CN109943507B - Preparation method and application of veterinary A-type clostridium perfringens toxin - Google Patents
Preparation method and application of veterinary A-type clostridium perfringens toxin Download PDFInfo
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Abstract
The invention discloses a preparation method and application of A-type clostridium perfringens toxin for livestock. The preparation method of the toxoid comprises the following steps: inoculating the clostridium perfringens type A production strain into a culture medium for fermentation culture to obtain a fermentation product, then inactivating and detoxifying the fermentation product under L-lysine, formaldehyde aqueous solution and pH6.8, and centrifuging supernatant of qualified bacteria liquid which is inactivated and detoxified to obtain toxoid. The culture medium comprises the following substances: casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4And glucose. The toxicity of the clostridium perfringens type A toxin prepared by the method can be improved to 12.5 times of the vaccine preparation standard, the primary-immune serum neutralization titer and the secondary-immune serum neutralization titer of the toxoid prepared by the clostridium perfringens type A toxin on domestic rabbits can be respectively improved to 4 times and 50 times of the traditional process, and the serum titer cost ratio can be improved to 48.8 times and 1216.4 times of the traditional process.
Description
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to a preparation method and application of A-type clostridium perfringens toxin for livestock.
Background
Clostridium perfringens (c. perfringens) old clostridium welchii (c. welchii) or clostridium perfringens (Bacillus perfringens) is one of the main pathogenic bacteria causing various necrotic enteritis and enterotoxemia in animals, food poisoning in humans and traumatic gas gangrene, and is widely distributed in soil and digestive tracts of animals. The strain can produce exotoxin with strong toxicity and some enzymes related to invasiveness, and some strains can also produce enterotoxin, hemolysin, hemagglutinin and the like. Traditionally, the 4 major exotoxins produced by them are classified as type 5A (. alpha.), B (. alpha.,. beta.,. epsilon.), C (. alpha.,. beta.), D (. alpha.,. epsilon.), E (. alpha.,. iota.) (L.,. M., Beijing: Chinese agricultural Press, 2013: 194. 196.). The clostridium perfringens alpha toxin (CPA) is produced by various bacteria, has the highest expression level of A bacteria, is the most important virulence factor basically, has the molecular mass of 42.528kDa and is zinc-dependent protease. Clostridium perfringens type a is an important pathogenic bacterium causing traumatic gas gangrene, and is one of potential toxin warfare agents. In addition, clostridium perfringens type a also poses a serious threat to the breeding industry of China, and the incidence rate of piglet red dysentery and necrotic enteritis or enterotoxemia of ruminants in various parts of the year is on an increasing trend. It has recently been found that clostridium perfringens type a is probably the main pathogenic bacterium in "sudden death" in livestock.
The biological products for preventing The disease in Europe and America are Clostridium perfringens type A toxoid (Animal and Plant Health infection Service, USDA.9 CFR Ch.I (1-1-07 Edition) [ S ]. Washington: U.S. GOVERNMENT PRINTING OFFICE,2007.3, British Pharmacopoeia (Veterinanry) [ S ]. London: The Stationery OFFICE, 2005.); the vaccine containing the component in China comprises: rabbit clostridium perfringens disease (type a) inactivated vaccine (about 271.4 ten thousand parts per year) and piglet clostridium perfringens disease bivalent inactivated vaccine (A, C type) (about 31.4 ten thousand parts per year). The preparation process of the culture medium prepared by the method is complicated, long in time consumption and high in manpower, and particularly, the phenomenon of unstable toxicity generation performance due to the fact that the quality of the raw materials is different frequently is important, so that the quality of the vaccine is influenced, and great waste and high cost are brought to the vaccine production. In addition, the virulence of pathogenic clostridium has obvious positive correlation with the immune effect. The clostridium tetani, the clostridium novyi and the clostridium botulinum have stronger toxin producing capability, the specified toxicity standards during vaccine preparation are at least 2000000MLD/mL, 2000MLD/mL and 50000MLD/mL respectively, and the specified toxicity attacking doses in the efficacy test are 300MLD, 50MLD and 10MLD respectively; the clostridium perfringens has relatively weak toxin producing capacity, and the toxin attacking dose specified by potency test in quality standard is 1 MLD. Among clostridium perfringens type A, B, C, D, clostridium perfringens type D has the strongest toxin-producing capability (1333-10000 MLD/mL), clostridium perfringens type A has the weakest toxin-producing capability (10-100 MLD/mL), and clostridium perfringens type A vaccine has the relatively worst immune protection effect (China veterinary drug Committee, China national Committee, animal pharmacopoeia, O.O.O.A.A.T.S.Beijing: China agricultural Press, 2011; agriculture department, animal biological product regulation Committee, China national Committee, animal biological product regulation, O.A.S.A.Beijing: chemical industry Press, 2000). Therefore, it is necessary and urgent to develop a preparation method of clostridium perfringens type a toxin with reliable quality, high toxicity, good stability and low cost, and a toxoid with good immune effect.
Disclosure of Invention
The invention aims to provide a preparation method and application of A-type clostridium perfringens toxin for livestock.
In a first aspect, the invention provides a clostridium perfringens type a fermentation medium.
The fermentation medium for clostridium perfringens type A provided by the invention comprises a solute; the solute consists of: casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4And glucose.
Further, the casein peptone, the yeast extract powder and the CaCl2Said ZnSO4·7H2O, said Na2HPO4·12H2O, said KH2PO4And the mass ratio of glucose may be (20-30): (10-15): (0.075-0.1): (0.0014-0.0028): (5-7.5): (0.5-0.75): 10.
further, the casein peptone, the yeast extract powder, the CaCl2Said ZnSO4·7H2O, said Na2HPO4·12H2O, said KH2PO4And the mass ratio of glucose is 30: 15: 0.1: 0.0014: 5: 0.5: 10.
in the culture medium, the culture medium further comprises a solvent; the solvent may specifically be water.
The culture medium comprises the casein peptone, the yeast extract powder and the CaCl2Said ZnSO4·7H2O, said Na2HPO4·12H2O, said KH2PO4And said glucose and water;
further, in the above-mentioned case,
the concentration of the casein peptone in the culture medium is 20-30 g/L;
the concentration of the yeast extract powder in the culture medium is 10-15 g/L;
the CaCl is2The concentration in the culture medium is 0.075-0.1 g/L;
the ZnSO4·7H2The concentration of O in the culture medium is 0.0014-0.0028 g/L;
the Na is2HPO4·12H2The concentration of O in the culture medium is 5-7.5 g/L;
the KH2PO4The concentration in the culture medium is 0.5-0.75 g/L;
the concentration of the glucose in the culture medium is 10-15 g/L.
In a still further aspect of the present invention,
the concentration of the casein peptone in the culture medium is 30 g/L;
the concentration of the yeast extract powder in the culture medium is 15 g/L;
the CaCl is2The concentration in the culture medium is 0.1 g/L;
the ZnSO4·7H2The concentration of O in the culture medium is 0.0014 g/L;
the Na is2HPO4·12H2The concentration of O in the culture medium is 5 g/L;
the KH2PO4The concentration in the culture medium is 0.5 g/L;
the concentration of the glucose in the medium was 10 g/L.
In the above culture medium, the pH value of the culture medium is 7.5-7.7.
In a second aspect, the present invention provides a method of preparing the above-described medium.
The method for preparing the culture medium comprises the following steps: mixing the rest components except glucose with water according to mass ratio, adjusting pH to 7.5-7.7, sterilizing, preparing glucose into 50% glucose aqueous solution, sterilizing, and adding to desired concentration before use to obtain culture medium.
Further, a 50% aqueous glucose solution was added before use to give a final glucose concentration of 0.5% in the medium.
In the above method for preparing the culture medium, the substance can be sufficiently dissolved and/or the dissolution can be accelerated by heating during the process of dissolving the solute.
In the above method for preparing the culture medium, the pH value can be specifically adjusted by using 10M sodium hydroxide solution.
In the above method for preparing the culture medium, the sterilization condition of the culture medium may be autoclaving at 116 deg.C for 30min, and the sterilization condition of the 50% glucose aqueous solution may be autoclaving at 110 deg.C for 40 min.
In the above method for preparing a culture medium, the water is preferably purified water.
The application of the culture medium or the method for preparing the culture medium in the preparation of the clostridium perfringens type A toxin and/or the clostridium perfringens type A toxoid also belongs to the protection scope of the invention.
In a third aspect, the present invention provides a process for the preparation of a clostridium perfringens type a toxin.
The method for preparing the clostridium perfringens type A toxin comprises the following steps: fermenting and culturing the clostridium perfringens type A production strain in the culture medium to obtain a fermentation product; and collecting the supernatant of the fermentation product to obtain the clostridium perfringens type A toxin.
In the above method for preparing a clostridium perfringens type a toxin, the clostridium perfringens type a production strain can be a clostridium perfringens type a C57-1 strain (CVCC number: 37) for veterinary use, and is purchased from the chinese veterinary medical science and technology center for culture collection of microorganisms (www.cvcc.org.cn, CVCC for short).
In the above method for preparing clostridium perfringens type a toxin, the conditions of the fermentation culture are as follows: fermenting and culturing at 35-37 deg.C for 5-6h, maintaining pH at 6.9 + -0.05 during fermentation, adding 50% glucose aqueous solution according to 1-1.2% of the total volume of the culture medium when fermenting and culturing for 4-5h, and controlling rotation speed at 25-30rpm during fermentation.
In the above method for preparing a clostridium perfringens type a toxin, the clostridium perfringens type a production strain further comprises the following steps before fermentation culture: preparing seed liquid of a clostridium perfringens type A production strain.
Further, the preparation of seed liquid of the clostridium perfringens type a production strain can be carried out according to the following method:
a-1) dissolving a freeze-dried strain with good vacuum degree by using a sterile strain culture medium (without glucose), inoculating the dissolved freeze-dried strain to the strain culture medium (without glucose) according to the inoculation amount of 1%, and culturing at 37 ℃ for 12-14 hours to obtain first-grade seeds;
a-2) inoculating the first-stage seeds into a strain culture medium according to the inoculation amount of 1-2% (before inoculation, adding a glucose aqueous solution with the mass fraction of 50% to ensure that the final concentration of glucose is 0.9%), and culturing at 37 ℃ for 9-11 hours to obtain second-stage seeds, namely the seed liquid of the clostridium perfringens type A production strain.
Further, the inoculation amount of the clostridium perfringens type a production strain seed liquid can be 4-5% (volume fraction).
The formula of the strain culture medium is as follows: the solvent is water; the solute and the concentration are as follows: 30g/L casein peptone, 15g/L yeast extract powder and 10mM Na2CO3、40mM Na2HPO4·12H2O、10mM KH2PO4The pH was 7.5.
In the above method for preparing a clostridium perfringens type a toxin, collecting the supernatant of the fermentation product is to centrifuge the fermentation product, collect the supernatant, filter the supernatant to obtain a filtrate, and if the filtrate is inoculated with anaerobic liver broth for aseptic growth, the filtrate is clostridium perfringens type a toxin (if the filtrate grows aseptically, the filtrate is filtered again until the filtrate grows aseptically).
Further, the centrifugation condition is 10000r/min for 60 min; the filtration was performed with a 0.22 μm filter.
The clostridium perfringens type A toxin prepared by the method also belongs to the protection scope of the invention.
In a fourth aspect, the invention provides a process for preparing a type a perfringolysin.
The method for preparing the type A clostridium perfringens toxin comprises the following steps: and (3) inactivating and detoxifying the A type clostridium perfringens toxin to obtain the A type clostridium perfringens toxoid.
Further, the method for preparing the type A perfringtonin comprises the following steps:
1) fermenting and culturing the clostridium perfringens type A production strain in the culture medium to obtain a fermentation product;
2) and (3) uniformly mixing the fermentation product with L-lysine and formaldehyde aqueous solution, and inactivating and detoxifying for 7-8 days at 36-37 ℃ under the condition that the pH value is 6.8-6.9 to obtain the inactivated and detoxified bacterial liquid.
In a still further aspect of the present invention,
in the 1), the conditions of the fermentation culture are as follows: fermenting and culturing at 35-37 deg.C for 5-6h, maintaining pH at 6.9 + -0.05 during fermentation, adding 50% glucose aqueous solution according to 1-1.2% of the total volume of the culture medium when fermenting and culturing for 4-5h, and controlling rotation speed at 25-30rpm during fermentation.
In the step 2), adding L-lysine into the fermentation product to ensure that the mass fraction of the L-lysine in the fermentation product is 0.7%; after dissolving and mixing evenly, adding formaldehyde aqueous solution (40 percent) according to the volume fraction of 0.5 percent; adding 10M sodium hydroxide to adjust pH to 6.8, inactivating and detoxicating at 37 deg.C for 7 days.
And 2) detecting the inactivation and detoxification effects of the inactivated and detoxified bacterium liquid.
The inactivation effect can be detected according to the following method: respectively inoculating the inactivated and detoxified bacterial liquid into anaerobic pork liver soup, common broth and common agar slant, culturing at 37 ℃, observing aseptic growth for 5 days, and indicating complete inactivation.
The detoxification effect can be detected according to the following method: centrifuging the inactivated and detoxified bacterial liquid for 30min at 3000r/min, removing thallus precipitate, leaving supernatant, filtering the supernatant with a 0.22-micron filter membrane, collecting filtrate, injecting 2 mice each with 0.4ml of 16-18g ICR into tail vein, and observing for 3 days to ensure that the bacteria are completely detoxified.
The clostridium perfringens type A toxoid prepared by the method also belongs to the protection scope of the invention.
In a fifth aspect, the invention provides any of the following applications:
(I) the use of a clostridium perfringens type a toxoid as defined above for the manufacture of a product for the treatment and/or prevention of a disease caused by a clostridium perfringens type a;
(II) the use of a Clostridium perfringens type A toxoid as defined above for the manufacture of a Clostridium perfringens type A toxin vaccine.
In the above applications, the diseases caused by clostridium perfringens type a include (but are not limited to) rabbit clostridium perfringens disease, piglet clostridium perfringens disease, necrotic enteritis of chickens, sudden death of cattle and the like.
In the above application, the clostridium perfringens type a toxin toxoid vaccine is specifically selected from (but not limited to) at least one of the following: (1) inactivated vaccine for clostridium perfringens disease (type a) of rabbit; (2) a piglet clostridium perfringens bivalent inactivated vaccine (type A, C); (3) the triple inactivated vaccine for the rabbit hemorrhagic disease, the pasteurellosis and the clostridium perfringens type A disease.
In a sixth aspect, the invention provides a clostridium perfringens type a toxoid vaccine.
The active component of the clostridium perfringens type A toxoid vaccine provided by the invention is the clostridium perfringens type A toxoid.
Further, the A-type clostridium perfringens toxin vaccine consists of the A-type clostridium perfringens toxoid and an adjuvant.
Further, the adjuvant is an alumina gel adjuvant.
In a seventh aspect, the present invention provides a method of preparing a type a perfringens toxin vaccine.
The method for preparing the clostridium perfringens type A toxin vaccine comprises the following steps:
1) fermenting and culturing the clostridium perfringens type A production strain in the culture medium to obtain a fermentation product;
2) uniformly mixing the fermentation product with L-lysine and formaldehyde aqueous solution, and inactivating and detoxifying at 36-37 ℃ for 7-8 days under the condition that the pH value is 6.8-6.9 to obtain inactivated and detoxified bacterial liquid;
3) centrifuging the inactivated and detoxified bacterial liquid, and collecting supernatant; and concentrating the supernatant, and adding an alumina gel adjuvant to obtain the clostridium perfringens type A toxoid vaccine.
Further, in the above-mentioned case,
in the 1), the conditions of the fermentation culture are as follows: fermenting and culturing at 35-37 deg.C for 5-6h, maintaining pH at 6.9 + -0.05 during fermentation, adding 50% glucose aqueous solution according to 1-1.2% of the total volume of the culture medium when fermenting and culturing for 4-5h, and controlling rotation speed at 25-30rpm during fermentation.
In the step 2), adding L-lysine into the fermentation product to ensure that the mass fraction of the L-lysine in the fermentation product is 0.7%; after dissolving and mixing evenly, adding formaldehyde aqueous solution (40 percent) according to the volume fraction of 0.5 percent; adding 10M sodium hydroxide to adjust pH to 6.8, inactivating and detoxicating at 37 deg.C for 7 days.
In the step 3), the centrifugation condition is 3000r/min for 30 min;
the method for adding the alumina gel adjuvant after concentrating the supernatant can be carried out according to the following steps: concentrating the supernatant by 10 times by using a 5kDa ultrafiltration membrane to obtain a concentrated clostridium perfringens type A toxoid; adding the concentrated clostridium perfringens type A toxoid into a bottle filled with autoclaved aluminum gel, adding normal saline to the bottle until the volume is required, adjusting the pH value to 6.8, and enabling the final concentration of the aluminum gel to be 20% (v/v) and the content of the clostridium perfringens type A toxoid to be 1600 MLD/mL.
In the above method for preparing a clostridium perfringens type a toxin vaccine, a step of detecting the inactivation and detoxification effects of the inactivated and detoxified bacterium solution is further included between 2) and 3).
The invention obtains the toxigenic culture medium based on the mass ratio of the following components (calculated according to the finished product of the 1000mL culture medium) by screening: 30g/L casein peptone, 15g/L yeast extract powder and 0.1g/L CaCl2、0.0014g/L ZnSO4·7H2O、5g/L Na2HPO4·12H2O、0.5g/L KH2PO410g/L glucose. The culture medium has strong toxin production capability in fermentation tank culture, stable toxin production capability, controllable quality, convenient preparation and use and low price.
The invention uses commercial casein peptone, yeast extract powder and other finished products as raw materials to replace the raw materials of beef, beef liver and the like with uncontrollable original quality, and ensures that the toxin production capacity of the culture medium reaches or even exceeds the original regulation standard and has better repeatability by screening the culture medium formula and optimizing the toxin production culture conditions.
The invention evaluates the effect of the vaccine on rabbits. As a result, the A-type clostridium perfringens toxoid vaccine prepared by the method can protect rabbits from being attacked by the toxin, and the serum neutralization titer also exceeds the corresponding standard of Chinese veterinary pharmacopoeia and the vaccine prepared by the traditional process.
The invention has the following advantages: the preparation method of the culture medium used by the invention is simple (only the required 28 hours for digesting the soup by the enzyme of the raw meat liver and stomach is reduced to 5 hours), and the cost is low (the comprehensive cost is 0.37 times of that of the original traditional process). The toxicity of the A-type clostridium perfringens toxin prepared by the culture medium according to the method can be improved to 12.5 times of the standard of Chinese veterinary biological product regulation, the primary-immune serum neutralization potency and the secondary-immune serum neutralization potency of the toxoid prepared by the culture medium on domestic rabbits can be respectively improved to 4 times and 50 times of the prior art, and the serum potency cost ratio can be improved to 48.8 times and 1216.4 times of the prior art. The invention has wide prospect for replacing the existing meat liver and stomach enzyme digestion soup or anaerobic meat liver soup to be used for producing the inactivated vaccine of the clostridium perfringens type A.
Drawings
FIG. 1 is the electrophoresis picture of protein after inactivation and detoxification of C57-1 toxin. 1 modified inactivation method (10 times concentration); 2 is the traditional inactivation method (10 times concentration); 3 modified inactivation method (not concentrated); 4, traditional inactivation method (not concentrating); m is a protein Marker, and is 140kDa, 115kDa, 80kDa, 65kDa, 50kDa, 40kDa, 30kDa, 25kDa and 15kDa from top to bottom in sequence.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The method of the present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.
The strain for preparing clostridium perfringens type a toxin used in the following examples is clostridium perfringens C57-1 strain (CVCC number: 37) purchased from the chinese veterinary microbial culture collection management center (www.cvcc.org.cn, abbreviated as CVCC).
The compositions of the anaerobic liver soup used in the following examples are shown in table 1.
Table 1 shows the composition of anaerobic pork liver soup
| Beef | 250g |
| Liver (cattle, sheep or pig) | 250g |
| Peptone | 10g |
| Sodium chloride | 5g |
| Glucose | 2g |
| Distilled water is added to | 1000ml |
The preparation method of the anaerobic pork liver soup comprises the following steps:
1. removing fat and fascia from beef, mincing with meat mincer, mixing with sliced beef liver (about 100 g), adding distilled water, stirring, 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 pieces.
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 flannel, adding glucose, and stirring to melt.
5. Cleaning the cooked liver blocks, cutting into small blocks, washing with distilled water, and packaging into test tubes or neutral glass bottles in an amount of 1/10.
6. Subpackaging the filtrate in neutral container (such as test tube, and appropriate amount of liquid paraffin) containing liver block, and sterilizing at 116 deg.C for 30-40 min.
The anaerobic pork liver soup is used for culturing and detecting common anaerobic bacteria. When the strain is used for strain preservation, glucose is not added.
The components of the gelatin buffer used in the following examples are shown in Table 2.
Table 2 shows the gelatin buffer composition
| Distilled water | 1000ml |
| Gelatin | 2g |
| Na2HPO4·12H2O | 9.25g |
| NaH2PO4·2H2O | 8.34g |
The preparation method of the gelatin buffer solution comprises the following steps: melting gelatin vapor, mixing, boiling, filtering, and sterilizing at 116 deg.C for 30 min.
The general broth culture used in the following examples has two formulations, fresh medium and dry powder medium, either of which is optional. The culture medium is used for testing bacterial vaccines, and the specific formula and the preparation method are as follows:
the formula I is as follows: adding 10g of peptone, 5.0g of sodium chloride and 1000ml of beef soup, mixing, slightly heating for dissolving, then placing to room temperature, adjusting the pH value to 7.4-7.6, filtering, subpackaging, sterilizing at 116 ℃ for 30 minutes, and adjusting the pH value of the sterilized culture medium to 7.2-7.4.
And a second formula: adding 10g of peptone, 5.0g of beef extract powder, 5.0g of yeast extract powder, 5.0g of sodium chloride and purified water to 1000ml, mixing, slightly heating for dissolving, placing the heated culture medium to room temperature, adjusting the pH value to 7.4-7.6, subpackaging, sterilizing at 116 ℃ for 30 minutes, and adjusting the pH value of the sterilized culture medium to 7.2-7.4.
The formula and preparation method of the common agar culture medium adopted in the following examples are as follows: adding 10g of peptone, 5.0g of sodium chloride, 12g of agar and beef soup to 1000ml, mixing, and heating to dissolve. After the agar is completely dissolved, adjusting the pH value to 7.4-7.6 by using a sodium hydroxide solution. Removing precipitate by egg white clarification method or coagulation precipitation method. The culture medium is used for testing bacterial vaccines.
The specific steps of removing the precipitate by the egg white clarification method are as follows: (1) adding purified water with the same amount into 2 chicken proteins, stirring, adding into 1000ml culture medium at 50 deg.C, and stirring. (2) Placing in a circulating steam pan, heating for 1 hr to fully coagulate protein. (3) Taking out, and filtering with absorbent cotton or filter paper under steam heating.
The concrete steps of removing the precipitate by the coagulating sedimentation method are as follows: (1) and (3) putting the agar culture medium with the adjusted pH value into a circulating steam pot, introducing a small amount of steam, and gradually cooling the agar culture medium to be automatically solidified after about 1 hour. (2) After complete cooling, the agar was decanted and the bottom precipitate was cut off. (3) Heating and melting the upper transparent part, and packaging.
Example 1 cultivation of Clostridium perfringens type A and preparation of Clostridium perfringens type A toxin
Firstly, the culture of the clostridium perfringens type A and the preparation of the clostridium perfringens type A toxin
This example relates to the following strain media and fermentation media:
the solvent of the strain culture medium is purified water; the solute and the concentration are as follows: 30g/L casein peptone, 15g/L yeast extract powder and 10mM Na2CO3、40mM Na2HPO4·12H2O、10mM KH2PO4The pH was 7.5. Subpackaging into small tubes, and autoclaving at 110 deg.C for 30 min.
The solvent of the fermentation medium is purified water; the solute and the concentration are as follows: 30g/L casein peptone, 15g/L yeast extract powder and 0.1g/L CaCl2、0.0014g/L ZnSO4·7H2O、5g/L Na2HPO4·12H2O、0.5g/L KH2PO410g/L glucose, pH 7.5. Autoclaving at 116 deg.C for 30 min.
The preparation method of the fermentation medium comprises the following steps: mixing the rest components except glucose in the culture medium with purified water, adjusting pH to 7.5, autoclaving at 116 deg.C for 30min, preparing glucose into 50% glucose aqueous solution, autoclaving at 110 deg.C for 40min, and adding to desired concentration before use to obtain the fermentation culture medium.
1. Preparation of seed liquid
Taking a freeze-dried strain with good vacuum degree, dissolving the freeze-dried strain by using a sterile strain culture medium (without glucose), inoculating the strain culture medium (without glucose) according to the inoculation amount of 1%, and performing static culture at 37 ℃ for 12 hours to obtain a first-class seed; inoculating the first-stage seed with a strain culture medium (adding 50% glucose aqueous solution before inoculation to make final concentration of glucose be 0.9%), and standing at 37 deg.C for 9 hr to obtain second-stage seed.
2. Fermentation culture of bioreactor
After completion of step 1, a sterilized 50% by weight aqueous glucose solution was added to the fermentor containing the previously sterilized fermentation medium (without glucose) to a final glucose concentration of 0.5% and the pH was adjusted to 6.9. And then inoculating the second-stage seeds prepared in the step 1 according to 5% of the total amount of the culture medium, wherein the fermentation temperature is 37 ℃, the pH value is maintained to be 6.9 by automatically supplementing alkali (5M NaOH), and the stirring speed is set to be 25 rpm. When the culture is carried out for 5 hours, a glucose aqueous solution with the mass fraction of 50 percent is supplemented, so that the final concentration of glucose is 0.5 percent. After fermentation culture for 6h, the fermentation product is harvested.
3. Preparation of clostridium perfringens type a toxins
And (3) after the step 2 is finished, centrifuging the fermentation product prepared in the step 2 for 60min at 10000r/min, discarding thalli precipitates to leave supernatant, filtering the supernatant by using a 0.22-micrometer filter membrane, inoculating anaerobic pork liver soup to the filtered filtrate for aseptic growth, and subpackaging the filtered filtrate as clostridium perfringens type A toxin (if bacteria grow, filtering again until the bacteria grow aseptically) into 1mL of small parts, freezing and storing at-80 ℃, and taking 1 small part of the small parts each time for cold water bath melting for virus detection.
4. Determining the toxicity of the toxin in the bacterial liquid to the white mouse after the culture
And (3) after the step (3) is finished, taking the type A clostridium perfringens toxin obtained in the step (3) to melt in a cold water bath, and diluting the type A clostridium perfringens toxin with a gelatin buffer solution according to the method shown in the following table 3 to obtain a toxin diluent.
Toxin dilutions were injected tail vein into clean grade 16-18g ICR mice, 2 per titer, 0.2mL per injection, and mice were observed for mortality within 72h post-injection. The minimum amount of toxin that can cause 2/2 death in mice is the MLD of clostridium perfringens type a toxin in mice. Virulence assays were performed on each of the toxins produced in 3 batches.
TABLE 3 dilution of toxins with gelatin buffer
| Pipe number | Gelatin buffer | Toxins | Content of toxin stock solution in 0.2mL of |
|
| 1 | 9.0mL | Stock solution 1.0mL | 0.02 |
|
| 2 | 1.0 | Tube | 1 liquid 1.0mL | 0.01 |
| 3 | 3.0 | Tube | 1 liquid 1.0mL | 0.005 |
| 4 | 4.0 | Tube | 1 liquid 1.0mL | 0.004mL |
| 5 | 5.1 | Tube | 1 liquid 0.9mL | 0.003mL |
| 6 | 9.0 | Tube | 1 liquid 1.0mL | 0.002mL |
The results are shown in Table 4. As can be seen from the results, the virulence of all 3 experiments far exceeds the virulence standard of the Clostridium perfringens type A C57-1 strain specified in the national institute of people's republic of China veterinary biologicals code of second O year edition, wherein 1 mouse MLD is 0.05-0.01mL of toxin stock solution of the Clostridium perfringens type A.
Table 4 summarizes the toxicity results of the fermenter culture
| Number of test batches | Minimal lethal dose (mL) for |
| Batch | |
| 1 | 0.005 |
| |
0.004 |
| |
0.004 |
Secondly, culturing A-type clostridium perfringens by using traditional culture medium and culture process and preparing A-type clostridium perfringens toxin
A clostridium perfringens type A C57-1 strain (CVCC number: 37) for animals is used as a clostridium perfringens type A production strain, a traditional culture medium and a traditional culture process are used for culturing the clostridium perfringens type A, and clostridium perfringens type A toxin is prepared. The traditional culture medium and culture process are used for culturing the clostridium perfringens type A and the preparation method of the clostridium perfringens type A toxin, which are compiled by Committee on veterinary biological product regulations of Ministry of agriculture, veterinary biological product regulations of the people' S republic of China, two O edition [ S ]. Beijing: chemical industry Press, 2000, "pp. 101-102.
The toxicity results in the conventional culture medium (pork liver and stomach enzyme digestion soup or anaerobic pork liver soup) are summarized in Table 5. The result shows that compared with the traditional culture medium and culture process, the fermentation culture medium and the fermentation process have strong toxin production capacity and good repeatability, and can be used as the first-choice toxin production culture medium for preparing the seedlings of the clostridium perfringens type A.
Table 5 summarizes the results of the toxicity in the conventional medium (ROUGANXIE-DISINFECTION SOURCE or AROMATIC-TOUGANY SOURCE)
| Number of test batches | Culture medium | Minimal lethal dose (mL) for |
| 1 | Meat liver and stomach enzyme digestion soup | ＞0.01 |
| 2 | Meat liver and stomach enzyme digestion soup | ＞0.05 |
| 3 | Meat liver and stomach enzyme digestion soup | 0.025 |
| 4 | Meat liver soup for anerobic reaction | 0.025 |
| 5 | Meat liver and stomach enzyme digestion soup | 0.02 |
| 6 | Meat liver and stomach enzyme digestion soup | ＞0.05 |
| 7 | Meat liver soup for anerobic reaction | ＞0.05 |
| 8 | Meat liver and stomach enzyme digestion soup | 0.05 |
| 9 | Meat liver and stomach enzyme digestion soup | 0.05 |
| 10 | Meat liver and stomach enzyme digestion soup | 0.025 |
| 11 | Meat liver and stomach enzyme digestion soup | 0.06 |
Example 2 preparation of a Clostridium perfringens type A toxin and a Clostridium perfringens type A toxoid vaccine
1. Inactivation of clostridium perfringens type a and detoxification of toxins
Traditional inactivation methods: adding a formaldehyde aqueous solution (40%) into the fermentation product obtained in the first step of example 1 according to the volume fraction of 0.5%, and inactivating and detoxifying at 37 ℃ for 7 days to obtain an inactivated and detoxified bacterial liquid.
An improved inactivation method comprises the following steps: adding L-lysine into the fermentation product obtained in the first step of the embodiment 1 to ensure that the mass fraction of the L-lysine in the fermentation product is 0.7%, dissolving and uniformly mixing, adding a formaldehyde aqueous solution (40%) according to the volume fraction of 0.5%, adjusting the pH value to 6.8 by using 10M sodium hydroxide, and inactivating and detoxifying at 37 ℃ for 7 days to obtain the inactivated and detoxified bacterial liquid.
2. Detection of inactivation and detoxification effects of clostridium perfringens type A toxin
(1) Respectively inoculating the inactivated and detoxified bacterial liquid obtained in the step 1 into anaerobic pork liver soup, common broth and common agar slant, culturing at 37 ℃, observing aseptic growth for 5 days, and indicating complete inactivation.
(2) Centrifuging the inactivated and detoxified bacterial liquid obtained in the step 1 for 30min at 3000r/min, removing the bacterial precipitate, leaving supernatant, filtering the supernatant by using a 0.22-micron filter membrane, collecting filtrate, injecting 2 ICR mice with 16-18g of ICR through tail vein, wherein each mouse is injected with 0.4ml of filtered stock solution, and the complete detoxification is shown by observing the live health for 3 days.
3. SDS-PAGE electrophoresis detection of inactivated and detoxified bacterial liquid
Centrifuging 3000r/min for 30min the inactivated and detoxified bacterial liquid obtained after the inactivation and the detoxification of the traditional inactivation method and the improved inactivation method in the step 1, removing the bacterial precipitate, and leaving the supernatant; and adding protein electrophoresis loading buffer solutions into the supernatant respectively, boiling for 10 minutes, and performing SDS-PAGE electrophoresis. The results are shown in FIG. 1, where the modified inactivation method retained more protein antigen than the conventional inactivation method.
4. Preparation of clostridium perfringens type a toxoid
And (3) centrifuging the completely inactivated and detoxified bacterial liquid obtained in the step (2) for 30min at 3000r/min, discarding the bacterial precipitate, leaving the supernatant to obtain the type A clostridium perfringens toxoid, concentrating the type A clostridium perfringens toxoid by 10 times by using a 5kDa ultrafiltration membrane to obtain the concentrated type A clostridium perfringens toxoid, and storing at 2-8 ℃ for later use.
5. Preparation of clostridium perfringens type a toxoid vaccine
And (3) adding the concentrated clostridium perfringens type A toxoid obtained in the step (4) into a bottle filled with autoclaved aluminum gel (a product of PI Pharma company, the product number is VAC 20 HA), adding normal saline to a required volume, adjusting the pH value to 6.8, and enabling the final concentration of the aluminum gel to be 20% (v/v) and the content of the toxoid to be 1600 MLD/mL. Shaking and mixing uniformly, and storing in a refrigerator at the temperature of 2-8 ℃ for later use to obtain the clostridium perfringens type A toxoid vaccine.
Example 3 detection of the immunological Effect of Clostridium perfringens type A toxoid vaccine on rabbits
The clostridium perfringens type a toxoid vaccine prepared in example 2, the vaccine prepared by the conventional process (the vaccine prepared according to page 101-102 of the second o year edition of the chinese veterinary biological product code), the commercial vaccine (inactivated vaccine of clostridium perfringens disease (type a) of rabbit, Shandong Huahong bioengineering Co., Ltd., 201801001 batches) were injected subcutaneously into the neck and back of 4 rabbits, respectively immunized with different dosages (as shown in table 6), and 2 non-immunized rabbits were used as a control, and the second immunization was performed 21 days after immunization (the immunization dosage and the inoculation were the same as the first immunization). Before immunization, 21 days and 35 days after first immunization, blood is collected from the middle ear artery of each rabbit for 5mL, serum is separated, the neutralizing potency of the mixed serum of 4 rabbits in each group on the clostridium perfringens type A toxin is respectively determined according to the method of Chinese veterinary pharmacopoeia (concretely, the neutralizing potency refers to the number of mouse MLD (minimum lethal dose) which can be neutralized by 0.1mL of serum in China national veterinary drug 2015 edition three S. After 38 days of first immunization, the rabbits of each immunization group and the control group are detoxified, 2A type clostridium perfringens C57-1 strains of MLD rabbits are injected into the ear vein of each rabbit, and 10 days of observation are carried out.
The result of the measurement of the serum of the rabbit before immunization shows that: neutralizing titer to clostridium perfringens type A toxin is less than 1, and the neutralizing titer is antibody negative rabbits. After the rabbits were immunized with the vaccines of different groups, the neutralizing potency of the mixed serum of 4 rabbits per group against clostridium perfringens type a toxin was determined. The results show that: for the clostridium perfringens type A toxin, the primary immune serum titer of the seedlings and the commercial seedlings in the traditional process does not reach 1, and the secondary immune serum titer is 1; the titer of the primary immune serum of the vaccine of the invention is 4 at most, and the titer of the secondary immune serum is greatly increased to 50, which are both remarkably improved compared with the traditional process vaccine or commercial vaccine (Table 6). After two immunizations, 2 rabbits were challenged with an lethal dose of clostridium perfringens type a toxin by otic intravenous injection, and all rabbits of the vaccine groups were 100% protected.
Table 6 shows the results of the serum titer determination and the immune challenge protection of the Clostridium perfringens type A toxoid vaccine on rabbits
Example 4 comparison of the Process of the invention with conventional Process
According to the method for preparing the meat liver and stomach enzyme digestion soup adopted in the conventional process in table 9, the meat liver and stomach enzyme digestion soup is prepared. The traditional vaccine is prepared by utilizing meat liver and stomach enzyme digestion soup. The traditional vaccine preparation method is described in detail in the Chinese veterinary biological product code, second O year edition page 101-102.
Compared with the traditional process, the method has the advantages of better stability, shorter time consumption and lower cost, particularly greatly enhanced immune effect, greatly improved serum titer cost ratio, and detailed results shown in tables 7-10.
Table 7 shows the comparison of the parameters of the present invention with those of the conventional process
Table 8 shows the cost accounting of the method of the present invention
Table 9 shows the cost accounting of the conventional process (meat liver and stomach enzyme digestion soup)
Table 10 shows the ingredients of anaerobic meat, liver and stomach enzyme digestion soup used in the conventional process
| Beef | 200g |
| Liver (cattle, sheep, pig) | 50g |
| Pepsin (1: 3000) | 3～4g |
| Hydrochloric acid | 10～11ml |
| Peptone | 10g |
| Glucose | 10g |
| Distilled water is added to | 1000ml |
The preparation method of the anaerobic meat liver and stomach enzyme digestion soup used in the traditional process comprises the following steps:
1. adding hydrochloric acid and minced beef and liver into warm water at about 65 ℃, fully stirring, adding pepsin, fully stirring, and mixing at 56-58 ℃.
2. Digesting for 22-24 hours at 53-55 ℃. The mixture was stirred well 1 time per hour for the first 10 hours.
3. Extracting supernatant, heating to 80 ℃, adding peptone, boiling, and adjusting the pH to 7.6-7.8. Boiling for 10 min, filtering or precipitating, collecting supernatant, adding glucose, dissolving, and packaging.
4. Sterilizing at 116 deg.C for 40 min.
Claims (11)
1. A clostridium perfringens type a fermentation medium comprising solutes; the solute consists of: casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4And glucose; casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4And glucose in a mass ratio of (20-30): (10-15): (0.075-0.1): (0.0014-0.0028）：（5-7.5）：（0.5-0.75）：10。
2. The culture medium according to claim 1, wherein: casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4And glucose in a mass ratio of 30: 15: 0.1: 0.0014: 5: 0.5: 10.
3. the culture medium according to claim 1 or 2, characterized in that: the medium further comprises a solvent.
4. The culture medium of claim 3, wherein: the solvent is water; the culture medium is composed of casein peptone, yeast extract powder, CaCl2、ZnSO4·7H2O、Na2HPO4·12H2O、KH2PO4Glucose and water; the concentration of the casein peptone in the culture medium is 20-30 g/L; the concentration of the yeast extract powder in the culture medium is 10-15 g/L; CaCl2The concentration in the culture medium is 0.075-0.1 g/L; ZnSO4·7H2The concentration of O in the culture medium is 0.0014-0.0028 g/L; na (Na)2HPO4·12H2The concentration of O in the culture medium is 5-7.5 g/L; KH (Perkin Elmer)2PO4The concentration in the culture medium is 0.5-0.75 g/L; the concentration of glucose in the culture medium is 10-15 g/L.
5. The culture medium according to claim 4, wherein: the concentration of casein peptone in the medium was 30 g/L; the concentration of the yeast extract powder in the culture medium is 15 g/L; CaCl2The concentration in the culture medium is 0.1 g/L; ZnSO4·7H2The concentration of O in the culture medium is 0.0014 g/L; na (Na)2HPO4·12H2The concentration of O in the culture medium is 5 g/L; KH (Perkin Elmer)2PO4The concentration in the culture medium is 0.5 g/L; concentration of glucose in the culture mediumThe degree is 10 g/L; the pH value of the culture medium is 7.5-7.7.
6. A method of preparing a culture medium according to any one of claims 1 to 5, comprising the steps of: mixing the components except glucose in the culture medium of any one of claims 1-5 with water according to mass ratio, adjusting pH to 7.5-7.7, sterilizing, preparing glucose into 50% glucose aqueous solution, sterilizing, and adding to desired concentration before use to obtain the culture medium.
7. Use of a medium according to any one of claims 1 to 5 or a process according to claim 6 for the preparation of a clostridium perfringens type a toxin and/or a clostridium perfringens type a toxoid.
8. A process for preparing a clostridium perfringens type a toxin comprising the steps of: fermenting and culturing a clostridium perfringens type A production strain in the culture medium of any one of claims 1-5 to obtain a fermentation product; collecting the supernatant of the fermentation product to obtain the clostridium perfringens type A toxin;
or, a process for preparing a type a perfringens toxoid comprising the steps of: fermenting and culturing a clostridium perfringens type A production strain in the culture medium of any one of claims 1-5 to obtain a fermentation product; inactivating and detoxifying the fermentation product to obtain clostridium perfringens type A toxoid; the inactivation and detoxification method comprises the following steps: mixing the fermentation product with L-lysine and formaldehyde aqueous solution, inactivating and detoxifying at 36-37 deg.C for 7-8 days under the condition of pH value of 6.8-6.9.
9. The method of claim 8, wherein: the conditions of the fermentation culture are as follows: fermenting and culturing at 35-37 deg.C for 5-6h, maintaining pH at 6.9 + -0.05 during fermentation, adding 50% glucose aqueous solution according to 1-1.2% of the total volume of the culture medium when fermenting and culturing for 4-5h, and controlling rotation speed at 25-30rpm during fermentation.
10. Use of a clostridium perfringens type a toxoid produced by the process of claim 8 or 9 in the manufacture of a product for the treatment and/or prevention of a disease caused by clostridium perfringens type a;
or, use of a clostridium perfringens type a toxoid produced by the process of claim 8 or 9 in the manufacture of a clostridium perfringens type a toxin vaccine.
11. A process for preparing a clostridium perfringens type a toxin vaccine comprising the steps of:
1) fermenting and culturing a clostridium perfringens type A production strain in the culture medium of any one of claims 1-5 to obtain a fermentation product;
2) uniformly mixing the fermentation product with L-lysine and formaldehyde aqueous solution, and inactivating and detoxifying at 36-37 ℃ for 7-8 days under the condition that the pH value is 6.8-6.9 to obtain inactivated and detoxified bacterial liquid;
3) centrifuging the inactivated and detoxified bacterial liquid, and collecting supernatant; and concentrating the supernatant, and adding an alumina gel adjuvant to obtain the clostridium perfringens type A toxoid vaccine.
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