CN105749270A - Rotavirus vaccine and preparation method thereof - Google Patents

Rotavirus vaccine and preparation method thereof Download PDF

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CN105749270A
CN105749270A CN201610122156.6A CN201610122156A CN105749270A CN 105749270 A CN105749270 A CN 105749270A CN 201610122156 A CN201610122156 A CN 201610122156A CN 105749270 A CN105749270 A CN 105749270A
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rotavirus
mrc
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张改梅
甘建辉
刘建凯
肖坚
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SHENZHEN KANGTAI BIOLOGICAL PRODUCTS CO Ltd
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SHENZHEN KANGTAI BIOLOGICAL PRODUCTS CO Ltd
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    • A—HUMAN NECESSITIES
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12351—Methods of production or purification of viral material

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Abstract

The invention discloses a rotavirus vaccine and a preparation method. The preparation method comprises the following steps: performing adaptive culture on the rotavirus on MRC-5 cells for continuous 10-12 generations; after recovery of the MRC-5 cells, when the MRC-5 cells grow to a compact monolayer, performing secondary culture for 3-4 days according to a ratio of 1:3 to1:4; adding a micro-carrier of which the concentration is 10-15g/L into a culture medium, and inoculating the MRC-5 cells with an initial density of 0.5-2*10<6>cells/ml after secondary culture to a bioreactor to ensure that the cells grow on the micro-carrier; when the MRC-5 cells grow to have the density of 1-3*10<7>cells/ml, inoculating serum type rotavirus activated by pancreatin to infect the MRC-5 cells; and when the CPE reaches 80% or above, after obtaining a virus solution, preparing the virus solution into a multi-valence rotavirus live vaccine. The invention also discloses a corresponding rotavirus vaccine. The preparation method disclosed by the invention can greatly improve the safety of the rotavirus vaccine, the virus harvest yield and titer, save the labor cost and time cost, and greatly reduce the production cost, and can be efficiently applied to large-scale production of multi-valence rotavirus vaccines.

Description

Rotavirus Vaccine and preparation method thereof
Technical field
The present invention relates to living biological cell pharmaceutical field, particularly relate to a kind of Rotavirus Vaccine and preparation side thereof Method.
Background technology
The children at rotavirus main infection 3 monthly age to 5 years old, its infection rate up to 30%-65%, wherein 6 Monthly age occurs moderate and severe infection most in the children of 2 years old.According to the World Health Organization (WHO) report, When 2008, the whole world there are about 45.3 ten thousand children and dies from rotavirus gastroenteritis (RVGE), accounts for whole death The 5% of children, the death rate of less than 5 years old children is 86/,100,000.Wherein, China's rotavirus diarrhea is dead Rate accounts for the whole world 8%.Additionally, each province of China, city all have been reported that the record of rotavirus eruption and prevalence.Colyliform is sick Not only harm human life is healthy for poison diarrhoea, also society and family is caused huge economic loss.Therefore, In 2009, WHO advised that RV vaccine is included in its children planning immunity procedure, especially by all member states It is that rate of death caused by diarrhoea accounts for less than 5 years old gross children mortality and reaches the country of more than 10%.
Owing to the Serotypes of rotavirus is various, and advantage epidemic strain is being continuously updated and is changing, no Effective cross-protection is lacked between homologous serotype.Although presently commercially available Rotavirus Vaccine can counterweight Disease diarrhoea plays a good protection, but RV can not be prevented well to infect.Therefore, one bag is developed The multivalence RV vaccine including the modal several Serotypes of HRV is current main flow direction.
In multivalence Rotavirus Vaccine R&D process, how to improve virus titer and increase virus harvest amount is to close Key point.General conventional animal cell carries out virus multiplication.The most conventional zooblast large-scale cultivation method There are the cultivation of spinner culture, cell factory and bioreactor culture etc..Rolling bottle technology is traditional attached cell Culture technique, spinner culture has simple in construction, small investment, technology maturation, amplifies and only need to simply increase The advantages such as rolling bottle quantity.But also having its shortcoming: labour intensity is big, take up an area space greatly, unit volume provides thin The surface area of intracellular growth is little, and easily by environmental pollution, cell density is low, difference and differences between batches between bottle More difficult control etc., thus it is difficult to industrialization or large-scale production, it is also easy to appearance dirty by bacterium or other virus The vaccine quality hidden danger contaminated and cause, relates to bio-safety and public health problem.
Cell factory (Cell Factory) is a kind of to make use of cultivation table to greatest extent in limited space Face, thus save substantial amounts of hall space, it is not necessary to carry out what any old factory rehabilitation can realize enhancing production capacities Purpose.The NUNC cell factory that NUNC company of Denmark produces is to apply more cell factory system at present System.Can be used for the commercial scales such as vaccine, monoclonal antibody or bio-pharmaceuticals such as to produce, be particularly suitable for adherent Cell, it can also be used to suspend and cultivate, will not change the dynamic of cell growth from laboratory scale when being amplified Mechanical condition, it is possible to provide the specification of 1,2,10 and 40 layers makes amplification become simple, low stain risk, Saving space, culture surface ensures after tested most beneficial for cell attachment and growth.But cell factory also has one A little inconveniences used: as during vitellophag owing to cannot be carried out piping and druming or using scraper auxiliary, it is more difficult to will Cell all digests；Product price is more expensive, and sterilization recycles after needing to irradiate with Co 60, produces into Originally it is greatly increased；Additionally, harvest yield is less, clean inconvenience.
Microcarrier Culture Techniques is to utilize sheet or spherical microcarrier to make cell be attached to its surface, at biological respinse The technology cultivated in device.Highdensity microcarrier considerably increases the surface area that cell is cultivated, thus realizes producing The expansion of energy.Microcarrier is suitable to the various cultivations such as shaking flask, rolling bottle, agitator tank and WAVE bioreactor System.Using microcarrier to cultivate attached cell is the most rising a kind of training mode, its advantage It is: specific surface area is big；Employing even suspension is cultivated, and cultivates the advantage with adhere-wall culture by suspending and merges, Simplify the Detection & Controling of environmental factor；Culture medium utilization rate is high；Easily amplify；Labour intensity is little；Training Foster system takes up room little；Reduce cost and avoid polluting.But, existing employing microcarrier biological respinse It is the most immature that Rotavirus Vaccine technology cultivated by device, it is difficult to realizes large-scale production.
Summary of the invention
Technical problem solved by the invention is to provide the preparation method of a kind of Rotavirus Vaccine, the method Can increase substantially Rotavirus Vaccine security, virus harvest amount and titre, save labor cost and time Between cost, be substantially reduced production cost, efficient application is in the large-scale production of multivalence Rotavirus Vaccine.
The present invention is solved the technical problem that to be to provide a kind of Rotavirus Vaccine further, and this vaccine can be big Amplitude improves Rotavirus Vaccine security, virus harvest amount and titre, saves labor cost and becomes with the time This, be substantially reduced production cost, and efficient application is in the large-scale production of multivalence Rotavirus Vaccine.
In order to solve above-mentioned technical problem, the invention discloses below scheme:
The preparation method of a kind of Rotavirus Vaccine, comprises the following steps:
Step 1, rotavirus carries out adaptability cultivation on MRC-5 cell, passes 10-12 generation continuously；
Step 2, after recovery MRC-5 cell, when it grows to fine and close individual layer, is passed on by 1:3-1:4 Cultivate 3-4 days；
Step 3, adding concentration in the medium is the microcarrier of 10-15g/L, and inoculation initial density is 0.5-2 ×106The MRC-5 cell through Secondary Culture of cells/ml is to bioreactor so that it is on described microcarrier Growth；
Step 4, treats that MRC-5 cell grows to 1-3 × 107During cells/ml, inoculate the blood after pancreatin activates Clear type rotavirus so that it is infect MRC-5 cell；
Step 5, when CPE reaches more than 80%, after results virus liquid, is made into multivalence rotavirus Live vaccine.
Preferably, described microcarrier uses Cytodex I microcarrier of GE company, and described bioreactor is adopted With 310 type 14L biological reactor for cell culture of NBS company of the U.S..
Preferably, in described step 3, keep described bioreactor control parameter be: pH value 7.0-7.8, Temperature 35 DEG C-37 DEG C, dissolved oxygen 40%-60%, stir speed (S.S.) 20-75rpm.
Preferably, in described step 4, according to MOI=0.001-0.05 ratio virus inoculation, will stirring speed Rate controls at 25rpm, make virus uniform adsorption in cell surface, after about 1-2 hour, then by stir speed (S.S.) control System is 50～75rpm.
Preferably, described rotavirus carries out pancreatin activation by following steps:
Activator is added in virus liquid so that pancreatin final concentration of 10～30 μ g/ml, be placed in 37 DEG C of water-baths Acting on 70-90 minute, period rocks 2-3 time.
Preferably, in described step 3, continuous perfusion type is used to cultivate.
Preferably, described medium component includes the little ox blood of BME, Glu, sodium acid carbonate and 10% Clearly.
Preferably, in above steps, with MRC-5 cell described in Vero cell replacement；With M199, DMEM, sodium acid carbonate and 10% calf serum substitute described medium component.
Preferably, during described rotavirus is G1, G2, G3, G4, G9 type rotavirus arbitrarily the most at least A kind of.
Correspondingly, the invention also discloses a kind of Rotavirus Vaccine, prepare according to method as above.
The invention has the beneficial effects as follows:
Embodiments of the invention become fiber by using the microcarrier cultivation human embryo lung (HEL) that combines with bioreactor (MRC-5) cell proliferation rotavirus, thus increase substantially Rotavirus Vaccine security, virus receipts The amount of obtaining and titre, save labor cost and time cost, be substantially reduced production cost, and efficient application is in many In the large-scale production of valency Rotavirus Vaccine.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to enforcement In example or description of the prior art, the required accompanying drawing used is briefly described, it should be apparent that, describe below In accompanying drawing be only some embodiments of the present invention, for those of ordinary skill in the art, do not paying On the premise of going out creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 be the Rotavirus Vaccine of the present invention preparation method first embodiment in rotavirus at MRC-5 Adaptability on cell cultivates titre change schematic diagram.
Fig. 2 be the Rotavirus Vaccine of the present invention preparation method first embodiment in microcarrier suspension culture MRC-5 cell is at the growth conditions schematic diagram of different time.Wherein, A is MRC-5 cell attachment microcarrier 24h；B is MRC-5 cell attachment microcarrier 48h；C is MRC-5 cell attachment microcarrier 72h；D For MRC-5 cell attachment microcarrier 96h；E is MRC-5 cell attachment microcarrier 120h；F is MRC-5 Cell attachment microcarrier 144h.
Fig. 3 be the Rotavirus Vaccine of the present invention preparation method first embodiment in microcarrier suspension culture The growth curve schematic diagram of MRC-5 cell proliferation rotavirus infection different time；Wherein, A is that Vero is thin Born of the same parents attach microcarrier 24h；B is Vero cell attachment microcarrier 48h；C is Vero cell attachment microcarrier 72h；D is Vero cell attachment microcarrier 96h；E is Vero cell attachment microcarrier 120h；F is Vero Cell attachment microcarrier 144h.
Fig. 4 be the Rotavirus Vaccine of the present invention preparation method the second embodiment in microcarrier suspension culture Vero cell is at the growth conditions schematic diagram of different time；Wherein, A is cell after inoculation rotavirus 24h State；B is cell state after inoculation rotavirus 48h；C is cell state after inoculation rotavirus 72h； D is cell state after inoculation rotavirus 96h.
Fig. 5 be the Rotavirus Vaccine of the present invention preparation method the second embodiment in inoculate after rotavirus not With Vero cytopathy view under the time.
Fig. 6 be the Rotavirus Vaccine of the present invention preparation method the second embodiment in microcarrier suspension culture wheel Shape virus infects the growth curve schematic diagram of different time.
Detailed description of the invention
The first embodiment of the preparation method of the Rotavirus Vaccine that the present invention provides, this reality are described below in detail Execute example to realize flow process prepared by Rotavirus Vaccine and mainly comprise the steps that
In step 1, rotavirus carries out adaptability cultivation on MRC-5 cell, passes 10-12 generation continuously；
In step 2, after recovery MRC-5 cell, when it grows to fine and close individual layer, enter by 1:3-1:4 Row Secondary Culture 3-4 days；
In step 3, adding concentration in the medium is the microcarrier of 10-15g/L, and inoculation initial density is 0.5-2×106The MRC-5 cell through Secondary Culture of cells/ml is to bioreactor so that it is in described micro-load Grow on body；
In step 4, treat that MRC-5 cell grows to 1-3 × 107During cells/ml, inoculate and activate through pancreatin After each serotype rotavirus so that it is infect MRC-5 cell；
In steps of 5, when CPE reaches more than 80%, after results virus liquid, it is made into multivalence colyliform Viral lived vaccine.
Preferably, described rotavirus carries out pancreatin activation by following steps:
Activator is added in virus liquid so that pancreatin final concentration of 10～30 μ g/ml, be placed in 37 DEG C of water-baths Acting on 70-90 minute, period rocks 2-3 time.
Preferably, in described step 3, continuous perfusion type is used to cultivate.
When implementing, described medium component can include BME, Glu, sodium acid carbonate and 10% Calf serum.
When implementing, described rotavirus can be the various serotypes wheels such as G1, G2, G3, G4, G9 type Shape virus.
When implementing, described microcarrier can use Cytodex I microcarrier of GE company.CytodexTM Being that microcarrier is exclusively used in all kinds of zooblasts of cultivation, its volume of culture can be from more than several milliliters to 6000 liters. Application Cytodex microcarrier, it is possible to achieve the simple suspending cultivation of attached cell, every milliliter of nutrient solution Available millions of cells.
When implementing, described bioreactor can use 310 type 14L cells of NBS company of the U.S. to cultivate Bioreactor.BLOFLO310 type 14L biological reactor for cell culture be at present the most state-of-the-art dynamic, Plant cell culture systems, is characterized in: be suitable for adhere-wall culture cell and suspension cell；It is suitable for various animals Cell, insect cell and plant cell etc.；The incubation of perfusion type；Low (nothing) shearing force stirs, nothing Bubbles for aeration；Cell concentration is detected in real time by persistently detecting oxygen uptake rate；Its perfusion type is cultivated and is belonged to training continuously Supporting, cell is retained in reactor assembly, is continuously added fresh culture while results nutrient solution.Perfusion The major advantage that formula is cultivated is that the culture medium of continuous pouring can provide sufficient nutritional labeling, and takes away metabolism Product.Meanwhile, cell retains in the reactor, can reach the highest cell density.With additive method phase Ratio, the virus yield that perfusion type is cultivated can improve an order of magnitude, it is possible to is substantially reduced disappearing of labour Consumption.
Further, in described step 3, keep described bioreactor control parameter be: pH value 7.0-7.8, Temperature 35 DEG C-37 DEG C, dissolved oxygen 40%-60%, stir speed (S.S.) 20-75rpm.
Further, in described step 4, according to MOI=0.001-0.05 ratio virus inoculation, will stir Mix speed and control at 25rpm, make virus uniform adsorption in cell surface, after about 1-2 hour, then speed will be stirred Rate controls 50～75rpm.
As seen from Figure 1, rotavirus is Secondary Culture on MRC-5 cell, along with the increase of generation, Show the trend that virulence strengthens with multiplication capacity.
As seen from Figure 3, microcarrier suspension culture MRC-5 cell amplification rotavirus, virus titer with The increase of infection time and strengthen, and, have higher security, having becomes Rotavirus Vaccine and produces Cytostromatic application prospect.
The various serotype rotavirus related to the present embodiment below in conjunction with some examples are described in detail.
Example 1
Microcarrier suspension culture MRC-5 cell proliferation rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 10 that born of the same parents inoculate the initial density of bioreactor5Cells/ml, samples observation of cell every 24h after inoculation Upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Example 2
Microcarrier bioreactor suspends and cultivates MRC-5 cell proliferation G1 type rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 105cells/ml that born of the same parents inoculate the initial density of bioreactor, observes thin every 24h sampling after inoculation Born of the same parents' upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Example 3
Microcarrier bioreactor suspends and cultivates MRC-5 cell proliferation G2 type rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 10 that born of the same parents inoculate the initial density of bioreactor5Cells/ml, samples observation of cell every 24h after inoculation Upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Example 4
Microcarrier bioreactor suspends and cultivates MRC-5 cell proliferation G3 type rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 10 that born of the same parents inoculate the initial density of bioreactor5Cells/ml, samples observation of cell every 24h after inoculation Upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Example 5
Microcarrier bioreactor suspends and cultivates MRC-5 cell proliferation G4 type rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 10 that born of the same parents inoculate the initial density of bioreactor5Cells/ml, samples observation of cell every 24h after inoculation Upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Example 6
Microcarrier bioreactor suspends and cultivates MRC-5 cell proliferation G9 type rotavirus
(1) rotavirus adaptability on MRC-5 cell is cultivated, and passed for 10 generations continuously, detects its titre Change.
(2) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(3) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(4) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 5～10g/L, carefully It is 2～5 × 10 that born of the same parents inoculate the initial density of bioreactor5Cells/ml, samples observation of cell every 24h after inoculation Upgrowth situation on microcarrier.
(5) 1～3 × 10 are grown into when cell6During cells/ml, inoculate each in MOI=0.001～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(6) regulation bioreactor control parameter: pH be 7.2～7.6, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(7) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
Below with reference to Fig. 4-Fig. 6 describe in detail the present invention provide Rotavirus Vaccine preparation method second Embodiment, the present embodiment is substantially the same as in the previous example, and it differs primarily in that, in above steps, With MRC-5 cell described in WHO-Vero cell replacement, correspondingly, its medium component can use M199, DMEM, sodium acid carbonate and 10% calf serum substitute.
The various serotype rotavirus related to the present embodiment below in conjunction with some examples are described in detail.
Example 7
Microcarrier bioreactor suspension Cultivation of Vero propagation G1 type rotavirus
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～20 μ g/ml, the effect of being placed in 37 DEG C of water-baths 60～80min, period rocks 2～3 times.
(3) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 12～15g/L, The initial density of cell inoculation bioreactor is 0.5～2 × 106Cells/ml, sees every 24h sampling after inoculation Examine cell upgrowth situation on microcarrier.
(4) 1～3 × 10 are grown into when cell7During cells/ml, inoculate each in MOI=0.01～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(5) regulation bioreactor control parameter: pH be 7.0～7.8, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, centrifugal, concentrate, Mix the steps such as addition protective agent with other serotype liquid by a certain percentage and make multivalence rotavirus epidemic disease alive Seedling.
Example 8
Microcarrier bioreactor suspension Cultivation of Vero propagation G2 type rotavirus
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 20～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 60～75min, period rocks 2～3 times.
(3) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 12～15g/L, The initial density of cell inoculation bioreactor is 0.5～2 × 106Cells/ml, sees every 24h sampling after inoculation Examine cell upgrowth situation on microcarrier.
(4) 1～3 × 10 are grown into when cell7During cells/ml, inoculate in MOI=0.004～0.008 ratio Various serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(5) regulation bioreactor control parameter: pH be 7.0～7.8, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, centrifugal, concentrate, Mix the steps such as addition protective agent with other serotype liquid by a certain percentage and make multivalence rotavirus epidemic disease alive Seedling.
Example 9
Microcarrier bioreactor suspension Cultivation of Vero propagation G3 type rotavirus
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～20 μ g/ml, the effect of being placed in 37 DEG C of water-baths 60～75min, period rocks 2～3 times.
(3) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 12～15g/L, The initial density of cell inoculation bioreactor is 0.5～2 × 106Cells/ml, sees every 24h sampling after inoculation Examine cell upgrowth situation on microcarrier.
(4) 1～3 × 10 are grown into when cell7During cells/ml, inoculate each in MOI=0.008～0.02 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(5) regulation bioreactor control parameter: pH be 7.0～7.8, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, centrifugal, concentrate, Mix the steps such as addition protective agent with other serotype liquid by a certain percentage and make multivalence rotavirus epidemic disease alive Seedling.
Example 10
Microcarrier bioreactor suspension Cultivation of Vero propagation G4 type rotavirus
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 20～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 60～80min, period rocks 2～3 times.
(3) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 12～15g/L, The initial density of cell inoculation bioreactor is 0.5～2 × 106Cells/ml, sees every 24h sampling after inoculation Examine cell upgrowth situation on microcarrier.
(4) 1～3 × 10 are grown into when cell7During cells/ml, inoculate each in MOI=0.001～0.04 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(5) regulation bioreactor control parameter: pH be 7.0～7.8, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, centrifugal, concentrate, Mix the steps such as addition protective agent with other serotype liquid by a certain percentage and make multivalence rotavirus epidemic disease alive Seedling.
Example 11
Microcarrier bioreactor suspension Cultivation of Vero propagation G9 type rotavirus
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to.
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～20 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times.
(3) microcarrier selected is the Cytodex I of GE company, adds in culture medium by 12～15g/L, The initial density of cell inoculation bioreactor is 0.5～2 × 106Cells/ml, sees every 24h sampling after inoculation Examine cell upgrowth situation on microcarrier.
(4) 1～3 × 10 are grown into when cell7During cells/ml, inoculate each in MOI=0.01～0.05 ratio Plant serotype rotavirus, make virus infected cell, sample observation of cell pathology situation every 24h.
(5) regulation bioreactor control parameter: pH be 7.0～7.8, temperature be 35 DEG C～37 DEG C, dissolved oxygen 40%～60%, stir speed (S.S.) is 20～50rpm.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, centrifugal, concentrate, Mix the steps such as addition protective agent with other serotype liquid by a certain percentage and make multivalence rotavirus epidemic disease alive Seedling.
Reference examples
It is respectively adopted 3L rolling bottle, 2 layer cell factory and 14L microcarrier bioreactor, rotavirus is expanded Increasing and cultivate, its contrast situation is shown in Table 3.
(1) carry out passing on and breeding of cell with rolling bottle: after recovery cell, treat that cell grows to fine and close individual layer Time, carry out Secondary Culture by 1:3～1:4, cultivate 3～4 days.The T225 cell bottle of 2 long to fine and close individual layers 1 3L rolling bottle can be passed to, afterwards, be seeded to 3L rolling bottle, 2 layer cell factory and microcarrier respectively In (12～15g/L) 14L bioreactor, adjust cell density to 5.0 × 105cells/ml。
(2) rotavirus inoculation pre-treatment: activator is added in virus liquid and make pancreatin final concentration of 10～30 μ g/ml, the effect of being placed in 37 DEG C of water-baths 70～90min, period rocks 2～3 times (by G1 type rotavirus Test as test sample).
(3) when cell confluency degree reaches more than 90%, various blood are inoculated in MOI=0.001～0.05 ratio Clear type rotavirus, makes virus infected cell, samples observation of cell pathology situation every 24h.
(6) results virus liquid when CPE reaches more than 80%, multigelation for several times after, sampling carries out titre Measure.
The different culture technique of table 3 cultivates rotavirus contrast situation
As can be seen from Table 3, using microcarrier suspension culture colyliform disease degree, it produces poison amount and is greatly improved.
The Rotavirus Vaccine that the present invention provides, i.e. prepares according to the method described in foregoing embodiments, no Repeat again.
Above content is to combine concrete preferred embodiment further description made for the present invention, no Can assert the present invention be embodied as be confined to these explanations.Common for the technical field of the invention For technical staff, without departing from the inventive concept of the premise, it is also possible to make some simple deductions or replace Change, all should be considered as belonging to protection scope of the present invention.

Claims (10)

1. the preparation method of a Rotavirus Vaccine, it is characterised in that comprise the following steps:
Step 1, rotavirus carries out adaptability cultivation on MRC-5 cell, passes 10-12 generation continuously；
Step 2, after recovery MRC-5 cell, when it grows to fine and close individual layer, is passed on by 1:3-1:4 Cultivate 3-4 days；
Step 3, adding concentration in the medium is the microcarrier of 10-15g/L, and inoculation initial density is 0.5-2 ×106The MRC-5 cell through Secondary Culture of cells/ml is to bioreactor so that it is on described microcarrier Growth；
Step 4, treats that MRC-5 cell grows to 1-3 × 107During cells/ml, inoculate the blood after pancreatin activates Clear type rotavirus so that it is infect MRC-5 cell；
Step 5, treats that CPE reaches more than 80%, after results virus liquid, is made into multivalence rotavirus and lives Vaccine.
2. the method for claim 1, it is characterised in that described microcarrier uses GE company Cytodex I microcarrier, described bioreactor uses 310 type 14L cells of NBS company of the U.S. to cultivate Bioreactor.
3. method as claimed in claim 2, it is characterised in that in described step 3, keep described life Thing reactor controls parameter: pH value 7.0-7.8, temperature 35 DEG C-37 DEG C, dissolved oxygen 40%-60%, stirring speed Rate 20-75rpm.
4. method as claimed in claim 3, it is characterised in that in described step 4, according to MOI=0.001-0.05 ratio virus inoculation, controls stir speed (S.S.) at 25rpm, make virus uniform adsorption in Cell surface, after about 1-2 hour, then controls stir speed (S.S.) 50～75rpm.
5. the method as according to any one of claim 1-4, it is characterised in that described rotavirus passes through Following steps carry out pancreatin activation:
Activator is added in virus liquid so that pancreatin final concentration of 10～30 μ g/ml, be placed in 37 DEG C of water-baths Acting on 70-90 minute, period rocks 2-3 time.
6. the method as according to any one of claim 1-4, it is characterised in that in described step 3, Continuous perfusion type is used to cultivate.
7. the method as according to any one of claim 1-4, it is characterised in that described medium component bag Include BME, Glu, sodium acid carbonate and 10% calf serum.
8. method as claimed in claim 7, it is characterised in that in above steps, replace with Vero cell For described MRC-5 cell, replace described training with M199, DMEM, sodium acid carbonate and 10% calf serum Support based component.
9. the method for claim 1, it is characterised in that described rotavirus is G1, G2, G3, In G4, G9 type rotavirus any at least one.
10. a Rotavirus Vaccine, it is characterised in that according to the side according to any one of claim 1-9 Method prepares.
CN201610122156.6A 2016-03-03 2016-03-03 Rotavirus vaccine and preparation method thereof Pending CN105749270A (en)

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CN114410592A (en) * 2021-12-30 2022-04-29 苏州百源基因技术有限公司 Rotavirus culture medium and application thereof
CN114410592B (en) * 2021-12-30 2024-05-24 苏州百源基因技术有限公司 Rotavirus culture medium and application thereof

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