CN1323718C - Process for preparing viral and bacterial vaccine for animals utilizing vegetable oil adjuvant - Google Patents
Process for preparing viral and bacterial vaccine for animals utilizing vegetable oil adjuvant Download PDFInfo
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- CN1323718C CN1323718C CNB2004100089261A CN200410008926A CN1323718C CN 1323718 C CN1323718 C CN 1323718C CN B2004100089261 A CNB2004100089261 A CN B2004100089261A CN 200410008926 A CN200410008926 A CN 200410008926A CN 1323718 C CN1323718 C CN 1323718C
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Abstract
The present invention relates to a method for preparing an animal virus and bacterium vaccine by using a vegetable oil adjuvant, which is characterized in that the method comprises the following steps: (1) antigen agent manufacture: manufactured sprout poison(bacterium) strains are copied by sensitive cells or a culture medium, antigens are obtained after inactivation, and the antigens are manufactured into the antigen agent; (2) vegetable oil adjuvant manufacture; (3) uniformly mixing: the antigen agent and the vegetable oil adjuvant are proportionally mixed and homogenized to manufacture the vaccine. The antigen agent is a mixture of the antigens and disinfected Tween-80, and the vegetable oil adjuvant is a mixture of vegetable oil and span-80. In the method, the novel adjuvant is adopted to prepare the animal virus and bacterium vaccine, so the method not only does not influence the immune efficacy of the vaccine but also can reduce the adverse reaction of the vaccine in use; meanwhile, the method is favorable for producing safe food meat. Compared with the prior art, the method has the obvious advantages. The method is suitable for various animal virus and bacterium vaccines.
Description
Technical field:
The present invention relates to utilize the vegetable oil adjuvant to produce the method for vaccine, particularly utilize the vegetable oil adjuvant to prepare the method for zoosis toxicity, bacterial vaccine.
Background technology:
Adjuvant promotes immunoreactive material as a kind of antigen of assisting, and can help antigen slowly to discharge in the injection site, to increase immunostimulating effect.In existing technology, the mineral oil adjuvant is comparatively general in the application of animal vaccine, but in recent years in the immunoprophylaxis of animal mineral oil Adjuvanted vaccines, because it is excessive to occur immunoreation often, cause part vaccine injected animal a couple of days hyperpyrexia, subtract food, useless food, miscarriage and fertility performance descend, and reduces young animal weightening finish untoward reaction such as slow down as the dairy stock milk yield, especially few animals causes death because of anaphylaxis takes place, and is unfavorable for Developing of Animal Industry.In addition, mineral oil (white oil) is a kind of material that utilizes chemical method to abstract from crude oil, and it is injected in the animal body as vaccine adjuvant, and slowly metabolism, even minute quantity composition for a long time is trapped in the part, has both influenced the animal meat quality, influences health again.Therefore studying a kind of novel adjuvant, make its immune efficacy that neither influences vaccine, can reduce the untoward reaction of vaccine again, help the meat product of production safety simultaneously again, then is the important topic of pendulum in face of us.
The content of invention
The object of the present invention is to provide a kind of method of utilizing the vegetable oil adjuvant to prepare zoosis toxicity, bacterial vaccine, to solve the above-mentioned problem that exists, thereby obtain neither to influence the immune efficacy of vaccine, can reduce the untoward reaction of vaccine again, help the animal vaccine of the meat product of production safety simultaneously again.
The objective of the invention is to realize by following technical scheme.The present invention utilizes the vegetable oil adjuvant to prepare the method for zoosis toxicity, bacterial vaccine, it is characterized in that it may further comprise the steps:
(1) makes antigen-agent, seedling systeming virus (bacteria) strain is duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen; And described antigen made antigen-agent; Can prepare by industry conventional method.
(2) make the vegetable oil adjuvant;
(3) mixing mixes described antigen-agent in proportion with described vegetable oil adjuvant, homogenizing is made vaccine.
In step (1),
Described seedling systeming virus (bacteria) strain is arbitrary in following or two kinds: O type foot and mouth disease virus Akesu/58 strain, OMIII strain and OZ strain, rabies virus-CVS strain, transmissible gastroenteritis (TEGV-TS strain), epidemic diarrhea virus (PEDV-QD strain), subtype avian influenza strain H5N1, subtype avian influenza strain H9N2, Pullorum Disease Salmonella; In addition, also can adopt other zoosis toxicity, bacterial vaccine.
Described sensitive cells is any in following: BHK-21 cells and IB-RS-2 cell, Vero cell, PK-cell, 293 cells, ST-cell, Hela-cell; Also can adopt other sensitive cells.
Described culture medium is any in following: LB, sodium thiosulfate agar, ordinary broth, plain agar plate.Also can adopt other culture medium.
In step (1),
Described antigen-agent is the tween 80 mixture of antigen and sterilization;
In step (2),
Described vegetable oil adjuvant is the mixture of vegetable oil and Arlacel-80.
Described vegetable oil is any in following: Oleum Brassicae campestris, Oleum Glycines, Oleum Arachidis hypogaeae semen, Oleum Ricini, linum wet goods vegetable oil.Also can adopt other edible oil to the person poultry harmless.
In described step (1), described antigen is made antigen-agent be: the tween 80 of antigen and sterilization is pressed 10-40: 1 mixed, make antigen-agent;
In described step (2), making vegetable oil adjuvant is: vegetable oil and Arlacel-80 are pressed 5-15: 1 mixed, through sterilization, make the vegetable oil adjuvant;
In described step (3), described antigen-agent is mixed into described vegetable oil adjuvant in proportion: described antigen-agent and vegetable oil adjuvant are pressed 0.5-1.5: 1 mixed, stir, and form uniform colostrum; Described homogenizing is: with high pressure homogenizer described colostrum is carried out high speed shear and emulsifying homogenizing.
In described step (3), when described antigen-agent mixes with the vegetable oil adjuvant, earlier antigen-agent partly is added in whole vegetable oil adjuvants, the limit edged stirs, and makes it form uniform colostrum; Again remaining antigen-agent is joined described uniformly first Ruzhong.
The present invention adopts the component of vegetable oil as adjuvant, makes the vegetable oil adjuvant.Vegetable oil is a kind of natural materials that extracts from plant kernel, it is injected in the animal body as adjuvant neither influences the animal food quality, do not influence the people's of edible this animal product health again, simultaneously have the performance of mineral oil adjuvant again: 1. it can increase antigenic surface area, is easily engulfed by macrophage; 2. prolong antigen retaining the phase in vivo, increase the opportunity that contacts with immunocyte; 3. bring out the inflammatory reaction of antigen injection site and regional nodes thereof, help the proliferation function of immune stimulatory cell.Therefore, the vegetable oil adjuvant in the developing vaccines process, will be the best substitute of mineral oil adjuvant.
In sum, the present invention adopts novel adjuvant to prepare zoosis toxicity, bacterial vaccine, when it is used, neither influences the immune efficacy of vaccine, can reduce the untoward reaction of vaccine again, helps the food meat of production safety simultaneously again; Compared with prior art, has tangible advantage.The present invention is suitable for various zoosis toxicity, bacterial vaccine uses.
The specific embodiment
Embodiment 1
Present embodiment is zoosis toxicity subtype avian influenza H5N1 and the H9N2 vegetable oil adjuvant deactivation mixed vaccine that utilizes the preparation of vegetable oil adjuvant, and its preparation method may further comprise the steps:
(1) makes antigen-agent, the seedling strain is duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen and described antigen is made antigen-agent; Wherein:
1) strain: seedling epidemic isolates H5N1 and H9N2 (by 1: 1 proportioning), the biological characteristics of seedling strain: Embryo Gallus domesticus median infective dose (EID
50) be 8.25log2/ml, the viral level of promptly planting poison is that every ml contains 8.25 EID
50Embryo Gallus domesticus mean time to death (MDT) for allantoic cavity inoculation 0.1ml after 72 hours; The preceding HA 〉=8log2 of the chick embryo allantoic liquid deactivation that blood clotting (HA) titre is promptly gathered in the crops to the 1%SPF chicken red blood cell; Pure degree is no any exogenous virus and germ contamination.
2) duplicating and deactivation of virus: the SPF Embryo Gallus domesticus 0.1ml of allantoic cavity inoculation 9-11 age in days, HA tests in 72 hours detect the HA titre of chick embryo allantoic liquid, and the chick embryo allantoic liquid of results HA 〉=8log2 adds formalin, and 37 ℃ of deactivations form antigen.
The culture medium that antigen inoculation after the deactivation is suitable (plain agar plate) is carried out the check of antibacterial, mycete and mycoplasma, and the allantoic fluid 0.1ml after the deactivation is through allantoic cavity inoculation 9-11 day instar chicken embryo, continuously 3 generations of blind passage.
3) allantoic fluid after the deactivation and autoclaved tween 80 are made antigen-agent by 25: 1 mixed,
(2) make the vegetable oil adjuvant;
With vegetable oil (Oleum Glycines) and the mixed emulsifying of Arlacel-80 by 10: 1, through 1.5Mpa, the 30min autoclave sterilization is made the vegetable oil adjuvant;
(3) described antigen-agent is mixed with described vegetable oil adjuvant in proportion, homogenizing is made vaccine.With described antigen-agent and vegetable oil adjuvant mixed by 1: 1, in the present embodiment, earlier the antigen-agent after the 40-60% deactivation is added in the vegetable oil adjuvant, the limit edged stirs, make it form uniform colostrum, making on the basis of water in oil emulsion then, remaining antigen-agent is added in the vegetable oil adjuvant again, further emulsifying.And then through high speed shearing of high pressure homogenizer and emulsifying, making outward appearance is milky vaccine.
Embodiment 2
Present embodiment is the pig O type foot and mouth disease inactivated vaccine that utilizes the preparation of vegetable oil adjuvant, and its preparation method may further comprise the steps:
(1) makes antigen-agent, the seedling strain is duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen and described antigen is made antigen-agent; Wherein:
1) strain: the seedling strain is a pig O type foot and mouth disease virus OZ strain
The biological characteristics of seedling strain: the seedling strain should meet O type foot and mouth disease virus characteristic.The neonatal rat poison is fed newborn white mice with O type hoof-and-mouth disease pig blister skin poison by the 6-8 age in days, the neonatal rat muscle poison in 4-5 generation, phosphate buffer dilution with PH7.6-7.8, the subcutaneous injection 3-4 of nape portion age in days neonatal rat 0.2ml/ only, each titre is no less than 4, observe judgement in 5 days, its LD50 must not be lower than 8.5.
The rejuvenation of seedling seed culture of viruses: the cell seed culture of viruses answers rejuvenation to be no less than for 3 generations, answers conformance with standard.Quantity-produced algebraically must not surpass for 14 generations.Pure degree is no any exogenous virus and germ contamination.
2) duplicating and deactivation of virus: with the 3-14 seedling cell seed culture of viruses in generation, inoculation has formed the BHK-21 cells and the IB-RS-2 cell of good monolayer, collects the cell venom.Measure the standard compliant deactivation of carrying out through steriling test and malicious valency.Be added in the venom of removing cell debris by certain amount with inactivator BEI, be warming up to 30 ℃, disconnected with the hypo solution group of degerming immediately after deactivation 28 hours, deactivation stop, form antigen, put under 5 ℃ the condition and preserve.
Antigen inoculation meat soup after the deactivation carries out antibacterial, mycete and mycoplasma check.Antigen cervical region subcutaneous injection 3-4 age in days neonatal rat 4-5 after the deactivation, 0.2ml/,, establish 2 of healthy blank Mus, observed 7 days, all foot and mouth disease symptom and morbidity death all must not appear in neonatal rats.
3) antigen after the deactivation and autoclaved tween 80 are made antigen-agent by 25: 1 mixed.
(2) make the vegetable oil adjuvant;
With vegetable oil (Oleum Glycines) and the mixed emulsifying of Arlacel-80 by 10: 1, through 1.5Mpa, the 30min autoclave sterilization is made the vegetable oil adjuvant;
(3) described antigen-agent is mixed with described vegetable oil adjuvant in proportion, homogenizing is made vaccine.
With described antigen-agent and vegetable oil adjuvant mixed by 1: 1, in the present embodiment, earlier the antigen-agent after the 40-60% deactivation is added in the vegetable oil adjuvant, the limit edged stirs, make it form uniform colostrum, making on the basis of water in oil emulsion then, remaining antigen-agent is added in the vegetable oil adjuvant again, further emulsifying.And then through high speed shearing of high pressure homogenizer and emulsifying, making outward appearance is milky vaccine.
Embodiment 3
Present embodiment is the cattle O type foot and mouth disease inactivated vaccine that utilizes the preparation of vegetable oil adjuvant, and its preparation method may further comprise the steps:
(1) makes antigen-agent, seedling systeming virus (bacteria) strain is duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen and described antigen is made antigen-agent; Wherein:
1) strain: the seedling strain is the biological characteristics of cattle O type foot and mouth disease virus Akesu/58 strain seedling strain: the seedling strain should meet O type foot and mouth disease virus characteristic.Seed culture of viruses is carried out 1: 1000 times of dilution with PBS (phosphate buffer), go up 2-4 year cattle 2ml that pure cut and lingual surface are embrocated no foot and mouth disease O type serum antibody, in 24-48 hour, the test cattle typical foot and mouth disease should occur and time send out the sexually transmitted disease (STD) damage.
The rejuvenation of seedling seed culture of viruses: the cell seed culture of viruses answers rejuvenation to be no less than for 3 generations, answers conformance with standard.Quantity-produced algebraically must not surpass for 14 generations.Pure degree is no any exogenous virus and germ contamination.
2) duplicating and deactivation of virus: with the 3-14 seedling cell seed culture of viruses in generation, inoculation has formed the BHK-21 cells and the IB-RS-2 cell of good monolayer, collects the cell venom.Measure the standard compliant deactivation of carrying out through steriling test and malicious valency.Be added in the venom of removing cell debris by certain amount with inactivator BEI, be warming up to 30 ℃, disconnected with the hypo solution group of degerming immediately after deactivation 28 hours, deactivation stop, form antigen, put under 5 ℃ the condition and preserve.
Antigen inoculation plain agar plate after the deactivation carries out antibacterial, mycete and mycoplasma check.Antigen cervical region subcutaneous injection 3-4 age in days neonatal rat 4-5 after the deactivation, 0.2ml/,, establish 2 of healthy blank Mus, observed 7 days, all foot and mouth disease symptom and morbidity death all must not appear in neonatal rats.
3) antigen after the deactivation and autoclaved tween 80 are made antigen-agent by 25: 1 mixed,
(2) make the vegetable oil adjuvant;
With vegetable oil (Oleum Glycines) and the mixed emulsifying of Arlacel-80 by 10: 1, through 1.5Mpa, the 30min autoclave sterilization is made the vegetable oil adjuvant;
(3) described antigen-agent is mixed with described vegetable oil adjuvant in proportion, homogenizing is made vaccine.
With described antigen-agent and vegetable oil adjuvant mixed by 1: 1, in the present embodiment, earlier the antigen-agent after the 40-60% deactivation is added in the vegetable oil adjuvant, the limit edged stirs, make it form uniform colostrum, making on the basis of water in oil emulsion then, remaining antigen-agent is added in the vegetable oil adjuvant again, further emulsifying.And then through high speed shearing of high pressure homogenizer and emulsifying, making outward appearance is milky vaccine.
Embodiment 4
Present embodiment is the pig O type foot and mouth disease inactivated vaccine (concentrated type) that utilizes the preparation of vegetable oil adjuvant, and its preparation method may further comprise the steps:
(1) makes antigen-agent, seedling systeming virus (bacteria) strain is duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen and described antigen is made antigen-agent; Wherein:
1) strain: the seedling strain is a pig O type foot and mouth disease virus OZ strain
The biological characteristics of seedling strain: the seedling strain should meet O type foot and mouth disease virus characteristic.The neonatal rat poison is fed newborn white mice with O type hoof-and-mouth disease pig blister skin poison by the 6-8 age in days, the neonatal rat muscle poison in 4-5 generation, phosphate buffer dilution with PH7.6-7.8, the subcutaneous injection 3-4 of nape portion age in days neonatal rat 0.2ml/ only, each titre is no less than 4, observe judgement in 5 days, its LD50 must not be lower than 8.5.
The rejuvenation of seedling seed culture of viruses: the cell seed culture of viruses answers rejuvenation to be no less than for 3 generations, answers conformance with standard.Quantity-produced algebraically must not surpass for 14 generations.Pure degree is no any exogenous virus and germ contamination.
2) duplicating and deactivation of virus: with the 3-14 seedling cell seed culture of viruses in generation, inoculation has formed the BHK-21 cells and the IB-RS-2 cell of good monolayer, and the addition of virus-culturing fluid is collected the cell venom by every square centimeter of cell monolayer 0.1-0.3ml.Measure the standard compliant deactivation of carrying out through steriling test and malicious valency.Be added in the venom of removing cell debris by certain amount with inactivator BEI, be warming up to 30 ℃, disconnected with the hypo solution group of degerming immediately after deactivation 28 hours, deactivation stop, form antigen, put under 5 ℃ the condition and preserve.
The culture medium that antigen inoculation after the deactivation is suitable (meat soup) is carried out antibacterial, mycete and mycoplasma check.Antigen cervical region subcutaneous injection 3-4 age in days neonatal rat 4-5 after the deactivation, 0.2ml/,, establish 2 of healthy blank Mus, observed 7 days, all foot and mouth disease symptom and morbidity death all must not appear in neonatal rats.
3) antigen after the deactivation and autoclaved tween 80 are made antigen-agent by 25: 1 mixed.
(2) make the vegetable oil adjuvant;
With vegetable oil (Oleum Arachidis hypogaeae semen) and the mixed emulsifying of Arlacel-80 by 10: 1, through 1.5Mpa, the 30min autoclave sterilization is made the vegetable oil adjuvant;
(3) described antigen-agent is mixed with described vegetable oil adjuvant in proportion, homogenizing is made vaccine.With described antigen-agent and vegetable oil adjuvant mixed by 1: 1, in the present embodiment, earlier the antigen-agent after 50% deactivation is added in the vegetable oil adjuvant, the limit edged stirs, make it form uniform colostrum, making on the basis of water in oil emulsion then, remaining antigen-agent is added in the vegetable oil adjuvant again, further emulsifying.And then through high speed shearing of high pressure homogenizer and emulsifying, making outward appearance is milky vaccine.
Claims (1)
1, a kind of method of utilizing the vegetable oil adjuvant to prepare zoosis toxicity and/or bacterial vaccine is characterized in that it may further comprise the steps:
(1) makes antigen-agent, seedling strain and/or bacterial strain are duplicated with sensitive cells or culture medium, after the deactivation, obtain antigen; And described antigen made antigen-agent;
(2) make the vegetable oil adjuvant;
(3) mixing mixes described antigen-agent in proportion with described vegetable oil adjuvant, homogenizing is made vaccine;
In described step (1), described antigen-agent is the tween 80 mixture of antigen and sterilization;
In described step (2), described vegetable oil adjuvant is the mixture of vegetable oil and Arlacel-80;
In described step (1), described antigen is made antigen-agent be: the tween 80 of antigen and sterilization is pressed 10-40: 1 mixed, make antigen-agent;
In described step (2), making vegetable oil adjuvant is: vegetable oil and Arlacel-80 are pressed 5-15: 1 mixed, through sterilization, make the vegetable oil adjuvant;
In described step (3), described antigen-agent is mixed into described vegetable oil adjuvant in proportion: described antigen-agent and vegetable oil adjuvant are pressed 0.5-1.5: 1 mixed, stir, and form uniform colostrum; Described homogenizing is: with high pressure homogenizer described colostrum is carried out high speed shear and emulsifying homogenizing;
In described step (3), when described antigen-agent mixes with the vegetable oil adjuvant, earlier the described antigen-agent of part is added in whole described vegetable oil adjuvants, the limit edged stirs, and makes it form uniform colostrum; Again all the other described antigen-agents are joined described uniformly first Ruzhong.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US9950061B2 (en) | 2014-04-03 | 2018-04-24 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
Families Citing this family (2)
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CN103301248A (en) * | 2013-06-27 | 2013-09-18 | 罗寿康 | Traditional Chinese medicine for treating rabies |
CN103751775A (en) * | 2013-12-31 | 2014-04-30 | 浙江大学 | Ginsenoside-containing vegetable oil adjuvant and preparation method and application thereof |
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CN1270838A (en) * | 1999-01-29 | 2000-10-25 | 辉瑞产品公司 | Adjuvant used in vaccine |
WO2003064475A1 (en) * | 2002-01-30 | 2003-08-07 | Ovita Limited | Novel monoclonal antibody and nematode larval antigens |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1270838A (en) * | 1999-01-29 | 2000-10-25 | 辉瑞产品公司 | Adjuvant used in vaccine |
WO2003064475A1 (en) * | 2002-01-30 | 2003-08-07 | Ovita Limited | Novel monoclonal antibody and nematode larval antigens |
Non-Patent Citations (3)
Title |
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中等毒力新城活疫苗对鸡的免疫效果 李自力 摘,毕丁仁 校,国外医学.预防.诊断.治疗用生物制品分册,第23卷第2期 2000 * |
新城疫植物油乳剂苗的研究 谢芝勋,等,中国预防兽医学报,第22卷第4期 2000 * |
新城疫植物油乳剂苗的研究 谢芝勋,等,中国预防兽医学报,第22卷第4期 2000;中等毒力新城活疫苗对鸡的免疫效果 李自力 摘,毕丁仁 校,国外医学.预防.诊断.治疗用生物制品分册,第23卷第2期 2000 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9950061B2 (en) | 2014-04-03 | 2018-04-24 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
US10702601B2 (en) | 2014-04-03 | 2020-07-07 | Boehringer Ingelheim Animal Health USA Inc. | Porcine epidemic diarrhea virus vaccine |
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