CN101967458B - Method for preparing bacteria liquid for Riemerella anatipestifer vaccine - Google Patents
Method for preparing bacteria liquid for Riemerella anatipestifer vaccine Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 24
- 239000007788 liquid Substances 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 18
- 229960005486 vaccine Drugs 0.000 title claims abstract description 17
- 241001478212 Riemerella anatipestifer Species 0.000 title abstract description 10
- 239000002609 medium Substances 0.000 claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
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- 235000015097 nutrients Nutrition 0.000 claims abstract description 11
- 239000006916 nutrient agar Substances 0.000 claims abstract description 10
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- 210000002966 serum Anatomy 0.000 claims description 16
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- 238000011081 inoculation Methods 0.000 claims description 11
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- 229910019142 PO4 Inorganic materials 0.000 claims description 3
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- 235000015278 beef Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000011156 evaluation Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing bacteria liquid for Riemerella anatipestifer vaccine, which comprises the propagation and identification of the first-level seed, and the propagation and fermentation of the second-level seed; the propagation and identification of the first-level seeds comprises the following steps of: inoculating freeze-dry basic microbial strain into the nutrient broth containing 10 percent of fetal bovine serum, and culturing the nutrient broth for 24h at the temperature of 37 DEG C; inoculating the obtained product onto the nutrient agar plate containing 10 percent of fetal bovine serum by streaking, then culturing the obtained product in the environment containing 5 to 10 percent of CO2 or candle jar for 24h to 48h at the temperature of 37 DEG C; after that, selecting several typical colonies, inoculating the typical colonies onto the nutrient agar slant containing 10 percent of fetal bovine serum, culturing the product in the environment containing 5 to 10 percent of CO2 or candle jar for 24h at the temperature of 37 DEG C, and obtaining first-level seeds; the propagation of the second-level seeds comprises the following steps of: inoculating the first-level seeds into the nutrient broth containing 10 percent of fetal bovine serum; and culturing the nutrient broth for 24h at the temperature of 37 DEG C; sampling the obtained product for pureness test; and if obtained product is tested to be qualified, keeping the product at the temperature of 2 to 8 DEG C, wherein the service life thereof is not more than five days, the culture medium used in the fermentation is the N-J synthetic medium containing one percent of lysed whole blood, the fermenting microbial strain is inoculated in an amount which is 2 percent based on that of the second-level seed liquid, the fermentation manner is liquid fermentation and culture in a culture tank, and the culture condition is continuously culturing 24h at the temperature of 37 DEG C.
Description
Technical field
The present invention relates to biologic product technology for animals field, particularly a kind of method of producing the Riemerlla anatipestifer vaccine with bacterium liquid.
Background technology
Infectious serositis of duck is a kind of contagious disease that betides tame duck, goose, turkey and multiple bird, claim riemerella anatipestifer disease, pest of duck Bacillus pasteurii disease, duck septicemia, pest of duck syndromes, pest of duck septicemia and new duck disease again, its cause of disease be riemerella anatipestifer (Riemerella anatipestifer, RA).This disease is acute sepsis or chronic infection, mainly shows as cough clinically, breathes, diarrhea, an eye nasal discharge increase, ataxia and neck tremble, and symptoms such as neck is crooked appear in the chronic case of minority; Typical cytopathic is fibrinous pericarditis, serohepatitis, airsacculitis, meningitis and caseous salpingitis.This disease all can take place throughout the year, and is serious with the morbidity in winter, mainly propagation such as the feed through polluting, drinking-water, the spittle; The duck in age in 1-8 week is extremely sensitive, and is higher with the duckling sickness rate in 2-3 age in week especially, can reach 90%; Mortality ratio is subjected to influence of various factors, and difference is bigger, often is 10-20%, but also have up to 60-75%.Stressors such as stocking density be excessive, it is smooth to ventilate, sanitary condition is poor, abrupt change of climate etc., can bring out the generation of this disease, and mortality ratio improves greatly; Anti-duck excessively can show nervous symptoms, grows slowly, becomes thin, and the price of deed descends, and has a strong impact on the production achievement, causes serious economy loss.
Infectious serositis of duck is at present duck one of the most serious transmissible disease that already works the mischief to be supported in countries in the world, worldwide distributes, and almost adopting intensification to support countries that duck produces at all has discovery, support duck to the world and already caused enormous economic loss.China is a foster duck big country, support the duck industry and in the rural economy of China, be seized of consequence, also be people one of the important sources of required meat egg of living, infectious serositis of duck is geographic popular more serious each foster duck of China, be that China duckery (particularly commodity duck field) is difficult to tackle, cause supports duck one of the main transmissible disease of loss of helping already most, the resistance of RA strengthens gradually in addition, supports duck to China and has already caused the tremendous economic loss.
Because the sick provisions duck of RA already brings serious economy loss, relevant its immunoprophylaxis research has caused increasing concern.Although RA serotype is more; substantially there is not cross-protection between various; brought bigger difficulty for the development of vaccine; but, still caused veterinary biologics field scientific research personnel's extensive concern and thought the essential measure or the vaccination of silent Salmonella prevention in the pest of duck about the research of RA vaccine.At present the RA vaccine of research report mainly contain the RA inactivated vaccine (have only a kind of serotype the unit price inactivated vaccine, contain the multivalent inactivated vaccine of multiple serotype), RA and E.coli bivalent inactivated vaccine, RA attenuated live vaccines, extract the subunit vaccine that obtains RA subunit composition.Wherein the inactivated vaccine effect is certain, and just progress is a silent Salmonella prevention best choice in the pest of duck at present.It is the basis and one of key that riemerella anatipestifer inactivated vaccine is produced that large scale fermentation is produced the Salmonella bacterium liquid of writing from memory in the pest of duck, but the zymotechnique of batch production large scale fermentation production riemerella anatipestifer inactivated vaccine bacterium liquid is not arranged at present.
Summary of the invention
The object of the present invention is to provide a kind of method of producing the Riemerlla anatipestifer vaccine with bacterium liquid, particularly the batch production large scale fermentation is produced the technology of Riemerlla anatipestifer bacterium liquid.
The present invention is by the following technical solutions:
A kind of method of producing the Riemerlla anatipestifer vaccine with bacterium liquid comprises the breeding of first order seed and evaluation, the breeding of secondary seed, fermentation; The breeding of described first order seed be accredited as: freeze-drying basis bacterial classification is inserted the nutrient broth that contains 10% calf serum, cultivated 24 hours for 37 ℃; Streak inoculation is containing 5～10%CO containing on the 10% calf serum nutrient agar plate then
2Perhaps light up in the environment of cylinder, after cultivating in 37 ℃, 24～48 hours, select the colonies typical inoculation to contain some on 10% calf serum nutrient agar medium inclined-plane, put 37 ℃, 5-10%CO2 or candle cylinder environment and cultivated 24 hours down, as first order seed; Described secondary seed breeding is: get first order seed and be inoculated in the nutrient broth that contains 10% calf serum, put 37 ℃ and cultivated 24 hours, sampling is done purely after the assay was approved, puts 2～8 ℃ of preservations, and the usage period must not be above 5 days; Described fermentation, the used substratum that ferments is: the N-J synthetic medium that contains 1% cracking whole blood; The bacterial classification inoculation amount of fermentation is: by the 2% inoculation secondary seed solution of cultivating base unit weight; Fermentation mode is: the culture tank liquid fermentation and culture; Culture condition is: 37 ℃ of cultured continuously 24 hours.
The described production Riemerlla anatipestifer vaccine method of bacterium liquid, the described N-J synthetic medium that contains 1% cracking whole blood comprises basic medium and additive, the component of described basic medium and ratio are: Tryptones, 1.5-1.9%; Yeast extract, 0.2-0.7%; Beef extract, 0.2-0.7%; Lactoalbumin hydrolysate, 0.2-0.7%; Glucose, 0.1-0.4%; NaCl, 0.5%; Potassium primary phosphate, 0.1-0.7%; Dipotassium hydrogen phosphate, 0.1-0.7%; Na2HPO412H2O, 0.5-0.9%; (NH4) 2SO4,0.1-0.2%; NH4Cl, 0.01-0.03%; Wherein said each percentages of ingredients is a weight/volume percent; Described additive is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation 3 times before using; Cracking blood cell whole blood 1% adds in the basic medium by volume.
Batch production large scale fermentation of the present invention is produced technology laboratory detection, pilot scale and the production application of Riemerlla anatipestifer, shows to have good effect.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment 1
The preparation of 1% cracking blood cell whole blood N-J synthetic medium:
The 1% cracking blood cell whole blood N-J synthetic medium of configuration 100ml: Tryptones: 1.9g; Yeast extract: 0.7g; Beef extract: 0.7g; Lactoalbumin hydrolysate: 0.7g; Glucose: 0.4g; NaCl:0.5g; Potassium primary phosphate: 0.3g; Dipotassium hydrogen phosphate: 0.3g; Na
2HPO
412H
2O:0.9g; (NH
4)
2SO
4: 0.2g; NH
4Cl:0.03g; Water adds to 100mL; With 121 ℃ of sterilizations of above-mentioned each component 15 minutes, composition subject to sterilization was cooled to when being lower than 40 ℃, added additive cracking blood cell whole blood 1ml then in above-mentioned basic medium; The N-J synthetic medium gets product after shaking up.
Described additive cracking blood cell whole blood is taked healthy and strong ox blood of Pest-or disease-free area and the aseptic fibre that takes off for the method that adopts jugular vein blood sampling or carotid artery bloodletting is aseptic, in-20 ℃ and room temperature freeze thawing, behind the multigelation 3 times, be stored in-20 ℃ of standby or blood samplings and asepticly directly be stored in-20 ℃ after taking off fibre, take out multigelation before using and make for 3 times.
Embodiment 2
The preparation of infectious serositis of duck inactivated vaccine seedling bacterium liquid:
1 bacterial classification serum I type riemerella anatipestifer RA-CH-I strain.
2 substratum, 1% cracking blood cell whole blood N-J synthetic medium, 10% serum nutrient agar plate, cracking whole blood and check are produced with standby after the assay was approved by " Chinese veterinary drug allusion quotation " appendix with substratum is all strict.
3 methods
3.1 the breeding of first order seed and evaluation
Freeze dried serum I type riemerella anatipestifer RA-CH-I strain basis bacterial classification is inserted 5ml contain in the nutrient broth of 10% calf serum, cultivated 24 hours for 37 ℃; Streak inoculation contains on the 10% calf serum nutrient agar plate at 2 then, by standard [smooth surface, slightly projection, circle, be butyraceous bacterium colony, colony diameter 1.2～1.7mm; The bacterium smear can be seen thalline and be single (in pairs accidental or be thread), and size is 0.2～0.4 * 15 μ m.] select the colonies typical inoculation to contain 5 on 10% calf serum nutrient agar medium inclined-plane, put 37 ℃, 5-10%CO
2(cylinder of perhaps lighting up) environment was cultivated 24 hours down, was stored in 4 ℃ of refrigerators and (was no more than 7) as first order seed.
3.2 secondary seed breeding
Get 3 of first order seeds, the 2-3ml nutrient broth of whenever drawing washes, and merges to be inoculated in the nutrient broth that 350ml contains 10% calf serum, puts 37 ℃ and cultivates 24 hours, and sampling is done purely after the assay was approved, is stored in 4 ℃ of refrigerators and (is no more than 5) as first order seed.
3.3 seedling is cultivated with bacterium liquid
The employing culture tank is cultivated, the N-J synthetic medium of the 15L that in the 20L fermentor tank, packs into and an amount of foam killer, and the sterilization back adds cracking blood cell whole blood 150ml in 1% ratio simultaneously by the 2% inoculation secondary seed solution 300ml that cultivates base unit weight, cultivates 24 hours in 37 ℃.
3.4 pure check
After bacterium liquid was cultivated and finished, sampling was done pure check with 10% calf serum nutrient agar plate, should be pure.
3.5 the bacterium number is measured
With bacterium liquid quantitative sampling centrifugal after, with continuous normal saline centrifuge washing 3 times and do suitably dilution, measure the 560nm OD of place value down, according to formula: contain bacterium sum (CFU/ml)=OD in ultraviolet spectrophotometer
560nmValue * extension rate * 2 * 10
9CFU/ml calculates bacterium liquid bacteria containing amount, and every 1.00ml contains viable bacteria and is not less than 3.00 * 10
10CFU, reference when seedling is joined in the count results conduct.
4 results
Get zymocyte liquid 1ml, after 3000～5000 rev/mins of centrifugal 5-10 minutes, with continuous normal saline centrifugal (3000～5000 rev/mins, 5-10 minute) washing 3 times; With physiological saline 50ml dissolving and dilution precipitation (i.e. 50 times of dilutions); Measure the 560nm OD of place value (transferring 0) down in ultraviolet spectrophotometer, record OD with physiological saline
560nmBe 0.589; According to formula: contain bacterium sum (CFU/ml)=OD
560nmValue * extension rate * 2 * 10
9CFU/ml=0.589 * 50 * 2 * 10
9CFU/ml=5.89 * 10
10CFU/ml.
Be that the bacterium number that contains of riemerella anatipestifer is 58,900,000,000 in every milliliter of this fermented liquid.
According to this count results, fermented liquid promptly can be used for the bacterium liquid that the seedling of infectious serositis of duck inactivated vaccine is used, and makes the infectious serositis of duck inactivated vaccine at last through deactivation, emulsification etc.
Embodiment 3
5 batches of totally 10.8 ten thousand milliliters of laboratory system vaccines have been produced in laboratory trial-production, and lot number is respectively 2005001,2005002,2005003,2005004,2005005.In the production of vaccine process, strictness is tested by work in-process and inspection after construction regulation, and work in-process production and survey report the results are shown in Table 1, and final product quality check situation sees Table 2 (the original survey report of 5 batches of laboratory trial product finished products sees the back for details).
Table 1 infectious serositis of duck inactivated vaccine work in-process are produced and assay
Table 2 2005001-2005005 criticizes infectious serositis of duck inactivated vaccine laboratory inspection after construction result
Conclusion
5 batches of laboratory system vaccines (2005001,2005002,2005003,2005004,2005005) have been produced.Safe in whole process of production, smooth and easy.Each Interventions Requested of work in-process and finished product all reach on every index of " infectious serositis of duck inactivated vaccine quality standard ".Fully proved this production of vaccine process stabilizing, stable, safe and effective according to the vaccine quality that rules (draft) are formulated, can carry out large-scale production.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. a method of producing the Riemerlla anatipestifer vaccine with bacterium liquid is characterized in that, comprises the breeding of first order seed and evaluation, the breeding of secondary seed, fermentation; The breeding of described first order seed be accredited as: freeze-drying basis bacterial classification is inserted the nutrient broth that contains 10% calf serum, cultivated 24 hours for 37 ℃; Streak inoculation is containing 5～10%CO containing on the 10% calf serum nutrient agar plate then
2Perhaps light up in the environment of cylinder, select the colonies typical inoculation to contain some on 10% calf serum nutrient agar medium inclined-plane after cultivating in 37 ℃, 24～48 hours, put 37 ℃, 5-10%CO2 or candle cylinder environment were cultivated 24 hours down, as first order seed; Described secondary seed breeding is: get first order seed and be inoculated in the nutrient broth that contains 10% calf serum, put 37 ℃ and cultivated 24 hours, sampling is done purely after the assay was approved, puts 2～8 ℃ of preservations, and the usage period must not be above 5 days; Described fermentation, the used substratum that ferments is: the N-J synthetic medium that contains 1% cracking whole blood; The bacterial classification inoculation amount of fermentation is: by the 2% inoculation secondary seed solution of cultivating base unit weight; Fermentation mode is: the culture tank liquid fermentation and culture; Culture condition is: 37 ℃ of cultured continuously 24 hours; The described N-J synthetic medium that contains 1% cracking whole blood comprises basic medium and additive, and the component of described basic medium and ratio are: Tryptones, 1.5-1.9%; Yeast extract, 0.2-0.7%; Beef extract, 0.2-0.7%; Lactoalbumin hydrolysate, 0.2-0.7%; Glucose, 0.1-0.4%; NaCl, 0.5%; Potassium primary phosphate, 0.1-0.7%; Dipotassium hydrogen phosphate, 0.1-0.7%; Na
2HPO
412H
2O, 0.5-0.9%; (NH
4)
2SO
4, 0.1-0.2%; NH
4Cl, 0.01-0.03%; Wherein said each percentages of ingredients is a weight/volume percent; Described additive is a cracking blood cell whole blood, and cracking blood cell whole blood 1% adds in the basic medium by volume.
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| CN105695375A (en) * | 2016-04-25 | 2016-06-22 | 四川农业大学 | Novel riemerella anatipestifer culture medium and preparation method thereof |
| CN107513510A (en) * | 2017-09-04 | 2017-12-26 | 广东省农业科学院动物卫生研究所 | Riemerella anatipestifer disease attenuated live vaccines and preparation method thereof |
Citations (2)
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|---|---|---|---|---|
| WO2001004317A1 (en) * | 1999-07-14 | 2001-01-18 | Institute Of Molecular Agrobiology | Ompa gene for an outer membrane protein of riemerella anatipestifer and methods of use |
| CN101507816A (en) * | 2009-02-03 | 2009-08-19 | 福州大北农生物技术有限公司 | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001004317A1 (en) * | 1999-07-14 | 2001-01-18 | Institute Of Molecular Agrobiology | Ompa gene for an outer membrane protein of riemerella anatipestifer and methods of use |
| CN101507816A (en) * | 2009-02-03 | 2009-08-19 | 福州大北农生物技术有限公司 | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof |
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| Title |
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| 范忠军等.鸭传染性浆膜炎蜂胶灭活疫苗的研制.《首届中国兽药大会-兽医生物制品学、兽医微生物学学术论坛论文集》.2008,第325页左栏第1、2、3段. * |
| 蔡家利等.鸭疫里默氏杆菌培养特性的研究.《中国家禽》.2004,第8卷(第1期),全文. * |
| 邓舜洲等.鸭疫里氏杆菌（2型）灭活疫苗的研制.《江西农业大学学报》.2002,第24卷(第5期),全文. * |
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