CN108992674A - A kind of heat resisting protective and its application - Google Patents
A kind of heat resisting protective and its application Download PDFInfo
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- CN108992674A CN108992674A CN201810767282.6A CN201810767282A CN108992674A CN 108992674 A CN108992674 A CN 108992674A CN 201810767282 A CN201810767282 A CN 201810767282A CN 108992674 A CN108992674 A CN 108992674A
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- duck plague
- cell
- heat resisting
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides a kind of heat resisting protective and its applications, belong to technical field of biological product preparation.Heat resisting protective of the invention contains gelatin 1% ~ 4%, sucrose 5% ~ 15% or trehalose 1% ~ 5%, sorbierite 1% ~ 4% or mannitol 1% ~ 3%, tryptone 1% ~ 4% or defatted milk 2% ~ 6%, L-sodium 0.005% ~ 0.1% or arginine 0.5% ~ 3%, and solvent is PBS solution.Heat resisting protective of the invention is added in vaccine, so that live vaccine can save 24 months at 2-8 DEG C, viral level and potency are not reduced, and are required to reduce preservation, convenient for the storage and transport of live vaccine, are reduced production cost.
Description
Technical field
The present invention relates to technical field of biological product preparation, more particularly to a kind of heat resisting protective of vaccine.
Background technique
Duck plague (Duck Plague, DP), be the duck caused by duck plague virus (Duck Plague Virus, DPV), goose and
A kind of acute septic infectious disease of a variety of wild goose mesh birds.DPV is propagated rapidly, and popular morbidity and mortality are very high extensively,
Huge economic loss is caused to duck culturing industry, is to endanger one of infectious disease the most serious of duck culturing industry.Immunity inoculation duck plague disease
Malicious vaccine is one of the effective measures of anti-duck plague processed, and production duck plague virus vaccine needs to cultivate a large amount of duck plague virus liquid.China
Cell used in breeding duck plague virus is chick embryo fibroblast primary cell (CEF) at present.Although country has provided used chicken
Embryo must derive from SPF chicken group, but due to the hysteresis quality of detection and the complexity of feeding environment, so that production duck plague CEF
There are the security risks that exogenous virus pollutes for cell；In addition, with a large amount of chicken embryos, large labor intensity when production, while also increasing
CEF cell contaminated possibility.The currently reported host cell for showing chicken gizzard cancerous cell line and can be used as duck plague virus,
But chicken gizzard cancer cell pole is not easy to cultivate, and increases the production cost of duck plague virus culture.
DF-1 cell is a kind of chicken fibroblasts system that can be passed on, and derives from ELL chicken embryo tire, which is morphologically in
Threadiness.DF-1 cell line is a kind of stable cell line without oncogene, spontaneous infinite multiplication.Existing research table at present
Bright infectious bursal disease virus, chook MDV, newcastle disease virus can on DF-1 cell well-grown, but do not have
The report of DF-1 cell line culture duck plague virus method.
In the prior art the store method of Duck plague live vaccine be -15 DEG C or less save 24 months, the vaccine from production, examine
To factory, then the time through 2 months is at least needed from dealer to user, once be more than -15 DEG C this is " cold for storage temperature around here
Chain " will result in live virus content in vaccine and decline, influences the using effect of vaccine, or even cause immuning failure.It is this simultaneously
Preservation condition increases preservation cost to a certain extent, does not utilize transport and storage.If it is mild to provide a kind of preservation condition
Duck plague live vaccine, then advantageously reduce the preservation cost of vaccine, reduce because preservation condition change brought by vaccine quality
Decline.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the invention is to provide a kind of epidemic diseases that vaccine is effectively reduced and saves cost
Seedling heat resisting protective.
Present invention firstly provides a kind of heat resisting protective suitable for vaccine, the component including following proportion, gelatin 1% ~
4%, sucrose 5% ~ 15% or trehalose 1% ~ 5%, sorbierite 1% ~ 4% or mannitol 1% ~ 3%, tryptone 1% ~ 4% or defatted milk 2% ~
6%, L-sodium 0.005% ~ 0.1% or arginine 0.5% ~ 3%, solvent are PBS solution；The % be each component quality with it is heat-resisting
The mass volume ratio of protective agent total volume.
Further, heat resisting protective of the invention includes the component of following proportion: gelatin 1.5% ~ 4%, sucrose 8% ~ 12%
Or trehalose 2% ~ 5%, sorbierite 2% ~ 4% or mannitol 1.5% ~ 3%, tryptone 1.5% ~ 4% or defatted milk 2% ~ 6%, L- paddy ammonia
Sour sodium 0.005% ~ 0.05% or arginine 1% ~ 3%, solvent are the PBS solution of 0.01-0.05M.
Preferably, heat resisting protective of the invention includes the component of following proportion: gelatin 2% ~ 4%, sucrose 9% ~ 12% or sea
Algae sugar 2.5% ~ 5%, sorbierite 2.5% ~ 4% or mannitol 2% ~ 3%, tryptone 2% ~ 4% or defatted milk 2% ~ 5%, L-sodium
0.008% ~ 0.03% or arginine 1.5% ~ 3%, solvent is the PBS solution of 0.01-0.02M.
It is highly preferred that heat resisting protective of the invention includes the component of following proportion: gelatin 2%, sucrose 10%, sorbierite
4%, tryptone 3%, L-sodium 0.01%；
Or, gelatin 1%, sucrose 15%, sorbierite 4%, tryptone 4%, L-sodium 0.005%；
Or, gelatin 4%, sucrose 5%, sorbierite 2%, tryptone 1%, L-sodium 0.1%；
Or, gelatin 2%, trehalose 5%, sorbierite 3%, defatted milk 5%, arginine 1.5%；
Or, gelatin 2%, sucrose 10%, mannitol 3%, defatted milk 2%, L-sodium 0.1%；
Or, gelatin 4%, trehalose 1%, mannitol 2%, defatted milk 4%, arginine 3%；
The above proportion solvent is the PBS solution of 0.01M.
The present invention provides the preparation methods of above-mentioned heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin, sucrose (or trehalose of filtration sterilization) and tryptone (or defatted milk) are weighed by formula, is added in PBS solution
To being completely dissolved, sterilize；
(2) B liquid is prepared
Sorbierite (or mannitol) and L-sodium (arginine) are weighed by formula, is added in PBS solution to being completely dissolved, removes
Bacterium；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
Biological products containing heat resisting protective of the present invention belong to the scope of protection of the present invention.
Further, the biological products are vaccine.The present invention provides the epidemic diseases living of the duck plague containing above-mentioned heat resisting protective
Seedling, infectious bursal disease live-vaccine, live Newcastle disease vaccine, pseudorabies living vaccines or with high-pathogenicity porcine reproductive and breathing
Syndrome live vaccine.
Further, the duck plague virus of the Duck plague live vaccine is duck plague virus or the training of DF-1 cell of CEF cell culture
Feeding duck plague virus.
The present invention provides above-mentioned heat resisting protectives to extend vaccine storage life or improve the application in vaccine heat resistance.
Duck plague virus in Duck plague live vaccine described in the embodiment of the present invention is using chicken embryo fibroblasts system DF-1 as place
What main culture obtained.
The Duck plague live vaccine is prepared by following steps:
(1) overdose duck plague virus liquid is inoculated with DF-1 cell；
(2) it connects 5-10 days cell liquid of the harvest containing virus after poison, is inoculated with DF-1 cell again according to step (1) after multigelation,
Such blind passage mostly generation, to harvesting virus liquid when being inoculated with after duck plague virus in 120 hours CPE up to 75% or more；
(3) it is inoculated in DF-1 cell according to the volume ratio with DF-1 cell liquid 0.5%-5% after the virus liquid freeze thawing harvested, cultivated,
Duck plague virus liquid is harvested, viral level is not less than 10 in duck plague virus liquid7.0TCID50/ml；
(4) heat resisting protective is added according to virus liquid volume ratio 1:1 ~ 1:5.
Step (1) overdose is not less than 106.5TCID50The virus liquid of/ml according to DF-1 cell liquid volume ratio
3%-12% inoculum concentration.
Preferably, step (1) overdose is 106.5-107.5TCID50The virus liquid of/ml according to DF-1 cellular liquid
The 3%-12% inoculum concentration of product ratio.
It is highly preferred that step (1) overdose is 107.5TCID50The virus liquid of/ml according to DF-1 cell liquid volume
The 3%-8% inoculum concentration of ratio.In a preferred embodiment of the present invention, using 107.5TCID50The virus liquid of/ml is according to thin with DF-1
5% inoculum concentration of cytosol volume ratio.It will be appreciated by those skilled in the art that no matter using the above-mentioned any viral level enumerated or
Volume ratio, as long as the inoculum concentration of duck plague virus is sufficiently large to be suitable for the invention technical solution.
The SPF chick embryo allantoic liquid of 9-11 age in days is added in step (1) the DF-1 cell liquid.
The SPF chick embryo allantoic liquid of the 9-11 age in days of volume ratio 3%-8% is added in step (1) the DF-1 cell liquid.
In step (2), it is preferable that connect 6-9 days harvest cell liquid after poison.
Preferably, it is inoculated with DF-1 cell, such blind passage mostly generation, until 120 after inoculation again according to step (1) after multigelation
In hour CPE up to 75% or more when harvest virus liquid；It is highly preferred that harvest disease when CPE is up to 75% or more in 72 hours to after being inoculated with
Venom.
Chick embryo allantoic liquid is not added in step (3) the DF-1 cell liquid.
Viral level is not less than 10 in the every plumage part vaccine of Duck plague live vaccine of the invention3.5TCID50。
DF-1 cell culture fluid used in the embodiment of the present invention contains the DMEM/F12 liquid of 90%~92% percent by volume, resists
The cow's serum of raw element and 8%~10% percent by volume, the pH value of cell culture fluid are 7.0~7.2.The antibiotic is 100
The combination of IU/ml penicillin and 100 IU/ml streptomysins.
DF-1 cell maintenance medium used in the embodiment of the present invention contains DMEM/F12 liquid, the antibiosis of 95%~98% percent by volume
The cow's serum of element and 2%~5% percent by volume, the pH value of cell maintenance medium are 7.2~7.4.The antibiotic is 100IU/
The combination of ml penicillin and 100IU/ml streptomysin.
The present invention is not limited to the formula of the above-mentioned cell culture fluid enumerated and cell maintenance medium, those skilled in the art can be with
The method of DF-1 can be cultivated using common other known in this field to cultivate DF-1 cell line culture side according to the invention
Method can be realized technical effect expected from the present invention.
Through EDTA- pancreatin had digestive transfer culture when cell passes on, continue to cultivate with cell culture fluid, forms passage cell single layer.
The beneficial effects of the present invention are:
The first, the present invention is that duck plague virus breeding has searched out a kind of new host, with DF-1 cell culture duck plague virus, is avoided
It is easy to improve duck plague vaccine there are the security risk that exogenous virus pollutes using CEF cell culture and virus in the prior art
Safety.
The second, duck plague virus cultural method provided by the invention, can make duck plague virus good in DF-1 grown on cell lines,
Viral level is up to 107.4~108.0 TCID50/ ml, viral yield is higher than CEF primary cell, compared with the viral level of CEF cell culture
Improve 0.25-0.75 titre.
Third, since DF-1 cell is a kind of chicken fibroblasts system that can be passed on, it is on-demand, time saving and energy saving to have
Feature, therefore in duck plague virus liquid production process, it avoids in the prior art due to needing using CEF cell and by SPF
The limitation of chicken embryo supply and hatching age in days, greatly simplifies production technology and production cost.
4th, heat resisting protective of the invention preparation is simple, and at low cost, heat-resisting protective effect is good.Conventional Duck plague live vaccine
Preservation condition is -20 DEG C and saves 24 months, and heat resisting protective of the invention can make Duck plague live vaccine save 24 at 2-8 DEG C
Month, viral level and potency do not reduce, to reduce the preservation requirement of vaccine, are conducive to the storage and fortune of Duck plague live vaccine
It is defeated, reduce production cost, remarkable in economical benefits.
Detailed description of the invention
Fig. 1 is DF-1 control cell；Fig. 2 is that DPV F80 connects malicious DF-1 cell with 1% and connects after poison for 24 hours；Fig. 3 is DPV F80
Malicious DF-1 cell, which is connect, with 1% meets 48h after poison；Fig. 4 is that DPV F80 connects malicious DF-1 cell with 1% and meets 72h after poison.
Specific embodiment
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It should be understood that this
A little examples are only for illustrating the present invention and not for limiting the scope of the present invention, unmentioned specific experiment in the following example
Method is usually carried out according to routine experiment method.
All technical terms used herein are identical as the normally understood meaning of those skilled in the art.Institute herein
The technical term used is intended merely to the purpose of description specific embodiment, is not intended to and limits the scope of the invention.
In following embodiment, DF-1 cell is purchased from Ran Shun (Shanghai) Biotechnology Co., Ltd.Duck plague virus is chicken embryo
AV1222 plants of low virulent strain, it is purchased from China Veterinery Drug Inspection Office.Except there is a special instruction, various reagents, the original used in the present invention
Material is can commodity commercially or can the product as made from well known method.
The preparation of 1 duck plague virus liquid of embodiment
DF-1 cell is conventionally cultivated to single layer, is set up and is connect malicious group and blank control group.Experimental group is by volume
5%(, that is, 300ul) (the duck plague virus liquid DPV F65 generation poison of CEF cell Attenuation, viral level are inoculation DPV F65 generation poison
107.5TCID50/ ml), at the same by cell liquid volume 6ml add 5%(, that is, 300ul) 9 ~ 10 ages in days SPF chick embryo allantoic liquid, set 37
℃、5%CO2Under the conditions of adsorb 60min, during which gently shake cell bottle every 15min, maintaining liquid added after absorption, simultaneously
CEF cell inoculation DF-1 cell controls are set up as blank control group, inoculative proportion and amount and the duck plague virus for adding allantoic fluid
It is identical.
Daily observation cytopathy situation, will be connect if not occurring cytopathy still after culture 7 days poison group cell bottle and
Continue according to the above method after freeze thawing 1 time under the conditions of the cell bottle of blank control group sets -20 DEG C be inoculated with DF-1 cell, continuous blind passage,
It is harvested after being often commissioned to train feeding 7 days.Poison group DPV F66 ~ F69 generation (i.e. the 1-4 generation of DF-1 cell culture) is connect without obvious cytopathy
Become；There is obvious lesion, i.e. cell rounding in cell after DPV F70 is commissioned to train feeding 6 days, there is graininess missing, cell seine.With generation
Secondary increase, lesion time of occurrence are done sth. in advance, and lesion is more obvious, is more, and sick cell starts shedding off.48 hours after DPV F72 inoculation
There is cytopathy, 72 hours CPE are up to 75% or more.Blank control group does not occur cytopathy always.
DPV F75 starts for poison, connects poison amount and adjusts to 1%, while no longer adding the SPF chick embryo allantoic liquid of 9 ~ 10 ages in days.It connects
Lesion time retardation after poison occurs obvious lesion for 96 hours, and 120 hours CPE are up to 75% or more, as generation increases, when lesion
Between ahead of time, there is within 48 hours obvious lesion after DPV F77 inoculation, 72 hours CPE are up to 75% or more.The operation of blank control group with
Experimental group is identical, and what difference was blank control group inoculation is CEF cell.
48 hours or so after DPV F80 ~ F100 1% inoculation by volume, there is circle contracting in cell, there is graininess missing, lesion
Cell starts shedding off, and attached cell draws in the net, and occurs vacuole in endochylema, after inoculation 72 hours CPE up to 75% or more, see Fig. 1 ~
Fig. 4.
The measurement of 2 viral level of embodiment
DPV F80 ~ F100 difference generation virus liquid serum-free the M199 harvested in embodiment 1 work is serially diluted for 10 times, takes 4
A acceptable diluent degree, is inoculated with the 96 hole Microtitration plates of CEF cell for having grown up to good single layer respectively, and each dilution connects
6 holes of kind, every hole 0.1ml, while setting normal cell controls.Set 37 DEG C, containing 5% CO2After adsorbing 1 hour in incubator, every hole is mended
Add the M199 maintaining liquid 0.1ml containing 4% serum, observe 120~144 hours, records cytopathy (CPE) hole count.By Reed-
Muench method calculates TCID50, the results showed that DPV F80 ~ F100 stablizes for viral level, and viral level is up to 107.4~108.0
TCID50/ mL。
1 viral level testing result of table
DPV F80, F86, F95 generation malicious specificity identification of 3 DF-1 cell of embodiment production
F80, F86, F95 for harvesting in embodiment 1 are diluted to serum-free M199 containing 100TCID for virus liquid50 / 0.1ml,
After mixing with the anti-duck plague virus specific serum of equivalent, 37 DEG C are neutralized 1 hour, and inoculation 6 has grown up to the cell hole of CEF single layer
(48 porocyte plates), every hole 0.2ml, while setting virus control and each 6 hole of normal cell controls.Set 37 DEG C, containing 5% CO2Culture
Case culture simultaneously observes 120-144 hours, as a result neutralization group and the cell-free lesion of normal cell controls group, virus control group cell
There is cytopathy.
Anti- duck plague virus specific serum used in the present embodiment the preparation method comprises the following steps:
(1) preparation method of anti-duck plague virus specific serum
9 week old SPF chickens 40 are taken, the duck plague virus F80 of every intramuscular injection embodiment 1 harvest contains for virus liquid 0.5ml(virus
Amount is 107.6TCID50/ ml)；Every intramuscular injection duck plague virus F80 is for virus liquid 0.2ml and duck plague virus F80 generation poison after 14 days
The inactivated vaccine 1.0ml(inactivation provirus content of strain is 107.6TCID50/ ml)；Then it is inactivated again with duck plague virus F80 for strain
Vaccine booster immunization 2 times, every 1.0ml.4 immune rear 28 days acquisition serum, detect neutralize antibody titers.When antibody level reaches
It largely takes a blood sample when to peak value, separates serum.
(2) anti-duck plague virus specific serum standard
[character] faint yellow supernatant liquid.
[steriling test] tests by existing " Chinese veterinary pharmacopoeia " annex 3306, answers asepsis growth.
[mycoplasma inspection] tests by existing " Chinese veterinary pharmacopoeia " annex 3308, should be without mycoplasma contamination.
[specificity identification] newcastle disease, bird flu, aviadenovirus (having coagulation) are HI negative antibody；It is avian infectious
Bronchitis virus, reticuloendothiliosis virus, avian leukosis virus, chicken infectivity bursa of Fabricius virus, chicken pass
Metachromia anemia virus, Avianreovirus, avian infectious laryngotracheitis virus, avian encephalomyclitis virus are ELISA antibody yin
Property；Bird pox virus, chicken Marek's disease virus, aviadenovirus (I group)；Duck hepatitis, duck tembusu virus disease neutralizing antibody are negative.
The neutralize antibody titers of [neutralization titer measurement] and duck plague virus should be not less than 1:64.
Preservation below -70 DEG C of [storage and validity period], it is 24 months that storage life, which is fixed tentatively,.
4 DPV F80 of embodiment, the malicious exogenous virus detection of F86, F95 generation
(1) PCR method detects duck tembusu virus DTMUV, III type virus DHV-3 of duck hepatitis I type virus DHV-1 and duck hepatitis
DPV F80, F86, F95 difference generation virus liquid kit is taken to extract RNA, with reverse transcription reagent box reverse transcription at cDNA
PCR detection is carried out afterwards.Primer sequence is shown in Table 2.
Table 2
PCR reaction system: 10 μ L, 10 × PCR buffer of DNA profiling, 5 μ L, 2.5mmol/L dNTP, 2 μ L, upstream is drawn
1 μ L of object, 1 μ L, Ex Taq enzyme of downstream primer 1 μ L, ddH2O 30µL。
PCR response procedures:
(a) duck tembusu virus (DTMUV)
PCR response procedures are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 40 seconds, and 56 DEG C are annealed 40 seconds, and 72 DEG C extend 1 minute, altogether
35 circulations；72 DEG C extend 10 minutes.
(b) duck hepatitis I type is viral (DHV-1 type)
PCR response procedures are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 1 minute, and 50 DEG C are annealed 1 minute, and 72 DEG C extend 1 minute,
35 circulations；72 DEG C extend 10 minutes.
(c) III type of duck hepatitis is viral (DHV-3 type)
PCR response procedures are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 30 seconds, 55 DEG C anneal 30 seconds, 72 DEG C extend 1 minute, 30
A circulation；72 DEG C extend 5 minutes.
PCR testing result: F80, F86, F95 made from embodiment 1 generation poison (namely passed on DF-1 cell the 15th,
21,30 generations poison) three generations virus liquid through PCR detection duck tembusu virus, duck hepatitis I type virus and duck hepatitis III type virus
Antigen is feminine gender, is not expanded to three purpose bands in table 2, illustrate duck plague virus liquid made from 1 method of embodiment not by
To the pollution of Three Common duck exogenous virus.
(2) chicken inspection technique
DPV F80, F86, F95 difference generation virus liquid is taken to be diluted to every milliliter containing 10 with sterile saline3.5TCID50, often
A 10 week old SPF chicken 20 of generation virus liquid intramuscular injection, 1m/ is only.After 14 days, every eye droppings, collunarium inoculation 0.1ml(contain
104.5TCID50), intramuscular injection 1ml(contain 105.5TCID50).Observation 21 days after, according to the above method with dosage repeated inoculation 1 time.
It takes a blood sample within 21 days after 2nd inoculation, separates serum.In 56 days, should not there are caused by vaccine locally or systemically symptom or respiratory tract
Symptom or death.If there is dead chicken, pathological examination should be carried out, to prove whether caused by duck plague virus.
Newcastle disease virus, adenovirus (having coagulation), H5 hypotype fowl is measured to the isolated serum HI method of inspection to flow
Influenza Virus, H9 subtype avian influenza virus antibody；Avian infectious bronchitis virus, chicken infectivity throat gas are measured with ELISA method
Pipe inflammation virus, avian leukosis virus, fowl Reticuloendotheliosis Virus, chicken infectivity bursa of Fabricius virus, chicken infectious anemia
The antibody of virus, Avianreovirus and avian encephalomyclitis virus；The antibody inspection of chicken Marek's disease virus is carried out with AGP method
It surveys；Duck tembusu virus, duck hepatitis virus antibody are surveyed with neutralization test.
As a result: after the virus liquid of 3 generations is inoculated with chick twice respectively, taking a blood sample within 56th, carry out Serum Antibody Detection, antibody
Detection is feminine gender.In 56 days, locally or systemically symptom or respiratory symptom or death are not caused, see Table 3 for details.
3 exogenous virus detection method of table and result
Note: "-" indicates negative.
The comparison for the viral level that 5 DPV F80 of embodiment, F86, F95 generation poison are proliferated on CEF cell and DF-1 cell
By DPV F80 tamed on DF-1 cell in embodiment 1, F86, F95 generation poison and without the DPV of DF-1 cell domestication
F65 generation poison (i.e. embodiment 1 initially connect seed culture of viruses) respectively 1% inoculation by volume grown up to good single layer DF-1 cell and
Cell maintenance medium is added after sixty minutes and continues to set 37 DEG C, 5%CO for CEF cell, 37 DEG C of absorption2Culture, observes cytopathy daily,
Harvest virus liquid (CPE sentences standard up to 75% eventually) when cytopathy is up to 75% or so, the virus liquid of harvest sets -20 DEG C of freeze thawing 1
Sampling carries out the measurement of viral level with CEF cell after secondary, according to the above method by DPV F80, F86, F95 generation poison and DPV F65
Generation poison continuously bred for 3 generations on CEF cell and DF-1 cell respectively (per generation do 5 repetition test, take its average, be shown in Table 4),
Per generation sampling carries out the measurement of viral level with CEF cell.
TCID on the CEF cell and DF-1 cell of the different generation poison of table 4 respectively50Measurement result
Table 4 is the result shows that DPV F80, F86, F95 the generation poison tamed on DF-1 cell are proliferated in CEF cell and DF-1 cell
Well, the viral level being proliferated on DF-1 cell is slightly above CEF cell, about 0.25 ~ 0.75 titre；It is tamed and dociled on DF-1 cell
The virus of proliferation of DPV F80, F86, F95 generation poison of change with the DPV F65 generation poison without the domestication of DF-1 cell on CEF cell
Content and harvest time no significant difference；It can be in CEF for poison without the DPV F65 of DF-1 cell acclimation method provided by the invention
Cell Proliferation, but cannot be in DF-1 cell Proliferation.
The comparative test that embodiment 6 is different about heat-resisting protective agent prescription, dosage is different
In the present invention, the % for being related to heat-resisting protective agent prescription is the mass volume ratio of each component quality and heat resisting protective total volume.
Phosphate buffer (0.01mol/L PBS, pH value 7.2 ~ 7.4) formula is following:
Sodium chloride 8.0g
Potassium chloride 0.2g
Disodium hydrogen phosphate dodecahydrate 2.9g
Potassium dihydrogen phosphate 0.2g
Water for injection adds to 1000ml
PH value is adjusted to 7.2 ~ 7.4 with hydrochloric acid.
, formula 1 and its preparation duck plague vaccine in application
Heat resisting protective contains the component of following proportion: gelatin 2%, sucrose 10%, sorbierite 4%, tryptone 3%, Pidolidone
Sodium 0.01%.
The preparation method and application of above-mentioned heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin, sucrose and tryptone are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, goes out then at 121 DEG C
Bacterium 30min；
(2) B liquid is prepared
Sorbierite and L-sodium are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filtration sterilization；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique includes: that plate layer temperature is dropped to -38 DEG C in 1.5h and maintains 3
Plate layer temperature is risen to -6 DEG C by -38 DEG C and maintains 12 ~ 15h by ~ 6h, 3h, then is warming up to 0 DEG C, maintains 5 ~ 8h, then be warming up to 10
DEG C, 3 ~ 6h is maintained, then be warming up to 30 DEG C, maintains 4 ~ 6h.
, formula 2 and its preparation duck plague vaccine in application
Heat resisting protective contains the component of following proportion: gelatin 4%, sucrose 5%, sorbierite 2%, tryptone 1%, L-sodium
0.1%。
(1) A liquid is prepared
Gelatin, sucrose and tryptone are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, goes out then at 121 DEG C
Bacterium 30min；
(2) B liquid is prepared
Sorbierite and L-sodium are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filtration sterilization；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 3 and its preparation duck plague vaccine in application
Heat resisting protective includes the component of following proportion: gelatin 1%, sucrose 15%, sorbierite 4%, tryptone 4%, Pidolidone
Sodium 0.005%.
The preparation method and application of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin, sucrose and tryptone are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, goes out then at 121 DEG C
Bacterium 30min；
(2) B liquid is prepared
Sorbierite and L-sodium are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filtration sterilization；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 4 and its preparation duck plague vaccine in application
A kind of component of Duck plague live vaccine heat resisting protective: polyvinylpyrrolidone (PVP-K30) 3%, sucrose 10%, sorbierite
3%, tryptone 1%, L-sodium 0.01%.
The preparation method and application of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Sucrose and tryptone are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, sterilizes then at 121 DEG C
30min；
(2) B liquid is prepared
Polyvinylpyrrolidone (PVP-K30), sorbierite and L-sodium are weighed by formula, is added in the PBS solution of 0.01M
To being completely dissolved, filtration sterilization；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 5 and its preparation duck plague vaccine in application
Heat resisting protective includes: gelatin 2%, trehalose 5%, sorbierite 3%, defatted milk 5%, arginine monohydrochloride 1.5%.
The preparation method of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin and defatted milk are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, then at 121 DEG C of sterilizing 30min；
(2) B liquid is prepared
Trehalose, sorbierite and arginine monohydrochloride are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filters
Degerming；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 6 and its preparation duck plague vaccine in application
A kind of Duck plague live vaccine heat resisting protective contains the component of following proportion: gelatin 2%, sucrose 12%, mannitol 3%, defatted milk
2%, L-sodium 0.1%.
The preparation method of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin, sucrose and defatted milk are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, sterilizes then at 121 DEG C
30min；
(2) B liquid is prepared
Mannitol and L-sodium are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filtration sterilization；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 7 and its preparation duck plague vaccine in application
A kind of Duck plague live vaccine heat resisting protective contains the component of following proportion: gelatin 4%, trehalose 1%, mannitol 2%, degreasing
Milk 4%, arginine monohydrochloride 3%.
The preparation method of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin and defatted milk are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, then at 121 DEG C of sterilizing 30min；
(2) B liquid is prepared
Trehalose, mannitol and arginine monohydrochloride are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filters
Degerming；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique is referring to the lyophilized technique for being formulated 1.
, formula 8 and its preparation duck plague vaccine in application
A kind of Duck plague live vaccine heat resisting protective contains following components: gelatin 5%, trehalose 1%, mannitol 2%, tryptone 1%,
Arginine monohydrochloride 5%.
The preparation method of above-mentioned Duck plague live vaccine heat resisting protective, comprising the following steps:
(1) A liquid is prepared
Gelatin and tryptone are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, sterilizes then at 121 DEG C
30min；
(2) B liquid is prepared
Trehalose, mannitol and arginine monohydrochloride are weighed by formula, is added in the PBS solution of 0.01M to being completely dissolved, filters
Degerming；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
(4) by the above-mentioned heat resisting protective prepared and duck plague virus liquid, 1:1 mixes to obtain freeze-drying liquid by volume, then into
Duck plague live vaccine is made in row frozen dried.Wherein, lyophilized technique includes: to participate in the lyophilized technique of formula 1.
8 groups of heat resisting protective Duck plague live vaccines and use GPF (General Protection False agent (GPF (General Protection False agent 1:6% prepared by embodiment 6
Sucrose and 12% defatted milk；The gelatin of GPF (General Protection False agent 2:2% and 10% sucrose) it is living using duck plague made from conventional lyophilized technique
Vaccine compares its physical parameter and places different time restrovirus content situation under the conditions of 37 DEG C and under the conditions of 2 ~ 8 DEG C, single
Position is lgTCID50/ plumage part.As a result it is shown in Table 5 and table 6 respectively.
The different heat resisting protectives of table 5 are under the conditions of 37 DEG C to the protection result of vaccine
As shown in Table 5, duck plague virus is lyophilized with GPF (General Protection False agent and heat resisting protective, and freeze-drying loss is all very small
(in 0.25 titre), but heat-resisting restrovirus content loss difference is clearly.2 37 DEG C of GPF (General Protection False agent heat-resisting 10d diseases
Malicious content reduces by 1.0 ~ 1.25 titres；Heat-resisting protective agent prescription 4 is with formula 8 to the protection of duck plague virus outside the range of protection
Effect is not that very well, 37 DEG C of heat-resisting 10d viral levels reduce by 1.0 titres, and 37 DEG C of heat-resisting 30d viral levels are reduced to 0；Formula
1-3, the heat-resisting 10d viral level of 37 DEG C of 5-7 heat resisting protective of formula lose very little, reduce by 0.25 ~ 0.50 titre, 37 DEG C heat-resisting
30d viral level loses very little, reduces by 0.75 ~ 1.0 titre.
The different heat resisting protectives of table 6 are in 2 ~ 8 DEG C of protection results to vaccine
As shown in Table 6, duck plague virus is lyophilized with GPF (General Protection False agent and heat resisting protective, and freeze-drying loss is all very small
(in 0.25 titre).2 ~ 8 DEG C save 12 months, and formula 4, the duck plague virus of 8 and 2 GPF (General Protection False agent prescription freeze-dryings of formula are sick
Malicious content reduces by 0.4 ~ 0.5 titre, and formula 1-3, the viral level for being formulated 5-7 lose very small (0 ~ 0.25 titre
It is interior)；2 ~ 8 DEG C save 24 months, and the duck plague virus viral level that 8 and 2 formula 4, formula GPF (General Protection False agent prescriptions are lyophilized drops
Low 1.0 titres, formula 1-3, the viral level for being formulated 5-7 lose very small (in 0.25 ~ 0.5 titre).
Comparative test of the embodiment 7 about protective agent and virus liquid different proportion proportion
By the virus liquid (10 of harvest7.5TCID50/ ml) it is diluted to 10 respectively7.0TCID50/ml、106.75TCID50/ml、
106.5TCID50/ml、106.0TCID50Antibiotic is added by 1% volume ratio respectively in the virus liquid of this 5 various concentrations in/ml,
Then body is successively pressed according to viral level height with the prepared heat-resisting protective agent prescription 1 of embodiment 6 and GPF (General Protection False agent 1 respectively
Heat-resisting protective agent prescription 1 or GPF (General Protection False agent 1 is added than 1:1,1:2,1:3,1:4,1:5 in product, after mixing quantitative separating, warp
Vacuum freezedrying rear pressing cover labeling, the Duck plague live vaccine of preparation set 37 DEG C of preservations.Compare its physical parameter and in 37 DEG C of items
Different time restrovirus content situation, unit lgTCID are placed under part50/ml.It the results are shown in Table 7.
The different protective agents of table 7 and different content virus liquid different proportion match under the conditions of 37 DEG C to the protection knot of vaccine
Fruit
As shown in Table 7, heat-resisting protective agent prescription 1 and GPF (General Protection False agent 1 and different content virus liquid match freeze-drying in varing proportions
Heat-resisting different time is as the result is shown under the conditions of 37 DEG C afterwards: according to viral level difference, heat-resisting protective agent prescription 1 and virus liquid
Volume ratio 1:1 ~ 1:5 it is all very small to the loss of the viral level of duck plague virus in 37 DEG C of heat-resisting 10d, reduce by 0.25 ~ 0.35
Titre, 37 DEG C 30 days heat-resisting, reduces by 0.87 ~ 1.1 titre；And volume ratio 1:1 ~ 1:5 of GPF (General Protection False agent 1 and different virus liquid
It is bigger in viral level loss of 37 DEG C of heat-resisting 10d to duck plague virus, reduce by 1.0 ~ 1.25 titres, 37 DEG C of heat-resisting 30d, drop
It is more than low 3 titres.
The preparation and inspection of 8 Duck plague live vaccine of embodiment
F80 duck plague virus liquid made from embodiment 1 is inoculated with DF-1 cell monolayer, 37 DEG C of trainings by the volume ratio 1% of cell maintenance medium
It supports, when lesion is up to 75% or more, the cytopathy venom of harvest is as production seed culture of viruses.
The passage and culture of seedling cell: DF-1 is cultivated according to a conventional method after expiring single layer to cell, discards growth-promoting media, is used
The PBS that sterilizes is cleaned cell surface 1 time, through pancreas enzyme -EDTA digestive juice had digestive transfer culture, cell growth medium is added and continues to cultivate, is formed
When good single layer, for continuing passage or virus inoculation.
The preparation of seedling virus liquid: selecting well-grown DF-1 cell, discard cell culture fluid, connects poison amount by 1% and connects
Kind production seed culture of viruses, sets 37~38 DEG C of cultures.It connects after poison when lesion harvests virus liquid up to 75% or more, at -15 DEG C after freeze thawing 1 time
It saves backup.
With seedling, packing and freeze-drying: antibiotic is added by 1% volume ratio in the virus liquid of harvest, then matches with embodiment 6
The heat-resisting protective agent prescription 1 made 1:1 quantitative separating after mixing by volume.Chilled vacuum drying rear pressing cover labeling,
The Duck plague live vaccine of preparation sets 2 ~ 8 DEG C of preservations.
3 batches of (lot number 201701,201702,201703) Duck plague live vaccines, separately sampled progress are prepared according to the above method
Product inspection.
1, character inspection visually observes 3 batches of vaccines of observation, and appearance is Sponge Porosity agglomerate, is easily detached from, adds with bottle wall
It is dissolved rapidly after dilution.
2, steriling test, mycoplasma are examined, residual moisture measurement and vacuum degree measure
It tests by existing " Chinese veterinary pharmacopoeia " annex, 201701,201702,201703 batches former without bacterium, mould and branch
Body pollution, residual moisture and vacuum degree measurement are qualified.Concrete outcome is shown in Table 8.
The inspection result of 83 batches of products of table
3, exogenous virus is examined
3.1 PCR methods detect duck tembusu virus, duck hepatitis I type virus and III type virus of duck hepatitis referring to embodiment 4
PCR method, 3 batches of Lab Products are through PCR detection duck tembusu virus, duck hepatitis I type virus and III type of duck hepatitis as the result is shown
Viral antigen is feminine gender.
3.2 chicken inspection techniques (are not less than since the duck plague positive serum of preparation is unable to 10 plumage part of complete neutralization
104.5TCID50) vaccine, therefore use chicken method of inspection carry out:
Vaccine is diluted to 1 plumage part/ml with sterile saline, 9 week old SPF chicken 20 of every batch of vaccine intramuscular injection, and 1 plumage part/only,
After 14 days, every eye droppings, collunarium be inoculated with 0.1ml(10 plumage part containing vaccine), intramuscular injection 1ml(100 plumage part containing vaccine).Observation 21
In the future, according to the above method with dosage repeated inoculation 1 time.It takes a blood sample within 21 days after 2nd inoculation, separates serum, carry out related cause of disease
Serum antibody test.In 56 days, there should not be locally or systemically symptom or respiratory symptom or death caused by vaccine.If there is
Dead chicken should carry out pathological examination, to prove whether caused by duck plague virus.
Newcastle disease virus, adenovirus (having coagulation), H5 hypotype fowl is measured to the isolated serum HI method of inspection to flow
Influenza Virus, H9 subtype avian influenza virus antibody；Avian infectious bronchitis virus, chicken infectivity throat gas are measured with ELISA method
Pipe inflammation virus, avian leukosis virus, fowl Reticuloendotheliosis Virus, chicken infectivity bursa of Fabricius virus, Avianreovirus
With the antibody of avian encephalomyclitis virus；The antibody test of chicken Marek's disease virus is carried out with AGP method.
After 3 batches of vaccines (201701,201702,201703) are inoculated with chick twice as the result is shown, take a blood sample within 56 days, carry out serum
Antibody test, antibody test are feminine gender.In 56 days, 201701,201702,201703 batches of vaccines do not cause part or complete
Body symptom or respiratory symptom or death, concrete outcome are shown in Table 9.
9 exogenous virus detection method of table and result
Note: "-" indicates negative.
4, diagnostic test
3 batches of vaccines (201701,201702,201703) are diluted to 100TCID with serum-free M199 respectively50/ 0.1ml, with anti-duck
Pestivirus specific positive serum mixed in equal amounts, 37 DEG C neutralize 1 hour, and inoculation 6 has grown up to cell hole (48 holes of CEF single layer
Cell plates), every hole 0.2ml, while setting virus control and each 6 hole of normal cell controls.Set 37 DEG C, containing 5% CO2Incubator culture
And it observes 120-144 hours.As a result neutralization group and cell controls group do not neutralize virus control group and occur typically without CPE
CPE variation.
5, safety verification
Plumage part is indicated by label, vaccine is diluted to 10 plumage parts/ml with sterile saline, 2 monthly ages of the inoculation susceptible duck of health, 10
Only/group, intramuscular injection, 1ml/ only, separately take the identical susceptible duck of 10 conditions, and every intramuscular injection 1ml sterile saline is made
For control, isolated rearing.It is observed 14 after inoculation, records the health condition of duck, and cutd open on 14th and kill observation pathological change.Knot
Fruit shows that 3 batches of Lab Products are inoculated with the 2 monthly ages susceptible duck of health within the observation period on the 14th, and the state of mind, diet and excrement are just
Often, all ducks are strong lives.Dissect on the 14th observes each histoorgan and is visible by naked eyes lesion.It the results are shown in Table 10.
The safety examination result of 10 3 batches of Lab Products of table
6, efficacy test
The measurement of 6.1 viral levels indicates plumage part by label, and vaccine is diluted to every 0.1ml containing 1 plumage part with serum-free M199, then
Continue 10 times of work to be serially diluted, takes 10-2、10-3、10-4、10-5 4 acceptable diluent degree, are inoculated with have grown up to good single layer respectively
96 hole Microtitration plates of CEF cell, each dilution is inoculated with 6 holes, every hole 0.1ml, while setting 6 hole of normal cell controls.
Set 37 DEG C, containing 5% CO2After adsorbing 1 hour in incubator, the M199 maintaining liquid 0.1ml containing 4% serum is added in every hole, is cultivated and is seen
It examines 120~144 hours.According to CPE production, TCID is calculated by Reed-Muench method50。
6.2 take susceptible duck 10 of 2 monthly ages health with duck inspection, indicate plumage part by label, every intramuscular injection is inoculated with epidemic disease
1/50 plumage part of seedling, separately set 10 it is not immune compare, the isolated rearing under similarity condition.After 14 days, every duck intramuscular injection inspection
It tests with virulent (CVCC AV1221) 1.0ml(containing 1000 MLD), is observed 14 after attacking poison, the morbidity death knot of record test duck
Fruit.
The 6.3 vaccine every batch of of anti-aging test 3 batches extract 4 bottles every time, set after placing 7,10,30 days in 37 DEG C of incubators
The measurement of viral level is carried out by 6.1.
Efficacy test result: the viral level of the every plumage part of 3 batches of Lab Products is respectively 104.0TCID50、104.0TCID50、
104.25TCID50；After 3 batches of laboratory vaccine samples are placed 7,10 in 37 DEG C of incubators, the viral level of the every plumage part of vaccine is equal
103.5TCID50More than；After placing 30 in 37 DEG C of incubators, the viral level of the every plumage part of vaccine is 103.0TCID50With
On；It is immunized after 2 monthly ages susceptible duck with 1/50 plumage part and attacks within 14th poison, non-immunized controls duck is all dead, and 3 immune group ducks are all strong
Living, protective rate is 100%.As a result see Table 1 for details 1, table 12.
The viral level of 11 3 batches of Lab Products of table measures and the poison of attacking of immune duck protects result
Viral level after 12 3 batches, table, 37 DEG C of Lab Products are ageing-resistant measures (TCID50/ plumage part)
The Duck plague live vaccine of the invention of embodiment 9 and existing similar product safety and effect comparative test
Material: with DF-1 cell produce Duck plague live vaccine (F80 plant), lot number 201701, control vaccine for commercially available chicken embryo at
The Duck plague live vaccine (AV1222 plants) of fibrocyte (CEF) preparation, lot number 2017001.
Safety examination
By 201701 batches of Duck plague live vaccines (F80 plants) and 2017001 batches of Duck plague live vaccines (AV1222 plants), respectively intramuscular injection 60
The susceptible duck of ~ 70 ages in days 10, every 1ml(contain 10 plumage parts), while setting control duck 10.Isolated rearing is observed 14, is as a result seen
Table 13.
The safety comparison result of 13 201701 batches and 2017001 batches Duck plague live vaccines of table
Efficacy test: by 201701 batches of Duck plague live vaccines (F80 plants) and 2017001 batches of Duck plague live vaccines (AV1222 plants), respectively
The susceptible duck of 60 ~ 70 age in days of intramuscular injection 10, every 1ml(contain 1/50 plumage part), while setting injection dilution and compareing duck 10.Every
From raising, after being immunized 14, poison, intramuscular injection are attacked with virulent AV1221 plants of duck plague, 1ml/ only (contains 1000 MLD).It is seen after attacking poison
It examines 14, the symptoms such as spiritual depressed, anorexia of as a result non-immunized controls duck appearance in the 4th day after attacking poison, and starts death occur, until
7th day all dead.2 immune group ducks are all strong to live, and protective rate is 100%, and see Table 1 for details 4.
14 201701 batches and 2017001 batches Duck plague live vaccine immune efficacy comparative test results of table
9 heat resisting protective of embodiment is preparing the application in other vaccines
1 heat resisting protective of formula of embodiment 6 is applied respectively in Bursal Disease (K85 plants), newcastle disease (La
Sota plants), in pseudoabies (Bartha-K61 plants) and high-pathogenicity porcine reproductive and respiration syndrome (GDr180 plants), through 37 DEG C
Heat-resisting 7d, 10d, 30d, test result are shown in Table 15.
Application effect of the 15 heat-resisting protective agent prescription 1 of table on different vaccines
Above embodiment is only that preferred embodiments of the present invention will be described, is not limited the scope of the present invention
Fixed, without departing from the spirit of the design of the present invention, this field ordinary engineering and technical personnel does technical solution of the present invention
All variations and modifications out, should fall within the scope of protection determined by the claims of the present invention.
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Claims (10)
1. a kind of heat resisting protective, which is characterized in that the component including following proportion: gelatin 1% ~ 4%, sucrose 5% ~ 15% or seaweed
Sugar 1% ~ 5%, sorbierite 1% ~ 4% or mannitol 1% ~ 3%, tryptone 1% ~ 4% or defatted milk 2% ~ 6%, L-sodium 0.005% ~
0.1% or arginine 0.5% ~ 3%, solvent be PBS solution；The % is the mass body of each component quality and heat resisting protective total volume
Product ratio.
2. heat resisting protective according to claim 1, which is characterized in that the component including following proportion: gelatin 1.5% ~
4%, sucrose 8% ~ 12% or trehalose 2% ~ 5%, sorbierite 2% ~ 4% or mannitol 1.5% ~ 3%, tryptone 1.5% ~ 4% or defatted milk
2% ~ 6%, L-sodium 0.005% ~ 0.05% or arginine 1% ~ 3%, solvent are the PBS solution of 0.01-0.05M.
3. heat resisting protective according to claim 1, which is characterized in that the component including following proportion: gelatin 2% ~ 4%,
Sucrose 9% ~ 12% or trehalose 2.5% ~ 5%, sorbierite 2.5% ~ 4% or mannitol 2% ~ 3%, tryptone 2% ~ 4% or defatted milk 2% ~
5%, L-sodium 0.008% ~ 0.03% or arginine 1.5% ~ 3%, solvent are the PBS solution of 0.01-0.02M.
4. heat resisting protective according to claim 1, which is characterized in that the component including following proportion: gelatin 2%, sucrose
10%, sorbierite 4%, tryptone 3%, L-sodium 0.01%；
Or, gelatin 1%, sucrose 15%, sorbierite 4%, tryptone 4%, L-sodium 0.005%；
Or, gelatin 4%, sucrose 5%, sorbierite 2%, tryptone 1%, L-sodium 0.1%；
Or, gelatin 2%, trehalose 5%, sorbierite 3%, defatted milk 5%, arginine 1.5%；
Or, gelatin 2%, sucrose 10%, mannitol 3%, defatted milk 2%, L-sodium 0.1%；
Or, gelatin 4%, trehalose 1%, mannitol 2%, defatted milk 4%, arginine 3%；
The above proportion solvent is the PBS solution of 0.01M.
5. the preparation method of any heat resisting protective of claim 1-4, which comprises the following steps:
(1) A liquid is prepared
Gelatin, sucrose or the trehalose of filtration sterilization and tryptone or defatted milk are weighed by formula, is added in PBS solution to complete
Fully dissolved, sterilizing；
(2) B liquid is prepared
Sorbierite or mannitol and L-sodium or arginine are weighed by formula, is added in PBS solution to being completely dissolved, removes
Bacterium；
(3) by A liquid and B liquid, 1:1 is mixed by volume, and heat resisting protective is made.
6. any heat resisting protective of claim 1-4 is extending vaccine storage life or is improving answering in vaccine heat resistance
With.
7. a kind of biological products, which is characterized in that contain any heat resisting protective of claim 1-4.
8. the Duck plague live vaccine containing any heat resisting protective of claim 1-4.
9. Duck plague live vaccine as claimed in claim 8, which is characterized in that the duck plague virus of the Duck plague live vaccine is that CEF is thin
The duck plague virus of born of the same parents' culture or the duck plague virus of DF-1 cell culture.
10. Duck plague live vaccine as claimed in claim 9, which is characterized in that the Duck plague live vaccine is prepared by following steps
It obtains:
(1) overdose duck plague virus liquid is inoculated with DF-1 cell；
(2) it connects 5-10 days cell liquid of the harvest containing virus after poison, is inoculated with DF-1 cell again according to step (1) after multigelation,
Such blind passage mostly generation, to harvesting virus liquid when being inoculated with after duck plague virus in 120 hours CPE up to 75% or more；
(3) it is inoculated in DF-1 cell according to the volume ratio with DF-1 cell liquid 0.5%-5% after the virus liquid freeze thawing harvested, cultivated,
Duck plague virus liquid is harvested, viral level is not less than 10 in duck plague virus liquid7.0TCID50/ml；
(4) heat resisting protective is added in virus liquid, is lyophilized and duck plague virus live vaccine is made.
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| CN111518775B (en) * | 2020-06-18 | 2023-09-22 | 合肥铼科生物科技有限公司 | Preservation solution for preserving virus samples at normal temperature for long time and application thereof |
| CN113699120A (en) * | 2021-07-20 | 2021-11-26 | 罗益（无锡）生物制药有限公司 | Heat-resistant protective agent, application thereof and preservation method of live viruses capable of being preserved at room temperature |
| CN113730592A (en) * | 2021-09-03 | 2021-12-03 | 吉林正业生物制品股份有限公司 | Duck tembusu virus live vaccine freeze-drying protective agent, preparation method and application |
| CN113730592B (en) * | 2021-09-03 | 2023-12-19 | 吉林正业生物制品股份有限公司 | Duck tembusu virus live vaccine freeze-drying protective agent and preparation method and application thereof |
| CN116763914A (en) * | 2023-02-06 | 2023-09-19 | 广东永顺生物制药股份有限公司 | Swine fever and highly pathogenic porcine reproductive and respiratory syndrome bivalent heat-resistant protective agent live vaccine and preparation method thereof |
| CN116763914B (en) * | 2023-02-06 | 2024-02-23 | 广东永顺生物制药股份有限公司 | Swine fever and highly pathogenic porcine reproductive and respiratory syndrome bivalent heat-resistant protective agent live vaccine and preparation method thereof |
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