CN106957860A - The preparation method of H5 subtype avian influenza virus HA subunit vaccines - Google Patents
The preparation method of H5 subtype avian influenza virus HA subunit vaccines Download PDFInfo
- Publication number
- CN106957860A CN106957860A CN201710140542.2A CN201710140542A CN106957860A CN 106957860 A CN106957860 A CN 106957860A CN 201710140542 A CN201710140542 A CN 201710140542A CN 106957860 A CN106957860 A CN 106957860A
- Authority
- CN
- China
- Prior art keywords
- avian influenza
- influenza virus
- subtype avian
- preparation
- cells
- 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
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 56
- 229940031626 subunit vaccine Drugs 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 24
- 229910052760 oxygen Inorganic materials 0.000 claims description 24
- 239000001301 oxygen Substances 0.000 claims description 24
- 241000700605 Viruses Species 0.000 claims description 23
- 238000012216 screening Methods 0.000 claims description 22
- 238000004113 cell culture Methods 0.000 claims description 7
- 238000003306 harvesting Methods 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 230000035755 proliferation Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 241000701447 unidentified baculovirus Species 0.000 claims description 3
- 231100000572 poisoning Toxicity 0.000 claims description 2
- 230000000607 poisoning effect Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 150000002926 oxygen Chemical class 0.000 claims 1
- 230000002062 proliferating effect Effects 0.000 claims 1
- 230000001902 propagating effect Effects 0.000 claims 1
- 230000003362 replicative effect Effects 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 230000001988 toxicity Effects 0.000 claims 1
- 231100000419 toxicity Toxicity 0.000 claims 1
- 229960005486 vaccine Drugs 0.000 abstract description 25
- 238000004519 manufacturing process Methods 0.000 abstract description 24
- 210000002966 serum Anatomy 0.000 abstract description 11
- 241000287828 Gallus gallus Species 0.000 abstract description 8
- 241000238631 Hexapoda Species 0.000 abstract description 8
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000007812 deficiency Effects 0.000 abstract description 2
- 238000004114 suspension culture Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 111
- 231100000614 poison Toxicity 0.000 description 26
- 239000002574 poison Substances 0.000 description 26
- 238000000034 method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 239000012679 serum free medium Substances 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000002054 inoculum Substances 0.000 description 6
- 239000004017 serum-free culture medium Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 206010064097 avian influenza Diseases 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000003067 hemagglutinative effect Effects 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000003123 plant toxin Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 208000002979 Influenza in Birds Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 241000255993 Trichoplusia ni Species 0.000 description 2
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical compound [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 2
- 229940063655 aluminum stearate Drugs 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000013320 baculovirus expression vector system Methods 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 229960003971 influenza vaccine Drugs 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000013190 sterility testing Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001473385 H5N1 subtype Species 0.000 description 1
- 101150039660 HA gene Proteins 0.000 description 1
- 206010024769 Local reaction Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000256247 Spodoptera exigua Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229940124973 vaccine residual Drugs 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
-
- 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
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- 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
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to the preparation method of H5 subtype avian influenza virus HA subunit vaccines, belong to biological technical field.Utilize two kinds of insect cells of Sf9 and HiFi, kind respectively to H5 subtype avian influenza virus HA subunit vaccines is malicious and is inoculated with kind of a malicious vaccine progress high efficiently multiplying, so as to overcome the deficiency of production of vaccine technology currently based on chicken embryo tissue seedling, serum free suspension culture can be realized in the reactor, production cost is greatly reduced, growth cycle is shortened, can be with rapid expansion production scale, human resources are reduced, the yield and quality of vaccine is significantly improved.
Description
Technical field
The present invention relates to the preparation method of H5 subtype avian influenza virus HA subunit vaccines, belong to biological technical field.
Background technology
The development of H5 subtype avian influenza virus serious harms China aquaculture, threatens to public safety health.In portion
The area for dividing cultivation density higher, bird flu turns into the key factor of restriction local economic development.Especially break out in recent years
H5N1 subtype highly pathogenic avian influenza virus not only cause the large area of birds to be fallen ill, it is dead, more human health is constituted
It is serious to threaten.Prevent the inoculation that the most effective method of the disease is exactly vaccine, and the avian influenza vaccine of existing market production is equal
For chicken embryo tissue seedling.
Prepared using chicken embryo propagative viruses and be related to live virus operation, life that can be to environment in the production process of avian influenza vaccine
Thing produces safely threat.Limited simultaneously when bird flu is popular due to being supplied by chicken embryo, production of vaccine has the production cycle
The shortcomings of growing and yield poorly.
Therefore, human society need badly it is a kind of can fast reaction, the new H5 subtype avian influenza virus HA that largely produces it is sub-
The mode of production of subunit vaccine.
Insect rhabdovirus system (Baculovirus expression vector system, BEVS) is as a kind of
Carrier for expression of eukaryon system, big to exogenous gene cloning capacity with safe, recombinant virus is easy to screening, with complete
Post translational processing modification system and efficiently expressing exogenous gene ability the features such as, at present turn into genetic engineering four greatly express
One of system (i.e. baculoviral, Escherichia coli, yeast, mammalian cell expression system).Simultaneously because insect cell belongs to
Half adherent half suspension cell, for realizing that large-scale production virus or recombinant protein provide good condition.
But, not yet have been reported that open breed using insect rhabdovirus system produces H5 hypotypes in currently available technology
The method of avian influenza virus HA subunit vaccines.It would therefore be desirable to utilize insect rhabdovirus system propagation production vaccine
Implementation process in solve following technical problem：First, selecting which kind of cell line is used for H5 subtype avian influenza virus HA subunits
The production of vaccine；Secondly, how to obtain and be capable of high efficiently multiplying, meet the cell line of production of vaccine；Finally, we also need to seek
Suitable culture manufacturing condition is found, so as to ensure efficient, the safety of production of vaccine, is conducive to industrialization promotion and reality
Application.
The content of the invention
The goal of the invention of the present invention is to provide a kind of new utilization insect baculovirus propagation H5 subtype avian influenza virus HA
The method of subunit vaccine.
In order to realize this purpose, following technical scheme is we disclosed.
First, we disclose using HiFi cell culture propagation H5 subtype avian influenza virus HA subunit vaccines, and
Further, our open described H5 subtype avian influenza virus HA subunit vaccines are by expression recombination expression H5 hypotypes fowl stream
The baculoviral kind poison of Influenza Virus HA albumen, which is seeded in HiFi cells, replicates breeding.
Wherein, the baculoviral kind poison of preferred expression recombination expression H5 subtype avian influenza virus HA albumen is thin using Sf9
Born of the same parents replicate, breeding.
Described HiFi cells refer to cabbage looper gonad cell, belong to one kind of insect cell.Described Sf9 refers to grass
Ground Beet armyworm cell, is also one kind of insect cell.
Further, we have carried out monoclonal screening to HiFi cells and Sf9 cells, utilize this high efficiently multiplying mechanism
Screening obtain HiFi cells and Sf9 cells good matching relationship can be set up between virus so that breeding obtain
H5 hypotypes its influenza virus HA subunit vaccine hemagglutinative titers reach 212, the inactivation epidemic disease prepared using this subunit vaccine
Seedling antibody titer reaches 8.17log2, and effect is better than conventional vaccine.
Specifically, this screening mode is described as follows：Dispel the Sf9 cell droppers after serum-free is tamed are resuspended
Afterwards, carry out many wheel cells monoclonalizations using 96 orifice plates to screen, we indicate the row of 96 orifice plates with A-H, and row are indicated with 1-12,
Coordinate positioned at first hole in the upper left corner is labeled as A1, and each hole in 96 holes obtains a mark by that analogy
Mark.
Then the cell suspension of 200 μ l volumes is added in A1 holes by we, cell number 1000, then with 8 road liquid reliefs
Device adds 100 μ l cell culture fluids in all holes, is mixed above and below A1 holes and suctions out 100 μ l addition B1 holes, then above and below B1 holes
Mix and suction out 100 μ l addition C1 holes, by that analogy, one-level is carried out successively and is diluted to H1 holes；Then, respectively with 8 road liquid-transfering guns from
Mixed up and down in A1-H1 holes and suction out 100 μ l, be added in A2-H2 holes, by that analogy, carried out two grades successively and be diluted to A12-H12
Hole, thus obtains multiple slender hilums, when the cell in slender hilum covers with single hole, and it is homogeneous to select cellular morphology, raw
Long cell line amplification of good performance.
The cell line obtained to being screened through excessive wheel cells monoclonalization carries out H5 subtype avian influenza virus HA subunits epidemic disease
The propagation of seed poison, with baculoviral TCID50For examination criteria, it is sub- single that screening obtains the shaft-like H5 subtype avian influenza virus HA of propagation
The ability of position vaccine kind poison reaches 108.5TCID50/ mL cell line is as expressing recombination expression H5 subtype avian influenza virus
The Sf9 cells of the baculoviral kind poison of HA albumen are standby.
For HIFi cells, its mode screened is described as follows：HiFi cells after serum-free is tamed are used
Dropper is resuspended dispel after, carry out many wheel cells monoclonalizations using 96 orifice plates and screen, we indicate the row of 96 orifice plates with A-H,
Row are indicated with 1-12, and the coordinate positioned at first hole in the upper left corner is labeled as A1, by that analogy each hole in 96 holes
All obtain a mark number.
Then the cell suspension of 200 μ l volumes is added in A1 holes by we, cell number 1000, then with 8 road liquid reliefs
Device adds 100 μ l cell culture fluids in all holes, is mixed above and below A1 holes and suctions out 100 μ l addition B1 holes, then above and below B1 holes
Mix and suction out 100 μ l addition C1 holes, by that analogy, one-level is carried out successively and is diluted to H1 holes；Then, respectively with 8 road liquid-transfering guns from
Mixed up and down in A1-H1 holes and suction out 100 μ l, be added in A2-H2 holes, by that analogy, carried out two grades successively and be diluted to A12-H12
Hole, thus obtains multiple slender hilums, when the cell in slender hilum covers with single hole, and it is homogeneous to select cellular morphology, raw
Long cell line amplification of good performance.
The cell line obtained to being screened through excessive wheel cells monoclonalization carries out H5 subtype avian influenza virus HA subunits epidemic disease
The propagation of seedling, using culture HA potency as examination criteria, screening obtains the shaft-like H5 subtype avian influenza virus HA subunits of propagation
The ability of vaccine reaches 11-12log2 cell line as expressing recombination expression H5 subtype avian influenza virus HA subunits epidemic disease
The HiFi cells of seedling are standby.
Finally, we also disclosed production of vaccine craft condition, comprise the following steps,（1）Cultivate for breeding H5
The Sf9-HA cells of subtype avian influenza virus HA subunit vaccines poisoning；（2）Kind of a poison is inoculated in Sf9-HA cells, and cultivates increasing
Grow, harvest kind poison；（3）Cultivate the HiFi-HA cells for breeding H5 subtype avian influenza virus HA subunit vaccines；（4）It will plant
Poison is inoculated in HiFi-HA cells, and cultivates propagation, harvests H5 subtype avian influenza virus HA subunit vaccines.
Wherein, step（2）The middle inoculum concentration for planting poison is MOI0.1~1, step（4）The middle inoculum concentration for planting poison is MOI1~4.
Step（2）And step（4）In, the proliferation conditions of virus in the reactor are：Dissolved oxygen amount is satisfied for 40~60% solution
And oxygen dissolved；Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.
Specifically, this production of vaccine craft condition can be described as, and comprise the following steps：
(1) it is used for the culture for breeding the sf9-HA cells of H5 subtype avian influenza virus subunit vaccines kind poison：
a:Shaking flask breeds the method from suspension SF9-HA cells：The cell of recovery is resuspended in serum free medium,
From the culture that suspends in 100mL shaking flasks, after culture 96h, according to 1:4 ratio is passed on；
b:Method of the stirring reactor from the propagation SF9-HA cells that suspend：By the SF9-HA cells of amplification according to 1~2 ×
106Cell/mL initial density is seeded in fresh serum-free media；Dissolved oxygen amount is 40~60% solution saturation oxygen dissolved；
Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate after 72~96h, cell, which reaches, can be inoculated with H5
Subtype avian influenza virus subunit vaccine prepares kind of a requirement 2~4 × 10 for poison6cell/mL；
(2) propagation and harvest of poison are planted：After cell reaches that inoculation is required in step 1, follow the steps below：Poison will be planted according to MOI
0.1~1 inoculum concentration is entered in reactor by liquid supply device cultivates, while adding fresh serum-free media to original cultivates body
Long-pending 2 times, continue to cultivate；The condition of the propagation of virus in the reactor：The solution saturation oxygen dissolved of dissolved oxygen amount 40~60%；Stir
Speed is mixed for 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate after 72~96h, plant toxic effect valency and reach
108.5TCID50/ mL, reaches that the later stage connects the standard of poison, Cord blood is standby.
(3) it is used for the culture for breeding the HiFi-HA cells of H5 subtype avian influenza virus subunit vaccines：
Shaking flask breeds the method from suspension HiFi-HA cells：The cell of recovery is resuspended in serum free medium, in 100mL
From the culture that suspends in shaking flask, after culture 96h, according to 1:5 ratio is passed on；
Method of the stirring reactor from the propagation HiFi-HA cells that suspend：By the HiFi-HA cells of amplification according to 1~2 ×
106Cell/mL initial density is seeded in fresh serum-free media；Dissolved oxygen amount is 40~60% solution saturation oxygen dissolved；
Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate after 72~96h, cell, which reaches, can be inoculated with H5
2~4 × 106cell/mL of requirement of subtype avian influenza virus subunit vaccine kind poison；
The propagation and harvest of H5 subtype avian influenza virus subunit vaccines：After cell reaches that inoculation is required in step 3, carry out following
Step：Kind of poison is entered in reactor according to MOI 1~4 inoculum concentration by liquid supply device and cultivated, while addition is fresh without blood
Clear culture medium continues to cultivate to 2 times of former volume of culture；The condition of the propagation of virus in the reactor：Dissolved oxygen amount 40~60%
Solution saturation oxygen dissolved；Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate after 72~96h, H5
Subtype avian influenza virus subunit vaccine HA potency reaches 211~12, meet seedling requirement.
Wherein Sf9-HA cells and HiFi-HA cells refer to the thin of the excessive wheel cells monoclonalization screening acquisition of foregoing warp
Born of the same parents' strain.
The preparation method of H5 subtype avian influenza virus HA subunit vaccines disclosed in this invention is overcome currently with chicken embryo
The deficiency of production of vaccine technology based on Tissue vaccine, serum free suspension culture can be realized in the reactor, is greatly reduced
Production cost, shortens growth cycle, can be reduced human resources with rapid expansion production scale, significantly improved the yield of vaccine
And quality.
Meanwhile, the present invention, which is provided in the incubation of H5 subtype avian influenza virus subunit vaccines, need not add serum,
It is safe, in the absence of the possibility polluted by exogenous virus.
In addition, present invention application bioreactor carries out production of vaccine, with high degree of automation, simple production process is steady
It is fixed, reduce artificial operation.Yield is big, takes up an area small.
Meanwhile, the present invention provides H5 subtype avian influenza virus subunit vaccine preparation methods, and this method is simple, safety, height
Effect, obtained virus liquid potency is high, steady quality, is adapted to industry amplification culture.
Finally, prepared by the H5 subtype avian influenza virus subunit vaccine obtained after technical scheme disclosed in this invention
After vaccine inoculation, antibody titer is high, and the duration is long, can effectively protect inoculation animal.
Brief description of the drawings
Fig. 1 is antibody titer testing result after vaccine immunity.
Embodiment
In order to be better understood from the present invention, below we in conjunction with specific embodiments to the present invention further explained
State.
Unless there are specified otherwise, reagent, instrument used etc. are commercially available prod in the embodiment of the present invention.
Material explanation
According to open source literature《The expression of H5 HA Gene of H 9 Subtype AIV rhabdovirus systems and activity identification》（Herding and beast
Cure the 3rd phase of volume 46 in 2014：15-19）Disclosed in mode obtain H5 subtype avian influenza virus subunit vaccines kind poison.
Serum free medium for Sf9-HA cell culture is GIBCO production Sf-900 II SFM.
Serum free medium for HiFi-HA cell culture is GIBCO production Express FiveTM SFM。
The SF9 cell lines of the high efficiently multiplying H5 subtype avian influenza virus subunit vaccines kind of embodiment 1 poison（SF9-HA）'s
Screening
SF9-HA cell lines are screened from Spodopterafrugiperda (Spodoptera frugiperda) ovary cell line SF9 and obtained.
Specific screening step is as follows：
SF-9 cells are adapted to and tamed in the culture medium of serum-free, and serum free medium produces Sf- using GIBCO companies
900™ II SFM。
It is passaged to after 2 ~ 4 generations, obtains substantially adapting to the SF9 cell lines of free serum culture, dispel cell dropper is resuspended
Afterwards, many wheel cells monoclonalization screenings are carried out, monoclonal screening technique is as follows：The cell suspension of 200ul volumes is added into 96 holes
The hole of the upper left corner one of plate, i.e. A1 holes,（1000 cells of cell number）, then with 8 road pipettors in all Kong Zhongjia 100ul cells
Nutrient solution.Mixed above and below A1 holes and suction out 100ul, add B1 holes, by that analogy, one-level dilution is carried out successively；With 8 road pipettors
From A1 ~ H1 holes, mix suction out 100ul up and down, add A2 ~ H2 holes, by that analogy, two grades of dilutions are carried out successively；In theory can be with
Obtain multiple slender hilums.Treat that slender hilum cell covers with single hole, select that cellular morphology is homogeneous, the good cell line of growth performance
Expand, freeze, it is standby.
It will screen and obtain the propagation that cell line carries out H5 subtype avian influenza virus subunit vaccines kind poison, with baculoviral
TCID50For examination criteria, screening obtains the cell line for being capable of high efficiently multiplying H5 subtype avian influenza virus subunit vaccines kind poison.
Cell after the screening of three-wheel monoclonal is after test detection, and the new SF9 cell lines propagation of acquisition is shaft-like
The ability of H5 subtype avian influenza virus subunit vaccines kind poison is from original 107.5TCID50/ mL increases to 108.5TCID50/
ML, sets up screening candidate cell strain cell bank；
Shaking culture：Candidate cell plant weight is hanged into the fresh culture of free serum culture, shaking flask concussion and cultivate, condition of culture
For：Rotating speed is that 60~100rpm, temperature are 27 DEG C；
Method of the stirring reactor from the propagation SF9-HA cells that suspend：By the SF9-HA cells of amplification according to 1~2 ×
106cell/mL initial density is seeded in fresh serum-free media；Dissolved oxygen amount is 40~60% solution saturation oxygen dissolved；
Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.
The HiFi cell lines of the high efficiently multiplying H5 subtype avian influenza virus subunit vaccines of embodiment 2（HiFi-HA）Sieve
Choosing
HiFi-HA cell lines are from cabbage looper Trichoplusiani egg cells system BTI-Tn-5B1-4（HiFi cell lines）Carefully
Screening is obtained in born of the same parents system.Specific screening step is as follows：
HiFi cells are adapted to and tamed in the culture medium of serum-free, and serum free medium is the production of GIBCO companies
Express FiveTMSFM。
It is passaged to after 2~4 generations, obtains substantially adapting to the HiFi cell lines of free serum culture, blow cell dropper is resuspended
After dissipating, many wheel cells monoclonalization screenings are carried out, the acquisition pattern of monoclonal cell strain is identical with SF9-HA, selects cellular morphology
Homogeneous, the good cell line of growth performance expands, freezes, standby.
It will screen and obtain the propagation that cell line carries out H5 subtype avian influenza virus subunit vaccines, with culture HA potency
For examination criteria, screening obtains the HiFi cell lines for being capable of high efficiently multiplying H5 subtype avian influenza virus subunit vaccines.
Cell after the screening of three-wheel monoclonal is after test detection, the new HiFi-HA cell lines propagation of acquisition
The ability of shaft-like H5 subtype avian influenza virus subunit vaccine kind poison is improved to 11~12log2 from 9 original~10log2, is built
Vertical screening candidate cell strain cell bank；
Shaking culture：Candidate cell plant weight is hanged into the fresh culture of free serum culture, shaking flask concussion and cultivate, condition of culture
For：Rotating speed is that 60~100rpm, temperature are 27 DEG C；
Method of the stirring reactor from the propagation HiFi-HA cells that suspend：By the HiFi-HA cells of amplification according to 1~2 ×
106cell/mL initial density is seeded in fresh serum-free media；Dissolved oxygen amount is 40~60% solution saturation oxygen dissolved；
Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.
Embodiment 3 prepares the mode of production of H5 subtype avian influenza virus subunit vaccines on a large scale, comprises the following steps：
Prepare the passage and culture of kind of poison cell：Shaking culture, which suspends, cultivates SF9-HA cells, treats that cell density reaches 4~6
×106Sub-bottle passage is carried out during cell/mL, condition of culture is：Rotating speed is that 60~100rpm, temperature are 27 DEG C；Treat cell density
4~6 × 10 are reached again6During cell/mL, the culture that suspends certainly is carried out in bioreactor for continuing to pass on or be inoculated in, instead
Answer device condition of culture：1~2 × 106Cell/mL cell initial densities dissolved oxygen amount is 40~60% solution saturation oxygen dissolved；Stirring
Speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.
The propagation of cell kind poison：SF9-HA enters after bioreactor culture 24h, and recombinant virus is seeded in reactor.Will
Plant poison and culture in reactor is entered by liquid supply device according to MOI 0.1~1 inoculum concentration, while adding fresh serum free culture
Base continues to cultivate to 2 times of former volume of culture；The condition of the propagation of virus in the reactor：The solution of dissolved oxygen amount 40~60% is satisfied
And oxygen dissolved；Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate after 72~96h, plant toxic effect valency
Reach 108.5TCID50/ mL, reaches that the later stage connects the standard of poison, Cord blood is standby.
Produce the passage and culture of vaccine cell：Shaking culture, which suspends, cultivates HiFi-HA cells, treats that cell density reaches
4~6 × 106Sub-bottle passage is carried out during cell/mL, condition of culture is：Rotating speed is that 60~100rpm, temperature are 27 DEG C；Treat cell
Density reaches 4~6 × 10 again6During cell/mL, carried out for continuing to pass on or be inoculated in bioreactor from the training that suspends
Support, bioreactor culture condition：1~2 × 106Cell/mL cell initial densities dissolved oxygen amount is 40~60% solution saturation dissolved oxygen
Degree；Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.
The propagation of seedling venom：HiFi-HA enters after bioreactor culture, and recombinant virus is seeded in reactor.It will plant
Poison is entered in reactor according to MOI 0.1~1 inoculum concentration by liquid supply device to be cultivated, while adding fresh serum-free media
To 2 times of former volume of culture, continue to cultivate；The condition of the propagation of virus in the reactor：The solution saturation of dissolved oxygen amount 40~60%
Oxygen dissolved；Mixing speed is 60~100rpm；PH is 6.0~6.5；Temperature is 27 DEG C.Cultivate 72~96h after, connect after poison every
A period of time sampling observation, with microscope observation of cell lesion situation, and detects the HA potency of cell culture, treats the big portion of cell
Separate existing lesion（It is enlargement, broken）, and oxygen dissolving value is in obvious ascendant trend, stops reactor stirring, harvests antigen.
The inspection of semifinished product of embodiment 4 and seedling, product inspection：
a：The measure of HA-HI test：After the cell suspension multigelation harvested by more than 3 times, hemagglutinative titer is determined.Hemagglutinative titer >=
11log2。
b:Sterility testing：According to《People's Republic of China's veterinary biologicses quality standard》Annex progress of page 301, nothing
Bacterial growth.
c:The configuration of oil-adjuvant vaccine：Oil phase is prepared according to injection white oil（See《People's Republic of China's biology for animals
Quality of item standard》Annex page 343）96 parts, 4 parts of Si Ben -80, the proportional arrangement oil phase of 2 parts of aluminum stearate.Take aluminum stearate,
Mixed with a small amount of injection white oil, heating and melting is mixed to translucent, then with full dose Si Ben -80 and remaining injection white oil
It is even, through 121 DEG C of sterilizing 15min, room temperature is cooled to, it is standby.Aqueous phase, which prepares to learn from else's experience, examines qualified H5 subtype avian influenza virus sub-
Subunit vaccine, Tween-80 emulsification 1min is added according to the 4% of virus liquid.By aqueous phase and oil phase according to 1:3（Volume ratio）Ratio is entered
Row emulsification, 1000rpm × 3~5min.
The vaccine finished product detection of embodiment 5
a：Character outward appearance is white " milky " liquid；Formulation is in water-in-oil type；Stability 37 DEG C preserve 21 days or with 3000rpm ×
15min, for demulsification；The viscosity clean suction pipes of 1mL（It is upper it is intraoral through 2.7mm, under it is intraoral through 1.2mm）, the epidemic disease of 25 DEG C or so of absorption
Seedling 1mL, makes its vertical natural flow out, the time required to record outflow 0.4mL, within 8 seconds.
b：Sterility testing is pressed《People's Republic of China's veterinary biologicses quality standard》Annex progress of page 301, as a result：Nothing
Bacteria growing；
c:Safety verification is with 3 ~ 5 week old SPF（Specefic Pathogen Free, i.e. specific pathogen free）Chicken 10, subcutaneous,
The plumage part of intramuscular injection vaccine 2（0.4mL）, observe 14 days.Chicken all survives, cut open inspection injection site, without caused by vaccine injection
Serious local reaction, such as red and swollen, vaccine residual.
d：Effect is detected（Serum titer）
With 3~5 week old SPF（Specefic Pathogen Free, i.e. specific pathogen free）Chicken 10, subcutaneous, intramuscular injection epidemic disease
The plumage part of seedling 1（0.2mL), while setting up similar commercially available vaccine and each 10 of blank control.After 14 days, 21 days, 28 days, each group is gathered
Serum simultaneously determines Serum HI antibody potency.Concrete outcome is shown in Fig. 1.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710140542.2A CN106957860A (en) | 2017-03-10 | 2017-03-10 | The preparation method of H5 subtype avian influenza virus HA subunit vaccines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710140542.2A CN106957860A (en) | 2017-03-10 | 2017-03-10 | The preparation method of H5 subtype avian influenza virus HA subunit vaccines |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106957860A true CN106957860A (en) | 2017-07-18 |
Family
ID=59470715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710140542.2A Pending CN106957860A (en) | 2017-03-10 | 2017-03-10 | The preparation method of H5 subtype avian influenza virus HA subunit vaccines |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106957860A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1781553A (en) * | 2004-11-29 | 2006-06-07 | 中国农业科学院哈尔滨兽医研究所 | Subunit vaccine of H5N1 subtype bird flu virus gene ergineering and its preparing method |
| US20070184526A1 (en) * | 2003-07-11 | 2007-08-09 | Gale Smith | Functional influenza virus like particles (VLPs) |
| CN101816785A (en) * | 2010-04-29 | 2010-09-01 | 扬州优邦生物制药有限公司 | Preparation method and product of H9N2 subtype avian influenza inactivated vaccine |
-
2017
- 2017-03-10 CN CN201710140542.2A patent/CN106957860A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070184526A1 (en) * | 2003-07-11 | 2007-08-09 | Gale Smith | Functional influenza virus like particles (VLPs) |
| CN1781553A (en) * | 2004-11-29 | 2006-06-07 | 中国农业科学院哈尔滨兽医研究所 | Subunit vaccine of H5N1 subtype bird flu virus gene ergineering and its preparing method |
| CN101816785A (en) * | 2010-04-29 | 2010-09-01 | 扬州优邦生物制药有限公司 | Preparation method and product of H9N2 subtype avian influenza inactivated vaccine |
Non-Patent Citations (4)
| Title |
|---|
| 张焜 等: "《新兴产业和高新技术现状与前景研究丛书 生物制药及工程技术现状与应用前景》", 31 May 2015, 广州：广东经济出版社 * |
| 查国飞 等: "H5亚型禽流感病毒HA基因杆状病毒系统的表达及活性鉴定", 《畜牧与兽医》 * |
| 梁红: "《应用植物学》", 29 February 1996 * |
| 王新华等: "《禽流感诊断及防制手册》", 31 March 2004 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105311630B (en) | A kind of mammalian cell suspension culture prepares the method and application of vaccine | |
| CN101979519A (en) | Method for preparing pseudorabies vaccines | |
| CN102091329B (en) | Preparation method of inactivated porcine parvovirus vaccine and product thereof | |
| CN101869702B (en) | Vaccine produced by suspension microcarrier cell culture system and method thereof | |
| CN102406926A (en) | Virus preparation or vaccine production method by culturing cells with polyester fiber carrier | |
| CN110093307A (en) | The method for adapting to the BHK-21-SC cell strain of serum free suspension culture and preparing vaccine antigen with the cell strain | |
| CN101955915B (en) | Method for producing influenza virus vaccine | |
| CN104726392B (en) | A kind of method of the suspension mammalian cell system preparing free serum culture and its cell line prepared and application | |
| CN107904215A (en) | A kind of full suspension culture method of avian influenza virus | |
| CN114317405A (en) | Serum-free full-suspension culture type F81 cell line and construction method and application thereof | |
| CN108220227A (en) | A kind of method for the culture newcastle disease virus that suspended by the continuous cell line that suspends entirely | |
| CN108396006A (en) | A kind of BHK-21 cell strains and its cultural method and purposes | |
| CN104027798B (en) | Method for culturing and producing PVC 2 antigen through whole suspension cells | |
| CN104232564A (en) | Culture medium suitable for preparing hog cholera vaccines from ST cell and using method thereof | |
| CN104689311A (en) | Method for producing avian encephalomyelitis virus inactivated vaccine | |
| CN105274064B (en) | A kind of duck tembusu virus attenuated vaccine strain and its application | |
| CN102886043B (en) | Binary inactivated vaccine against Japanese encephalitis virus and porcine parvovirus and preparation method thereof | |
| CN105838683A (en) | Method for proliferation of mink canine distemper virus by applying novel cell microcarrier | |
| CN104004720A (en) | Method for producing porcine circovirus type 2 antigens in large scale with high density | |
| CN108421037A (en) | A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends | |
| CN108853489B (en) | A method of PEDV attenuated vaccine is produced using serum free medium | |
| CN106801031A (en) | Without Tumor formation mdck cell clone strain | |
| CN106957860A (en) | The preparation method of H5 subtype avian influenza virus HA subunit vaccines | |
| CN103320393B (en) | Method for culturing gosling plague virus by use of goose embryo continuous cell line and bioreactor | |
| CN105816872A (en) | Preparation method of mink parvoviral enteritis inactivated vaccine and vaccine prepared by using same |
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 |
Application publication date: 20170718 |
|
| RJ01 | Rejection of invention patent application after publication |