CN106822886A - The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 - Google Patents
The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 Download PDFInfo
- Publication number
- CN106822886A CN106822886A CN201710134514.XA CN201710134514A CN106822886A CN 106822886 A CN106822886 A CN 106822886A CN 201710134514 A CN201710134514 A CN 201710134514A CN 106822886 A CN106822886 A CN 106822886A
- Authority
- CN
- China
- Prior art keywords
- aviadenovirus
- type
- virus
- cloacal bursa
- seedling
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- 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
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10251—Methods of production or purification of viral material
-
- 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
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/10011—Birnaviridae
- C12N2720/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/10011—Birnaviridae
- C12N2720/10051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Mycology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of use LMH cell lines production infections chicken cloacal bursa and the method for the type bivalent inactivated vaccine of aviadenovirus 4, it is comprised the following steps：Respectively by infections chicken cloacal bursa and the type virus inoculation of aviadenovirus 4 in LMH cells, infections chicken cloacal bursa seedling virus liquid and the type seedling virus liquid of aviadenovirus 4 are obtained through steps such as culture, freeze thawing, centrifugation, concentrations, then after the seedling virus liquid of two kinds of viruses is inactivated, isometric mixing, emulsification is made emulsion-type inactivated vaccine.The bigeminy vaccine potency that the present invention is provided is high, has complete immanoprotection action to infections chicken cloacal bursa virus and the type of aviadenovirus 4 virus, and security is good, and stabilization is effective, and the protection period is long.
Description
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to one kind produces avian infectious method with LMH cell lines
The method of family name's capsule and the type bivalent inactivated vaccine of aviadenovirus 4.
Background technology
Bursal Disease is that a kind of chick that arc reovirus virus cause is acute, contagious disease.Due to the disease
Morbidity is suddenly, the course of disease is short, the death rate is high, and can cause chicken body immunosupress, be still at present poultry husbandry Infectious Diseases it
One.The immune of bursal disease virus has live vaccine and the class vaccine of inactivated vaccine two currently on the market.The weak immune effect of live vaccine virulence
Really poor, virulence is stronger to be had injury on the bursa of farbricius and influences immune effect；Inactivated vaccine remains complete virus structure not
Security performance is substantially increased while influence immune effect.
Fowl I groups of adenovirus are the common pathogen infections of poultry, in worldwide distribution, have now been found that the poultry of institute's has age
It is susceptible, simply in view of the pathogenicity that is formed of the kind of poultry, age is different.Have now been found that fowl I groups of adenovirus have to chicken
Strong pathogenic, major lesions show as inclusion body hepatitis and hydropericardium-hepatitis syndrome, and the disease can directly trigger chicken farm 20
There is more than 30% death rate in~40 day age chicken, in addition its clinically also usually with IBV, exhale intestines
The accompanying infections such as lonely virus, H9 subtype avian influenza virus, cause kind of laying hen an obvious respiratory diseases occur, arthritis, and serious
The problems such as egg drop reduction and lopsided egg.Therefore at present the disease have become have a strong impact on chicken farm production performance great epidemic disease it
One.
The type of aviadenovirus 4 (FAV-4) in fowl I groups of adenovirus is currently study hotspot in the world, and it possesses high causing a disease
Property, the hydropericardium-hepatitis syndrome (or Ankara disease) that can directly trigger, the disease is starting in Pakistan in 1987,
Less than broiler breeding enterprise of whole Pakistan is brought disaster in year, afterwards in Kuwait, Iran, the former Soviet Union, Japan, in
The ground such as South America and Mexico has and occurs, or even the disease is in pigeon also once large area outburst.At present it has been proved that fowl I groups of glands
Virus, effective prevention and control can be carried out especially for highly pathogenic FAV-4 types by inactivated vaccine, and wherein Pakistan takes sense
The formalin-killed vaccine prevention and control hydropericardium-hepatitis syndrome for contaminating the liver homogenate preparation of chicken obtains significantly success.
Prior art is to thin using CEF (CEF), chicken embryo kidney more than the fowl I groups of preparations of adenovirus inactivated vaccine
Born of the same parents (CEK), chick kidney cells (CK), 4 kinds of primary cells of Embryo liver cell (CEL) are separated and virus of proliferation.But primary liver kidney is thin
Born of the same parents prepare cumbersome, easy pollution, are difficult transfection, and are easily mixed into CEF in cell processes are prepared, and are unfavorable for experimental implementation.
Chicken gizzard cancerous cell line (LMH cell lines) was set up by a Japanese scholars in 1981.By using diethyl
Base nitrosamine is induced male Leghorn for a long time, causes chicken body to produce liver cancer, then separate cell from liver cancer tissue and obtain
Arrive.LMH is in typical characteristics of epithelial cells, and the cell line maintains a large amount of phenotypic characteristics of chicken liver cell differentiation, is mainly used in
The structure of recombined adhenovirus." chicken gizzard cancerous cell line is used as duck plague disease for the Chinese patent application of the A of Publication No. CN 105420198
The application of malicious host " is using chicken gizzard cancerous cell line as duck plague virus host, it was demonstrated that chicken gizzard cancerous cell line is applied to Virus culture.
Currently without the relevant report using the LMH cell lines production bursa of farbricius and the type bivalent inactivated vaccine of aviadenovirus 4.
The content of the invention
To solve technical problem present in prior art, given birth to LMH cell lines it is an object of the invention to provide one kind
The method for producing infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4.
The technical scheme that the present invention is provided is as follows：
The present invention provides a kind of LMH cell lines and produces infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4
Method, it is comprised the following steps：
(1) preparation of continuous cell line LMH cells：LMH cells are carried out into digestion dispersion with pancreas enzyme -EDTA digestive juice to pass
In generation, DMEM nutrient solutions are added in 37 DEG C, 5%CO2Cultivated in incubator, until form well-grown cell monolayer, then with nothing
Serum DMEM nutrient solutions cleaning cell 3 times, for infections chicken cloacal bursa virus and the type virus inoculation of aviadenovirus 4；
(2) poison breeding is planted：It is respectively 1 by final volume by infections chicken cloacal bursa virus and the type of aviadenovirus 4 virus seed culture of viruses:
50~1:500 be inoculated into step (1) preparation cell monolayers, and 37 DEG C absorption 30~60 minutes after inhale abandon virus liquid, add
Cell maintenance medium is in 37 DEG C, 5%CO2Individually cultivate 58 ± 2 hours, until cytopathy CPE harvests chicken respectively up to more than 80%
Infectious bursa of Fabricius cytopathy venom and the type cytopathy venom of aviadenovirus 4；
(3) collection virus, concentration and purifying：The infections chicken cloacal bursa cytopathy venom and aviadenovirus that will be harvested respectively
4 type cytopathy venom are placed in -20 DEG C of multigelations twice, 5000rpm, and supernatant is collected after 4 DEG C of centrifugation 10min, then distinguish
Concentrated using 50K hollow fiber columns, as infections chicken cloacal bursa seedling virus liquid and the type seedling of aviadenovirus 4 virus
Liquid；
(4) after the seedling virus liquid of above two virus is inactivated, isometric mixing, emulsification is made emulsion-type inactivation
Vaccine.
Further, in the DMEM nutrient solutions in above-mentioned steps (1) containing 5~10% (v/v) NBCSs, 1.0~
1.2% (v/v) be dual anti-and 0.5~2.5% (m/v) HEPES, it is described it is dual anti-be the mould containing 100IU/mL
The Streptomycin Solution of element and 10mg/mL.
Further, pancreas enzyme -EDTA digestive juice is the pancreatin containing 0.025% (m/v), 0.02% in above-mentioned steps (1)
(m/v) Hank ' the s solution of EDTA.
Further, the cell maintenance medium of above-mentioned steps (2) is to include 0.5~1.5% (v/v) NBCS, 0.2
~0.5% (m/v) glutamine, 1.0~1.2% (v/v) are dual anti-, 1~3% (v/v) liposome complex and 1~2% (m/v)
The DMEM nutrient solutions of HEPES, it is described it is dual anti-be penicillin containing 100IU/mL and 10mg/mL streptomysin
Solution.
Further, the cell maintenance medium of the step (2) also includes 0.05~0.2% (m/v) lipoic acid.
Wherein, above-mentioned liposome complex is the internal compound comprising catalase, described lipid bluk recombination
The preparation of thing is comprised the following steps：
(1) 0.2g phosphatid ylcholines, 0.08g cholesterol, 0.04g DSPEs are weighed, is mixed, be dissolved in
50mL ethanol, ultrasonically treated 5min；
(2) and then at 28 DEG C, rotary evaporation under conditions of 0.1MPa removes ethanol, stands 30min, adds 100mL pH
It is 7.4 PBS, hydration reaction about 1h is vibrated in 35 DEG C of water-baths, obtains liposome suspension；
(3) particle diameter of liposome suspension is adjusted to 150~200nm using syringe filter, obtain inside and include
The liposome of catalase.
Further, in above-mentioned steps (2) pH be 7.4 PBS in containing 0.2~0.6% (m/v) peroxide
Change hydrogen enzyme.
Further, the seedling virus liquid of (4) two kinds of viruses of above-mentioned steps needs to carry out viral level survey before being inactivated
It is fixed, specially：Seedling virus liquid is done into 10 times with DMEM nutrient solutions to be serially diluted, 10 are taken-5、10-6、10-7、10-8、10-95
Dilution factor is inoculated with 48 hole confluent monolayers LMH Tissue Culture Plates, and each dilution factor repeats 5 holes, while setting up negative control cell hole；
Per hole 0.1mL, after 37 DEG C of absorption 30min, cell maintenance medium 0.3mL is added in 37 DEG C, 5%CO2Culture 120 hours, observation is thin
Born of the same parents lesion CPE, calculates TCID50。
The present invention is used for the seedling virus liquid of two kinds of viruses for preparing the bursa of farbricius and the type bivalent inactivated vaccine of aviadenovirus 4
Titre must is fulfilled for following condition, specially：After two kinds of seedling virus liquids of virus mix in equal volume in step (4), make 0.1ml
Infections chicken cloacal bursa content >=10 in water phase8.0TCID50, type viral level >=10 of aviadenovirus 48.2TCID50。
Further, the inactivation of virus uses Formalin inactivation in the step (4), and the formalin is in seedling disease
Final concentration of the 0.1% of venom.
Further, vaccine antigen emulsification is made emulsion-type inactivated vaccine and is specially by the step (4)：
(1) prepared by oil phase：94 parts of high-quality injection white oil is taken, 2 parts of aluminum stearate, 4 parts of Arlacel-80 is first slow by white oil
Heating, add Arlacel-80 and aluminum stearate, heated when stirring, until aluminum stearate be fully dissolved to it is transparent untill, autoclaving
It is standby；
(2) water is mutually prepared：The infections chicken cloacal bursa antigen and each 48 parts of the type of aviadenovirus 4 after inactivation are taken, sterilizing is added
4 parts of Tween-80 afterwards, starts stirring, untill being completely dissolved Tween-80, is made water phase；
(3) emulsify：Water is added in oil phase, is emulsified using IKA emulsifying agents, 16000rpm, emulsification 5 minutes is
Can；
(4) dispense：The vaccine of preparation is dispensed according to every bottle of 250mL.
The inventors discovered that carrying out infections chicken cloacal bursa, the type seed culture of viruses of aviadenovirus 4 respectively in LMH cell monolayers connects poison
Afterwards, add conventional cell maintenance medium, LMH cells to occur death quickly, cause final obtained viral vaccine potency relatively low.This
Inventor is improved by substantial amounts of experiment and screening to the formula of cell maintenance medium, is found in low content NBCS
A certain amount of catalase liposome complex and lipoic acid are added in DMEM nutrient solutions, the life of LMH cells can be significantly improved
Long status, it is to avoid death occur too early in LMH cells, so as to obtain the seedling virus liquid of virus titer higher.It is thin after connecing poison
The content that born of the same parents cultivate virus in the bursal disease venom and the type virus liquid of aviadenovirus 4 for obtaining for 58 ± 2 hours can be stably reached
107.0-107.5/ 0.1mL, further it is concentrated after, virus content up to 108.0TCID50More than, achieve unexpected
Technique effect.
Additionally, the LMH cellular contexts for using understand, and without exogenous pathogen, easy proliferative, being applicable very much bioreactor is carried out
Large-scale culture, meets future vaccines Production trend, and cytoactive is high, and haptens is highly clarified, and easily carries out at concentration
Reason, is especially suitable for carrying out the high-quality inactivated vaccine production of antigen titre high.
Compared with prior art, advantage of the invention is that：
(1) infections chicken cloacal bursa virus and the type virus multiplication of aviadenovirus 4 are carried out respectively using LMH passage cells, profit
Both TCID are determined with Reed-Muench methods50, can stably reach 107.0-107.5/ 0.1mL, virus titer is conventional
More than 10 times of chick embryo method and primary cell method, such as carry out virus multiplication using bioreactor, and its virus titer when the time comes can be with
100 times of levels are reached, and the antigen for obtaining highly is clarified, and easily carries out concentration, is appropriate for the excellent of antigen titre high
Matter inactivated vaccine is produced.
(2) antibody is produced soon after the immune animal of the bivalent inactivated vaccine that the present invention is provided, and potency is high, reduces the hair of epidemic disease
Give birth to and spread, there is complete immanoprotection action, and security to infections chicken cloacal bursa virus and the type of aviadenovirus 4 virus
Well, there is not any adverse reaction, stabilization is effective, and the protection period is long.
(3) formula by optimizing cell culture fluid of the invention, creatively adds catalase liposome complex
And lipoic acid, make to carry out infections chicken cloacal bursa virus respectively and the type virus of aviadenovirus 4 connects the LMH cells after poison in low content
Remain to keep good growth conditions in the DMEM nutrient solutions of NBCS, it is to avoid death occurs too early in cell, so as to improve
The potency of the malicious vaccine of system, and in shorter incubation time 58 ± 2 hours, you can titre virus liquid higher is harvested, is greatlyd save
Production cost, improve production efficiency.
Specific embodiment
The present invention is further described below by way of specific embodiment, but the present invention is not limited only to following examples.
The preparation of the liposome complex of embodiment 1
(1) 0.2g phosphatid ylcholines, 0.08g cholesterol, 0.04g DSPEs are weighed, is mixed, be dissolved in
50mL ethanol, ultrasonically treated 5min；
(2) and then at 28 DEG C, rotary evaporation under conditions of 0.1MPa removes ethanol, stands 30min, adds 100mL pH
It is 7.4 PBSs containing 0.5% (m/v) catalase, hydration reaction about 1h is vibrated in 35 DEG C of water-baths, obtains fat
Plastid suspension；
(3) particle diameter of liposome suspension is adjusted to 150nm using syringe filter, obtain inside and include peroxidating
The liposome of hydrogen enzyme.
The preparation of the infections chicken cloacal bursa of embodiment 2 and the type seedling virus liquid of aviadenovirus 4 and viral level are determined
(1) preparation of seedling virus liquid：
1. the preparation of continuous cell line LMH cells：By the pancreatin -0.02% that LMH cell mass volume ratios are 0.025%
EDTA digestion dispersion passages, add containing 10% (v/v) NBCS, 1.0% (v/v) be dual anti-and 2.0% (m/v) ethoxy
The DMEM nutrient solutions of piperazine ethanesulfonic acid, in 37 DEG C, 5%CO2Cultivated in incubator, until well-grown cell monolayer is formed,
Cell is cleaned with serum-free DMEM nutrient solutions 3 times, be inoculated with for infections chicken cloacal bursa virus and the type of aviadenovirus 4 again.
2. poison breeding is planted：Infections chicken cloacal bursa seed culture of viruses is pressed into final volume 1 respectively:100 be inoculated into that 1. step prepared it is thin
Born of the same parents, final volume 1 is pressed by the type seed culture of viruses of aviadenovirus 4:200 are inoculated into the cell that 1. step is prepared, and after 37 DEG C of absorption 35 minutes
Virus liquid is abandoned in suction, add containing 1.0% (v/v) NBCS, 0.4% (m/v) glutamine, 1.0% (v/v) it is dual anti-,
The DMEM cultures of 2.5% (v/v) liposome complex, 0.15% (m/v) lipoic acid and 2% (m/v) HEPES
Liquid, in 37 DEG C, 5%CO2Individually cultivate 60 hours, until cytopathy CPE harvests avian infectious Fa Shi respectively up to more than 80%
Bladder cell virus liquid and the type cytopathy venom of aviadenovirus 4.
3. collection virus, concentration and purifying：The infections chicken cloacal bursa cytopathy venom and aviadenovirus 4 that will be harvested respectively
Type cytopathy venom is placed in -20 DEG C of multigelations twice, 5000rpm, and supernatant is collected after 4 DEG C of centrifugation 10min, then makes respectively
Concentrated with 50K hollow fiber columns, as infections chicken cloacal bursa seedling virus liquid and the type seedling virus liquid of aviadenovirus 4.
(2) viral level is determined
Infections chicken cloacal bursa seedling virus liquid and the type seedling virus liquid of aviadenovirus 4 are done 10 with DMEM nutrient solutions respectively
It is serially diluted again, takes 10-5、10-6、10-7、10-8、10-95 dilution factors are inoculated with 48 hole confluent monolayers LMH Tissue Culture Plates, often
Individual dilution factor repeats 5 holes, while setting up negative control cell hole；Per hole 0.1mL, after 37 DEG C of absorption 30min, cell maintenance is added
Liquid 0.3mL is in 37 DEG C, 5%CO2Culture 120 hours, observation of cell lesion CPE calculates TCID50。
The inactivation and inactivation inspection of the infections chicken cloacal bursa of embodiment 3 and the type seedling virus liquid of aviadenovirus 4
(1) inactivation of the type of infections chicken cloacal bursa/aviadenovirus 4：Virus liquid is imported in inactivation tank, formalin is added
Solution, is sufficiently mixed, and the ultimate density of formalin solution is 0.1%, and 37 DEG C of inactivations (reach 37 DEG C in 16 hours with temperature in tank
Start timing) take out afterwards, 6 DEG C of preservations are put, should be no more than 1 month.
(2) the type inactivation of virus of infections chicken cloacal bursa/aviadenovirus 4 inspection：The sample of inactivation inspection is taken, DMEM nutrition is used
Liquid is with 10-1、10-2、10-3Three dilution proportion measuring samples, while the inactivation provirus sample for setting up same dilution ratio is used for
Positive control, is inoculated in 48 hole LMH cell monolayer cells, every group of hole of repeated inoculation 5, per 37 DEG C of hole 0.4mL, 5%CO respectively2
Culture 96 hours.Do not occur cytopathy in inactivated samples each group dilution factor hole, it is thin in each dilution factor hole of positive controls
Born of the same parents' lesion is obvious, up to more than 80%, is judged to that inactivation inspection is qualified.
The preparation of the bivalent inactivated vaccine of embodiment 4
The present invention can be used for prepare bivalent inactivated vaccine condition be：Per 0.1mL infections chicken cloacal bursa virus content >=
108.0TCID50, per type viral level >=10 of 0.1mL aviadenovirus 48.2TCID50Can be used to prepare vaccine, the bigeminy inactivates epidemic disease
The preparation of seedling is specially：
(1) prepared by oil phase：94 parts of high-quality injection white oil is taken, 2 parts of aluminum stearate, 4 parts of Arlacel-80 is first slow by white oil
Heating, add Arlacel-80 and aluminum stearate, heated when stirring, until aluminum stearate be fully dissolved to it is transparent untill, autoclaving
It is standby；
(2) water is mutually prepared：Infections chicken cloacal bursa antigen and the type of aviadenovirus 4 after the obtained inactivation of Example 2 is each
48 parts, 4 parts of Tween-80 after sterilizing is added, start stirring, untill being completely dissolved Tween-80, be made water phase；
(3) emulsify：Water is added in oil phase, is emulsified using IKA emulsifying agents, 16000rpm, emulsification 5 minutes is
Can；
(4) dispense：The vaccine of preparation is dispensed according to every bottle of 250mL.
The preparation of the infections chicken cloacal bursa of comparative example 1,2,3 and the type seedling virus liquid of aviadenovirus 4 and viral level are determined
The preparation process of the infections chicken cloacal bursa of comparative example 1 and the type seedling virus liquid of aviadenovirus 4 and the basic phase of embodiment 2
Together, difference is that described step (2) is planted in poison breeding, and after LMH cells access seed culture of viruses, the cell maintenance medium of addition contained
Hydrogen oxide enzyme, rather than catalase liposome, will catalase be added directly into cell maintenance medium.
The preparation process of the infections chicken cloacal bursa of comparative example 2 and the type seedling virus liquid of aviadenovirus 4 and the basic phase of embodiment 2
Together, difference is that described step (2) is planted in poison breeding, and after LMH cells access seed culture of viruses, the cell maintenance medium of addition was free of
Hydrogen oxide enzyme liposome.
The preparation process of the infections chicken cloacal bursa of comparative example 3 and the type seedling virus liquid of aviadenovirus 4 and the basic phase of embodiment 2
Together, difference is, described step (2) is planted in poison breeding, after LMH cells access seed culture of viruses, the cell maintenance medium of addition not sulfur-bearing
Octanoic acid.
And the method observed as described in comparative example 1-3 prepares infections chicken cloacal bursa and the type seedling virus liquid of aviadenovirus 4,
The virus liquid TCID that different time is harvested50, and prepare infections chicken cloacal bursa and the type system of aviadenovirus 4 with the method for embodiment 2
Seedling diseases venom compares, and see the table below 1 and 2.
The infections chicken cloacal bursa virus liquid TCID that the different time of table 1 is harvested50
The type virus liquid TCID of aviadenovirus 4 that the different time of table 2 is harvested50
From comparative example 1, catalase is added directly into cell maintenance medium, LMH cells are within a short period of time
There is lesion, after culture 36h, infections chicken cloacal bursa CPE ratios reach and reach after 60%, 48h 90%, and the type of aviadenovirus 4
CPE ratios reach 95% after reaching 65%, 48h, and both viral levels are obtained containing for virus in virus liquid far below embodiment 2
Amount.From comparative example 2, catalase liposome is free of in cell maintenance medium, disease occur within the shortest time in LMH cells
Become, after culture 36h, infections chicken cloacal bursa and the type CPE of aviadenovirus 4 reach 85%, and both harvest the viral of virus liquid
Content is minimum.From comparative example 3, there is the time of lesion and comparative example 1 in not lipoic acid in cell maintenance medium, LMH cells
Quite, after culture 36h, infections chicken cloacal bursa and the type CPE of aviadenovirus 4 reach 60%, 48h and reach 85%, both
The content of virus in virus liquid is obtained far below embodiment 2 for viral level.Result above shows to be added in cell culture fluid
Hydrogen oxide enzyme liposome and lipoic acid can significantly improve the growth conditions after LMH cells connect poison, delay it cytopathy occur, carry
The potency of viral vaccine high.
The bivalent inactivated vaccine product inspection of embodiment 5
(1) proterties
1. outward appearance：It is milky emulsion.
2. formulation：Water-in-oil type.A cleaning suction pipe is taken, is drawn in a small amount of vaccine instillation cold water, in addition to first drips, do not expanded
Dissipate.
3. stability：Draw in 10 milliliters of addition centrifuge tubes of vaccine, be centrifuged 15 minutes with 3000rpm, the water that ttom of pipe is separated out
Accordingly≤0.5mL.
4. viscosity：With the 1.0mL suction pipes that exit inside diameter is 1.2mm, 25 DEG C or so 1.0mL are drawn, make its vertical natural stream
Go out, the time needed for record outflow 0.4mL, should be no more than 8 seconds.
(2) steriling test：Take finished product inoculation sulphur glycollate culture medium tubule and each two of peptone from casein agar, every 0.2mL,
One is put 37 DEG C of cultures, and one is put 25 DEG C of cultures, is observed 3~5, should be pure, asepsis growth.
(3) safety verification：With 7 age in days SPF chickens 10, every muscle or neck hypodermic injection vaccine 1mL are observed 14,
As a result test chicken is good for and is lived, without any locally and systemically adverse reaction.
(4) content of formaldehyde is determined：
1. the preparation of reference substance solution：Take the formalin demarcated appropriate, be made into every 1.0mL molten containing formaldehyde 1.0mg
Liquid, precision is measured during 5.0mL puts 50mL measuring bottles, adds water to scale, is shaken up, and is obtained final product.
2. the preparation of tested sample：Tested product 5.0mL is measured with 5.0mL measuring pipettes, is put in 50mL measuring bottles, told with 20%
- 80 ethanol solution 10mL of temperature, wash suction pipe by several times, and washing lotion is incorporated in 50mL measuring bottles, shakes up, and is diluted with water to scale, shakes strongly
Shake, static layering, if subnatant is not clarified, filtration discards just filtrate, takes clarification subsequent filtrate, obtains final product.
3. determination method：Precision draws reference substance solution and tested each 0.5mL of product solution, and acetic acid-ammonium acetate buffering is added respectively
Liquid 10mL, acetylacetone,2,4-pentanedione test solution 10mL, put 60 DEG C of waters bath with thermostatic control 15 minutes, and cold water is cooled down 5 minutes, after placing 20 minutes, by purple
Outward-visible spectrophotometer method, determines trap at the wavelength of 410nm, and calculating is obtained final product.Content of formaldehyde meets national standard,
It is qualified to check.
(5) loading quantity inspection：Test sample 3 is taken, room temperature is allowed to come to, notes avoiding loss during unlatching.With reference to loading amount inspection
Look into using reference table is measured, loading quantity inspection is carried out with the suction pipe through markization, syringe or graduated cylinder.
The Vaccine potency test of embodiment 6
Take 21 age in days SPF chickens 70, by four batches of infections chicken cloacal bursas and the type bivalent inactivated vaccine of aviadenovirus 4 respectively with
0.3mL/ chest muscle injection, 15 SPF chickens of every batch of inactivated vaccine, remaining 10 are only used as control group injecting normal saline.It is immune
7,14,21,28,35,42,49 days afterwards, taken a blood sample respectively together with control group, determine its infections chicken cloacal bursa NAT and
21 days, the 35 days and 49 days type serum NATs of aviadenovirus 4, as a result see the table below 3-4.
Different time infections chicken cloacal bursa antibody level after table 3 is immune
The type antibody level of different time aviadenovirus 4 after table 4 is immune
Result shows, using LMH cell carriers of the invention can turn out virus titer high infections chicken cloacal bursa and
The type of aviadenovirus 4；Knowable to the immuning effect test of SPF chickens, using the infections chicken cloacal bursa and fowl adenopathy of present invention production
The malicious immune SPF chickens of 4 type bivalent inactivated vaccines can not only produce high-level infections chicken cloacal bursa neutralizing antibody and can also produce
The type neutralizing antibody of high-caliber aviadenovirus 4, the bivalent inactivated vaccine effect that the present invention is provided is preferable.
The above is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred embodiment is not construed as
Limitation of the invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change
Enter and retouch and also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of method that use LMH cell lines produce infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4, its feature
It is to comprise the following steps：
(1) preparation of continuous cell line LMH cells：LMH cells are carried out into digestion dispersion passage with pancreas enzyme -EDTA digestive juice, plus
Enter DMEM nutrient solutions in 37 DEG C, 5%CO2Cultivated in incubator, until forming well-grown cell monolayer, then use serum-free
DMEM nutrient solutions cleaning cell 3 times, for infections chicken cloacal bursa virus and the type virus inoculation of aviadenovirus 4；
(2) poison breeding is planted：It is respectively 1 by final volume by infections chicken cloacal bursa virus and the type of aviadenovirus 4 virus seed culture of viruses:50~
1:500 cell monolayers for being inoculated into step (1) preparation, and inhaled after 30~60 minutes in 37 DEG C of absorption and abandon virus liquid, add cell
Maintaining liquid is in 37 DEG C, 5%CO2Individually culture 58 ± 2 hours, until cytopathy CPE is up to more than 80%, harvests chicken and infect respectively
Property bursa of farbricius cytopathy venom and the type cytopathy venom of aviadenovirus 4；
(3) collection virus, concentration and purifying：The infections chicken cloacal bursa cytopathy venom and the type of aviadenovirus 4 that will be harvested respectively
Cytopathy venom is placed in -20 DEG C of multigelations twice, 5000rpm, and supernatant is collected after 4 DEG C of centrifugation 10min, then uses respectively
50K hollow fiber columns are concentrated, as infections chicken cloacal bursa seedling virus liquid and the type seedling virus liquid of aviadenovirus 4；
(4) after the seedling virus liquid of above two virus is inactivated, isometric mixing, emulsification is made emulsion-type inactivation epidemic disease
Seedling.
2. use LMH cell lines production infections chicken cloacal bursa according to claim 1 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that in the DMEM nutrient solutions in the step (1) containing 5~10% (v/v) NBCSs, 1.0~
1.2% (v/v) be dual anti-and 0.5~2.5% (m/v) HEPES, it is described it is dual anti-be the mould containing 100IU/mL
The Streptomycin Solution of element and 10mg/mL.
3. use LMH cell lines production infections chicken cloacal bursa according to claim 1 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that pancreas enzyme -EDTA digestive juice is the pancreatin containing 0.025% (m/v) in the step (1),
Hank ' the s solution of 0.02% (m/v) EDTA.
4. use LMH cell lines production infections chicken cloacal bursa according to claim 1 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that the cell maintenance medium of the step (2) for include 0.5~1.5% (v/v) NBCS,
0.2~0.5% (m/v) glutamine, 1.0~1.2% (v/v) are dual anti-, 1~3% (v/v) liposome complex and 1~2%
(m/v) the DMEM nutrient solutions of HEPES.
5. use LMH cell lines production infections chicken cloacal bursa according to claim 4 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that described liposome complex is the internal compound comprising catalase.
6. use LMH cell lines production infections chicken cloacal bursa according to claim 5 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that described liposome complex is prepared as：
(1) 0.2g phosphatid ylcholines, 0.08g cholesterol, 0.04g DSPEs are weighed, is mixed, be dissolved in 50mL
Ethanol, ultrasonically treated 5min；
(2) and then at 28 DEG C, rotary evaporation under conditions of 0.1MPa removes ethanol, stands 30min, and it is 7.4 to add 100mL pH
PBS, in 35 DEG C of water-baths vibrate hydration reaction about 1h, obtain liposome suspension；
(3) particle diameter of liposome suspension is adjusted to 150~200nm using syringe filter, obtain inside and include peroxide
Change the liposome of hydrogen enzyme.
7. use LMH cell lines production infections chicken cloacal bursa according to claim 6 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that in the step (2) pH be 7.4 PBS in containing 0.2~0.6% (m/v) mistake
Hydrogen oxide enzyme.
8. use LMH cell lines production infections chicken cloacal bursa according to claim 4 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that the cell maintenance medium of the step (2) also includes 0.05~0.2% (m/v) lipoic acid.
9. use LMH cell lines production infections chicken cloacal bursa according to claim 1 and the type bigeminy of aviadenovirus 4 inactivate epidemic disease
The method of seedling, it is characterised in that after two kinds of seedling virus liquids of virus mix in equal volume in the step (4), make 0.1ml water phases
Middle infections chicken cloacal bursa content >=108.0TCID50, type viral level >=10 of aviadenovirus 48.2TCID50。
10. use LMH cell lines production infections chicken cloacal bursa according to claim 1 and the inactivation of the type bigeminy of aviadenovirus 4
The method of vaccine, it is characterised in that vaccine antigen emulsification is made emulsion-type inactivated vaccine and is specially by the step (4)：
(1) prepared by oil phase：94 parts of high-quality injection white oil is taken, 2 parts of aluminum stearate, 4 parts of Arlacel-80 first slowly adds white oil
Temperature, add Arlacel-80 and aluminum stearate, heated when stirring, until aluminum stearate be fully dissolved to it is transparent untill, autoclaving is standby
With；
(2) water is mutually prepared：The infections chicken cloacal bursa antigen and each 48 parts of the type of aviadenovirus 4 after inactivation are taken, after adding sterilizing
4 parts of Tween-80, starts stirring, untill being completely dissolved Tween-80, is made water phase；
(3) emulsify：Water is added in oil phase, is emulsified using IKA emulsifying agents, 16000rpm, emulsified 5 minutes；
(4) dispense：The vaccine of preparation is dispensed according to every bottle of 250mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710134514.XA CN106822886A (en) | 2017-03-08 | 2017-03-08 | The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710134514.XA CN106822886A (en) | 2017-03-08 | 2017-03-08 | The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106822886A true CN106822886A (en) | 2017-06-13 |
Family
ID=59138089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710134514.XA Pending CN106822886A (en) | 2017-03-08 | 2017-03-08 | The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106822886A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446892A (en) * | 2017-08-24 | 2017-12-08 | 乾元浩生物股份有限公司 | A kind of cultural method of LMH cells |
| CN108969760A (en) * | 2018-07-24 | 2018-12-11 | 广东渔跃生物技术有限公司 | A kind of method and vaccine with LMH cell line production 4 type vaccine of aviadenovirus |
| CN111000990A (en) * | 2019-12-19 | 2020-04-14 | 广东渔跃生物技术有限公司 | Duck tembusu virus and duck adenovirus bivalent inactivated vaccine and preparation method thereof |
| CN114231503A (en) * | 2021-11-15 | 2022-03-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Chicken infectious bursal disease virus and serum 4 type avian adenovirus bivalent inactivated vaccine as well as preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202378A (en) * | 1997-06-02 | 1998-12-23 | 财团法人化学及血清疗法研究所 | Process for preparing infectious bursal disease virus and avian reovirus using cell lines |
-
2017
- 2017-03-08 CN CN201710134514.XA patent/CN106822886A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1202378A (en) * | 1997-06-02 | 1998-12-23 | 财团法人化学及血清疗法研究所 | Process for preparing infectious bursal disease virus and avian reovirus using cell lines |
Non-Patent Citations (1)
| Title |
|---|
| 赵莉等: "Ⅰ型禽腺病毒 AV208 株在鸡肝癌细胞中增殖规律的研究", 《微生物学通报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446892A (en) * | 2017-08-24 | 2017-12-08 | 乾元浩生物股份有限公司 | A kind of cultural method of LMH cells |
| CN108969760A (en) * | 2018-07-24 | 2018-12-11 | 广东渔跃生物技术有限公司 | A kind of method and vaccine with LMH cell line production 4 type vaccine of aviadenovirus |
| CN111000990A (en) * | 2019-12-19 | 2020-04-14 | 广东渔跃生物技术有限公司 | Duck tembusu virus and duck adenovirus bivalent inactivated vaccine and preparation method thereof |
| CN114231503A (en) * | 2021-11-15 | 2022-03-25 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Chicken infectious bursal disease virus and serum 4 type avian adenovirus bivalent inactivated vaccine as well as preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106798918A (en) | A kind of method that use LMH cell lines produce aviadenovirus inactivated vaccine | |
| CN106729690A (en) | The preparation method of newcastle disease, avian influenza virus and aviadenovirus triple inactivated vaccine | |
| CN106822886A (en) | The preparation method of infections chicken cloacal bursa and the type bivalent inactivated vaccine of aviadenovirus 4 | |
| CN102617731B (en) | Porcine circovirus-resistant type 2 egg yolk antibody and preparation method and application thereof | |
| CN104530232B (en) | The preparation method of duck virus hepatitis refined yolk antibody | |
| CN109718370B (en) | A kind of duck reovirus vaccine and preparation method thereof | |
| CN105367654A (en) | Preparation method of I-type duck hepatitis refined yolk antibodies | |
| CN107875383A (en) | Kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2 | |
| CN102492661A (en) | Preparation method and application of immunization preparation for controlling novel duck reovirus | |
| CN105664150B (en) | A kind of newcastle disease virus, avian influenza virus and aviadenovirus triple inactivated vaccine | |
| CN105950564A (en) | Muscovy duck hepatitis virus and method for preparing muscovy duck hepatitis virus refined egg yolk antibody by adopting same | |
| CN105582533A (en) | Combined inactivated vaccine for avian influenza virus and fowl adenovirus | |
| CN105535958B (en) | A kind of newcastle disease virus, infective bronchitis, aviadenovirus triple inactivated vaccine | |
| CN107050448A (en) | A kind of preparation method of avian influenza virus, aviadenovirus bivalent inactivated vaccine | |
| CN109336971A (en) | The preparation method and products thereof of goose astrovirus Yolk antibody | |
| CN109692329B (en) | Duck astrovirus vaccine and preparation method thereof | |
| CN109207436A (en) | One plant of 4 type aviadenovirus strain of I group and its application | |
| CN102965344B (en) | Production of infectious bronchitis virus and vaccine from cell line | |
| CN108969760B (en) | A kind of method and vaccine with LMH cell line production 4 type vaccine of aviadenovirus | |
| CN104984337B (en) | A kind of newcastle disease, avian influenza antigen antibody complex inactivated vaccine and preparation method thereof | |
| CN106177938A (en) | Duck viral hepatitis bivalent inactivated vaccine and preparation method thereof | |
| CN104474542A (en) | Preparation method of bi-combined inactivated vaccine | |
| CN107137705B (en) | The production method of pseudorabies gE gene delection viral inactivation vaccines | |
| CN109207437A (en) | One plant of 8 type aviadenovirus strain of I group and its application | |
| CN106636013A (en) | Ankara virus strain FAdV-HB and preparation and application of inactivated vaccine of ankara virus strain FAdV-HB |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170613 |