CN102688484A - Production method for an inactivated vaccine for type-C Clostridial enteritis - Google Patents
Production method for an inactivated vaccine for type-C Clostridial enteritis Download PDFInfo
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Abstract
The invention relates to a production method for an inactivated vaccine for type-C Clostridial enteritis. The inactivated vaccine aiming at the Clostridial enteritis is not invented in China at present, whereas a hepatic and gastric enzyme digestion broth culture medium is usually adopted by the inactivated vaccine aiming at Clostridial diseases of other animals. The culture medium comprises the following main components: beef and liver and is greatly influenced by the varieties, the ages, the freshness and the digestion degrees of the animals, thus, the quality of the culture medium is not stable, the dispersion between each two batches is high and the manufacturing process is complicated, and further, the quality of the vaccine is influenced, especially the consistency of the vaccine, whereas a synthetized culture medium provided by the invention is capable of overcoming the defects, is a development tendency of performing bacterial high-cell density culture, has the advantages of simplicity in operation and stable quality and provides a powerful guarantee for the production of the high-quality inactivated vaccine for the type-C Clostridial enteritis.
Description
Technical field
The present invention relates to the production method of a kind of chicken necrotizing enterocolitis (C type) inactivated vaccine.Belong to veterinary biologics and make the field.
Background technology
Chicken necrotizing enterocolitis is called clostridium enteritis (Clostridial enteritis) again, unbalance disease of flora (dysbacteriosis) or intestinal bacteria overgrowth disease (SIBO).Be a kind of disease that is caused by bacillus perfringens (Clostridium perfringens) A type or C type, A type bacillus perfringens produces alpha toxin, and the C type produces α and β toxin, and these two kinds of toxin are the main toxin that cause necrotic enteritis.It is characteristic that the fowl that dies of illness has wheat bran appearance necrosis region with hepatomegaly, intestinal bleeding and intestinal mucosa; Normal chicken crowd's sickness rate is 1.3%～37.3%; The sickness rate of specific pathogen free flock can be up to 62% (Wang Zehua, Li Canheng. the diagnosis and treatment of chicken necrotizing enterocolitis, Chinese fowl industry guide; 2002,19 (14): 35-36).
At present domestic still not to the vaccine of chicken necrotizing enterocolitis, treatment should disease mainly be adopted antibiotic.What domestic other clostridium class production of vaccine adopted is meat liver gastric enzyme digestion soup culture medium; Its main component beef regulating liver-QI of this culture medium etc.; Receive animal varieties, age, influence fresh and digestible bigger; Cause the culture medium quality unstable, batch between dispersion big and manufacturing process is loaded down with trivial details, and then influence the homogeneity of the quality of vaccine, particularly vaccine.Large scale fermentation according to bacillus perfringens Nutrition and Metabolism characteristics designs, has added the somatomedin that promotes growing microorganism, improves protective antigen content with synthetic medium, has the poison of producing amount height, steady quality, advantage such as easy and simple to handle.Synthetic medium is the key factor of bacterial vaccine quality quality; In view of this; The present invention is intended to design a kind of suitable C type bacillus perfringens CVCC60102 strain growth, the product poison is measured synthetic medium high, that composition is confirmed, is easy to online detection, monitoring relatively and in time adjusts, preparation C type bacillus perfringens inactivated vaccine.
Summary of the invention
Being inoculated in pancreas casein peptone and yeast extract powder by the volume ratio 1%～2% of culture medium total amount C type bacillus perfringens CVCC 60102 strain seed liquor is in the liquid synthetic medium of main raw material(s); 37 ℃ of anaerobism cultivate/or leave standstill and cultivated 18 hours; Cultivate and finish after the centrifugal 30min of 3000r/min; Remove thalline; Ultrafiltration and concentration system with the 8Ku molecular cut off carries out ultrafiltration and concentration respectively, and after the formalin deactivation detoxification through 0.6% fully, the aluminium hydroxide gel that adds final concentration 20% is mixed with vaccine.
The present invention describes in detail
1. production of vaccine is used strain
(1) source bacillus perfringens CVCC60102 strain strain, former C59-2,5-2 identifies, takes care of and supply by China Veterinery Drug Inspection Office.This bacterium is to separate nineteen fifty-three from Inner Mongolia Autonomous Region to receive the struck Cor Caprae seu ovis blood of the right flag of alliance's new bus, virulent strain.
(2) strain properties
Virulence: the intravenous injection of meat liver gastric enzyme digestion soup culture supernatant, the 0.001ml white mice that causes death; The rabbit 0.05ml cause death, the 0.5ml sheep that can cause death.
Immunogenicity: 1.0ml aluminium glue Seedling immunizing rabbit can produce 75%～100% protection.
2 culture medium adopt the synthetic medium of the present invention's design
(1) prescription is as follows:
Peptone 3g, yeast powder 0.3g, sodium chloride 0.25g, dipotassium hydrogen phosphate 0.45g, glucose 0.5g, sodium sulfite 0.2g, cysteine hydrochloride 0.05g, dextrin 1g, Vc0.001g, deionized water adds to 100ml.
(2) synthetic medium compound method
Take by weighing each composition by prescription and be dissolved in the water for injection, regulate pH value to 8.0～8.4 with the sodium hydroxide of 2mol/L.116 ℃ of autoclaving 30min.
(3) synthetic medium check
1) character solution clear, be brown.
2) steriling test by People's Republic of China's veterinary drug allusion quotation (Chinese veterinary drug allusion quotation committee. 2010 editions three ones of People's Republic of China's veterinary drug allusion quotations. Chinese agriculture publishing house, 2011, to call " Chinese veterinary drug allusion quotation " in the following text) method of regulation carries out, and answers asepsis growth.
3) pH value should be 8.0～8.4.
4) growth test
⑴ seed liquor preparation
With C type bacillus perfringens CVCC 60102 strains (China Veterinery Drug Inspection Office provides) freeze-drying lactobacillus with after 1ml ordinary broth (China Veterinery Drug Inspection Office provides) dilution; Be inoculated in the 10ml meat liver gastric enzyme digestion soup; Put 37 ℃ and cultivated 18～24 hours, inoculate each 2 pipe of ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk simultaneously respectively, every pipe 0.2ml; Cultivated 48 hours, purely after the assay was approved as first order seed.2～8 ℃ of preservations, the operating period must not be above 15 days.Get first order seed 0.1ml inoculation 10ml meat liver gastric enzyme digestion soup; Cultivated 18 hours for 37 ℃; After the pure passed examination as secondary seed (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's veterinary biologics rules. Chemical Industry Press; 2001, to call " rules " in the following text).
⑵ bacterium liquid is cultivated and is judged
Seed liquor is inoculated into by 1% of culture medium total amount volume ratio in the 500ml triangular flask that the 200ml fluid medium is housed, and 37 ℃ leave standstill cultivation 18 hours, establish 1 simultaneously and do not inoculate negative control.The centrifugal 30min of 3000r/min that takes a sample respectively gets supernatant and carries out toxicity test (seeing note) by existing " rules ".Virulence should reach 500MLD/ml, and negative control is answered asepsis growth.
5) storage with effect duration fluid medium should join existing usefulness at present, place room temperature behind the autoclaving and use at once.
3 vaccine manufacturings
(1) seed liquor preparation
With C type bacillus perfringens CVCC 60102 strains (China Veterinery Drug Inspection Office provides) freeze-drying lactobacillus with after 1ml ordinary broth (China Veterinery Drug Inspection Office provides) dilution; Be inoculated in the 10ml meat liver gastric enzyme digestion soup; Put 37 ℃ and cultivated 18～24 hours, inoculate each 2 pipe of ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk simultaneously respectively, every pipe 0.2ml; Cultivated 48 hours, purely after the assay was approved as first order seed.2～8 ℃ of preservations, the operating period must not be above 15 days.Get first order seed 0.1ml inoculation 10ml meat liver gastric enzyme digestion soup; Cultivated 18 hours for 37 ℃; After the pure passed examination as secondary seed (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's veterinary biologics rules. Chemical Industry Press; 2001, to call " rules " in the following text).
(2) vaccine manufacturing
Being inoculated in pancreas casein peptone and yeast extract powder by 1% of culture medium total amount volume ratio seed liquor is in the liquid synthetic medium of main raw material(s), 37 ℃ of anaerobism cultivate/or leave standstill and cultivated 18 hours.Cultivate and finish the centrifugal 30min of back bacterium liquid 3000r/min; Get supernatant after the ultrafiltration and concentration system of 8Ku molecular cut off concentrates; Added 0.6% formalin deactivation detoxification immediately 4～6, inactivated vaccine is processed in the deactivation detoxification back aluminium hydroxide gel and 0.006% thimerosal that add final concentration 20% fully.
(3) product inspection
After these article of character left standstill, the upper strata was the yellowish-brown clear liquid, and lower floor is a pale precipitation, became even suspension after the jolting.
Steriling test is undertaken by existing " Chinese veterinary drug allusion quotation " note, answers asepsis growth.
10 of the broiler of safety verification subcutaneous vaccination 14 ages in days, every 2ml observed 10, any part and the systemic adverse reactions that are caused by vaccine should not occur.
5 of the broiler of efficacy test subcutaneous injection 14 ages in days, every 0.5ml is after 14 days; Together with 5 of the identical contrast broiler of condition, each intravenous injection 0.2ml (containing 1MLD) C type clostridium perfringens toxoid was observed 5; Immune group at least 4/5 protection, not immune matched group chicken 5/5 death.
Formaldehyde, thimerosal assay are undertaken by existing " Chinese veterinary drug allusion quotation " appendix, and the formaldehyde residual content should be no more than 0.5% in the goods, and the thimerosal residual content should be no more than 0.01%.
The present invention relates to the C type bacillus perfringens CVCC60102 strain that the information of microorganism the present invention relates to; From China Veterinery Drug Inspection Office China veterinary microorganism preservation administrative center (China Veterinery Drug Inspection Office China veterinary microorganism preservation administrative center writes. Chinese veterinary's strain catalogue second edition. Scientia Agricultura Sinica technology publishing house, 2002).
The present domestic chicken necrotizing enterocolitis inactivated vaccine that still do not have of positive effect of the present invention; And the common employing of other clostridium class vaccines is meat liver gastric enzyme digestion soup or lonely liver bouillon culture medium; Its main component beef regulating liver-QI of this culture medium etc. receive animal varieties, age, influence fresh and digestible bigger, cause the culture medium quality unstable, batch between dispersion big and manufacturing process is loaded down with trivial details; And then influence the homogeneity of the quality, particularly vaccine of vaccine.And synthetic medium provided by the present invention; Overcome above defective; Be the development trend of carrying out the antibacterial high-cell-density cultivation, have easy and simple to handle, stay-in-grade advantage, for high-quality chicken necrotizing enterocolitis inactivated vaccine production provides sound assurance.
The specific embodiment
Embodiment 1
Vaccine is made and check
(1) seed liquor preparation
With C type bacillus perfringens CVCC 60102 strains (China Veterinery Drug Inspection Office provides) freeze-drying lactobacillus with after 1ml ordinary broth (China Veterinery Drug Inspection Office provides) dilution; Be inoculated in the 10ml meat liver gastric enzyme digestion soup; Put 37 ℃ and cultivated 18～24 hours, inoculate each 2 pipe of ordinary broth, plain agar inclined-plane, lonely liver bouillon and litmus milk simultaneously respectively, every pipe 0.2ml; Cultivated 48 hours, purely after the assay was approved as first order seed.2～8 ℃ of preservations, the operating period must not be above 15 days.Get first order seed 0.1ml inoculation 10ml meat liver gastric enzyme digestion soup; Cultivated 18 hours for 37 ℃; After the pure passed examination as secondary seed (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's veterinary biologics rules. Chemical Industry Press; 2001, to call " rules " in the following text)
(2) vaccine manufacturing
Being inoculated in pancreas casein peptone and yeast extract powder by 1% of culture medium total amount seed liquor is in the liquid synthetic medium of main raw material(s), and 37 ℃ leave standstill cultivation 18 hours (or anaerobism was cultivated 18 hours).Cultivate and finish the centrifugal 30min of back bacterium liquid 3000r/min; Get supernatant after the millipore of 8Ku molecular cut off ultrafiltration and concentration system concentrates; Added 0.6% formalin deactivation detoxification immediately 4～6, inactivated vaccine is processed in the deactivation detoxification back aluminium hydroxide gel and 0.006% thimerosal that add final concentration 20% fully.
(3) vaccine product inspection
After these article of character left standstill, the upper strata was the yellowish-brown clear liquid, and lower floor is a pale precipitation, became even suspension after the jolting.
Steriling test carries out asepsis growth by existing " Chinese veterinary drug allusion quotation " note.
10 of the broiler of safety verification subcutaneous vaccination 14 ages in days, every 2ml observed 10, any part and the systemic adverse reactions that are caused by vaccine did not occur.
5 of the broiler of efficacy test subcutaneous injection 14 ages in days, every 0.5ml is after 14 days; Together with 5 of the identical contrast broiler of condition, each intravenous injection 0.2ml (containing 1MLD) C type clostridium perfringens toxoid was observed 5; Immune group 4/5 protection, not immune matched group chicken 5/5 death.
Formaldehyde, thimerosal assay are undertaken by existing " Chinese veterinary drug allusion quotation " appendix, and formaldehyde is residual all up to specification with the thimerosal residual content in the goods.
Embodiment 2
The research of C type bacillus perfringens CVCC60102 strain (C59-2 strain) synthetic medium
Filter out this synthetic medium prescription through leaving standstill cultivation C type bacillus perfringens CVCC60102 strain; Preparation condition and condition of culture to this synthetic medium are optimized; And can compare with the toxigenicity of meat liver stomach membrane digestion soup; The result should fill a prescription 37 ℃ to leave standstill and cultivate 18 hours toxigenicity and can reach 500～1000MLD/ml, produced malicious ability and met or exceeded meat liver stomach membrane digestion soup.
1 material
1.1 culture medium raw material
Pancreas casein peptone (lot number VM732731-644) is available from MERCK company; Yeast soaks powder (lot number 911948) available from OXOID company; Dipotassium hydrogen phosphate, glucose, iron chloride, cysteine hydrochloride, leucine, arginine, histidine, V
B1, V
K1, Vc, sodium sulfite, dextrin be the analytical pure chemical reagent, available from Beijing chemical reagents corporation.
1.2 meat liver stomach membrane digestion soup meat soup (lot number is 0130,0227,0307,0115,0119,0205), A type test tube use specification 25ml, triangular flask to use specification 500ml, basis set providing cultivated by China Veterinery Drug Inspection Office.
1.3 strain
Bacillus perfringens CVCC60102 strain, 0.3ml/ props up, and identifies, takes care of and supply by China Veterinery Drug Inspection Office.
1.4 animal
Mice: ICR system, 30～35 ages in days, body weight 16～20g, the SPF level is purchased and is provided by China Veterinery Drug Inspection Office's laboratory animal group.
2 methods and result
2.1 basic components screening
From the various combination prescription, through cultivating bacillus perfringens CVCC60102 strain, produce toxic effect and really compare, the screening basic components.
Take by weighing sodium chloride 2.5g, dipotassium hydrogen phosphate 4.5g, be dissolved in the 1000ml deionized water, using the sodium hydroxide adjust pH of 2mol/L is 8.0～8.4.Peptone and yeast powder are designed to 9 prescriptions by different proportion (W/V), are labeled as 1～9 (concrete ratio and numbering result see table 1), be settled to 100ml respectively, sterilized 30 minutes for 116 ℃, set up the contrast of meat liver gastric enzyme digestion soup simultaneously with above-mentioned buffer.By 1% adding seed liquor, 37 ℃ leave standstill cultivation 18 hours, get the centrifugal 30min of cultured bacterium liquid 3000r/min, get supernatant and survey poison.The result sees the following form 1.
9 groups of basic components toxigenicity of table 1 can be surveyed malicious result
* toxin is suitably diluted with the gelatin buffer,, in 24 hours, make all dead highly diluted multiple of mice 2/2 multiply by 5, be minimum lethal dose (MLD) number of every ml toxin 2 mices of diluent 0.2ml intravenous inoculation.As follows.
Prescription 5 can be higher with prescription 6 toxigenicity from table 1, near meat liver gastric enzyme digestion soup.
2.2 the screening of culture medium somatomedin
Choose toxigenicity and can process fluid medium, sterilized 30 minutes for 116 ℃, treat it and reduce to room temperature, divide to install in the A type test tube of sterilization near 2 groups of basic components of meat liver gastric enzyme digestion soup.In A type test tube, add somatomedin (the W/V ratio is seen table 2) such as iron chloride through 0.22 μ m filtration sterilization, cysteine hydrochloride, arginine, histidine, VB1, VK1, Vc, sodium sulfite, dextrin respectively, set up the contrast of meat liver gastric enzyme digestion soup simultaneously.By 1% adding seed liquor, 37 ℃ leave standstill cultivation 18 hours, get cultured bacterium liquid 3000rpm centrifugal 30 minutes, get supernatant and survey poison.The result sees the following form 2.
Table 2 culture medium somatomedin screening test is surveyed malicious result (MLD/ml)
Can find out that from table 2 result cysteine hydrochloride, sodium sulfite, Vc, dextrin all have the effect of certain promotion toxin producing.
2.3 synthetic medium combined sorting
Each production factor is designed to several prescriptions by various combination, and by 1% adding seed liquor, 37 ℃ leave standstill cultivation 18 hours, its toxigenicity ability of sampling and measuring.The result sees table 3.
Table 3 basic components adds combinations of. growth factors screening comparative test
The result finds out from table 3: the culture medium prescription (W/V) that is fit to is 3% peptone+0.3% yeast powder+0.5% glucose+0.2% sodium sulfite+0.05% cysteine hydrochloride+1% dextrin+0.001%Vc.
2.4 the optimization of C type synthetic medium preparation condition
Synthetic medium condition of high voltage, pH value are optimized, confirm that the righttest sterilising conditions is 116 ℃, 30min, optimum pH is 8.0～8.4.The result sees table 4, table 5.
The product of C type culture medium poison result (MLD/ml) under the different condition of high voltage of table 4
The product of C type culture medium poison result (MLD/ml) under table 5 condition of different pH
2.5 confirming of synthetic medium condition of culture
Synthetic medium formulations were prepared by six bottled 300ml medium, according to the amount of 1% were inoculated with strains of Clostridium perfringens CVCC60102 seed liquid, including a bottled separately in culture 6,8,10,12,14,16, 18,20,22 and 24h after sampling; additional preparation 5 bottled medium, respectively, at 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ and 40 ℃ cultured 22h after sampling, extraction of toxins after completion of the culture, and make poison force measured to determine the best time and toxigenic culture temperature, the results showed that 18 ~ 24h after training virulence reach 50MLD/ml, when the culture temperature at 36 ℃ ~ 37 ℃, the medium toxigenic best results.The result sees table 6, table 7.
The different incubation time toxigenicity of table 6 can be surveyed malicious result
Ψ represents 66 * dilution toxin, intravenous inoculation mice 0.2ml, and mice 1/2 is dead or all not dead, and other dilution factor does not detect, and therefore is decided to be < 330MLD/>ml.
Toxigenicity can be surveyed malicious result under the different cultivation temperature of table 7
2.6 the comparative test of synthetic medium and meat liver stomach membrane digestion soup is referring to table 8.
Table 8 synthetic medium and meat liver gastric enzyme digestion soup virulence comparative result
The result finds out from table 8, the alternative traditional culture medium of succeeding in developing of C type synthetic medium.
2.7 synthetic medium amplification culture test
C type culture medium is being cultivated on the successful basis in a small amount; Further amplification culture volume makes volume of culture reach 1000,2000,3000,4000,5000,6000,7000,8000,9000 and 10000ml respectively, respectively sampling in 18,20,22 and 24 hours; Toxigenicity to culture medium can be measured; The result shows that when volume of culture enlarges the toxigenicity of culture medium can be constant, and toxogenic virulence has all reached 500MLD/ml.The result sees table 9.
The different volume of culture of table 9 can be surveyed malicious result (MLD/ml) in the toxigenicity of different incubation times
3 conclusions
This synthetic medium prescription is peptone 3g, yeast powder 0.3g, and sodium chloride 0.25g, dipotassium hydrogen phosphate 0.45g, glucose 0.5g, sodium sulfite 0.2g, cysteine hydrochloride 0.05g, dextrin 1g, Vc0.001g, deionized water adds to 100ml.Toxigenicity can with meat liver stomach membrane digestion soup basically identical, can reach 500～1000ml/MLD, can substitute meat liver stomach membrane digestion soup.
Embodiment 3
Synthetic medium is cultivated the test of toxin concentration technology
The β toxin is that C type bacillus perfringens mainly excretes poison, and also is main immunogens, the about 28～34Kd of its molecular weight.With cultured C type bacillus perfringens CVCC60102 strain bacterium liquid; Through the centrifugal 30min of 3000r/min; Remove thalline; Millipore ultrafiltration and concentration system with PSPP carries out ultrafiltration and concentration respectively, concentrates forward and backward volume quantitative then, and gets and concentrate forward and backward and each 10ml of peritoneal effluent sample carries out toxicity test.The yield rate of 10Ku molecular cut off is 68%, its peritoneal effluent intravenous inoculation 0.2ml, mice 2/2 death; And the 8Ku yield rate reaches 80%, its peritoneal effluent peritoneal effluent intravenous inoculation 0.2ml, mice 0/2 death; Therefore the film bag of 8Ku molecular cut off suits to concentrate (the seeing table 10) of C type bacterium cultivation toxin.
The concentrated result of the film bag of table 10 different molecular interception
Embodiment 4
The test of chicken necrotizing enterocolitis (C type) inactivated vaccine immunogenicity
With cultured CVCC 60102 strain bacterium liquid; 3000r/min is centrifugal, and 30min gets supernatant; After the ultrafiltration and concentration system concentrated, the formalin deactivation detoxification with 0.6% added the aluminium hydroxide gel of final concentration 20% after the deactivation detoxification fully and 0.006% thimerosal is mixed with Seedling.5 of subcutaneous vaccination 14 age in days broiler, every 0.5ml, the C type toxin together with blank intravenous injection 0.2ml (containing 1MLD) after 14 days carries out counteracting toxic substances; Observed 5; Immune group has obtained 4/5 protection as a result, and not immune matched group chicken is all dead, shows that the immunogenicity of vaccine is good.Its result sees the following form 11.
Table 11 C type toxin is surveyed malicious result to 28 age in days broiler
| The toxin extension rate | The actual content of toxins of intravenous injection 0.2ml | The chicken death situation |
| 25 | 0.008ml | 0/2 |
| 22.2 | 0.009ml | 0/2 |
| 20 | 0.01ml | 0/2 |
| 10 | 0.02ml | 0/2 |
| 6.7 | 0.03ml | 1/2 |
| 5 | 0.04ml | 2/2 |
| 4 | 0.05ml | 2/2 |
Find out that from last table 11 C type toxin is that every 1ml contains 25MLD to the minimum lethal dose of 28 age in days broiler, i.e. 0.04ml/1MLD.
3 batches of vaccines of table 12 are to the immunoprotection result of the test of chicken
"-" representes not vaccination.
Can find out that from table 12 the inactivated vaccine immunogenicity of the C type bacillus perfringens CVCC60102 strain that synthetic medium is cultivated is good.
Embodiment 5
Chicken necrotizing enterocolitis (C type) inactivated vaccine safety testing
3 batches of chicken necrotizing enterocolitis (C type) inactivated vaccine with preparation; Difference subcutaneous injection 14 age in days broiler, 10 every group, every 2ml (containing 4 using dosages); Other establishes 5 as blank; Observed 10, and any part and systemic adverse reactions that vaccine causes did not occur, vaccine group and blank group average weight gain do not have significant difference.The result sees the following form 13.
3 batches of chicken necrotizing enterocolitis of table 13 (C type) inactivated vaccine safety testing result
3 batches of chicken necrotizing enterocolitis of table 14 (C type) inactivated vaccine safety testing body weight gains result
Note:
The method of toxin toxicity test: toxin is suitably diluted with the gelatin buffer; 2 of the white mice of each dilution factor tail vein injection body weight 16～20g; Every 0.2ml, the highly diluted multiple all dead with white mice in 24 hours 2/2 multiply by 5 contained minimum lethal dose (MLD) numbers for this every 1ml toxin.
Claims (2)
1. the production method of a chicken necrotizing enterocolitis (C type) inactivated vaccine; It is characterized in that being inoculated in pancreas casein peptone and yeast extract powder by the volume ratio 1%～2% of culture medium total amount C type bacillus perfringens CVCC60102 strain seed liquor is in the liquid synthetic medium of main raw material(s); 37 ℃ of anaerobism cultivate/or leave standstill and cultivated 18 hours; Cultivate and finish after the centrifugal 30min of 3000r/min removes thalline, the millipore ultrafiltration and concentration system with the 8Ku molecular cut off carries out ultrafiltration and concentration respectively; After the formalin deactivation detoxification through 0.6% fully, the aluminium hydroxide gel that adds final concentration 20% is mixed with vaccine.
2. the production method of chicken necrotizing enterocolitis as claimed in claim 1 (C type) inactivated vaccine is characterized in that wherein use with pancreas casein peptone and yeast extract powder as the prescription that the liquid synthetic medium of main raw material(s) closes is: peptone 3g, yeast powder 0.3g; Sodium chloride 0.25g, dipotassium hydrogen phosphate 0.45g, glucose 0.5g; Sodium sulfite 0.2g, cysteine hydrochloride 0.05g, dextrin 1g; Vc0.001g, deionized water adds to 100ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160555A (en) * | 2013-03-19 | 2013-06-19 | 武汉中博生物股份有限公司 | Culture medium, culture method and application of high-yield exotoxin of clostridium perfringens |
| CN109943507A (en) * | 2019-03-26 | 2019-06-28 | 中国兽医药品监察所 | A kind of preparation method and applications of A type C.perfringens toxoid for animals |
| CN116445373A (en) * | 2023-06-14 | 2023-07-18 | 金宇保灵生物药品有限公司 | C clostridium perfringens toxigenic culture medium and preparation method and application thereof |
| CN116445373B (en) * | 2023-06-14 | 2023-09-12 | 金宇保灵生物药品有限公司 | C clostridium perfringens toxigenic culture medium and preparation method and application thereof |
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