CN104531624B - A kind of method for cultivating mdck cell propagation restructuring H5N1 subtype avian influenza virus - Google Patents
A kind of method for cultivating mdck cell propagation restructuring H5N1 subtype avian influenza virus Download PDFInfo
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- CN104531624B CN104531624B CN201410810577.9A CN201410810577A CN104531624B CN 104531624 B CN104531624 B CN 104531624B CN 201410810577 A CN201410810577 A CN 201410810577A CN 104531624 B CN104531624 B CN 104531624B
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Abstract
The invention provides a kind of method for cultivating mdck cell propagation restructuring H5N1 subtype avian influenza virus, its step is：The preparation of chip carrier；Scraps of paper Vehicle element；Bioreactor sterilizes and preculture；Cell is inoculated with and the absorption on scraps of paper carrier；Cell is continuously cultivated；Avian influenza virus is inoculated with；Virus liquid harvests.The present invention has the beneficial effect that：It is that carrier cost is relatively low; the virus liquid HA-HI test (HA) of acquisition is high, and carrying out bioreactor pilot scale culture mdck cell propagation restructuring H5N1 subtype avian influenza virus using scraps of paper carrier is easier to realize the advantages that large-scale culture, production line occupy little space, production efficiency is high, used equipment, strong, reproducible stability.
Description
Technical field
The present invention relates to a kind of method for breeding avian influenza virus, especially a kind of scraps of paper carrier organism reactor scale
The method for cultivating mdck cell propagation avian influenza virus.
Background technology
Bird flu (Avian influenza, AI) is a kind of birds deadly infectious disease as caused by A type avian influenza virus,
A class infectious diseases are set to by International Office of Epizootics, it is also classified as a kind of Animal diseases by China.At present, vaccine immunity is anti-both at home and abroad
The major measure that bird flu breaks out is controlled, most of producers produce avian influenza vaccine, the technique by matrix of SPF chicken embryos at present in China
There is very big dependence to SPF chicken embryos；The virus liquid titre of institute's output is not high；Incubation conditional is not homogeneous enough, difference between batch
It is different larger；Because chicken embryo pollution condition is inevitable, causes endotoxin in vaccine higher, immune effect is made a big impact；
Chicken embryo production simultaneously causes substantial amounts of chicken embryo discarded object, to ecological environment and very big pollution.
Carrier large-scale culture animal cellular proliferation avian influenza virus is used in bioreactor, it has also become a kind of new life
The expanded application of production mode, but most producers carry out virus multiplication using microcarrier at present, and microcarrier is mostly the life of GE companies of the U.S.
Production, cost is very high, simultaneously because the addition of microcarrier limits in per unit nutrient solution, causes unit volume inner cell density not
Height, so as to which the antigen of high-titer can not be obtained.And the invention of the technique can not only overcome the drawbacks of being produced with chicken embryo, while again
Dependence of the bioreactor to microcarrier is changed, substantially reduces carrier cost, it is final to obtain high-titer antigen liquid, further
Production cost is reduced, has revolutionary innovation to whole avian influenza vaccine traditional processing technology.
The content of the invention
In order to solve the problems, such as exist with during spinner culture animal cellular proliferation avian influenza virus, the invention provides
A kind of process of scraps of paper carrier organism reactor large-scale culture mdck cell propagation restructuring H5N1 subtype avian influenza virus.
Method of the present invention, the titre of avian flu venom is remarkably improved, reduces mass discrepancy between batch, it is several to reduce microbiological contamination
Rate, endotoxin content is controlled, and this method is easily enlarged production scale, carries out intensive manufacture, available for significantly improving vaccine
Yield and quality.
The present invention provides a kind of scraps of paper carrier organism reactor large-scale culture mdck cell propagation restructuring H5N1 hypotype fowl
The method of influenza virus, it includes step in detail below：
1) preparation of chip carrier：The chip carrier can be a) by polyester fiber scraps of paper carrier by cutting out, and make
Long 10cm, wide 2cm waveform；B) scraps of paper carrier is done into growth 10cm, wide 2cm strip by cutting out；C) scraps of paper are carried
Body makes the one or more in diameter 2cm circle by cutting out.
2) scraps of paper Vehicle element：The sodium hydroxide for the scraps of paper carrier 0.5mol/L-1.5mol/L that step 1) is obtained
Solution soaks 12-36h, and it is 7.2-7.6 then to be cleaned with water for injection to pH, bleeds off water for injection, adds the immersion of PBS liquid and protects
Deposit.
3) bioreactor sterilizing and preculture：The scraps of paper carrier of step 2) processing will be passed through according to 15-30g/L use
Amount is added in bioreactor, adds the PBS liquid of submergence scraps of paper carrier, is steamed online after connecting all pipelines of bioreactor
Vapour sterilizes：121 DEG C, 30min, cell culture fluid is added after the completion of sterilizing and carries out preculture 12-24h.
4) cell inoculation and the absorption on the scraps of paper：It is 1 × 106-2 by cell density after reactor preculture is without exception
The cell suspension of × 107cells/g scraps of paper carriers is added in bioreactor by sterile pipes, and regulation motor rotating speed is 20-
40rpm, be advantageous to cell attachment on scraps of paper carrier.Adjusting culture parameters simultaneously is：PH7.0-7.4,37 ± 0.2 DEG C of temperature,
DO50%-80%, attaching time of the cell on scraps of paper carrier are 3-6h.
5) cell amplification cultivation：The cell of step 4) enters normal continuous cultivation conditions, adjustment after the attaching of scraps of paper carrier
Bioreactor culture parameter is：Rotating speed 60-90rpm, 37 ± 0.2 DEG C of temperature, pH7.0-7.4, DO 30%-70%.Because the scraps of paper carry
The surface area of body is larger, and during amplification cultivation, cell quantity can be in quantity multiplication by stages, so needing not timing in incubation
Sampling, the residual concentration of glucose in cell growth medium is determined, the sugar consumption rate in cell growth process is calculated, according to the consumption of cell
Sugared speed calculates the upgrowth situation of cell, in time adds glucose or changes fresh medium.
6) avian influenza virus inoculation and breeding：After the cell density of step 5) reaches requirement, bleed off thin in reactor
Born of the same parents' nutrient solution, avian influenza virus inoculation liquid and serum-free maintaining liquid are added by sterile pipes, adjustment bioreactor culture parameter is：
Rotating speed 60-90rpm, 35 DEG C -37 DEG C of temperature, pH7.0-7.4, DO 30%-70%, carry out virus multiplication culture.Inoculum concentration according to
The 1%-2% inoculations of total working volume.
7) virus liquid harvests：After step 6) inoculation avian influenza virus 12h, sampled every 6h, determine the HA effects of virus liquid
Valency, harvested after needed for its HA potency reaches, it is qualified after testing to be used afterwards as seedling antigen liquid.
Avian flu venom HA titre >=1 obtained by the inventive method:1024.
The scraps of paper carrier is polyester fiber class scraps of paper carrier.
Wherein, seed cell is supplied as：With rolling bottle amplification cultivation MDCK- α -2,3Gal cells, form health, life are selected
Cell suspension is made in long good cell pancreatin digestion, and carries out cell count, the kind as scraps of paper carrier organism reactor
Daughter cell.
Beneficial effects of the present invention are：
The present invention uses scraps of paper carrier large-scale culture animal cellular proliferation avian influenza virus in bioreactor, and cell expands
Increase comparatively fast, unit volume inner cell quantity is big, and output virus liquid titre is higher；Parameter in incubation can monitor, whole training
It is homogeneous to support process condition, the cell moment is in optimum growh state, differences between batches are small；Simultaneously because bioreactor respectively connects
Pipeline is closed conduit, and the probability of microbiological contamination is small, the quality of easily controllable product, substantially increases suitable production, the applicability of product
And biological safety.
Embodiment
Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments and
Purpose is only used for enumerating the present invention, not forms any type of any restriction to the real protection scope of the present invention, more non-to incite somebody to action
Protection scope of the present invention is confined to this.
Embodiment 1
A kind of method for cultivating mdck cell propagation restructuring H5N1 subtype avian influenza virus, it comprises the following steps：
Seed cell is supplied：With 7 liters of microcarrier bioreactor amplification cultivation MDCK- α -2,3Gal cells, form is selected
Cell suspension is made in healthy, well-grown cell pancreatin digestion, and carries out cell count, is reacted as scraps of paper carrier organism
The seed cell of device.
1) preparation of chip carrier：By polyester fiber scraps of paper carrier by cutting out as long 10cm, wide 2cm waveform paper
Piece.
2) scraps of paper Vehicle element：The chip carrier that step 1) obtains soaks 16h with 0.5mol/L sodium hydroxide solution,
Then it is 7.6 to be cleaned with water for injection to pH, and after bleeding off water for injection, it is stand-by to add the immersion of PBS liquid.
3) bioreactor sterilizing and preculture：The scraps of paper carrier that step 2) processing will be passed through is added in bioreactor,
The fresh PBS liquid that can submerge scraps of paper carrier is added, reactor is installed and carries out online steam after connecting all pipelines
Sterilizing, sterilizing are arranged to 121 DEG C, 25min.Sterilizing is completed, and question response device pot temperature passes through baiting valve after dropping to less than 40 DEG C
PBS liquid is bled off, adding cell culture fluid by inlet valve carries out preculture, and it is 35 DEG C to set preculture temperature, and incubation time is
20h。
4) cell inoculation and the absorption on the scraps of paper：After reactor preculture sampling detection nutrient solution, determine it is without exception after,
The seed cell that 1.6 × 107 cel ls/g scraps of paper carriers will be counted as is added in bioreactor by sterile pipes, regulation
Motor speed is 20rpm, is advantageous to cell attachment on scraps of paper carrier.The control of reactor all conditions, regulation training are opened simultaneously
Foster parameter is：PH7.2,37 ± 0.2 DEG C of temperature, DO 60%.
5) cell amplification cultivation：Under the slow-speed of revolution after the cell of step 4) attaches 3h on scraps of paper carrier, into normally connecting
Continuous cultivation conditions, adjustment bioreactor culture parameter are：Rotating speed 60rpm, 37 ± 0.2 DEG C of temperature, pH7.2, DO 60%.Amplification training
During supporting, not timing sampling, the glucose content in nutrient solution is determined, according to the Expenditure Levels of sugar, more renewed after cultivating 46h
Fresh nutrient solution.
6) avian influenza virus is inoculated with：After the cell growth of step 5) reaches certain density, the cell in reactor is bled off
Nutrient solution, recombinant fowl influenza virus H5N1 hypotype Re-6 strains production kind of poison and a serum-free maintaining liquid are added by sterile pipes,
The malicious usage amount of kind is the 1.5% of reactor total working volume, and adjustment bioreactor culture parameter is：Rotating speed 60rpm, 36 DEG C of temperature,
PH7.2, DO 60%, carry out avian influenza virus Multiplying culture.
7) virus liquid harvests：After step 6) inoculation avian influenza virus 12h, sampled every 6h, determine the HA effects of virus liquid
Valency, 36h virus liquid HA potency reaches 1 after connecing poison:Harvested after 1024, " recombinant fowl influenza virus (H5N1 Asias are announced according to the Ministry of Agriculture
Type) inactivated vaccine (cell source, Re-6 strains) manufactures and inspection Trial Regulation " stand-by as antigen for vaccine liquid after the assay was approved.
Embodiment 2
Diameter 2cm circular paper carrier is soaked into 12h with 1.0mol/L sodium hydroxide solution, then uses water for injection
Cleaning is 7.4 to pH, bleeds off water for injection, is added in bioreactor, while add PBS liquid.Connect bioreactor institute
Have and carry out 121 DEG C after pipeline, 30min steam sterilizings, sterilizing completion, to PBS liquid is bled off after less than 40 DEG C, adds carefully after tank temperature drop
Born of the same parents' nutrient solution, it is 36 DEG C of preculture 16h that regulation reactor, which controls temperature,.Select form health, growth after microcarrier amplification cultivation
Good MDCK- α -2,3Gal cells, the cell that density is 1.9 × 107 cells/gram scraps of paper carrier is made with pancreatin digestion and hangs
Liquid, it is inoculated in bioreactor without exception after preculture, regulation reactor rotating speed is 35rpm, while opens reactor control
Condition processed, regulation culture parameters are：PH7.3,37 ± 0.2 DEG C of temperature, DO 50%.After cell attaches 4h on scraps of paper carrier, adjust
It is rotating speed 60rpm, 37 ± 0.2 DEG C of temperature, pH7.3, DO 50% to save bioreactor culture parameter, into continuous cultivation conditions.Culture
During timing sampling measure nutrient solution in glucose content, change fresh medium after 40h after inoculation.
After cell amplification reaches certain density, the cell culture fluid in reactor is bled off, is inoculated with recombinant fowl influenza virus
H5N1 hypotype Re-5 strains production kind of a poison, while serum-free cell maintaining liquid is added, virus inoculation amount is reactor total working body
Long-pending 1.0%.It is rotating speed 80rpm, 36 DEG C of temperature, pH7.3, DO 50% to adjust bioreactor culture parameter, carries out virus multiplication training
Support.During virus amplification, timing sampling carries out the HA detections of virus liquid, and 48h virus liquid HA potency reaches 1 after connecing poison:
Harvested after 1024, through being used after the assay was approved as seedling antigen liquid.
Embodiment 3
Diameter about 2cm circular paper carrier is soaked into 12h with 0.5mol/L sodium hydroxide solution, then uses injection
It is 7.6 that water, which is cleaned to pH, bleeds off water for injection, is added in bioreactor, while add PBS liquid.Connect bioreactor
Carry out 121 DEG C after all pipelines, 30min steam sterilizings, sterilizing completion after tank temperature drop to bleeding off PBS liquid after less than 40 DEG C, addition
Cell culture fluid, it is 36 DEG C of preculture 24h that regulation reactor, which controls temperature,.Form is healthy, raw after selecting microcarrier amplification cultivation
Long good MDCK- α -2,3Gal cells, the cell that density is 2.0 × 107 cells/gram scraps of paper carrier is made with pancreatin digestion
Suspension, it is inoculated in bioreactor without exception after preculture, regulation reactor rotating speed is 40rpm, while opens reactor
Control condition, regulation culture parameters are：PH7.4,37 ± 0.2 DEG C of temperature, DO 50%.After cell attaches 4h on scraps of paper carrier,
It is rotating speed 70rpm, 37 ± 0.2 DEG C of temperature, pH7.4, DO 50% to adjust bioreactor culture parameter, into continuous cultivation conditions.Training
Glucose content during supporting in timing sampling measure nutrient solution, fresh medium is changed after inoculation after 42h.
After density needed for cell amplification reaches, the cell culture fluid in reactor is bled off, is inoculated with recombinant fowl influenza virus
H5N1 hypotype Re-4 strains production kind of a poison, while serum-free cell maintaining liquid is added, virus inoculation amount is reactor total working body
Long-pending 1.2%.It is rotating speed 90rpm, 36 DEG C of temperature, pH7.4, DO 50% to adjust bioreactor culture parameter, carries out virus multiplication training
Support.During virus amplification, timing sampling carries out the HA detections of virus liquid, 36h and 42h virus liquid HA potency reaches after connecing poison
1:Harvested after 1024, through being used after the assay was approved as seedling antigen liquid.
Claims (2)
- A kind of 1. method for cultivating mdck cell propagation restructuring H5N1 subtype avian influenza virus, it is characterised in that including following step Suddenly：1) preparation of chip carrier：The carrier can be prepared as, a) by polyester fiber scraps of paper carrier by cutting out, being growth 10cm, Wide 2cm waveform；B) scraps of paper carrier is done into growth 10cm, wide 2cm strip by cutting out；C) scraps of paper carrier is passed through Cut out, make diameter 2cm circle；2) scraps of paper Vehicle element：The sodium hydroxide solution for the scraps of paper carrier 0.5mol/L-1.5mol/L that step 1) is obtained 12-36h is soaked, it is 6.8-7.6 then to be cleaned with water for injection to pH, bleeds off water for injection, adds the immersion of PBS liquid and preserves；3) bioreactor sterilizing and preculture：Scraps of paper carrier by step 2) processing is added according to 15-30g/L usage amount Enter in bioreactor, add the PBS liquid of submergence scraps of paper carrier, online steam is carried out after connecting all pipelines of bioreactor Sterilizing：121 DEG C, 30min, cell culture fluid is added after the completion of sterilizing and carries out preculture 12-24h；4) cell inoculation and the absorption on scraps of paper carrier：By seed cell with 2 × 105-2×107Cells/g scraps of paper carriers Amount is added in scraps of paper carrier organism reactor by sterile pipes, and seed cell is loaded into after bioreactor and adjusts culture ginseng Number, rotating speed 20-40rpm, pH7.0-7.4,37 ± 0.2 DEG C of temperature, DO 50%-80%, is advantageous to cell and is pasted on scraps of paper carrier It is attached；5) cell is continuously cultivated：The cell that step 4) obtains enters normal continuous cultivation conditions, adjustment after scraps of paper carrier adsorption Bioreactor culture parameter is：Rotating speed 60-90rpm, 37 ± 0.2 DEG C of temperature, pH7.0-7.4, DO 30%-70%；6) avian influenza virus is inoculated with：After the requirement that the cell density of step 5) reaches inoculation avian influenza virus, reactor is bled off Interior cell culture fluid, adds avian influenza virus inoculation liquid and serum-free maintaining liquid, and adjustment bioreactor culture parameter is：Rotating speed 60-90rpm, 35 DEG C -37 DEG C of temperature, pH7.0-7.4, DO 30%-70%, carry out virus multiplication culture；7) virus liquid harvests：Virus liquid is harvested after potency needed for the virus liquid of step 6) reaches, it is qualified rear as system after testing Seedling antigen liquid uses.
- 2. the method as described in claim 1, it is characterised in that gained avian flu venom HA titre >=1:1024.
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| CN102861329A (en) * | 2012-09-19 | 2013-01-09 | 武汉中博生物股份有限公司 | Production method of canine parvovirus inactivated vaccine by utilizing bioreactor |
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| CN101875917B (en) * | 2009-04-28 | 2012-07-04 | 北京清大天一科技有限公司 | Method for producing swine fever vaccines by using animal cells cultured by micro-carriers of bio-reactor |
| CN102205116A (en) * | 2010-03-29 | 2011-10-05 | 杭州安普生物工程有限公司 | Method for producing vaccine by virtue of culturing animal cells |
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| CN102861329A (en) * | 2012-09-19 | 2013-01-09 | 武汉中博生物股份有限公司 | Production method of canine parvovirus inactivated vaccine by utilizing bioreactor |
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| 应用昆虫细胞表达预防性人乳头瘤病毒疫苗和应用填充床生物反应器表达H1N1流感病毒疫苗的研究;孙博;《中国博士学位论文全文数据库》;20130315(第2013/03期);E059-30 * |
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