CN110904007B - Animal clostridium novyi exotoxin, preparation method thereof, toxigenic culture medium and application - Google Patents

Abstract

The invention discloses clostridium novyi exotoxin, a preparation method thereof, a toxigenic culture medium and application. Wherein each 1000ml of the toxigenic culture medium is prepared from the following raw materials in parts by weight: 15-25g of peptone, 5-10g of tryptone and 5-10g of casein peptone; yeast extract powder 1-3g, naCl 1-3g, KH ₂ PO ₄ 5-10g, 5-10g of dextrin or maltose, 95-105g of stainless iron nails or scrap iron and the balance of water for injection. The toxicity of the clostridium novyi exotoxin liquid prepared by the method can be up to 1MLD which is less than or equal to 0.00008ml, and the toxicity is higher than the standard 6.25 times. And the neutralizing titer of the sheep black epidemic and fast epidemic bivalent inactivated vaccine prepared by the method in the corresponding serum of the rabbit and sheep is also up to or higher than the relevant rule standard.

Description

Animal clostridium novyi exotoxin, preparation method thereof, toxigenic culture medium and application

Technical Field

The invention belongs to the field of veterinary biological products, and in particular relates to a toxin production culture medium of veterinary clostridium novyi exotoxins, a method for preparing the veterinary clostridium novyi exotoxins by using the culture medium, and application of the veterinary clostridium novyi exotoxins in preparation of veterinary vaccines.

Background

Clostridium ovine disease (Clostridial Diseases of Sheep) is a type of disease caused by Clostridium microorganisms. Including fast sheep epidemic (caused by clostridium putrefum cl. Septicum), enterotoxemia (Enteromorpha, caused by clostridium welchii cl. Perfringens, also known as clostridium perfringens type D), sudden sheep sniping (Struck, caused by clostridium welchii cl. Perfringens type C), lamb dysentery (lamb dysentery, caused by clostridium welchii type B. Perfringens), black disease (Black disease, caused by clostridium novinai Clostridium novyi), botulism (Botulism, caused by clostridium botulinum type C, type D Clostridium botulinum), sudden sheep death (caused by clostridium perfringens type a).

Wherein Clostridium novyi is a gram positive E.coli, a strict anaerobe. The bacteria are widely present in the soil. When sheep feed on forage grass and feed contaminated with Clostridium novinarum spores, the spores enter the liver from the gastrointestinal wall. Normal liver is unfavorable for germination to become propagule due to high oxidation-reduction potential, and still remains in the liver in spore form. When the liver is damaged by immature wandering fasciola hepatica and its oxidation-reduction potential is reduced, the spore existing in the place can obtain proper growth condition, and can quickly grow and reproduce to produce toxin, and can be introduced into blood circulation to produce toxemia so as to damage neuron and other cells, and can result in acute shock and death. The strain is characterized by causing liver necrosis in sheep bodies and blood vessels under the skin of the sheep dying from illness to be engorged with blood, thereby blackening the epidermis, and being more obvious especially at hairless places. So that it is called "sheep black epidemic" or "infectious necrotic hepatitis of sheep".

The disease mainly occurs in low-lying humid areas where fasciola hepatica prevails in spring and summer rainy seasons, and is endemic, and sheep and goats can be infected by the disease. Most of the sheep with disease are fat sheep with good nutrition, and sheep aged 2-4 years are most susceptible to infection. Goats can also develop infections, lambs and 1 year old sheep are not normally infected, and cattle can also be infected.

The sick sheep usually develop an acute pattern, i.e., they are suddenly ill. Most of the sheep die suddenly without any clinical symptoms, so that the sheep cannot be found, the duration of the disease is short, and only a few of the sheep can last for 1-2 days. The sick sheep mainly shows mental depression, inappetence, independent and outlier, unstable walking, and no force of limbs in the later period, can only lie on the ground without rising, and has obviously raised body temperature which can reach about 41.5 ℃. Congestion occurs in some conjunctiva, the heartbeat is accelerated, and dyspnea occurs. Some sick sheep have abdominal pain, a small amount of foam flows out from the corners of the mouth, falls into comatose in prone position, and dies in situ without struggling before dying. Some sick sheep showed everything normal in the evening, but were dead in the colony house the next early morning. The sick sheep usually die after 2 hours of onset, and the maximum rate of illness is only 24 hours, and the death rate can reach 100%. The sick sheep rarely generates chronic type, and the chronic type mainly shows listlessness, weak constitution, obvious enlargement caused by accumulation of a large amount of ascites in the abdomen, obvious jaundice and longer duration of the disease course.

The sheep dying of illness is examined by a dissecting examination, and most subcutaneous veins are highly engorged, gall bladder is enlarged and full, a large amount of dark green thick bile is contained in the sheep, the liver edge is round, grass yellow or yellowish necrotic foci with clear limit and different sizes exist on the surface and deep part, and congestion occurs around the foci to be red. A large amount of reddish serous exudates accumulate in the thoracic cavity and abdominal cavity. The pericardium is obviously enlarged and effusion is opened in a spray shape. The interior is filled with grass yellow liquid, and is quickly solidified after meeting air, and is in amber-like peptone, and other organs have no obvious lesions. The duodenum of a few sick sheep can also cause congestion and bleeding, the real gastric mucosa has bleeding, the kidney surface is congested, and the essence is soft and brittle.

The inventor walks to the epidemic area, the highest death rate of a large group can reach 25 percent, and the highest death rate is about 10 percent generally, so that huge loss is brought to the local sheep raising industry. Because the success rate of treatment is very low, the important point of the disease is prevention, the prevention and treatment of fasciolopsis hepatica are enhanced, and the loss caused by the disease can be effectively reduced by combining with large-scale immunization. At present, vaccines for preventing the epidemic disease in China comprise a sheep black epidemic dry powder inactivated vaccine, a sheep black epidemic and fast epidemic combined inactivated vaccine and a sheep clostridial disease multiple dry powder inactivated vaccine, and the vaccines are prepared by using Clostridium novyi toxin.

At present, the preparation of the clostridium novyi exotoxin is commonly carried out by preparing anaerobic meat liver and stomach enzyme digestion soup, fish liver and stomach enzyme digestion soup culture medium and the like by using animal raw materials such as beef, fish, beef and the like, and the animal raw material culture medium is often unstable in toxigenic performance due to uneven quality of raw materials and large batch-to-batch difference, especially in recent years, due to the development of domestic dairy industry, the feeding amount of dairy cows is rapidly increased, and a large amount of obsolete dairy beef is filled in the market to further cause the quality reduction of raw materials for preparing vaccine culture medium; meanwhile, the residual medicines in beef and beef liver can often inhibit bacterial growth, so that the toxicity is low, the quality of the vaccine is seriously influenced under the above conditions, and the serious increase of the epidemic of the clostridium in the beef and the sheep in recent years also reflects the reduction of the quality of the vaccine. In addition, the traditional culture medium has the disadvantages of complicated preparation process, long time consumption, more manpower and high price, and the prepared culture medium has low toxicity production efficiency, thus bringing great waste and high cost to vaccine production and 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

In view of one or more of the problems of the prior art, one aspect of the present invention provides a clostridium novyi toxigenic medium, each 1000ml of the medium being made from the following raw materials in parts by weight:

the pH value of the culture medium is 7.6-7.8.

In another aspect, the invention provides a method for preparing a clostridium novyi toxigenic medium, comprising the steps of: the raw materials of peptone, tryptone, casein peptone, yeast extract, naCl and KH for preparing the culture medium are mixed ₂ PO ₄ Adding 70-80% of total volume of water for injection according to weight proportion, dissolving, adjusting pH value to 7.6-7.8, adding dextrin or maltose according to weight proportion, fixing volume by water for injection, adding rust-free iron nails or iron filings according to weight proportion, and sterilizing under high pressure to obtain the culture medium; preferably, the pH value is adjusted by adopting 2mol/L sodium hydroxide solution; the autoclaving condition is that the autoclaving is carried out for 30min at 116 ℃. The clostridium novyi toxigenic culture 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 of the solution obtained at the moment has certain buffer capacity after substances except dextrin or maltose in the raw materials are dissolved, the dextrin or the maltose is added after the pH value is regulated, and the pH value of the finally obtained culture medium is basically not changed by constant volume, so that the accuracy of the final proportioning volume can be ensured, and the accurate regulation of the pH value is facilitated; meanwhile, as the dextrin and the maltose belong to starch reagents and have an adhesive effect on the pH meter, the pH is adjusted before the dextrin or the maltose is added, so that the pH meter is protected.

In another aspect, the present invention provides a method for preparing clostridium novyi exotoxin, comprising the steps of:

seed liquid culture: inoculating Clostridium novyi into sugar-free anaerobic liver soup, and standing at 36-37deg.C for 60-72 hr to obtain Clostridium novyi seed solution;

fermentation culture: inoculating the seed solution into the clostridium novyi toxigenic culture medium for culture, collecting the culture, centrifuging, collecting supernatant and filtering the supernatant, wherein the obtained filtrate is clostridium novyi exotoxin.

The culture conditions in the above fermentation culture are 36-37deg.C for 60-72 hr, preferably 66-72 hr. According to experimental data, the toxicity of the clostridium novyi toxin is less than or equal to 0.00025ml when the clostridium novyi toxin is fermented and cultured for 60 hours, and the toxicity of the clostridium novyi toxin is less than or equal to 0.0001ml when the clostridium novyi toxin is fermented and cultured for 66-72 hours, so that the toxicity of the toxin obtained by the fermentation and the culture for 66-72 hours is obviously higher than the toxicity of the clostridium novyi toxin obtained by the fermentation and the culture for 60 hours.

The inoculation amount of the Clostridium novyi inoculation and the inoculation amount of the seed solution inoculation are both 5-6% (v/v).

The above-mentioned fermentation culture is a culture in a glass bottle or a fermenter.

The rotational speed of the centrifugation is 3000-4000rpm, and the time is 20-30min.

The filtration was performed with a 0.22 μm filter.

The clostridium novyi exotoxin prepared by the method also belongs to the content of the invention, and the clostridium novyi inactivated vaccine and the combined inactivated vaccine for the sheep black epidemic and the fast epidemic which are prepared by inactivating and detoxication the clostridium novyi exotoxin also belong to the content of the invention. The inactivated vaccine of the clostridium novyi is prepared by uniformly mixing inactivated and detoxified clostridium novyi exotoxin, purified and concentrated with aluminum hydroxide gel. The sheep black epidemic disease and fast epidemic combined inactivated vaccine is prepared by respectively purifying and concentrating inactivated and detoxified clostridium novyi exotoxin and inactivated and detoxified putrefying clostridium exotoxin, mixing the purified and concentrated clostridium novyi exotoxin with aluminum hydroxide glue according to the volume ratio of 5:1, and uniformly mixing the purified and concentrated clostridium novyi exotoxin and the inactivated and detoxified putrefying clostridium novyi exotoxin.

In the sheep black epidemic and fast epidemic combined inactivated vaccine, the virulence of the clostridium novyi exotoxin is less than or equal to 0.0001ml, and the virulence of the clostridium putref exotoxin is less than or equal to 0.005ml.

By adopting the scheme, the clostridium novyi toxigenic culture medium prepared by the invention has stable toxigenic performance, does not contain medicine and animal material residues carried by the traditional culture medium raw materials, has no inhibition effect on culturing toxigenic bacteria, is convenient for toxoid purification and concentration, and provides possibility for preparing toxoid multi-joint inactivated vaccine with low immune dose. Meanwhile, the preparation method is simple, the materials are convenient to obtain, the quality is excellent and the cost is low, the toxin virulence effect of the clostridium novyi exotoxin obtained by the method is more stable compared with that of the toxin prepared by the traditional culture medium, and the quality of the vaccine prepared by the method is higher, so that all the problems of the invention are solved. 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 uses the culture medium formula and the optimal content of each component to optimize the toxigenic culture condition, so that the toxigenic capacity of the culture medium for culturing the animal clostridium novyi exotoxin reaches or even exceeds the original regulation standard and almost has no batch-to-batch difference.

(2) The toxin-producing culture medium and the preparation method thereof have the advantages of convenient material acquisition and simple preparation, save the digestion processes of the anaerobic-meat-liver-stomach enzyme digestion soup and the fish-liver-stomach enzyme digestion soup, greatly shorten the preparation time and reduce the cost to below 30 percent of the original anaerobic-meat-liver-stomach enzyme digestion soup.

(3) The culture medium obtained by screening has stronger toxin producing capability no matter being cultured by a 500ml bottle or a fermentation tank, and has stable toxin producing capability and controllable quality. The toxin virulence of the clostridium novyi exotoxin prepared by the method can be up to more than 6.25 times of the seedling making standard of China veterinary biological product code.

(4) The invention evaluates the effect of the clostridium novyi inactivated vaccine on rabbits and sheep, and the serum neutralization titer of the sheep black epidemic and fast epidemic combined inactivated vaccine prepared by the invention can be 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 during purification and concentration can be reduced.

In conclusion, the invention has wide prospect in replacing the existing anaerobic-meat-liver-stomach enzyme digestion soup, fish-liver-meat-stomach enzyme digestion beef broth culture medium and the production of clostridium perfringens multi-linked inactivated vaccine.

Detailed Description

Aiming at the defect that animal raw materials are used for culture mediums such as anaerobic meat liver gastric enzyme digestion soup and the like suitable for clostridium novyi in the prior art, through experimental finding, the invention provides a toxin production culture medium suitable for clostridium novyi culture, which has the advantages of convenient material taking, simple and quick preparation method, low cost, high toxin toxicity and stable toxin production effect of clostridium novyi obtained by fermentation culture, and can be widely popularized and used.

Meanwhile, based on the clostridium novyi toxigenic culture medium provided by the invention, the clostridium novyi exotoxin with higher virulence level is provided, and the clostridium novyi inactivated vaccine is obtained based on the exotoxin, and the serum neutralization titer of the clostridium novyi inactivated vaccine also reaches or even exceeds the corresponding standard of the regulations.

The present invention will be described in further detail with reference to specific examples.

The following disclosure provides many different embodiments, or examples, for implementing different aspects of the invention. Examples of various specific processes and materials are provided, but one of ordinary skill in the art will recognize the application of other processes and/or the use of other materials.

The present invention is described in detail below with reference to specific examples. Examples detailed embodiments and specific operation procedures are given on the premise of the technical scheme of the present invention, but the disclosure of the present invention is not limited to the following examples.

The methods used in the examples described below are conventional methods unless otherwise specified.

The clostridium novyi production strain adopted in the invention is clostridium novyi C61-4 strain for animals, the strain number is CVCC60281, and the production date is as follows: and purchased from China veterinary microbiological culture collection center (China center for type culture collection) at 14 days 04 and 2015.

The pharmaceutical crude dextrins, biological agent dextrins, biochemical agent maltoses used in the examples are all commercially available.

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 composition is shown in the following table 1:

table 1: composition of liver soup for treating anaesthesia

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 of the dilutions (peptone water) used in the examples and the preparation method "China veterinary biological preparation protocol" are shown in Table 2 below:

table 2: peptone water component

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 code, and the composition is shown in the following table 3:

table 3: composition of anaerobic meat liver and stomach enzyme digestion soup

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 (when culturing Clostridium novyi, adding iron nails or iron filings), 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.

The examples are the same as the above materials used in the comparative examples.

Comparative example: evaluation of culture effect of clostridium novyi in traditional anaerobic meat liver gastric enzyme digestion soup

1. Strain culture and toxin preparation:

the ampoule containing freeze-dried clostridium novyi 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 fungus blocks, the freeze-dried fungus blocks are inoculated into a 6-branch tube of sugar-free anaerobic liver soup, the inoculation amount is 4-5% (v/v), the culture is placed at 36-37 ℃ for static culture for 60-72h, and pure detection is carried out on the culture, thus obtaining pure first-grade seed liquid.

Inoculating the first-stage seed solution into a 350ml/500ml bottle of anaerobic liver and stomach enzyme digestion soup culture medium according to the inoculum size of 5-6% (v/v), standing at 36-37 ℃ for culturing for 60-72h, collecting the culture, and centrifuging at 3000rpm for 30min; collecting supernatant, filtering the supernatant with 0.22 μm filter membrane, and sampling the obtained filtrate to obtain qualified Clostridium novinarum toxin.

2. Toxin Effect assay

The inventor prepares traditional anaerobic meat liver gastric enzyme digestion soup by using the formula of the table 3, prepares the clostridium novyi toxin by using the digestion soup as a culture medium, and performs 10 parallel experiments in total, and the toxin measuring effect is shown in the following table 4:

table 4: toxin detection result of clostridium novyi in traditional anaerobic liver and stomach enzyme digestion soup

As can be seen from Table 4 above, the preparation of Clostridium novyi toxin by conventional anaerobic, meat, liver and stomach enzyme digestion broth by detoxification was unstable and even resulted in non-growing development batches. In 10 experiments conducted by the inventor, 4 times of experiments do not meet the requirement of the regulation seedling preparation: 1MLD is less than or equal to 0.0005ml, only 1 times of toxicity can reach 1MLD is less than or equal to 0.0001ml, and 2 times of toxicity can reach 1MLD is less than or equal to 0.00015ml. Proved by the experiment, the traditional anaerobic meat liver gastric enzyme digestion soup culture medium has poor toxin production effect and is unstable. Meanwhile, the process for preparing the traditional anaerobic liver-stomach enzyme digestion soup culture medium is very complex and tedious, and a large amount of manpower, material resources and energy sources are needed. And is often influenced by the quality of animal raw materials such as beef, beef liver and the like and experience of operators.

Example 1: screening of clostridium novyi toxigenic culture medium

1. Preparation of Clostridium novinarum toxigenic Medium

Formula 1: preparation of clostridium novyi toxigenic culture medium

1.1 injection water 800mL, add peptone 20g, tryptone (from Oboc Biotechnology Co., ltd.) 5g, casein peptone (from Oboc Biotechnology Co., ltd.) 5g, yeast extract (from OXOID) 2.5g, sodium chloride 2.5g, KH2PO4 5g, and dissolve.

1.2. The pH was adjusted to 7.6-7.8 with 2mol/L NaOH.

1.3. 10g of crude dextrin (from Shandong chat Anhua pharmaceutical Co., ltd.) was added and shaken well.

1.4. 100g of stainless steel nails or scrap iron is added after cleaning.

1.5. The volume of the water for injection is fixed to 1000ml. Sterilizing with high pressure steam at 116 deg.C for 30min.

Formula 2: preparation of clostridium novyi toxigenic culture medium

2.1. 800mL of water for injection, 20g of peptone, 5g of tryptone, 5g of casein peptone, 2.5g of yeast extract powder, 2.5g of sodium chloride and KH are added ₂ PO ₄ 5g, and dissolving.

2.2. The pH was adjusted to 7.6-7.8 with 2mol/L NaOH.

2.3. 10g of dextrin (biological reagent, obock Biotechnology Co., ltd.) was added and shaken well.

2.4. 100g of stainless steel nails or scrap iron is added after cleaning.

2.5. The volume of the water for injection is fixed to 1000ml. Sterilizing with high pressure steam at 116 deg.C for 30min.

Formula 3: preparation of clostridium novyi toxigenic culture medium

3.1. 800mL of water for injection, 20g of peptone, 5g of tryptone, 5g of casein peptone, 2.5g of yeast extract powder, 2.5g of sodium chloride and KH are added ₂ PO ₄ 5g of maltose (national medicine group chemical Co., ltd.) 10 g.

3.2. The pH was adjusted to 7.6-7.8 with 2mol/L NaOH.

3.3. 100g of stainless steel nails or scrap iron is added after cleaning.

3.4. The volume of the water for injection is fixed to 1000ml. Sterilizing with high pressure steam at 116 deg.C for 30min.

In the preparation process of clostridium novyi toxigenic culture medium, raw materials of peptone, tryptone, casein peptone, yeast extract powder, naCl and KH in the culture medium are firstly mixed ₂ PO ₄ Adding the components into water for injection with the total volume of 80% according to the weight ratio for dissolution, adjusting the pH value to 7.6-7.8, then adding dextrin (biological reagent) or maltose or crude dextrin according to the weight ratio, and cleaning the components without rust or iron filings, thus the preparation and sample addition sequence effectively ensures complete dissolution of nutrient substances in a visible state; dissolving substances except dextrin (biological reagent) or maltose or crude dextrin and stainless iron nails or scrap iron in the raw materials, regulating the pH value of the solution to a certain buffer capacity, adding dextrin (biological reagent) or maltose or crude dextrin, and using water for injection to fix the volume. The pH value of the finally obtained culture medium is not changed basically, so that the accuracy of the volume of the final proportion can be ensured, and the accurate adjustment of the pH value is facilitated; meanwhile, as the dextrin, the maltose or the crude dextrin belongs to starch reagents and has an adhesive effect on the pH meter, the pH is adjusted before the dextrin, the maltose or the crude dextrin is added, so that the pH meter is protected.

2. Bacterial culture and toxin preparation

The ampoule containing freeze-dried clostridium novyi 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 fungus blocks, and the freeze-dried fungus blocks are inoculated into a 6-branch tube of sugar-free anaerobic meat liver soup and are placed at 37 ℃ for static culture for 60-72 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 the Clostridium novyi toxigenic culture medium prepared in the first step in a bottle with 350ml/500ml according to the inoculum size of 5-6% (v/v), standing at 37deg.C for 60-72 hr, collecting culture, centrifuging at 3000rpm for 30min; collecting supernatant, and filtering the supernatant by adopting a 0.22 mu m filter membrane, wherein the obtained filtrate is sampled and the toxin is detected to be qualified, namely the clostridium novyi exotoxin.

3. The results of comparison of the toxic effects of the three Clostridium novinarum toxic culture media are shown in Table 5 below.

Table 5: comparison of the toxin production effects of three Clostridium novinarum toxin production culture mediums

As can be seen from the data in Table 5, the crude dextrin composition of formula 1 is not sufficiently stable, and only one fermentation toxicity reaches the standard of the veterinary biological product code: 1 MLD.ltoreq.0.0005 ml. Dextrin (biological reagent) group of formula 2 has stable fermentation virulence and meets and exceeds the virulence standard of veterinary biological preparation procedure: 1MLD is less than or equal to 0.0005ml, the toxicity can reach 1MLD is less than or equal to 0.00025ml, and the toxicity is 2 times higher than the standard. The maltose group in the formula 3 has the most stable fermentation toxicity and far exceeds the toxicity standard of the veterinary biological product code: 1MLD is less than or equal to 0.0005ml, the highest toxicity can reach 1MLD is less than or equal to 0.00008ml, and the toxicity is 6.25 times higher than the standard. Compared with the results of the comparison example and the formula 1, the culture medium formula 2 and the formula 3 have stable toxin production effect, especially the culture medium of the formula 3 has good toxin production effect, the embodiment widens the thought in the selection of the raw materials for preparing the culture medium, and does not need to be limited to the raw materials which are difficult to store, have high cost and inconvenient to obtain, such as beef, beef liver, pepsin and the like used in the traditional anaerobic liver and stomach enzyme digestion soup culture medium, and the embodiment selects the finished products which are easy to obtain in the market as the raw materials for preparing the clostridium novyi toxin production culture medium, so that the raw materials are easy to obtain, the preparation process is simple and convenient, the time for preparing the culture medium is greatly saved, and the cost is reduced to 1/3 of the traditional anaerobic liver and stomach enzyme digestion soup. In addition, the toxicity effect in three experiments is achieved and exceeds the toxicity standard of the veterinary biological product code according to the data of the table 5, compared with the formula 3 using the dextrin (biological reagent), the effect of the maltose group in the formula 3 is relatively good, and the maltose is determined to be a preferred embodiment.

Example 2: preparing a clostridium novyi toxigenic culture medium

2.1 preparing a Clostridium novyi toxigenic culture medium (biological agent dextrin group)

The same formulation as formulation 2 in example 1 was used to prepare respective clostridium novyi toxigenic media according to the formulations listed in table 6 below, and the toxigenic effects were evaluated.

Table 6: novickers clostridium toxigenic culture medium formula (1000 ml)

In this example, the inventors prepared Clostridium novyi toxigenic medium for use by the method described in example 1 according to the formulation set forth in Table 6 above, then opened a vacuum-well ampoule containing lyophilized Clostridium novyi species by aseptic burst, extracted an appropriate amount of sterile nutrient broth to dissolve the lyophilized pellet, inoculated into a sugar-free anaerobic meat liver broth 6-branch tube, and incubated at 37℃for 60-72 hours. Meanwhile, the pure detection is carried out, and the pure seed liquid is the first-level seed liquid.

Inoculating the first-level seed liquid into a clostridium novyi toxigenic culture medium prepared according to formulas 21, 22 and 23 in a 350ml/500ml bottle according to the inoculum size of 5-6% (v/v), standing at 37 ℃ for culturing for 60-72 hours, sampling and testing to obtain the clostridium novyi exotoxin, and evaluating the toxigenic effect. The results show that the toxicity producing effects of the culture mediums prepared by the culture medium formulas 21, 22 and 23 listed in the table 6 can reach and exceed the toxicity standard of the veterinary biological product code, and the toxicity producing effects are stable.

2.2 preparing Clostridium novyi toxigenic Medium (malt sugar group)

The same formulation as in formulation 3 of example 1 was used to prepare respective clostridium novyi toxigenic media according to the formulations listed in table 7 below, and the toxigenic effects were evaluated.

Table 7: novickers clostridium toxigenic culture medium formula (1000 ml)

In this example, the inventors prepared clostridium novyi toxigenic medium for use by the method described in example 1 according to the formulation set forth in table 7 above, then opened a vial containing lyophilized clostridium novyi species with good vacuum, opened by aseptic burst, extracted an appropriate amount of sterile nutrient broth to dissolve the lyophilized pellet, inoculated in a sugar-free anaerobic liver broth 6-branch tube, and incubated at 37 ℃ for 60-72 hours. Meanwhile, the pure detection is carried out, and the pure seed liquid is the first-level seed liquid.

Inoculating the first-level seed liquid into a clostridium novyi toxigenic culture medium prepared according to formulas 31, 32 and 33 in a 350ml/500ml bottle according to the inoculum size of 5-6% (v/v), standing at 37 ℃ for culturing for 60-72 hours, sampling and testing to obtain the qualified clostridium novyi toxigenic, and evaluating the toxigenic effect. The results show that the toxicity producing effects of the culture mediums prepared by the culture medium formulas 31, 32 and 33 listed in the table 7 can reach and exceed the toxicity standard of the veterinary biological product code as shown in the table 5, and the toxicity producing effects are stable.

Example 3: preparation of clostridium novyi exotoxin and evaluation of toxin production effect

In this example, clostridium novyi was fermented using the medium of formula 3 in example 1.

3.1 preparation of the two-stage seed liquid exotoxin of the clostridium novyi and evaluation of the toxin production effect

Inoculating the first-stage seed solution into 350ml/500ml bottle of anaerobic liver soup according to the inoculum size of 5-6% (v/v), standing at 37deg.C for 72 hr, and taking the qualified product as the second-stage seed solution.

Inoculating qualified second-level seed solution into 12000ml of Clostridium novinarum toxigenic culture medium according to the inoculum size of 5-6% (v/v), standing at 37deg.C for 60-72 hr.

Sampling after culturing for 60h, 66h and 72h respectively, centrifuging the sample bacterial liquid at 3000rpm/min for 30min, extracting the supernatant, filtering with a 0.22 μm filter to obtain the clostridium novyi exotoxin, and measuring the toxin after dilution.

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 below, and 16-20gKM mice were injected into the tail vein, 2 mice per titer, 0.2mL each, and the death of the mice within 72 hours after injection was observed. The minimum toxin amount capable of enabling the mice to die by 2/2 is MLD of clostridium novyi exotoxin to the mice. The results of the 1 st and 2 nd experiments are shown in tables 9 and 10 below, which0/2 of ⁺ Indicating that 2 mice were injected and 0 had died; 1/2 ⁺ Meaning that 2 mice were injected and 1 died. 2/2 ⁺ Meaning that 2 mice were injected and 2 had died.

Table 8: toxin dilution route

Table 9: toxicity results of experiment 1 (12000 ml Scale)

As can be seen from table 9 above: culturing for 60h, sampling and measuring virulence to be less than or equal to 0.00025ml of 1 MLD; culturing for 66h, sampling and measuring virulence to be less than or equal to 0.00008ml of 1 MLD; the samples were cultured for 72 hours to determine that the virulence was 1 MLD.ltoreq.0.00008 ml, and no endpoint was detected.

Table 10: toxicity results of experiment 2 (12000 ml Scale)

As is clear from Table 10, the virulence was 1 MLD.ltoreq.0.00025 ml after 60 hours of incubation and sampling; culturing for 66h, sampling and measuring virulence to be less than or equal to 0.00008ml of 1 MLD; the samples were cultured for 72 hours to determine that the virulence was 1 MLD.ltoreq.0.00008 ml, and no endpoint was detected.

3.2 preparation of three-stage seed liquid exotoxin of clostridium novyi and evaluation of toxin production effect

Inoculating 350ml/500ml bottle of anaerobic meat liver soup into the first-stage seed solution according to the inoculation amount of 5-6%, standing at 37 ℃ for 72 hours, and taking the qualified product as the second-stage seed solution after pure inspection.

Inoculating the second-level seed liquid into 8000mL/10000mL bottle of anaerobic meat liver soup according to the inoculation amount of 5-6%, standing at 37 ℃ for 72 hours, and taking the qualified product as the third-level seed liquid after pure inspection.

Inoculating qualified tertiary seed liquid into 400000ml of clostridium novyi toxigenic culture medium according to the inoculum size of 5-6%, and standing at 37 ℃ for culturing for 60-72h.

Sampling after culturing for 60h, 66h and 72h respectively, centrifuging sample bacterial liquid at 3000rpm/min for 30min, collecting supernatant, filtering with 0.22 μm filter to obtain toxin, and measuring toxin after dilution.

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 72 hours after injection. The minimum toxin amount that can cause 2/2 death of mice is MLD of Clostridium novinarum toxin to mice. The results of the 3 rd experiment are shown in Table 11 below.

Table 11: toxicity results of experiment 3 (400000 ml scale)

As is clear from Table 11, the virulence 1MLD was measured by culturing for 60 hours and sampling to 0.00025ml or less; culturing for 66h, sampling and measuring virulence 1MLD less than or equal to 0.00008ml; culturing for 72h, sampling and measuring virulence 1MLD is less than or equal to 0.00008ml, and no endpoint is detected.

As can be seen from the results in tables 9 to 11, the invention adopts the clostridium novyi toxigenic culture medium (the culture medium of the formula 3 in the example 1) to carry out fermentation culture on clostridium novyi through 3 times of repeated expansion culture, and when the fermentation culture time is 60 hours, 66 hours and 72 hours, the toxicity of the clostridium novyi toxigenic obtained by the fermentation culture can reach 1MLD which is less than or equal to 0.00025ml and exceeds the toxicity standard of 0.0005ml of the clostridium novyi toxigenic standard in the Chinese veterinary biological product code (hereinafter referred to as code) in 2000 edition of the national regulation of "sheep black epidemic and fast epidemic combined inactivated vaccine". When the fermentation culture time is 66-72h, the result of three times of expansion culture tests is that 1MLD is less than or equal to 0.00008ml, and no end point is detected. Is 6.25 times of standard 1MLD of clostridium novyi vaccine-making toxin which is less than or equal to 0.0005ml in 'sheep black epidemic and fast epidemic combined inactivated vaccine' specified in the 2000 edition of the rule. Therefore, the fermentation culture time is preferably 66-72h when the clostridium novyi is fermented and cultured, the toxicity standard specified in the rule can be exceeded, the toxicity effect of the clostridium novyi toxin-producing medium is stable, the obtained clostridium novyi exotoxin is stable and strong, and the clostridium novyi exotoxin can be effectively used for preparing clostridium novyi inactivated vaccines.

Example 4: preparation and efficacy evaluation of sheep black epidemic and fast epidemic combined inactivated vaccine

4.1, preparing a 'sheep black epidemic and fast epidemic combined inactivated vaccine':

taking a clostridium putrefying bacteria culture (specifically clostridium putrefying bacteria C55-1 strain for animals, with a strain number CVCC60021C55-1 2011.01.25, which is purchased from China center for veterinary microbiological culture collection, and is prepared according to the manufacturing and inspection rules of two 000 year edition of "sheep black epidemic and fast epidemic combined inactivated vaccine" of the animal biological product rules), adding 0.8 percent formaldehyde solution (40 percent concentration) according to the volume, and inactivating and detoxifying for 3-4 days (stirring for 4 times/day) at 37 ℃. The cultured Clostridium novinarum culture (see example 3) was taken, 0.5% formaldehyde solution (40% concentration) was added by volume, and the mixture was inactivated at 37℃for 3 to 4 days (stirring 4 times/day). Inoculating the inactivated detoxified bacterial liquid into a large anaerobic meat liver soup tube, a small TSB tube and a casein peptone agar slant tube, culturing at 37 ℃ for 72 hours, and observing the aseptic growth for 3 days. Taking a 72-hour culture anaerobic meat liver soup big tube, transplanting a T.G small tube, a TSB small tube and a tyrosol agar inclined surface small tube, culturing for 72 hours at 37 ℃, and observing the sterile growth for 3 days. Indicating complete inactivation; and simultaneously taking inactivated bacterial liquid, centrifuging at 3000rpm/min for 30min, discarding bacterial precipitate, sucking the supernatant, filtering the supernatant by using a 0.22 mu m filter membrane, injecting 2 mice of 16-20gKM mice into tail veins, injecting 0.4ml each, and observing the average health care activity for 3-5 days to show that the detoxification is complete.

And (3) taking the inactivated and detoxified bacterial liquid, filtering the bacterial liquid in a sterilization container filled with 200-mesh copper yarns, and storing the bacterial liquid at the temperature of 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 completely detoxified clostridium putrefying bacteria (C55-1) bacterial liquid and clostridium novyi (C61-4) bacterial liquid which are stored at 2-8 ℃ respectively into sterilized glass bottles, wherein before the detoxication, the clostridium putrefying bacteria are inactivated, 1MLD is less than or equal to 0.005ml, 1MLD is less than or equal to 0.0001ml, and the pH is adjusted to 6.8-7.2 by using 2mol/L sterile sodium hydroxide solution. According to clostridium putrefying: clostridium novyi=1:1 was added to sterilized aluminum hydroxide vials. The ratio of the bacterial liquid to the aluminum gel is 5:1. Shaking and mixing uniformly to prepare the sheep black epidemic and fast epidemic combined inactivated vaccine. And storing at 2-8 deg.c for further use.

4.2, safety and efficacy evaluation of a "sheep black disease and fast epidemic bivalent inactivated vaccine":

14 white rabbits of Beijing big ear (1.5-2 kg) were prepared, of which 4 were used for collecting negative control serum and the other 10 were used for safety and efficacy evaluation.

4.2.1 safety test in white rabbits in Beijing big ear:

taking the prepared sheep black epidemic and fast epidemic combined inactivated vaccine, and intramuscular injecting 1.5-2 kg Beijing big ear white rabbits (2, 5 ml/rabbit). The results are shown in Table 12 below, after 14-21 days of observation.

Table 12: safety evaluation result of sheep black epidemic and fast epidemic combined inactivated vaccine

As can be seen from table 12 above, 2 white rabbits of beijing big ear injected were healthy and alive at 21 days of observation, and the injection site did not undergo necrosis reaction, judging that: the safety inspection of the prepared sheep black epidemic and fast epidemic combined inactivated vaccine meets the regulations.

4.2.2, evaluation of efficacy of "sheep black epidemic and fast epidemic combined inactivated vaccine" on white Beijing white rabbits at 3 ml/rabbit immunization dose:

taking 8 prepared sheep black epidemic and fast epidemic combined inactivated vaccine, and intramuscular injecting 1.5-2 kg Beijing big ear white rabbits and 3 ml/rabbit on the inner side of the thigh. The ear artery blood collection and serum separation were carried out on immunized rabbits and negative rabbits 21 days after immunization, 4 rabbit serum was randomly collected to prepare a mixed serum, 0.4ml of the mixed serum was respectively collected according to a serum neutralization method, and the serum neutralization titers of clostridium novyi and clostridium putref toxins (the number of MLD in 0.1ml of serum-neutralized mice) were measured. The results are shown in Table 13 below.

Table 13: efficacy evaluation result of sheep black epidemic and fast epidemic combined inactivated vaccine on white Beijing white rabbits at 3 ml/rabbit immunization dose

According to the regulations of biological products for China animal in 2000 edition: and (5) the neutralizing titer of the clostridium novyi serum reaches 5 mice MLD, and the mice are judged to be qualified. From the data in table 13 above, it can be seen that in the "sheep black epidemic, fast epidemic bigeminal inactivated vaccine" prepared by using the clostridium novyi inactivated bacterial liquid obtained by fermenting and culturing the toxigenic medium in this example, the neutralization titer of rabbit serum can reach 10 mice MLD, and no endpoint is detected, so that the standard of "chinese veterinary biological product protocol" is met or exceeded: serum neutralization titer is not less than 5 mice MLD. The culture medium has low cost, stable toxicity, and reduced or eliminated unqualified bacterial liquid in large-scale production.

4.2.3 evaluation of efficacy of "sheep black epidemic and fast epidemic combined inactivated vaccine" on sheep at 3 ml/sheep immunization dose:

6-12 months old, 30-40Kg healthy sheep without corresponding neutralizing antibody are intramuscular injected into neck and shoulder, 3 ml/sheep are immunized for 21 days, 4 sheep serum is randomly extracted and mixed after the sheep is subjected to jugular vein blood sampling to prepare serum, 0.4ml of mixed serum is respectively taken according to a serum neutralization method, and the serum neutralization titers of clostridium novyi and clostridium putref toxins (the number of MLD of 0.1ml serum neutralization mice) are measured. The results are shown in Table 14 below.

Table 14: efficacy evaluation of "sheep black disease, fast-epidemic bivalent inactivated vaccine" on sheep at 3 ml/sheep immunization dose:

the data in table 14 above shows that in the "combined inactivated vaccine for sheep black disease and fast epidemic" prepared from the inactivated bacteria solution of clostridium novyi obtained by fermenting and culturing in the toxigenic medium in this example, the neutralization titer of sheep serum can reach 8 mice MLD, and no endpoint is detected. Meets or exceeds the standard of China veterinary biological product code: serum neutralization titer is not less than 5 mice MLD. Proved by the invention, the clostridium novyi toxigenic culture medium has stronger toxigenic effect, and the vaccine prepared by the clostridium novyi toxigenic culture medium has higher quality, can be effectively used for large-scale vaccine production and application, and has stable and controllable quality.

Meanwhile, the preparation method of the clostridium novyi toxigenic medium of the present invention has advantages as shown in the following table 15 compared with the conventional preparation method of the medium, wherein the comparison is made by taking 10000ml of the medium as an example.

Table 15: the method of the invention is compared with the related parameters of the traditional technology (10000 ml)

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A clostridium novinaii toxigenic culture medium, which is characterized in that each 1000ml of the culture medium is prepared from the following raw materials in parts by weight:

15-25g of peptone;

5-10g of tryptone;

5-10g of casein peptone;

1-3g of yeast extract powder;

NaCl 1-3g；

KH ₂ PO ₄ 5-10g；

5-10g of maltose;

95-105g of stainless iron nails or scrap iron;

the balance of water for injection.

2. The clostridium novyi toxigenic medium of claim 1 wherein the pH of the medium is 7.6-7.8.

3. A method of formulating a clostridium novyi toxigenic medium according to claim 1 or 2, comprising the steps of:

the raw materials of peptone, tryptone, casein peptone, yeast extract, naCl and KH for preparing the culture medium are mixed ₂ PO ₄ Adding 70-80% of total volume of water for injection according to weight ratio, dissolving, regulating pH value to 7.6-7.8, adding maltose according to weight ratio, using water for injection to make constant volume, adding rust-free iron nails or iron filings according to weight ratio, and high-pressure sterilizing so as to obtain the invented culture medium.

4. A formulation process according to claim 3, wherein the pH adjustment is carried out using 2mol/L sodium hydroxide solution; the autoclaving condition is that the autoclaving is carried out for 30min at 116 ℃.

5. A method for preparing clostridium novinarum exotoxin, comprising the steps of:

seed liquid culture: inoculating Clostridium novyi into sugar-free anaerobic liver soup, and standing at 36-37deg.C for 60-72 hr to obtain Clostridium novyi seed solution;

fermentation culture: inoculating the seed solution into the clostridium novyi toxigenic culture medium according to claim 1 or 2 for culture, collecting the culture, centrifuging, collecting supernatant and filtering the supernatant, wherein the obtained filtrate is clostridium novyi exotoxin.

6. The method for producing an exotoxin according to claim 5, wherein the condition of the culture in the fermentation culture is 36 to 37℃for 60 to 72 hours.

7. The method for producing an exotoxin according to claim 6, wherein the condition of the culture in the fermentation culture is 36 to 37℃for 66 to 72 hours.

8. The method for producing a clostridium novyi exotoxin according to any one of claims 5 to 7 wherein the inoculum size of clostridium novyi inoculation and the inoculum size of seed liquid inoculation are both 5 to 6% (v/v); and/or

In the fermentation culture, the culture is cultured in a glass bottle or a fermentation tank; and/or

The rotational speed of the centrifugation is 3000-4000rpm, and the time is 20-30min; and/or

The filtration was carried out with a 0.22 μm filter.