CN105833261A - Method for producing combined inactivate vaccine of escherichia coli disease and pasteurellosis in yak - Google Patents
Method for producing combined inactivate vaccine of escherichia coli disease and pasteurellosis in yak Download PDFInfo
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- CN105833261A CN105833261A CN201610219981.8A CN201610219981A CN105833261A CN 105833261 A CN105833261 A CN 105833261A CN 201610219981 A CN201610219981 A CN 201610219981A CN 105833261 A CN105833261 A CN 105833261A
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- yak
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0258—Escherichia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a method for producing a combined inactivate vaccine of escherichia coli disease and pasteurellosis in yak. The method disclosed by the invention, which makes use of a self-prepared culture medium, is simple in production process and low in cost; safety tests and immunity tests on the yak prove that the combined inactivate vaccine disclosed by the invention has an immunizing effect on the escherichia coli disease and pasteurellosis; the combined inactivate vaccine is more convenient to use than a single vaccine, and the combined inactivate vaccine is capable of preventing the two diseases just by immunizing once, so that the workload of immunization is reduced, and immunity paralysis and immunity failure caused by frequent immunization are avoided; and the combined inactivate vaccine has the advantages of being good in safety, high in immunizing efficacy, long in shelf life and the like.
Description
Present disclosure relates to a kind of method producing colibacillosis and pasteurellosis bacillus bivalent inactivated vaccine, belongs to field of biology, particularly veterinary biologics field.
Background technology
Yak is the important domestic animal of one that Tibet herdsman depends on for existence, the existing yak in Tibet about 50,000,000.And yak aquaculture industry in yak colibacillosis serious harm.Current yak colibacillosis sickness rate is 10%, and mortality rate is 25%.The death of yak, brings bigger economic loss to peasants and herdsmen.Research to yak colibacillosis vaccine a few days ago only has yak colibacillosis aluminium glue adjuvant inactivated vaccine.This disease betides Yak cow the earliest, with lactational Yak cow easy infection, is secondly 1-4 monthly age calf, and public yak takes second place.Ill and the yak that carries disease germs is the main source of infection, and yak passes through digestive tract infection.The primary disease annual 4-6 month is onset peak period, is typically to distribute or endemicity, and hemorrhagic or water sample edema with small intestinal are characterized.Ill Adult Yak height is warm, shiver, appetite is useless absolutely, dyspnea；Have shows toxic neuropathy symptom, death of going into a coma afterwards.Yak Bacillus pasteurii disease is also called yak hueppe's disease, is called for short yak hemorrhagic septicemia, is a kind of septic infectious disease of the yak caused by pasteurella multocida.This disease is acute to be changed in septic through out-of-date, chronic through out-of-date, shows as the limitation suppurative inflammation of subcutaneous tissue, joint, each internal organs.This disease is about 2% at yak producing region sickness rate, but fatality rate about 90%, therefore endangers very big, is one of the important diseases of current yak compulsory immunization.Yak hemorrhagic septicemia is sporadic or endemicity, the most all can occur, but autumn and winter season morbidity is more, and the prime of life yak morbidity of more than a year is more.
In China's veterinary biological product, seedling bacterium solution produces and uses traditional natural medium, such as ordinary broth, Ma Dingtang, meat liver stomach membrane digestion soup etc., makes loaded down with trivial details.Its main component meat extract is affected greatly by the fresh and digestible degree in kind, age and the meat source of animal, and the quality causing vaccine is unstable.Large scale fermentation self-made medium designs according to bacteriotrophy metabolic characteristic, with the addition of the somatomedin promoting growing microorganism, improving protective antigen content, has breeding bacteria height, steady quality, the advantage such as easy and simple to handle.Self-made medium is the key factor of antibacterial freeze-dried live vaccine quality.And in prior art, not about the bigeminy vaccine of preventing and treating yak infectious disease.In view of this, it is an object of the invention to provide a kind of pasteurellosis bacillus, colibacillosis bivalent inactivated vaccine, self-made medium used is suitable for the pathogenic E of yak, pasteurellosis bacillus strain growth, composition relatively determine, be prone to on-line checking, monitoring and adjust in time.Vaccine has the immunization to colibacillosis and pasteurellosis bacillus, more easy to use than each single Seedling, only need immunity once, just can prevent two kinds of diseases, alleviate immunity inoculation workload, avoid the immunological paralysis because frequently immunity is caused and immuning failure, there is the advantages such as safety good, immune efficacy is high, long shelf-life.
Summary of the invention
Colon bacillus is purchased from Tibet Agricultural and Animal Husbandry College.These numbered Tibetan-9903 of yak colon bacillus, the deposit number of this bacterial strain is CCTCC NO:M 208216.This Pseudomonas is in known bacterial strain, the most disclosed.Pasteurellosis bacillus (cvcc393) is purchased from veterinary medicament inspection institute of China.The standard of strain and the regulation of store method same " People's Republic of China's regulations ".
Yak colon bacillus (hiding-9903) seed liquor is inoculated in the liquid self-made medium with pancreas casein peptone, agar powder and yeast extract powder as main raw material(s) by 1%～the 2% of culture medium total amount, 30-45 DEG C of fermentation or aerobic culture 10～36h, adding appropriate mass concentration according to pH rising condition in incubation is that the sterile dextrose solution of 30% is with control ph.After cultivation terminates, add the pasteurellosis bacillus seed bacterium solution after concentrating immediately, add 1.5% gelatin and 5% sucrose, add through sterilization treatment and be preheated to the conventional freeze drying protectant of 37 DEG C, subpackage lyophilizing.Every part viable count is no less than 3 × 109CFU。
The detailed description of the present invention
1. preparation seedling bacterium solution
(1) 0 DEG C of yak colon bacillus vacuum lyophilization bacterial strain 1 preserved is taken, it is incubated in fluid medium after lyophilizing bacterial strain is melted, take liquid culture respectively to rule 2 secondary plain agar flat boards, select circle, smooth, neat in edge, each 5 of the bacterium colony being of moderate size, after 37 DEG C are cultivated 20h, it is inoculated in the peptone yak broth bouillon of 2.5%, put 37 DEG C 140～160r/min shaking and cultivate after 24h microscopy and purely after inspection, in the culture medium of the big two jiaos of bottles of peptone yak meat soup of inoculation 2.5%, put 37 DEG C 140～24h is cultivated in 160r/min shaking.
(2) cultivate propagation after the recovery of pasteurellosis bacillus strain being passed on, cultivate or after flat bottle solid culture through submerged aerobic fermentation, collect bacterium solution, and concentrate standby.
2. culture medium uses the self-made medium of present invention design
(1) formula (W/V) is as follows:
Pancreas casein peptone 3%～5%, yeast extract powder 1%～2%, sodium chloride 0.08%, potassium dihydrogen phosphate 0.007%, disodium hydrogen phosphate 0.09% and glucose 0.1%, 1.5% gelatin and 5% sucrose.
(2) self-made medium compound method
Weighing medium component successively by above-mentioned formula and join in water for injection, regulating pH value with the sodium hydroxide solution of 1mol/L after fully dissolving is about 7,120 DEG C of sterilizing 45min.Facing the used time, sterile working adds 50% glucose solution of final concentration 1%, mixing.
30% glucose solution (W/V) weighs 30g glucose, is dissolved in water for injection, is settled to 100ml, 120 DEG C of sterilizing 45min, 3～7 DEG C of preservations.
The sodium hydroxide solution of 1mol/L weighs 4g sodium hydroxide, is dissolved in water for injection, and is settled to 100ml, 120 DEG C of sterilizing 45min, 3～7 DEG C of preservations.Prepare 1.5% gelatin and 5% sucrose gelatin is standby with sucrose solution 100ml.
3 vaccine manufactures
Yak colon bacillus seed liquor is inoculated in the liquid self-made medium with pancreas casein peptone, agar powder and yeast extract powder as main raw material(s) by the 1.5% of culture medium total amount, 35 DEG C of fermentations or aerobic culture 10～36h, adding appropriate mass concentration according to pH rising condition is that the sterile dextrose solution of 30% is with control ph.Cultivating and add the pasteurellosis bacillus seed bacterium solution after concentrating after terminating immediately, add 1.5% gelatin and 5% sucrose, add through sterilization treatment and be preheated to the freeze drying protectant of 37 DEG C, fully after mixing subpackage, vacuum lyophilization forms.Every part viable count is no less than 3 × 109CFU。
Conventional freeze drying protectant for the present invention can be the most commercially available freeze drying protectant, preferably gelatin, sucrose, glucose, albumin, Polyethylene Glycol.
The positive effect of the present invention
Yak infects colon bacillus, pasteurellosis bacillus epidemic disease present situation more seriously, brings the biggest loss to Tibetan area herdsman.The bigeminy vaccine of the preventing and treating yak infectious disease of the present invention was not the most reported.And non-multiple vaccines generally uses broth medium in prior art, making various factors poor stability in loaded down with trivial details, preparation process, its culture medium made is criticized a quality and is differed greatly, and then affects the homogeneity of vaccine.And self-made medium provided by the present invention, it being by the development trend of antibacterial high-cell-density cultivation, there is advantage easy and simple to handle, stay-in-grade, colon bacillus, pasteurellosis bacillus bigeminy vaccine for high-quality produce and provide sound assurance.The present invention uses self-made medium, production technology is simple, with low cost, confirm through yak safety testing and challenge experiments, the vaccine of the present invention has the immunization to colibacillosis and pasteurellosis bacillus, more easy to use than each single Seedling, only need immunity once, it is possible to prevent two kinds of diseases, alleviate immunity inoculation workload, avoid the immunological paralysis because frequently immunity is caused and immuning failure, there is the advantages such as safety good, immune efficacy is high, long shelf-life.
Embodiment 1
1. preparation seedling bacterium solution
(1) 0 DEG C of yak colon bacillus preserved (hiding-9903) vacuum lyophilization bacterial strain 1 is taken, it is incubated in fluid medium after lyophilizing bacterial strain is melted, take liquid culture respectively to rule 2 secondary plain agar flat boards, select circle, smooth, neat in edge, each 5 of the bacterium colony being of moderate size, after 37 DEG C are cultivated 20h, it is inoculated in the peptone yak broth bouillon of 2.5%, put 37 DEG C of 160r/min shakings and cultivate after 24h microscopy and purely after inspection, in the culture medium of the big two jiaos of bottles of peptone yak meat soup of inoculation 2.5%, put 37 DEG C of 160r/min shakings and cultivate 24h.
(2) cultivate propagation after the recovery of pasteurella multocida strain being passed on, cultivate or after flat bottle solid culture through submerged aerobic fermentation, collect bacterium solution, and concentrate standby.
2. culture medium uses the self-made medium of present invention design
(1) formula (W/V) is as follows:
Pancreas casein 5%, yeast extract powder 2%, sodium chloride 0.08%, potassium dihydrogen phosphate 0.007%, disodium hydrogen phosphate 0.09% and glucose 0.1%, 1.5% gelatin and 5% sucrose.
(2) self-made medium compound method
Weighing medium component successively by above-mentioned formula and join in water for injection, regulating pH value with the sodium hydroxide solution of 1mol/L after fully dissolving is about 7,120 DEG C of sterilizing 45min.Facing the used time, sterile working adds 50% glucose solution of final concentration 1%, mixing.
50% glucose solution (W/V) weighs 50g glucose, is dissolved in water for injection, is settled to 100ml, 120 DEG C of sterilizing 45min, 4 DEG C of preservations.
The sodium hydroxide solution of 1mol/L weighs 4g sodium hydroxide, is dissolved in water for injection, and is settled to 100ml, 120 DEG C of sterilizing 45min, 4 DEG C of preservations.Prepare 1.5% gelatin and 5% sucrose gelatin is standby with sucrose solution 100ml.
3 vaccine manufactures
Yak colon bacillus seed liquor is inoculated in the liquid self-made medium with pancreas casein peptone, agar powder and yeast extract powder as main raw material(s) by the 1.5% of culture medium total amount, 35 DEG C of fermentations or aerobic culture 24h, adding appropriate mass concentration according to pH rising condition is that the sterile dextrose solution of 30% is with control ph.Cultivating and add the pasteurellosis bacillus seed bacterium solution after concentrating after terminating immediately, add 1.5% gelatin and 5% sucrose, add through sterilization treatment and be preheated to the gelatin of 37 DEG C, fully after mixing subpackage, vacuum lyophilization forms.Every part viable count is no less than 3 × 109CFU。
4 product inspections
Character milky Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding diluent.
The method that purely inspection is specified by " Chinese veterinary pharmacopoeia " is tested, and purely inspection is qualified.
Count plate indicates head part by label, does count plate (method specified by " Chinese veterinary pharmacopoeia " is carried out) with plain agar flat board.Every part is no less than 3 × 10 containing viable count9CFU。
Safety verification indicates head part by label, vaccine ordinary broth or peptone water is diluted, subcutaneous injection body weight 1.5～2.0kg rabbit 2, each 1.0ml (containing 2 parts), observes 21, survival.
Residual moisture measures the method specified by " Chinese veterinary pharmacopoeia " and is measured, less than 4.0%.
Vacuum measures the method specified by " Chinese veterinary pharmacopoeia " and is measured, and meets regulation.
Embodiment 2
Safety testing
Select the Tibet health yak calf 20 at 1-3 monthly age, be randomly divided into 5 groups, often group 4.1-4 group is test group, often organizes 1 batch of yak colon bacillus of each inoculation and pasteurellosis bacillus inactivated vaccine, every inoculation 2mL；1,3 groups of yaks are in cervical region subcutaneous vaccination；2,4 groups of yaks inoculate at buttocks muscles.Yak inoculate latter 14 days, with the same area repeated inoculation vaccine 4mL inoculated first.5 groups is matched group, the dosage of every yak injection meat soup and the same test group of method.After after test group and matched group primary vaccination and again inoculating, all observed results were to 14 days.
Observing yak after after test group primary vaccination and again inoculating all strong alive, body temperature, breathing, heart sound etc. are front with inoculation identical, the most normally.Matched group yak is all strong to live.
Embodiment 3
Immuning effect test
Select the Tibet health yak calf 20 at 1-3 monthly age, be randomly divided into 5 groups, often group 4.1-4 group is test group, often organizes 1 batch of yak colon bacillus of each inoculation and pasteurellosis bacillus inactivated vaccine, every inoculation 2mL；1,3 groups of yaks are in cervical region subcutaneous vaccination；2,4 groups of yaks inoculate at buttocks muscles.Yak inoculate latter 14 days, with the same area repeated inoculation vaccine 2mL inoculated first.5 groups is matched group, the dosage of every yak injection meat soup and the same test group of method.After again inoculating 24 hours, by the bacterium solution 2M LD (2 times of minimum lethal doses) of 1-2 group yak intramuscular injection yak colon bacillus respectively, observe 14 days.By the bacterium solution 1M LD (minimum lethal dose) of 3-5 group yak intramuscular injection yak colon bacillus respectively, observe 14 days.
After 1 to 4 group yak counteracting toxic substances, 16 yaks all survive；After 5 groups of yak counteracting toxic substances, all dead.
By the bacterium solution 2M LD (2 times of minimum lethal doses) of above-mentioned 1-2 group yak intramuscular injection yak pasteurellosis bacillus respectively, observe 14 days.By the bacterium solution 1M LD (minimum lethal dose) of 3-4 group yak intramuscular injection yak pasteurellosis bacillus respectively, observe 14 days.Choose the health yak calf left side, Tibet at 4 1-3 monthly ages to be 5 groups is matched group simultaneously, the dosage of every yak injection meat soup and the same test group of method, the bacterium solution 1M LD (minimum lethal dose) of intramuscular injection yak pasteurellosis bacillus, observes 14 days.
After 1 to 4 group yak counteracting toxic substances, 16 yaks all survive；After 5 groups of yak counteracting toxic substances, all dead.
Embodiment 4
Immune period test
Carrying out immune period test with the 5 batch vaccines produced, every batch of vaccine divides 5 groups, often organizes the Tibet health yak calf at each 5 1-3 monthly ages of inoculation, and inoculation method, with embodiment 3, sets up 5 nonimmune yaks as blank simultaneously.Through immunity 30 days, 60 days, 90 days, 120 days, 180 days, 240 days, 300 days, after 360 days; make protest test; its result yak colon bacillus immunoprotection is 100% always, and 180 days immunoprotections of Bacillus pasteurii disease still have the protective rate of 80%, and immunoprotection reached 75% on 360th.
Embodiment 5
Storage life is tested
By vaccine under the preservation condition of 2-8 DEG C; storage life can reach 24 months; with 1 using dosage 2mL (preserve and expire 24 months), healthy susceptible yak is carried out immunity, within 14,21 days after immunity, carry out protest test respectively respectively, all protected.Vaccine quality does not change.
Claims (6)
1. produce yak colibacillosis and a method for pasteurellosis bacillus bivalent inactivated vaccine, it is characterized in that by
Yak colon bacillus seed liquor is inoculated in by 1%～the 2% of culture medium total amount with pancreas casein peptone, agar
In powder and liquid self-made medium that yeast extract powder is main raw material(s), 30-45 DEG C of fermentation or aerobic culture
10～36h, adding appropriate mass concentration according to pH rising condition in incubation is the sterile dextrose of 30%
Solution, with control ph, is cultivated after terminating, and adds the pasteurellosis bacillus seed bacterium solution after concentrating immediately, adds
1.5% gelatin and 5% sucrose, add freeze drying protectant, subpackage lyophilizing.
2. production yak colibacillosis as claimed in claim 1 and the method for pasteurellosis bacillus bivalent inactivated vaccine,
One or more during wherein freeze drying protectant is gelatin, sucrose, glucose, albumin or Polyethylene Glycol.
3. use yak colibacillosis and pasteurellosis bacillus bigeminy that method described in claim 1 prepares
Inactivated vaccine.
4. a method for immune yak, including yak is used the vaccine described in claim 2.
5. production yak colibacillosis as claimed in claim 1 or 2 and pasteurellosis bacillus bivalent inactivated vaccine
Method, wherein freeze drying protectant add before, need through sterilization treatment and be preheated to 37 DEG C.
6. production yak colibacillosis as claimed in claim 1 and the side of pasteurellosis bacillus bivalent inactivated vaccine
Method, wherein yak colon bacillus seed liquor is to obtain as follows: take 0 DEG C of yak preserved big
Intestinal Escherichia vacuum lyophilization bacterial strain 1, is incubated in fluid medium after being melted by lyophilizing bacterial strain,
Take liquid culture respectively to rule 2 secondary plain agar flat boards, select circular, smooth, neat in edge, size suitable
In each 5 of bacterium colony, cultivate after 20h, be inoculated into the peptone yak broth bouillon of 2.5% for 37 DEG C
In, put 37 DEG C 140～160r/min shaking and cultivate after 24h microscopy and purely after inspection, the egg of inoculation 2.5%
In the culture medium of the white big two jiaos of bottles of peptone yak meat soup, put 37 DEG C 140～24h is cultivated in 160r/min shaking.
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Cited By (1)
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Application publication date: 20160810 |