CN102973934B - Preparation method for Newcastle disease and avian influenza bi-combined inactivated vaccine - Google Patents

Preparation method for Newcastle disease and avian influenza bi-combined inactivated vaccine Download PDF

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CN102973934B
CN102973934B CN201210501862.3A CN201210501862A CN102973934B CN 102973934 B CN102973934 B CN 102973934B CN 201210501862 A CN201210501862 A CN 201210501862A CN 102973934 B CN102973934 B CN 102973934B
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gallus domesticus
embryo gallus
deactivation
avian influenza
venom
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CN102973934A (en
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江兴华
林志坤
陈玉文
陆奕滨
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Zhaofenghua Biotechnology Fuzhou Co ltd
Zhaofenghua Biotechnology Nanjing Co ltd
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FUZHOU DA BEI NONG BIOTECH Co Ltd
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Abstract

The invention relates to a preparation method for Newcastle disease and avian influenza bi-combined inactivated vaccine. The preparation method comprises the following steps: inoculating, chick embryo post-incubation, concentration of virus liquid, inactivation and incubation of virus liquid, thereby obtaining the Newcastle disease and avian influenza bi-combined inactivated vaccine. According to the preparation method, a continuous syringe and a magnetic stirrer are matched for inoculation, so that the efficiency is improved, and the loss of virus seed titer can be reduced; milipore quick tangential flow concentration equipment is used for concentration, so that the concentration efficiency is improved, and the titer loss in the concentration process can be decreased; and beta-propiolactone adopted as inactivator has no residuals and hazard, and has no untoward effect after being used.

Description

The preparation method of a kind of newcastle, bird flu bivalent inactivated vaccine
Technical field
The present invention relates to a kind of preparation method of birds inactivated vaccine, particularly relate to the preparation method of a kind of newcastle, bird flu bivalent inactivated vaccine.
Background technology
Newcastle is commonly called as fowl plague, is a kind of high degree in contact sexually transmitted disease caused by newcastle disease virus, and mainly through respiratory tract and digestive tract infection, the chicken at various age all can infect, and the most serious with chickling, M & M can reach more than 80%; Bird flu is infection/or the disease syndrome of a kind of birds caused by influenza A.Metainfective drylot feeding fowl comprises that poultry can show clinical symptom, respiratory system disease, egg production decline, the very high acute hemorrhagic disease of fatality rate, also can show as sub-clinical symptom, light respiratory system infection to multiple popular form such as asymptomatic band are malicious.The difference pathogenic according to bird flu, can be divided into high pathogenic avian influenza, low pathogenicity bird flu and no pathogenicity bird flu by bird flu.The important viral disease of these two kinds harm poultry husbandries development, spreads widely all over the world, causes huge economic loss to poultry husbandry.
For newcastle, bird flu, now existing corresponding individual event inactivated vaccine and dyad inactivated vaccine.
The preparation method of these newcastles existing, bird flu bivalent inactivated vaccine, although solve some difficult problems, also main exist following a few point defect:
1) inoculation adopts Bacillus Calmette-Guérin syringe, need inoculate by the absorption venom manually carried out repeatedly, artificial absorption venom is time-consuming, and efficiency is low, and seed culture of viruses bottle is exposed to the long period in the environment of room temperature simultaneously, can only the craft of variable interval shake all, the uniformity of seed culture of viruses can not be ensured on the one hand, long-time in the environment of room temperature on the other hand, plant tiring and being affected of poison, often cause decline of tiring, thus the antigen valence produced is reduced.
2) when concentrated employing filter post concentrates, once concentration amount is little, and efficiency is low, time-consuming, and concentration time is long reduces antigen valence, affects concentrated effect.
3) inactivator adopted is formaldehyde, and formaldehyde is carcinogen, has strong impulse, when formaldehyde acts on virus coat protein, easily make protein that crosslinked or virion gathering occurs, can not remake for the nucleic acid in glutelin, like this, the antigenicity of pathogen protein can be seriously damaged, and pathogen may be had to survive, there is the halfway risk of deactivation, free formaldehyde residual in vaccine, if after injecting body with vaccine, easily produce irritative response.
Summary of the invention
The object of the present invention is to provide some link of improvement of production process, improve and produce tiring of antigen, reduce the loss of tiring in concentration process, put forward highly enriched efficiency and effect, improve the immune effect of vaccine, reduce a kind of newcastle of immune side reaction, the preparation method of bird flu bivalent inactivated vaccine.
To achieve these goals, the present invention adopts following technical scheme:
The preparation method of this newcastle, bird flu bivalent inactivated vaccine, it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 4-10 5be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 9-10 age in days susceptible chick embryo allantoic cavity respectively with continuous injector, every embryo 0.1mL;
2) hatch after Embryo Gallus domesticus: postvaccinal Embryo Gallus domesticus is put after cultivating 96-120h respectively in the greenhouse of 37 DEG C, receive with vacuum-pump line and to survive in the stipulated time after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively and the Embryo Gallus domesticus liquid of death, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL;
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.025-0.1%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 4-6h; Be hydrolyzed and the virus liquid of deactivation be placed in 2-8 DEG C of preservation respectively afterwards;
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-806 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:2-3 mixing and emulsifying, get product.
Further, step 2) described in Embryo Gallus domesticus after concrete operation step in hatching be: Embryo Gallus domesticus every day of inoculation Newcastle Disease seed culture of viruses poison according to egg once, Embryo Gallus domesticus dead before discarding 60h; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 4-8h shines egg once, is taken out in time by dead Embryo Gallus domesticus and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 96-120h, take out the Embryo Gallus domesticus of remaining survival, is placed in 2-8 DEG C of cooling 4-24h respectively for results after the Embryo Gallus domesticus taking-up of survival.
The present invention adopts above technical scheme, joins magnetic stirring apparatus and inoculates, raise the efficiency by adopting continuous injector when inoculating, and reduces kind of a loss for toxic effect valency; The quick slipstream concentrator of concentrated employing Mi Libo, improves thickening efficiency, reduces the loss of tiring in concentration process; Inactivator adopts beta-propiolactone, noresidue, nonhazardous, the inactivator had no adverse reaction after using.
Detailed description of the invention
Technical scheme of the present invention is:
The preparation method of this newcastle, bird flu bivalent inactivated vaccine, it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 4-10 5be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 9-10 age in days susceptible chick embryo allantoic cavity respectively with continuous injector, every embryo 0.1mL;
2) hatch after Embryo Gallus domesticus: postvaccinal Embryo Gallus domesticus is put after cultivating 96-120h respectively in the greenhouse of 37 DEG C, receive with vacuum-pump line and to survive in the stipulated time after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively and the Embryo Gallus domesticus liquid of death, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL;
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.025-0.1%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 4-6h; Be hydrolyzed and the virus liquid of deactivation be placed in 2-8 DEG C of preservation respectively afterwards;
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-806 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:2-3 mixing and emulsifying, get product.
Further, step 2) described in Embryo Gallus domesticus after concrete operation step in hatching be: Embryo Gallus domesticus every day of inoculation Newcastle Disease seed culture of viruses poison according to egg once, Embryo Gallus domesticus dead before discarding 60h; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 4-8h shines egg once, is taken out in time by dead Embryo Gallus domesticus and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 96-120h, take out the Embryo Gallus domesticus of remaining survival, is placed in 2-8 DEG C of cooling 4-24h respectively for results after the Embryo Gallus domesticus taking-up of survival.
The present invention inoculates by adopting continuous injector to join magnetic stirring apparatus when inoculating, and raises the efficiency, and reduces kind of a loss for toxic effect valency; The quick slipstream concentrator of concentrated employing Mi Libo, improves thickening efficiency, reduces the loss of tiring in concentration process; Inactivator adopts beta-propiolactone, noresidue, nonhazardous, the inactivator had no adverse reaction after using.
Below in conjunction with specific embodiment, the present invention is further described:
Embodiment 1
The preparation method of this newcastle, bird flu bivalent inactivated vaccine, it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 4be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 9 age in days susceptible chick embryo allantoic cavities respectively with continuous injector, every embryo 0.1mL; Table 1 and table 2 are use the present invention and existing Bacillus Calmette-Guérin syringe to draw kind of malicious inoculation method respectively to producing Newcastle Disease venom and avian influenza venom docks the kind time and antigen valence compares, each inoculation 10 batches of antigens, the antigen valence often criticizing 3000 pieces of Embryo Gallus domesticus times used of inoculation and production contrasts;
Table 1 Newcastle Disease venom
Table 2 avian influenza venom
Can find out that from table 1 and table 2 the present invention draws kind of the method inoculation time used of poison than Bacillus Calmette-Guérin syringe short, virus liquid is tired height.
2) hatch after Embryo Gallus domesticus: put by postvaccinal Embryo Gallus domesticus in the greenhouse of 37 DEG C and cultivate respectively, the Embryo Gallus domesticus of inoculation Newcastle Disease seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 60h every day; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 4h shines egg once, is taken out in time by dead Embryo Gallus domesticus and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 96h, take out the Embryo Gallus domesticus of remaining survival, is placed in 2-8 DEG C of cooling 4-24h respectively for results after the Embryo Gallus domesticus taking-up of survival; Receive the Embryo Gallus domesticus liquid of surviving after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively with vacuum-pump line, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL; Table 3 and table 4 are respectively the production efficiency and concentrated effect and conventional contrast of filter post concentrator that to be concentrated by Newcastle Disease venom of the present invention and concentrate with avian influenza venom, and two kinds of methods all adopt 3 times to concentrate, use 3 × 10 5the antigen of mL concentrates;
Table 3 Newcastle Disease venom of the present invention concentrates and production efficiency and the concentrated effect of filtering post concentrator
Table 4 avian influenza venom of the present invention concentrates and production efficiency and the concentrated effect of filtering post concentrator
Can find out that from table 3 and table 4 the present invention more saves time than filter post concentrator, tiring, it is more to improve.
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.025%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 4h; Be hydrolyzed and the virus liquid of deactivation be placed in 2-8 DEG C of preservation respectively afterwards; Table 5 is that the inactivating efficacy of the present invention and formalin-inactivated agent contrasts.
The inactivating efficacy of table 5 the present invention and formalin-inactivated agent contrasts
The present invention's all batches of deactivations are all thorough as can be seen from Table 5, and use formaldehyde to occur two groups of halfway phenomenons of deactivation for inactivator.
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-806 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:2 mixing and emulsifying, get product.Table 6 uses the contrast of tiring for the present invention and former preparation method to finished product.
Table 6 the present invention and former preparation method use the contrast of tiring to finished product
note: be newly newcastle in table, flow for bird flu.
The present invention's antibody horizontal compared with former preparation method is high as can be seen from Table 6, and immune side reaction is little, non-stress.
Embodiment 2
The preparation method of this newcastle, bird flu bivalent inactivated vaccine, it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 5be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 10 age in days susceptible chick embryo allantoic cavities respectively with continuous injector, every embryo 0.1mL; Table 7 and table 8 are use the present invention and existing Bacillus Calmette-Guérin syringe to draw kind of malicious inoculation method respectively to producing Newcastle Disease venom and avian influenza venom docks the kind time and antigen valence compares, each inoculation 10 batches of antigens, the antigen valence often criticizing 3000 pieces of Embryo Gallus domesticus times used of inoculation and production contrasts;
Table 7 Newcastle Disease venom
Table 8 avian influenza venom
Can find out that from table 7 and table 8 the present invention draws kind of the method inoculation time used of poison than Bacillus Calmette-Guérin syringe short, virus liquid is tired height.
2) hatch after Embryo Gallus domesticus: put by postvaccinal Embryo Gallus domesticus in the greenhouse of 37 DEG C and cultivate respectively, the Embryo Gallus domesticus of inoculation Newcastle Disease seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 60h every day; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 8h shines egg once, is taken out in time by dead Embryo Gallus domesticus and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 120h, take out the Embryo Gallus domesticus of remaining survival, is placed in 2-8 DEG C of cooling 4-24h respectively for results after the Embryo Gallus domesticus taking-up of survival; Receive the Embryo Gallus domesticus liquid of surviving after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively with vacuum-pump line, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL; Table 9 and table 10 are respectively Newcastle Disease venom of the present invention to concentrate and concentrate production efficiency and concentrated effect and conventional contrast of filter post concentrator with avian influenza venom, and two kinds of methods all adopt 3 times to concentrate, use 3 × 10 5the antigen of mL concentrates;
Table 9 Newcastle Disease venom of the present invention concentrates and production efficiency and the concentrated effect of filtering post concentrator
Table 10 avian influenza venom of the present invention concentrates and production efficiency and the concentrated effect of filtering post concentrator
Can find out that from table 9 and table 10 the present invention more saves time than filter post concentrator, tiring, it is more to improve.
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.1%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 6h; Be hydrolyzed and the virus liquid of deactivation be placed in 2-8 DEG C of preservation respectively afterwards; Table 11 is that the inactivating efficacy of the present invention and formalin-inactivated agent contrasts.
The inactivating efficacy of table 11 the present invention and formalin-inactivated agent contrasts
The present invention's all batches of deactivations are all thorough as can be seen from Table 11, and use formaldehyde to occur two groups of halfway phenomenons of deactivation for inactivator.
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-806 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:3 mixing and emulsifying, get product.Table 12 uses the contrast of tiring for the present invention and former preparation method to finished product.
Table 12 the present invention and former preparation method use the contrast of tiring to finished product
Note: be newly newcastle in table, flow for bird flu.
The present invention's antibody horizontal compared with former preparation method is high as can be seen from Table 12, and immune side reaction is little, non-stress.
Embodiment 3
The preparation method of this newcastle, bird flu bivalent inactivated vaccine, it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 4.5be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 9.5 age in days susceptible chick embryo allantoic cavities respectively with continuous injector, every embryo 0.1mL; Table 13 and table 14 are use the present invention and existing Bacillus Calmette-Guérin syringe to draw kind of malicious inoculation method respectively to producing Newcastle Disease venom and avian influenza venom docks the kind time and antigen valence compares, each inoculation 10 batches of antigens, the antigen valence often criticizing 3000 pieces of Embryo Gallus domesticus times used of inoculation and production contrasts;
Table 13 Newcastle Disease venom
Table 14 avian influenza venom
Can find out that from table 13 and table 14 the present invention draws kind of the method inoculation time used of poison than Bacillus Calmette-Guérin syringe short, virus liquid is tired height.
2) hatch after Embryo Gallus domesticus: put by postvaccinal Embryo Gallus domesticus in the greenhouse of 37 DEG C and cultivate respectively, the Embryo Gallus domesticus of inoculation Newcastle Disease seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 60h every day; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 6h shines egg once, is taken out in time by dead Embryo Gallus domesticus and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 102h, take out the Embryo Gallus domesticus of remaining survival, is placed in 2-8 DEG C of cooling 4-24h respectively for results after the Embryo Gallus domesticus taking-up of survival; Receive the Embryo Gallus domesticus liquid of surviving after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively with vacuum-pump line, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL; Table 15 and table 16 are respectively Newcastle Disease venom of the present invention to concentrate and concentrate production efficiency and concentrated effect and conventional contrast of filter post concentrator with avian influenza venom, and two kinds of methods all adopt 3 times to concentrate, use 3 × 10 5the antigen of mL concentrates;
Table 15 Newcastle Disease venom of the present invention and production efficiency and the concentrated effect of filtering post concentrator
Table 16 avian influenza venom of the present invention and production efficiency and the concentrated effect of filtering post concentrator
Can find out that from table 15 and table 16 the present invention more saves time than filter post concentrator, tiring, it is more to improve.
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.06%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 5h; Be hydrolyzed and the virus liquid of deactivation put 2-8 DEG C of preservation respectively afterwards; Table 17 is that the inactivating efficacy of the present invention and formalin-inactivated agent contrasts.
The inactivating efficacy of table 17 the present invention and formalin-inactivated agent contrasts
The present invention's all batches of deactivations are all thorough as can be seen from Table 17, and use formaldehyde to occur two groups of halfway phenomenons of deactivation for inactivator.
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-806 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:2.5 mixing and emulsifying, get product.Table 18 uses the contrast of tiring for the present invention and former preparation method to finished product.
Table 18 the present invention and former preparation method use the contrast of tiring to finished product
Note: be newly newcastle in table, flow for bird flu.
The present invention's antibody horizontal compared with former preparation method is high as can be seen from Table 18, and immune side reaction is little, non-stress.

Claims (1)

1. a preparation method for newcastle, bird flu bivalent inactivated vaccine, is characterized in that: it comprises the following steps:
1) inoculate: respectively Avian pneumo-encephalitis virus and avian influenza seed culture of viruses poison are diluted 10 with sterile saline 4-10 5be placed in kind of a malicious bottle respectively doubly, during inoculation, plant malicious bottle and be placed on magnetic stirring apparatus, inoculate in 9-10 age in days susceptible chick embryo allantoic cavity respectively with continuous injector, every embryo 0.1mL;
2) hatch after Embryo Gallus domesticus: the Embryo Gallus domesticus of inoculation Newcastle Disease seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 60h every day; The Embryo Gallus domesticus of inoculation avian influenza seed culture of viruses poison shines egg once, Embryo Gallus domesticus dead before discarding 24h every day; Remaining Embryo Gallus domesticus respectively every 4-8h shines egg once, dead Embryo Gallus domesticus is taken out in time and is placed in 2-8 DEG C of cooling for results, continue to be cultured to 96-120h, take out the Embryo Gallus domesticus of remaining survival, 2-8 DEG C of cooling 4-24h is placed in respectively for results after the Embryo Gallus domesticus taking-up of survival, postvaccinal Embryo Gallus domesticus is put after cultivating 96-120h respectively in the greenhouse of 37 DEG C, receive with vacuum-pump line and to survive in the stipulated time after Seedling device gathers in the crops inoculation Avian pneumo-encephalitis virus and bird flu virus respectively and the Embryo Gallus domesticus liquid of death, obtain Newcastle Disease venom and avian influenza venom;
3) virus liquid is concentrated: by two-strain liquid under sterile conditions, concentrates, until Newcastle Disease venom is concentrated into viral level>=10 respectively with the quick slipstream concentrator of Mi Libo 8.7eID 50/ 0.1mL, avian influenza venom is concentrated into viral level>=10 8.3eID 50/ 0.1mL;
4) deactivation of virus liquid: the two-strain liquid after concentrated, adds the beta-propiolactone solution of virus liquid volume fraction 0.025-0.1%, respectively respectively at deactivation in deactivation tank; At deactivation pot liquid temperature 4 DEG C of deactivation 24h, be then warming up to 37 DEG C of hydrolysis 4-6h; Be hydrolyzed and the virus liquid of deactivation be placed in 2-8 DEG C of preservation respectively afterwards;
5) emulsifying: the Newcastle Disease venom of deactivation must be mixed sterilizing blastochyle with avian influenza venom mixed in equal amounts, get the sterilizing of tween 80 4 parts of loading container mesohighs, add mixing sterilizing blastochyle 96 parts after being cooled to room temperature, fully shake is until tween 80 dissolves completely, obtains aqueous phase; Get injection white oil 94 parts, Si Ben-80 6 parts, after mixing, add aluminium stearate 2 parts, limit edged stirs, and carries out autoclaving, obtain oil phase in tank body; By described aqueous phase and oil phase with volume ratio 1:2-3 mixing and emulsifying, get product.
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