens bacterial liquid and the clostridium perfringens bacterial liquid into a sterilizing glass bottle, and respectively adjusting pH to 6.8-7.2 by using 2mol/L sterile sodium hydroxide solution. Adding the mixture into a glass bottle filled with sterilized aluminum hydroxide gel according to the proportion of D to C to S=1 to 1. The ratio of the bacterial liquid to the aluminum gel is 5:1. Shaking and mixing uniformly to prepare the triple inactivated vaccine for the epidemic, sudden sniping and enterotoxemia of sheep. And (5) storing at 2-8 ℃ for standby.
2. Safety and efficacy evaluation of sheep fast epidemic, sudden sniping and enterotoxemia triple inactivated vaccine:
16 white rabbits of 1.5-2 kg Beijing big ear were prepared, of which 4 were used for collecting negative control serum and the other 12 were used for safety and efficacy evaluation.
Safety test in Beijing white rabbits:
and (3) taking 4 prepared triple inactivated vaccines, and subcutaneously injecting 1.5-2 kg of Beijing big ear white rabbits at the neck and back, wherein the ratio is 5 ml/rabbit. The results are shown in Table 12 below, after 10 days of observation.
Table 12 safety evaluation results of triple inactivated vaccine for fast epidemic, sudden sniping and enterotoxemia of sheep
From table 12 above, 4 white rabbits with Beijing big ears injected are healthy and alive within 10 days of observation, and necrosis reaction is not found at the injection part, which indicates that the safety test of the prepared triple inactivated vaccine for the fast epidemic, the sudden sniping and the enterotoxemia meets the regulations.
Evaluation of efficacy of sheep fast epidemic, sudden sniping and enterotoxemia triple inactivated vaccine on white Beijing big ear rabbits at 3 ml/immunization dose:
and taking 8 prepared triple inactivated vaccines, and subcutaneously injecting 1.5-2 kg of Beijing big ear white rabbits at the neck and back, wherein the ratio is 3 ml/rabbit. 4 negative control rabbits and immunized rabbits were randomly collected from their middle ear artery for 4 animals 21 days after immunization, and an equal amount of mixed serum was prepared, and 0.4ml of the mixed serum was obtained, and the serum neutralization titers (0.1 ml of serum neutralization mice MLD) of the immunized rabbits against each type of clostridium perfringens toxin and clostridium putref toxin were measured per 0.1ml of the mixed serum. The negative control group was tested by taking 0.6ml of the mixed serum. The results are shown in Table 13 below.
Table 13 triple inactivated vaccine for sheep fast epidemic disease, sudden sniping and enterotoxemia at 3 ml/immunization dose
Evaluation results of efficacy on white rabbits in Beijing
According to the regulations of biological products for China animal in 2000 edition: the neutralizing titer of the immune rabbits per 0.1ml of mixed serum on clostridium perfringens type C toxins reaches 1 mouse MLD, and the immune rabbits are judged to be qualified. According to the data in Table 13, it is shown that the three-combined inactivated vaccine for epidemic, sudden sniping and enterotoxemia of sheep prepared by using the C clostridium perfringens inactivated bacterial liquid obtained by fermenting and culturing the toxigenic culture medium in the embodiment has the highest neutralization titer of rabbit serum reaching 3 mice MLD and reaches or exceeds the standard of China veterinary biological product code. The culture medium has low cost, stable toxicity, and reduced or eliminated unqualified bacterial liquid in large-scale production.
Evaluation of efficacy of sheep fast epidemic, sudden sniping and enterotoxemia triple inactivated vaccine on sheep at 3 ml/immunization dose:
10 healthy sheep with the age of 6-12 months and the weight of 30-40kg and without corresponding neutralizing antibodies are selected, wherein 4 of the healthy sheep are used for collecting negative control serum, and the other 6 healthy sheep are used for immune injection, neck intramuscular injection and 3 ml/healthy sheep. 21 days after immunization, 4 negative control sheep and 4 randomly extracted immunized sheep were subjected to jugular vein blood collection respectively, equal amounts of mixed serum were prepared, 0.4ml of the mixed serum was taken respectively, and neutralization titers (0.1 ml of serum neutralization mice MLD) of each type of clostridium perfringens toxin and clostridium putrefsum serum were determined for each 0.1ml of the mixed serum of the immunized sheep. The negative control group was tested by taking 0.6ml of the mixed serum. The results are shown in Table 14 below.
Table 14 triple inactivated vaccine for sheep fast epidemic disease, sudden sniping and enterotoxemia at 3 ml/immunization dose
Efficacy evaluation results in sheep
The data in Table 14 above shows that the neutralizing titer of the immunized sheep per 0.1ml of the mixed serum against clostridium perfringens type C and clostridium putref toxin serum is not less than 1 MLD, and the neutralizing titer against clostridium perfringens type D toxin serum is not less than 3MLD.
According to the regulations of biological products for China animal in 2000 edition: and (3) the triple inactivated vaccine for the rapid epidemic disease, the sudden sniper and the enterotoxemia of sheep is 5 ml/dose, and the neutralizing titer of type C and type S serum is not lower than 1 MLD, and the type D serum is not lower than 3MLD, so that the vaccine is qualified. According to the data in the table 14, it can be known that the vaccine prepared by the clostridium perfringens type C inactivated bacteria liquid prepared by the clostridium perfringens type C attenuated bacteria liquid of the present invention has fast epidemic, sudden sniper and enterotoxemia triple inactivated vaccine, and the 3 ml/immune dose reaches or exceeds the rule standard, which proves that the clostridium perfringens type C attenuated bacteria liquid prepared by the present invention has strong toxin producing effect, and the vaccine prepared by the clostridium perfringens type C attenuated bacteria liquid has high quality, can be effectively used for large-scale vaccine production and application, and has stable and controllable quality.
Evaluation of efficacy of sheep fast epidemic, sudden sniping and enterotoxemia triple inactivated vaccine on sheep at 5 ml/immunization dose:
10 healthy sheep with the age of 6-12 months and the weight of 30-40kg and without corresponding neutralizing antibodies are selected, wherein 4 of the healthy sheep are used for collecting negative control serum, and the other 6 healthy sheep are used for immune injection, neck intramuscular injection and 5 ml/healthy sheep. 21 days after immunization, 4 negative control sheep and 4 randomly extracted immunized sheep were subjected to jugular vein blood collection respectively, equal amounts of mixed serum were prepared, 0.4ml of the mixed serum was taken respectively, and neutralization titers (0.1 ml of serum neutralization mice MLD) of each type of clostridium perfringens toxin and clostridium putrefsum serum were determined for each 0.1ml of the mixed serum of the immunized sheep. The negative control group was tested by taking 0.6ml of the mixed serum. The results are shown in Table 15 below.
Table 15 triple inactivated vaccine for sheep fast epidemic disease, sudden sniping and enterotoxemia at 5 ml/immunization dose
Efficacy evaluation in sheep
The data in Table 15 above shows that the neutralizing titer of the immunized sheep per 0.1ml of the mixed serum against clostridium perfringens type C toxin serum is not less than 1MLD, the neutralizing titer against clostridium putref toxin serum is not less than 1MLD, and the neutralizing titer against clostridium perfringens type D toxin serum is not less than 3MLD.
According to the regulations of biological products for China animal in 2000 edition: and (3) the triple inactivated vaccine for the rapid epidemic disease, the sudden sniper and the enterotoxemia of sheep is 5 ml/sheep immune dose, and the neutralization titer of type C and type S serum is not lower than 1MLD, and the type D serum is not lower than 3MLD, so that the vaccine is qualified. As can be seen from the data in the table 15, the vaccine prepared from the clostridium perfringens C inactivated bacteria liquid prepared by the toxigenic medium of the invention has the advantages of fast epidemic, sudden sniping and enterotoxemia combined inactivated vaccine, and the immune dosage of 5 ml/vaccine reaches or exceeds the rule standard. The culture medium has low cost, stable toxicity, and reduced or eliminated unqualified bacterial liquid in large-scale production. The C-type clostridium perfringens toxin-producing culture medium has better toxin-producing effect, and the vaccine prepared by the C-type clostridium perfringens toxin-producing culture medium has higher quality, can be effectively used for large-scale vaccine production application, and has stable and controllable quality.
Meanwhile, the preparation method of clostridium perfringens toxigenic medium of the present invention also has advantages as shown in table 16 below compared with the preparation method of medium in the prior art, wherein the comparison is made by taking 10000ml of medium as an example.
TABLE 16 comparison of parameters related to the conventional process for the inventive process (10000 ml)
Comparative example 1
The research shows that the addition of maltose in the culture of anaerobic bacteria is favorable to fermentation production. Further, the comparative example provides a C clostridium perfringens toxigenic culture medium and effect verification thereof. The formulation and preparation method of the specific clostridium perfringens type C toxigenic medium were substantially the same as the formulation 2 described in example 1, except that glucose was replaced with maltose. The culture medium was used for strain culture and toxin preparation by the method described in example 3, and the toxin production effect was tested. The final results are shown in Table 17.
TABLE 17 toxicity results (350 ml/500ml bottle Scale)
As can be seen from table 17 above: culturing for 10h to determine virulence 1MLD >0.01ml; culturing for 12h, sampling and measuring virulence 1MLD less than or equal to 0.0025ml; culturing for 14h, sampling and measuring virulence 1MLD less than or equal to 0.01ml; culturing for 16h, sampling and measuring virulence 1MLD >0.01ml.
Comparative example 2
This comparative example provides a clostridium perfringens toxigenic medium and validation of its effects. The formulation and preparation method of the specific clostridium perfringens type C toxigenic medium are substantially the same as the formulation 2 described in example 1, except that the component B, a glucose solution, is not contained. The culture medium was used for strain culture and toxin preparation by the method described in example 3, and the toxin production effect was tested. The final results are shown in Table 18.
TABLE 18 toxicity results (350 ml/500ml bottle Scale)
As can be seen from table 18 above: samples were taken for 10h, 12h, 14h, 16h and assayed for virulence 1MLD >0.01ml.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A C clostridium perfringens toxin-producing culture medium is characterized by comprising water, monosaccharide, peptone, yeast extract powder, naCl and Na 2 HPO 4 ·12H 2 O、KH 2 PO 4 And a polysaccharide; the monosaccharide is glucose, and the polysaccharide is dextrin or crude dextrin.
2. Clostridium perfringens type C toxigenic medium according to claim 1 wherein said clostridium perfringens type C toxigenic medium consists of component a and component B, each 1000ml of said component a being made of the following raw materials in weight ratio:
15-25g of peptone; 2-4g of yeast extract powder; naCl 2-4g；Na 2 HPO 4 ·12H 2 O 5-10g；KH 2 PO 4 5-10g; 5-10g of dextrin or crude dextrin; the balance being water;
the component B is an aqueous solution of glucose, and the final concentration of the glucose in the clostridium perfringens type C toxigenic culture medium is 0.9-1.1%.
3. Clostridium perfringens toxigenic medium according to claim 2 wherein per 1000ml of said component a is made from the following raw materials in weight ratio:
20g of peptone; 3g of yeast extract powder; 3g of NaCl; na (Na) 2 HPO 4 ·12H 2 O 10g；KH 2 PO 4 10g; 10g of dextrin; the balance being water; the component B is an aqueous solution of glucose with the concentration of 50%.
4. A clostridium perfringens type C toxigenic medium according to any of claims 1-3 wherein said clostridium perfringens type C toxigenic medium has a pH value of 7.8-8.2.
5. A method of preparing the clostridium perfringens type C toxigenic medium of any one of claims 1-4 comprising: peptone, yeast extract, naCl, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 Mixing with water with the total volume of 80% of the formula, adjusting pH value, mixing with polysaccharide, adding the rest water to fix volume, and sterilizing to obtain a culture medium component A; preparing monosaccharide into aqueous solution, and sterilizing to obtain culture medium component B.
6. The method of claim 5, wherein the pH is adjusted with sodium hydroxide;
and/or, the sterilization conditions of the culture medium component A are as follows: sterilizing with high pressure steam at 116 ℃ for 30 minutes; the sterilization conditions of the culture medium component B are as follows: and sterilizing with steam at 110deg.C for 30 min.
7. A method for preparing clostridium perfringens type C exotoxin, characterized in that the clostridium perfringens type C fermentation is performed with clostridium perfringens type C toxigenic medium according to any one of claims 1-4 or clostridium perfringens type C toxigenic medium prepared by the method of claim 5 or 6.
8. The method of claim 7, wherein the fermentation is inoculated in an amount of 1-1.5% (v/v), provided that: culturing at 36-37deg.C for 12-14 hr.
9. The method according to claim 8, wherein the fermentation is performed using a primary seed solution, a secondary seed solution or a tertiary seed solution of clostridium perfringens C as the fermentation bacteria; the preparation of the first-stage seed liquid adopts sugar-free anaerobic meat liver soup as a culture medium, the preparation of the second-stage seed liquid adopts anaerobic meat liver soup as a culture medium, and the preparation of the third-stage seed liquid adopts C-type clostridium perfringens toxin-producing culture medium according to any one of claims 1-4 or C-type clostridium perfringens toxin-producing culture medium prepared by the method of claim 5 or 6 as a culture medium;
and/or, the method further comprises the steps of centrifuging the obtained fermentation liquor after fermentation is completed, and filtering the centrifugated supernatant; the centrifugation conditions were 3000-4000rpm for 20-30min, and the filtration was performed with a 0.22 μm filter.
10. Use of the method of any one of claims 7-9 for the preparation of a vaccine comprising clostridium perfringens type C exotoxin.
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