CN112156182A - Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine - Google Patents
Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine Download PDFInfo
- Publication number
- CN112156182A CN112156182A CN202011249980.0A CN202011249980A CN112156182A CN 112156182 A CN112156182 A CN 112156182A CN 202011249980 A CN202011249980 A CN 202011249980A CN 112156182 A CN112156182 A CN 112156182A
- Authority
- CN
- China
- Prior art keywords
- virus
- newcastle disease
- avian influenza
- volume
- parts
- 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
- 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
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
- C12N2760/18134—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
- C12N2760/18151—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a complete suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine and a preparation method thereof, belonging to the field of avian virus vaccines. The method of the invention comprises the following steps: 1) culturing newcastle disease virus using AGE1 cells in suspension; 2) culturing the avian influenza virus in a suspension using MDCK cells; 3) performing ultrafiltration concentration on the viruses obtained in the step 1) and the step 2), inactivating the viruses, and adding pharmaceutically acceptable immunologic adjuvant and other auxiliary components to prepare the vaccine. The preparation method of the full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine can obtain high-titer viruses; the bivalent vaccine prepared by the method can generate higher antibodies than chick embryos and provide stronger protection.
Description
Technical Field
The present invention belongs to the field of avian virus vaccine.
Background
Newcastle Disease Virus (NDV) belongs to the family Paramyxoviridae, a single-stranded negative-strand RNA virus. Newcastle Disease (ND) is one of the most harmful viral infectious diseases to the poultry industry, has extremely high lethality rate and causes great loss to the poultry industry of all countries in the world. Currently, the primary measure for controlling ND is vaccination.
Avian Influenza Virus (AIV) is very prevalent around the world, particularly H9 subtype AIV, although the pathogenicity is low, the secondary infection caused by the AIV often brings great loss to the poultry industry, and data show that H9 subtype AIV gene can be recombined into highly pathogenic AIV to cause variation of the highly pathogenic AIV, thereby endangering the health of poultry and even human beings. Therefore, the development of safe and efficient H9 subtype AIV vaccine is a necessary choice.
The poultry vaccine is mainly produced by chick embryos at present, and the inactivated vaccine is produced by low-immunity hatching eggs or non-immunity hatching eggs, so that pathogens of vertical transmission diseases can be polluted in the vaccine, the diseases are transmitted along with the application of the vaccine, and the problem of insufficient chick embryos during large-scale production is also solved. The virus culture is directly related to the quality of antigen and the titer level, so the virus culture technology is one of the important factors influencing the quality of vaccines. The vaccine prepared by using the cells as the matrix has the advantages of no foreign factor pollution, easy large-scale production, capability of better maintaining the stability of virus antigens and the like.
At present, part of reports also adopt EB66 cells to produce the newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine, but the method adopts a second-order culture method, namely, part of cells are inoculated and then are subjected to adsorption culture for 1H, and then part of virus production culture medium is supplemented for culture, so that the operation steps are multiple, the consumed time is long, and the pollution is easily caused.
Disclosure of Invention
The invention aims to solve the technical problem of providing a novel chicken newcastle disease and avian influenza bivalent inactivated vaccine and a preparation method thereof.
The technical scheme of the invention comprises the following steps:
a preparation method of a complete suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine comprises the following steps:
1) culturing newcastle disease virus using AGE1 cells in suspension;
2) culturing the avian influenza virus in a suspension using MDCK cells;
3) inactivating the viruses obtained in the step 1) and the step 2), ultrafiltering and concentrating, and adding pharmaceutically acceptable immunologic adjuvant and other auxiliary components to prepare the vaccine.
The preparation method comprises the following steps of 3):
a. preparation of an aqueous phase: and (3) performing inactivation, ultrafiltration and concentration on the newcastle disease virus and the avian influenza virus according to the ratio of 1: (0.5-2) to obtain a mixed antigen, taking 94-98 parts by volume of the mixed antigen, adding 802-6 parts by volume of tween-and fully stirring until tween-80 is completely dissolved to obtain a water phase;
b. preparing an oil phase: mixing 92-96 parts by volume of white oil and 92-804-8 parts by volume of span-804-8 parts by volume to obtain an oil phase;
c. emulsification: mixing the water phase and the oil phase according to a volume ratio of 1: (1.5-2.5), mixing, and emulsifying.
The preparation method comprises the following steps of step 3) and step a:
the volume ratio of the newcastle disease virus to the avian influenza virus is 1: 1;
and/or the volume ratio of the mixed antigen to the Tween-80 is 96: 4.
The preparation method comprises the following steps of step 3) and step b:
94 parts by volume of white oil and 6 parts by volume of span-80.
The preparation method comprises the following steps of 3) and c:
the volume ratio of the water phase to the oil phase is 1:2.
The preparation method comprises the following steps of 1):
AGE1 cells were expanded to a cell density of 8.0X 106Inoculating the newcastle disease virus, adding 10-20% serum-free DMEM medium at the same time, adding pancreatin with the final concentration of 10-20 mu g/ml, culturing at 33-37 ℃, and harvesting virus liquid when the HA titer is detected to be not less than 8log 2.
Further, the volume ratio of serum-free DMEM medium is 15%;
further, the final concentration of pancreatin was 15. mu.g/ml;
further, the culture temperature was 35 ℃.
The preparation method comprises the following steps of 2):
MDCK cells were expanded to a cell density of 6.0X 106Inoculating avian influenza virus at a concentration of more than 8-16 μ g/ml, adding pancreatin at a final concentration of 8-16 μ g/ml, culturing at 35-37 deg.C, and collecting the venom when HA titer is not less than 8log 2.
Further, the final concentration of pancreatin was 12. mu.g/ml.
Further, the culture temperature was 37 ℃.
The newcastle disease and avian influenza bivalent inactivated vaccine prepared by the method.
The invention has the following beneficial effects:
1) the method has simple virus culture steps, and does not need second-order culture;
2) the invention can obtain high-titer newcastle disease virus and avian influenza virus, which exceeds the level of the prior art;
3) the bivalent vaccine prepared by injecting the method can generate higher antibodies than the bivalent vaccine prepared by injecting the chick embryo vaccine, and the vaccine has better protection effect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
EXAMPLE 1 preparation of a bivalent vaccine according to the invention
1 propagation of the Virus on cells
1.1 proliferation of Newcastle disease Virus on suspension cells
And (3) carrying out amplified passage on the AGE1 cells to a bioreactor of 7L, wherein the volume of a culture solution is 4-5L, the pH value is set to be 7.15, the dissolved oxygen value is set to be 60%, the rotating speed is set to be 75r/min, and the cells are cultured at 37 ℃. When the cell density is 8.0X 106When the concentration is more than one ml, inoculating aSG10 strains of Newcastle disease virus according to 0.0001MOI, simultaneously adding 15% serum-free DMEM culture medium by volume ratio, adding pancreatin solution with the final concentration of 15 mu g/ml at one time, adjusting the culture temperature to 35 ℃, culturing for 64-80 h, sampling to detect HA titer, and harvesting virus liquid when the HA titer is not less than 8log 2. A total of 3 batches of newcastle disease virus were prepared and their HA titers and virus content were determined. The results are shown in Table 1.
TABLE 1 cultivation of Newcastle disease Virus in a 7L bioreactor
1.2 propagation of avian influenza Virus on suspension cells
And (2) carrying out amplified passage on the MDCK cells to a 7L bioreactor, wherein the volume of a culture solution is 4-5L, the pH value is set to 7.0, the dissolved oxygen value is set to 50%, the rotating speed is set to 65r/min, and the MDCK cells are cultured at 37 ℃. When the cell density is 6.0X 106And (3) when the concentration is more than one ml, inoculating the avian influenza G strain virus according to 0.01MOI, adding pancreatin solution with the final concentration of 12 mu G/ml at one time, culturing for 42-54 h, sampling to detect the HA titer, and harvesting virus liquid when the HA titer is not less than 8log 2. 3 batches of avian influenza viruses were prepared in total and their HA titers and virus contents were determined. The results are shown in Table 2.
TABLE 2 cultivation of avian influenza G Strain Virus in a 7L bioreactor
2 concentration of the virus solution
3000ml of each of 3 NDV and H9 AIV virus solutions were concentrated to 1/2 volume by using an ultrafiltration membrane with a molecular weight cut-off of 100kD using the American Pall Corporation ultrafiltration system.
Inactivation of viral fluids
3.1 inactivation of Newcastle disease Virus solution 3 batches of 2-fold concentrated NDV aSG10 strain virus solution were added with 0.1% final concentration of formaldehyde, heated in water bath to 37 deg.C, timed, inactivated at 37 deg.C for 20h, shaken up every 2 h, after inactivation, the virus solution was taken to measure its HA titer, and subjected to inactivation test. The HA titer is not reduced after inactivation, and the inactivation test result shows that the inactivation is complete. The results are detailed in Table 3.
TABLE 3 inactivation of NDV aSG10 strain in virus fluid
3.2 liquid inactivation of avian influenza virus 3 batches of 2-fold concentrated H9 AIV strain G virus liquid are respectively added into formaldehyde with the final concentration of 0.2%, water bath heating is carried out to 37 ℃, timing is started, constant-temperature inactivation at 37 ℃ is carried out for 18H, shaking is carried out once every 2H, the virus liquid is taken after inactivation to measure the HA titer, and inactivation inspection is carried out. The HA titer is not reduced after inactivation, and the inactivation test result shows that the inactivation is complete. The results are detailed in Table 4.
TABLE 4 inactivation of AIV G strain virus liquid of subtype H9
4 preparation of vaccine
4.1 preparation of aqueous phase 3 batches of inactivated NDV and AIV antigens are mixed in equal volume, 96 volume parts of the mixed antigens are taken, sterilized and cooled Tween-804 volume parts are added, and the mixture is fully stirred until Tween-80 is completely dissolved to prepare an aqueous phase.
4.2 preparation of oil phase 94 parts by volume of white oil for injection and 806 parts by volume of span are taken and stirred uniformly, sterilized and cooled for standby.
4.3 according to the volume ratio of 1:2, the oil phase is firstly input into an emulsification tank and stirred, then the water phase is slowly added, the stirring is continued to fully mix the oil phase and the water phase, and then the mixture is emulsified on line by a shearing machine to prepare 3 batches of vaccines with the batch numbers of 01, 02 and 03 respectively.
EXAMPLE 2 preparation of the bivalent vaccine according to the invention
1 propagation of the Virus on cells
1.1 proliferation of Newcastle disease Virus on suspension cells
And (3) carrying out amplified passage on the AGE1 cells to a bioreactor of 7L, wherein the volume of a culture solution is 4-5L, the pH value is set to be 7.15, the dissolved oxygen value is set to be 60%, the rotating speed is set to be 75r/min, and the cells are cultured at 37 ℃. When the cell density is 8.0X 106When the concentration is more than one milliliter, inoculating aSG10 strains of Newcastle disease virus according to 0.0001MOI, simultaneously adding 15 percent of serum-free DMEM medium by volume ratio, adding 10 microgram/ml pancreatin solution at one time, adjusting the culture temperature to 33 ℃, culturing for 64-80 hours, sampling and detecting HA effectAnd (3) harvesting the virus liquid when the HA titer is not lower than 8log 2.
1.2 propagation of avian influenza Virus on suspension cells
And (2) carrying out amplified passage on the MDCK cells to a 7L bioreactor, wherein the volume of a culture solution is 4-5L, the pH value is set to 7.0, the dissolved oxygen value is set to 50%, the rotating speed is set to 65r/min, and the MDCK cells are cultured at 37 ℃. When the cell density is 6.0X 106And (3) when the concentration is more than one ml, inoculating the avian influenza G strain virus according to 0.01MOI, adding pancreatin solution with the final concentration of 8 mu G/ml at one time, culturing for 42-54 h, sampling to detect the HA titer, and harvesting virus liquid when the HA titer is not less than 8log 2.
2 concentration of the virus solution
3000ml of each of NDV and H9 AIV virus solutions were concentrated to 1/2 volume by using an ultrafiltration membrane with a molecular weight cut-off of 100kD using the Pall Corporation ultrafiltration system.
Inactivation of viral fluids
3.1 inactivation of Newcastle disease Virus solution 2 times concentrated NDV aSG10 strain virus solution was added to 0.1% final concentration formaldehyde, heated to 37 ℃ in a water bath, timed, and inactivated at 37 ℃ for 20h at constant temperature, shaken up every 2 h, after inactivation, the virus solution was taken to determine its HA titer, and subjected to inactivation test.
3.2 liquid inactivation of avian influenza virus H9 AIV strain G virus liquid concentrated 2 times is added into formaldehyde with final concentration of 0.2%, the mixture is heated to 37 ℃ in a water bath, timing is started, constant-temperature inactivation at 37 ℃ is carried out for 18H, shaking is carried out once every 2H, the virus liquid is taken after inactivation to measure the HA titer, and inactivation inspection is carried out.
4 preparation of vaccine
4.1 preparation of aqueous phase the inactivated NDV and AIV antigens are mixed according to the volume ratio of 2:1, 94 parts by volume of the mixed antigens are taken, sterilized and cooled Tween-806 parts by volume are added, and the mixture is fully stirred until Tween-80 is completely dissolved to prepare the aqueous phase.
4.2 preparation of oil phase 92 parts by volume of white oil for injection and span-808 parts by volume are taken and stirred uniformly, sterilized and cooled for standby.
4.3, inputting the oil phase into an emulsification tank, stirring, slowly adding the water phase, continuously stirring to fully and uniformly mix the oil phase and the water phase, and then carrying out online emulsification through a shearing machine according to the volume ratio of the emulsified water phase to the oil phase of 1: 1.5.
EXAMPLE 3 preparation of the bivalent vaccine according to the invention
1 propagation of the Virus on cells
1.1 proliferation of Newcastle disease Virus on suspension cells
And (3) carrying out amplified passage on the AGE1 cells to a bioreactor of 7L, wherein the volume of a culture solution is 4-5L, the pH value is set to be 7.15, the dissolved oxygen value is set to be 60%, the rotating speed is set to be 75r/min, and the cells are cultured at 37 ℃. When the cell density is 8.0X 106When the concentration is more than one ml, inoculating aSG10 strains of Newcastle disease virus according to 0.0001MOI, simultaneously adding 20% serum-free DMEM culture medium by volume ratio, adding pancreatin solution with the final concentration of 20 mu g/ml at one time, adjusting the culture temperature to 37 ℃, culturing for 64-80 h, sampling to detect HA titer, and harvesting virus liquid when the HA titer is not less than 8log 2.
1.2 propagation of avian influenza Virus on suspension cells
And (2) carrying out amplified passage on the MDCK cells to a 7L bioreactor, wherein the volume of a culture solution is 4-5L, the pH value is set to 7.0, the dissolved oxygen value is set to 50%, the rotating speed is set to 65r/min, and the MDCK cells are cultured at 37 ℃. When the cell density is 6.0X 106And (3) when the concentration is more than one ml, inoculating the avian influenza G strain virus according to 0.01MOI, adding pancreatin solution with the final concentration of 16 mu G/ml at one time, culturing for 42-54 h, sampling to detect the HA titer, and harvesting virus liquid when the HA titer is not less than 8log 2.
2 concentration of the virus solution
3000ml of each of NDV and H9 AIV virus solutions were concentrated to 1/2 volume by using an ultrafiltration membrane with a molecular weight cut-off of 100kD using the Pall Corporation ultrafiltration system.
Inactivation of viral fluids
3.1 inactivation of Newcastle disease Virus solution 2 times concentrated NDV aSG10 strain virus solution was added to 0.1% final concentration formaldehyde, heated to 37 ℃ in a water bath, timed, and inactivated at 37 ℃ for 20h at constant temperature, shaken up every 2 h, after inactivation, the virus solution was taken to determine its HA titer, and subjected to inactivation test.
3.2 liquid inactivation of avian influenza virus H9 AIV strain G virus liquid concentrated 2 times is added into formaldehyde with final concentration of 0.2%, the mixture is heated to 37 ℃ in a water bath, timing is started, constant-temperature inactivation at 37 ℃ is carried out for 18H, shaking is carried out once every 2H, the virus liquid is taken after inactivation to measure the HA titer, and inactivation inspection is carried out.
4 preparation of vaccine
4.1 preparation of aqueous phase two antigens of inactivated NDV, AIV mix according to the volume ratio 1:2, get 98 parts of mixed antigen, add Tween-802 parts after sterilizing and cooling, stir fully until Tween-80 is totally dissolved, prepare into aqueous phase.
4.2 preparation of oil phase 96 parts by volume of white oil for injection and span-804 parts by volume are taken and stirred uniformly, sterilized and cooled for standby.
4.3, inputting the oil phase into an emulsification tank, stirring, slowly adding the water phase, continuously stirring to fully and uniformly mix the oil phase and the water phase, and then carrying out online emulsification through a shearing machine according to the volume ratio of the emulsified water phase to the oil phase of 1: 2.5.
Experimental example 1 immunogenicity study of cell-derived vaccine
And selecting 1 batch of chicken embryo source newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine and 3 batches of cell source produced newcastle disease and avian influenza bivalent inactivated vaccines obtained in example 1 to perform immunogenicity test.
The preparation method of the chick embryo source vaccine comprises the following steps:
NDV and AIV concentrated inactivated antigen solution produced by 1 batch of chick embryos is taken and prepared into a newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine with the batch number of J01 according to the method of the section 4 of the embodiment 1.
1 immunization and toxin counteracting of Newcastle disease part
40 SPF chickens of 30-60 days old are divided into 4 groups and 10 chickens/group, 4 batches of vaccine of 20 mu l/group are respectively injected subcutaneously at the neck, and other 5 chickens are taken as non-immune control. On 21-28 days after immunization, each chicken is respectively subjected to blood collection, serum is separated, and HI antibody titer is determined by using chicken Newcastle disease virus (aSG10 strain) hemagglutination inhibition test antigen. The average level of ND partial antibodies of the cell source in 28 days after immunization is 7.3-7.7 log2, which is far higher than the standard 6log2, while the average value of the antibody of the chick embryo source is 6.3log2, which is lower than that of the cell source.
0.2ml of Newcastle disease virulent SG10 strain (containing 10 percent) is injected into each chicken muscle after blood collection5.0ELD50) And observing for 14 days, collecting chicken cloaca swabs on the 5 th day after the virus challenge, inoculating 10-day-old SPF (specific pathogen free) chicken embryos, and performing virus isolation. After the challenge, all control chickens die within 5 days, no newcastle disease clinical symptoms exist in an immune group, the separation of the chicken viruses in a cell-derived immune group is 10/10 negative, the separation of the chicken viruses in an embryo-derived immune group is 9/10 negative, and the cell-derived protection effect is better. The results are detailed in Table 5.
TABLE 5 effectiveness test results of Newcastle disease parts of chickens
Note: "/" indicates the absence of such an item.
2 immunization and challenge of avian influenza
40 SPF chickens of 21-35 days old are divided into 4 groups and 10 chickens/group, 4 batches of vaccine of 0.25 ml/group are respectively injected subcutaneously into the neck, and other 5 chickens are taken as non-immune control. And (3) collecting blood of each chicken 21-28 days after inoculation, separating serum, and measuring the HI antibody titer by using avian influenza virus (G strain) hemagglutination inhibition test antigen. The average level of AI partial antibodies of cell sources after 21 days of immunization is 9.1-9.5 log2, which is far higher than the standard 7log2, while the average value of antibodies of chick embryo sources is 8.8log2, which is lower than that of cell sources.
After blood collection, each chicken is injected with 0.2ml (containing 10) of H9 subtype avian influenza virus G strain by vein6.0EID50). And on the 5 th day after the challenge, respectively collecting throat cotton swabs and cloaca cotton swabs of each chicken, inoculating 10-day-old SPF (specific pathogen free) chick embryos through an allantoic cavity after mixing, and performing virus separation, wherein 10/10 virus separation of all chickens in the cell-derived immune group is negative, 9/10 virus separation of the chick embryo-derived immune group is negative, 5/5 virus separation of the chicken in the control group is positive, and the cell-derived protection effect is better. The results are detailed in Table 6.
Table 6 avian influenza fraction efficacy test results
Antibody detection and challenge test results show that the antibody titers of two inactivated vaccines of the chick embryo source and the cell source meet the regulations, the challenge protection test can achieve the protection effect, but the level of the antibody generated by the cell source is higher than that of the chick embryo source, the protection effect is stronger, and the batch difference of the three batches of vaccine test results is small, which indicates that the production process is stable and controllable in quality, and can be used for large-scale production.
In conclusion, the preparation method of the complete suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine can obtain high-titer viruses; the bivalent vaccine prepared by the method can generate higher antibodies than chick embryos and provide stronger protection.
Claims (10)
1. A preparation method of a complete suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine is characterized by comprising the following steps:
1) culturing newcastle disease virus using AGE1 cells in suspension;
2) culturing the avian influenza virus in a suspension using MDCK cells;
3) performing ultrafiltration concentration on the viruses obtained in the step 1) and the step 2), inactivating the viruses, and adding pharmaceutically acceptable immunologic adjuvant and other auxiliary components to prepare the vaccine.
2. The method of claim 1, wherein the step 3) comprises:
a. preparation of an aqueous phase: and (3) performing inactivation, ultrafiltration and concentration on the newcastle disease virus and the avian influenza virus according to the ratio of 1: (0.5-2) to obtain a mixed antigen, taking 94-98 parts by volume of the mixed antigen, adding 802-6 parts by volume of tween-and fully stirring until tween-80 is completely dissolved to obtain a water phase;
b. preparing an oil phase: mixing 92-96 parts by volume of white oil and 92-804-8 parts by volume of span-804-8 parts by volume to obtain an oil phase;
c. emulsification: mixing the water phase and the oil phase according to a volume ratio of 1: (1.5-2.5), mixing, and emulsifying.
3. The method of claim 2, wherein in step a of step 3):
the volume ratio of the newcastle disease virus to the avian influenza virus is 1: 1;
and/or the volume ratio of the mixed antigen to the Tween-80 is 96: 4.
4. The method of claim 2, wherein in step b of step 3):
94 parts by volume of white oil and 6 parts by volume of span-80.
5. The method of claim 2, wherein in step c of step 3):
the volume ratio of the water phase to the oil phase is 1:2.
6. The method according to any one of claims 1 to 5, wherein the step 1) is:
AGE1 cells were expanded to a cell density of 8.0X 106Inoculating the newcastle disease virus, adding 10-20% serum-free DMEM medium at the same time, adding pancreatin with the final concentration of 10-20 mu g/ml, culturing at 33-37 ℃, and harvesting virus liquid when the HA titer is detected to be not less than 8log 2.
7. The method of claim 6, wherein:
the volume ratio of serum-free DMEM medium addition is 15%;
and/or the final concentration of pancreatin is 15 mug/ml;
and/or the culture temperature is 35 ℃.
8. The method according to any one of claims 1 to 5, wherein the step 2) is:
MDCK cells were expanded to a cell density of 6.0X 106Inoculating avian influenza virus at a concentration of more than 8-16 μ g/ml, adding pancreatin at a final concentration of 8-16 μ g/ml, culturing at 35-37 deg.C, and collecting the venom when HA titer is not less than 8log 2.
9. The method of claim 8, wherein:
the final concentration of pancreatin is 12 μ g/ml;
and/or the incubation temperature is 37 ℃.
10. A newcastle disease and avian influenza bivalent inactivated vaccine prepared by the method of any one of claims 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011249980.0A CN112156182A (en) | 2020-11-10 | 2020-11-10 | Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011249980.0A CN112156182A (en) | 2020-11-10 | 2020-11-10 | Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112156182A true CN112156182A (en) | 2021-01-01 |
Family
ID=73865890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011249980.0A Pending CN112156182A (en) | 2020-11-10 | 2020-11-10 | Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112156182A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129281A (en) * | 2019-05-15 | 2019-08-16 | 成都天邦生物制品有限公司 | A kind of method and application using suspension cell culture newcastle disease virus |
| CN113122508A (en) * | 2021-03-30 | 2021-07-16 | 成都天邦生物制品有限公司 | Infectious bronchitis cell adaptive virus and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491672A (en) * | 2009-02-24 | 2009-07-29 | 乾元浩生物股份有限公司 | Chicken new castle disease-bird influenza dual oil-emulsion inactivated vaccine and preparation method thereof |
| WO2011134163A1 (en) * | 2010-04-29 | 2011-11-03 | 扬州优邦生物制药有限公司 | Preparation method for inactivated vaccine of h9n2 subtype avian influenza and the product thereof |
| CN108159412A (en) * | 2018-01-12 | 2018-06-15 | 华农（肇庆）生物产业技术研究院有限公司 | It is a kind of to produce cell source newcastle disease, method of bird flu bivalent inactivated vaccine and products thereof |
| CN108753737A (en) * | 2018-05-31 | 2018-11-06 | 吉林冠界生物技术有限公司 | A kind of method and its application being proliferated avian influenza virus on the full suspension cells of MDCK |
| CN110129281A (en) * | 2019-05-15 | 2019-08-16 | 成都天邦生物制品有限公司 | A kind of method and application using suspension cell culture newcastle disease virus |
-
2020
- 2020-11-10 CN CN202011249980.0A patent/CN112156182A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101491672A (en) * | 2009-02-24 | 2009-07-29 | 乾元浩生物股份有限公司 | Chicken new castle disease-bird influenza dual oil-emulsion inactivated vaccine and preparation method thereof |
| WO2011134163A1 (en) * | 2010-04-29 | 2011-11-03 | 扬州优邦生物制药有限公司 | Preparation method for inactivated vaccine of h9n2 subtype avian influenza and the product thereof |
| CN108159412A (en) * | 2018-01-12 | 2018-06-15 | 华农（肇庆）生物产业技术研究院有限公司 | It is a kind of to produce cell source newcastle disease, method of bird flu bivalent inactivated vaccine and products thereof |
| CN108753737A (en) * | 2018-05-31 | 2018-11-06 | 吉林冠界生物技术有限公司 | A kind of method and its application being proliferated avian influenza virus on the full suspension cells of MDCK |
| CN110129281A (en) * | 2019-05-15 | 2019-08-16 | 成都天邦生物制品有限公司 | A kind of method and application using suspension cell culture newcastle disease virus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129281A (en) * | 2019-05-15 | 2019-08-16 | 成都天邦生物制品有限公司 | A kind of method and application using suspension cell culture newcastle disease virus |
| CN113122508A (en) * | 2021-03-30 | 2021-07-16 | 成都天邦生物制品有限公司 | Infectious bronchitis cell adaptive virus and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106924727B (en) | Preparation method of avian influenza virus H9 subtype inactivated vaccine | |
| CN112156182A (en) | Full-suspension cell source newcastle disease and avian influenza bivalent inactivated vaccine | |
| CN102258777A (en) | Method for preparing inactivated vaccine and combined vaccine by breeding infectious bursal disease virus (IBDV) by chicken embryo source cell line | |
| CN104001167A (en) | Process for preparing avian influenza inactivated vaccine by full suspended culture cells and product | |
| CN108721615B (en) | A kind of method and its vaccine preparing duck tembusu virus inactivated vaccine | |
| CN107050448B (en) | Preparation method of avian influenza virus and avian adenovirus bivalent inactivated vaccine | |
| CN102133398A (en) | Method for industrially producing animal influenza vaccine by using bioreactor | |
| US11607448B2 (en) | Whole avian-origin reverse genetic system and its use in producing H7N9 subtype avian influenza vaccine | |
| CN105349562A (en) | Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain | |
| WO2011134163A1 (en) | Preparation method for inactivated vaccine of h9n2 subtype avian influenza and the product thereof | |
| CN108452298A (en) | A kind of technique producing yellow fever attenuated live vaccine with SPF chick-embryo cells | |
| CN104611299B (en) | H9N2 avian flus strain, preparation method, vaccine combination and its application of a kind of artificial recombination | |
| CN109692329B (en) | Duck astrovirus vaccine and preparation method thereof | |
| CN111714626A (en) | Method for producing avian influenza vaccine by using MDCK cell line and product thereof | |
| CN107320721B (en) | Vaccine composition and preparation method thereof | |
| CN107537032A (en) | A kind of tetravalence influenza virus subunit vaccine and preparation method thereof | |
| CN116162601A (en) | Preparation method of influenza virus split vaccine | |
| CN105833263B (en) | Bivalent vaccine of avian metapneumovirus and H9 subtype avian influenza virus | |
| CN108159412A (en) | It is a kind of to produce cell source newcastle disease, method of bird flu bivalent inactivated vaccine and products thereof | |
| CN104357406A (en) | Rabies virus SNK-CTN strain and application thereof | |
| CN112080478B (en) | Efficient propagation method and application of H5 subtype avian influenza virus | |
| CN108261543B (en) | The preparation method and applications of subculture cell source ND, IB, AI triple inactivated vaccine | |
| CN110467671B (en) | Method for preparing TW1 type avian infectious bronchitis virus positive serum by SPF chicken | |
| CN103007271A (en) | Chicken subunit four-combination vaccine and preparation and application thereof | |
| CN107137705B (en) | The production method of pseudorabies gE gene delection viral inactivation vaccines |
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 | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant after: Chengdu Shiji biopharmaceutical Co.,Ltd. Applicant after: Shiji Biotechnology (Nanjing) Co.,Ltd. Address before: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant before: CHENGDU TECBOND BIOLOGICAL PRODUCTS CO.,LTD. Applicant before: Shiji Biotechnology (Nanjing) Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20221208 Address after: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant after: Chengdu Shiji biopharmaceutical Co.,Ltd. Address before: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant before: Chengdu Shiji biopharmaceutical Co.,Ltd. Applicant before: Shiji Biotechnology (Nanjing) Co.,Ltd. |