CN105400743A - Preparation method of TGEV - Google Patents
Preparation method of TGEV Download PDFInfo
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- CN105400743A CN105400743A CN201510897095.6A CN201510897095A CN105400743A CN 105400743 A CN105400743 A CN 105400743A CN 201510897095 A CN201510897095 A CN 201510897095A CN 105400743 A CN105400743 A CN 105400743A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- 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
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- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention provides a preparation method of TGEV (transmissible gastroenteritis virus). According to the method, in a bioreactor, an ST cell is adopted as the host cell, microcarrier culture is employed for proliferation of TGEV, and at the same time efficient condition parameters are designed. The culture efficiency is significantly improved, the cell density is enhanced by 3-5 times than spinner bottles, the virus titer is high, and is increased from the 10<7.0-7.3>TCID50/ml of spinner bottle technique to 10<7.7>-10<8.5>TCID50/ml, and the titer is enhanced by 5-10 times. At the same time, a high titer antigen needs a high dilution factor during use, so that the content of heterologous protein in a unit volume antigen is reduced indirectly, thereby lowering the side reaction incidence of vaccines prepared with heterologous protein as the antigen. The technology is low in cost, and compared with the practice of selecting a traditional basic medium, the harvest time is greatly shortened, the interassay difference is small, and the process is stable.
Description
Technical field
The present invention relates to cell engineering field, be specifically related to a kind of TGEV virus preparation method.
Background technology
Transmissible gastroenteritis of swine (Transmissiblegastroenteritis, TGE) be a kind of high degree in contact intestinal tract disease caused by transmissible gastro-enteritis virus (TGEV), to vomit, severe diarrhea and dehydration be main clinical characteristics.The equal easy infection of pig of different ages and kind, wherein in 2 week age, piglet case fatality rate can reach 100%, is to cause piglet morbidity, dead Infectious Diseases.This disease mainly occurs in winter and early spring, and many countries and regions are widely current in the world, can with porcine rotavirus disease, porcine epizootic diarrhea polyinfection.Fashion trend strengthens gradually in recent years, and TGEV is still and causes one of piglet morbidity and main causes of death, and cause very big puzzlement to pig industry, therefore the epidemic prevention of this disease just seems even more important.
The production of current transmissible gastroenteritis of swine vaccine mainly adopts traditional rolling bottle to produce, and is inoculated in the good monolayer cell of growth conditions by transmissible gastro-enteritis virus, disposable results TGEV virus after cultivating.This technique uses many decades in China vaccine industry, ripe and stable.But rolling bottle is produced also exists certain shortcoming, as low in cell density, cell debris is many, vaccine difference is large, labour intensity is large, production efficiency is low, virus titer is low, high intracellular toxin etc., is more and more not suitable with the requirement of current vaccines scale operation.The static spinner culture technology of current ST cell has been widely used in propagation transmissible gastro-enteritis virus, though technology is simple, adds the labour intensity in workshop and microbiological contamination has a big risk.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, provides a kind of TGEV virus preparation method, with the technical problem that the TGEV virus titer prepared by the related manufacturing processes solving prior art is lower.
Another technical problem that the present invention will solve is the related manufacturing processes complex operation, consuming time longer of prior art.
The technical problem again that the present invention will solve is the related manufacturing processes unstable product quality of prior art.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of TGEV virus preparation method, comprises the following steps:
1) get microcarrier and be placed in PBS damping fluid immersion treatment, then sterilizing, discards PBS damping fluid, adds in the MEM cell growth medium containing 8 ~ 10% (w/w) serum, obtain mixed culture medium for subsequent use, in described mixed culture medium, the density of microcarrier is 2 ~ 10g/L;
2) in bio-reactor to step 1) base inoculation (0.4 ~ 2) × 10 in described mixed culture
6the ST cell of Cells/mL, then cultivates under pH6.6 ~ 7.5, temperature 35 ~ 38 DEG C, dissolved oxygen 40 ~ 60%, mixing speed 35 ~ 80rpm condition;
3) when cell density reaches (2 ~ 12) × 10
6during Cells/mL, discard the cell growth medium in mixed culture medium, add viral maintenance medium, then inoculate the transmissible gastro-enteritis virus of 1.5%-3% wherein, temperature 36 ~ 37 DEG C, cultivate under dissolved oxygen 40 ~ 60%, mixing speed 35 ~ 80rpm condition, wherein said viral maintenance medium is the MEM cell growth medium containing 1 ~ 3% serum;
4), when cultivating 10 ~ 16h after virus inoculation, results nutrient solution, namely obtains transmissible gastro-enteritis virus liquid after freeze thawing.
Preferably, step 1) described immersion treatment be microcarrier is placed in PBS damping fluid in 37 DEG C keep 2h; On this basis further preferably: step 1), microcarrier being placed in PBS damping fluid keeps the operation of 2h to repeat 3 times in 37 DEG C, then performs sterilizing again.
Preferably, step 1) described sterilizing is autoclaving under 121 DEG C of environment.
Preferably, described microcarrier is Cytodex series microcarrier.
Preferably, step 2) described in bio-reactor volume be 14L.
Preferably, step 2) in nutrient solution pH be 6.8 ~ 7.2.
Preferably, step 2) in culture temperature be 37 ~ 38 DEG C.
Preferably, step 3) in when discarding in mixed culture medium cell growth medium the accumulative incubation time of ST cell be 72 ~ 120h.
Preferably, step 3) described in the pH of viral maintenance medium be 7.0 ~ 7.4.
Preferably, step 4) in results nutrient solution time, the ST cell quantity come off mutually with microcarrier reaches more than 70% of ST cell total amount.
Preferably, step 4) described in freeze thawing, repeat 3 times.
In above technical scheme, described Cytodex series microcarrier, be only defined in by GE Company, model is a series of microcarrier products of Cytodex.In addition to the operation described above, can also introduce viral level further and measure link, such as, can be following steps: every for ST cell suspension hole 100 μ L is joined incubated overnight in 96 porocyte culture plates to the method; By the MEM maintenance medium containing 1-3% serum, transmissible gastro-enteritis virus is carried out 10 times of dilutions, each extent of dilution is got 100 μ L and is joined in 96 orifice plates of incubated overnight ST cell, put 37 DEG C, the cultivation of 5%CO2 incubator, record cytopathic hole count after 3-5 day and calculate viral TCID according to Reed-Muench method
50.
The present invention utilizes microcarrier to expand surface-area, and therefore the cell yield of unit volume nutrient solution is high; Suspension culture and adherent culture are merged, has both advantages concurrently; Available simple microscope observing cell, at the upgrowth situation of bead surface, simplifies the detection and control of the various environmental factors of Growth of Cells, favorable reproducibility; Cell amplifies easily, and substratum utilising efficiency is high, and cell harvesting process is uncomplicated; Culture systems floor space and space little, labour intensity is little.
Although Microcarrier Cell Culture Techniques has been widely used in cell engineering field, but when utilizing its preparation virus, different cell strain, different strain, need to design specific culture condition.Therefore, the present invention devises and utilizes the specific culture condition of ST cells produce TGEV on the basis selecting microcarrier culture system, and culture efficiency is significantly promoted, and cell density comparatively rolling bottle improves 3 ~ 5 times, and virus titer is high, by 10 of rolling bottle technique
7.0-7.3tCID
50/ ml is increased to 10
7.7-10
8.5tCID
50/ ml, tires and improves 5-10 doubly.Meanwhile, because high-titer antigen needs higher extension rate in use, therefore indirectly decrease heterologous protein content in unit volume antigen, thus reduce with the rate of side effects of its vaccine prepared by antigen.This explained hereafter cost is lower, and select traditional basic medium, harvest time shortens greatly, and differences between batches are little, process stabilizing.
Embodiment
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
The approximating language used in following examples can be used for quantitative expression, shows to allow quantity to have certain variation when not changing basic function.Therefore, this exact value itself is not limited to the numerical value that the language such as " approximately ", " left and right " is revised.In certain embodiments, " approximately " represents and allows its numerical value revised to change in the positive and negative scope of 10 (10%), such as, and any numerical value that what " about 100 " represented can be between 90 to 110.In addition, in the statement of " about first numerical value is to second value ", revise the first and second numerical value two numerical value approximately simultaneously.In some cases, approximating language may be relevant with the precision of surveying instrument.
Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent; Described experimental technique, if no special instructions, is ordinary method; Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged; % in following examples, if no special instructions, is mass percentage.
Embodiment 1
Bio-reactor: German Bei Lang company 14L bio-reactor.
Microcarrier: Cytodex-1 (purchased from GE company).
Transmissible gastro-enteritis virus: TGEVHB08 strain.
Cell growth medium: the MEM containing volumetric concentration being 8% new-born calf serum;
Virus maintenance medium: the MEM containing volumetric concentration being 2% new-born calf serum;
The microcarrier suspension culture of ST cell: in 14L bio-reactor, adds the microcarrier Cytodex-1 handled well according to the concentration of 2g/L, inoculation ST cell, cell implantation concentrations is 400,000/mL.During cell cultures, best setup parameter: the pH7.25 of reactor, temperature 37 DEG C, dissolved oxygen 55%, stirring velocity are 35rpm/min.Sample at regular intervals after the inoculation of ST cell, in basis of microscopic observation cell state, and carry out cell counting, measure the consumption of glucose.According to glucose consumption situation and Growth of Cells and dropping situations, about 48h carries out changing liquid, and continuing culturing cell subsequently to 120h, is that cell density before connecing poison reaches 3,000,000/more than ml.
The breeding of virus liquid and results: when cell density reaches 3,000,000/ml, change viral maintenance medium, by 1.5% volume Pigs Inoculated Transmissible gastroenteritis virus; Best setup parameter: the pH7.0 of reactor of viral proliferation, temperature 36.5 DEG C, dissolved oxygen 45%, stirring velocity are 35rpm/min.After TGEV virus inoculation, timing sampling, observation of cell pathology.According to cytopathy situation, 10-16h after virus inoculation, comes off when the cell on microcarrier reaches more than 70%, stops cultivating, and results virus liquid, freeze thawing three times, obtains transmissible gastro-enteritis virus (TGEV).
The mensuration of TGEV viral level: measuring TGEV viral level is 10
7.8tCID
50/ ml.
Embodiment 2
Bio-reactor: German Bei Lang company 14L bio-reactor.
Microcarrier: Cytodex-1 (purchased from GE company).
Transmissible gastro-enteritis virus: TGEVHB08 strain.
Cell growth medium: the MEM containing volumetric concentration being 10% new-born calf serum;
Virus maintenance medium: the MEM containing volumetric concentration being 3% new-born calf serum;
The microcarrier suspension culture of ST cell: in 14L bio-reactor, adds the microcarrier Cytodex-1 handled well according to the concentration of 8g/L, inoculation ST cell, cell implantation concentrations is 1,000,000/mL.During cell cultures, best setup parameter: the pH7.0 of reactor, temperature 37 DEG C, dissolved oxygen 55%, stirring velocity are 80rpm/min.Sample at regular intervals after the inoculation of ST cell, in basis of microscopic observation cell state, and carry out cell counting, measure the consumption of glucose.According to glucose consumption situation and Growth of Cells and dropping situations, about 24h carries out changing liquid, partly changes liquid subsequently at 48h and 72h, and cell counting is carried out in timing, and final cell density reaches 7,000,000/ml.
The breeding of virus liquid and results: when cell density reaches 7,000,000/ml, change viral maintenance medium, inoculates the transmissible gastro-enteritis virus of 3% volume.Best setup parameter: the pH7.0 of reactor of viral proliferation, temperature 37 DEG C, dissolved oxygen 50%, stirring velocity are 80rpm/min.After TGEV virus inoculation, timing sampling, observation of cell pathology.According to cytopathy situation, 10-13h after virus inoculation, comes off when the cell of microcarrier reaches more than 70%, stops cultivating, and results virus liquid, freeze thawing three times, obtains transmissible gastro-enteritis virus (TGEV).
The mensuration of TGEV viral level: measuring TGEV viral level is 10
8.25tCID
50/ ml.
Embodiment 3
A kind of TGEV virus preparation method, comprises the following steps:
1) get microcarrier and be placed in PBS damping fluid immersion treatment, then sterilizing, discards PBS damping fluid, adds in the MEM cell growth medium containing 9% (w/w) serum, obtain mixed culture medium for subsequent use, in described mixed culture medium, the density of microcarrier is 4g/L;
2) in bio-reactor to step 1) base inoculation 0.6 × 10 in described mixed culture
6the ST cell of Cells/mL, then cultivates under pH6.6, temperature 35 DEG C, dissolved oxygen 40%, mixing speed 35rpm condition;
3) when cell density reaches 5 × 10
6during Cells/mL, discard the cell growth medium in mixed culture medium, add viral maintenance medium, then inoculate the transmissible gastro-enteritis virus of 1.5% wherein, temperature 36 DEG C, cultivate under dissolved oxygen 40%, mixing speed 35rpm condition, wherein said viral maintenance medium is the MEM cell growth medium containing 1% serum;
4), when cultivating 10h after virus inoculation, results nutrient solution, namely freeze thawing obtains transmissible gastro-enteritis virus liquid three times.
On the basis of above technical scheme, meet the following conditions:
Step 1) described immersion treatment be microcarrier is placed in PBS damping fluid in 37 DEG C keep 2h, this step repeats 3 times, then performs sterilizing again.
Described microcarrier is Cytodex series microcarrier.
Step 2) described in bio-reactor volume be 14L.
Step 3) in when discarding in mixed culture medium cell growth medium the accumulative incubation time of ST cell be 72h.
Step 3) described in the pH of viral maintenance medium be 7.0 ~ 7.4.
Step 4) in results nutrient solution time, the ST cell quantity come off mutually with microcarrier reaches more than 70% of ST cell total amount.
Embodiment 4
A kind of TGEV virus preparation method, is characterized in that comprising the following steps:
1) get microcarrier and be placed in PBS damping fluid immersion treatment, then sterilizing, discards PBS damping fluid, adds in the MEM cell growth medium containing 10% (w/w) serum, obtain mixed culture medium for subsequent use, in described mixed culture medium, the density of microcarrier is 10g/L;
2) in bio-reactor to step 1) base inoculation 2 × 10 in described mixed culture
6the ST cell of Cells/mL, then cultivates under pH7.5, temperature 38 DEG C, dissolved oxygen 60%, mixing speed 80rpm condition;
3) when cell density reaches 11 × 10
6during Cells/mL, discard the cell growth medium in mixed culture medium, add viral maintenance medium, then inoculate the transmissible gastro-enteritis virus of 3% wherein, temperature 37 DEG C, cultivate under dissolved oxygen 60%, mixing speed 80rpm condition, wherein said viral maintenance medium is the MEM cell growth medium containing 3% serum;
4), when cultivating 16h after virus inoculation, results nutrient solution, namely freeze thawing obtains transmissible gastro-enteritis virus liquid twice.
On the basis of above technical scheme, meet the following conditions:
Described immersion treatment microcarrier is placed in PBS damping fluid to keep 2h in 37 DEG C.
Step 3) in when discarding in mixed culture medium cell growth medium the accumulative incubation time of ST cell be 120h.
Step 3) described in the pH of viral maintenance medium be 7.4.
Step 4) in results nutrient solution time, the ST cell quantity come off mutually with microcarrier reaches more than 70% of ST cell total amount.
Embodiment 5
A kind of TGEV virus preparation method, is characterized in that comprising the following steps:
1) get microcarrier and be placed in PBS damping fluid immersion treatment, then sterilizing, discards PBS damping fluid, adds in the MEM cell growth medium containing 8.5% (w/w) serum, obtain mixed culture medium for subsequent use, in described mixed culture medium, the density of microcarrier is 6g/L;
2) in bio-reactor to step 1) base inoculation 0.7 × 10 in described mixed culture
6the ST cell of Cells/mL, then cultivates under pH7, temperature 37 DEG C, dissolved oxygen 50%, mixing speed 60rpm condition;
3) when cell density reaches 8 × 10
6during Cells/mL, discard the cell growth medium in mixed culture medium, add viral maintenance medium, then inoculate the transmissible gastro-enteritis virus of 2% wherein, temperature 36.5 DEG C, cultivate under dissolved oxygen 50%, mixing speed 60rpm condition, wherein said viral maintenance medium is the MEM cell growth medium containing 2% serum;
4), when cultivating 13h after virus inoculation, results nutrient solution, namely obtains transmissible gastro-enteritis virus liquid after freeze thawing.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a TGEV virus preparation method, is characterized in that comprising the following steps:
1) get microcarrier and be placed in PBS damping fluid immersion treatment, then sterilizing, discards PBS damping fluid, adds in the MEM cell growth medium containing 8 ~ 10% (w/w) serum, obtain mixed culture medium for subsequent use, in described mixed culture medium, the density of microcarrier is 2 ~ 10g/L;
2) in bio-reactor to step 1) base inoculation (0.4 ~ 2) × 10 in described mixed culture
6the ST cell of Cells/mL, then cultivates under pH6.6 ~ 7.5, temperature 35 ~ 38 DEG C, dissolved oxygen 40 ~ 60%, mixing speed 35 ~ 80rpm condition;
3) when cell density reaches (2 ~ 12) × 10
6during Cells/mL, discard the cell growth medium in mixed culture medium, add viral maintenance medium, then inoculate the transmissible gastro-enteritis virus of 1.5%-3% wherein, temperature 36 ~ 37 DEG C, cultivate under dissolved oxygen 40 ~ 60%, mixing speed 35 ~ 80rpm condition, wherein said viral maintenance medium is the MEM cell growth medium containing 1 ~ 3% serum;
4), when cultivating 10 ~ 16h after virus inoculation, results nutrient solution, namely obtains transmissible gastro-enteritis virus liquid after freeze thawing.
2. preparation method according to claim 1, is characterized in that step 1) described immersion treatment be microcarrier is placed in PBS damping fluid in 37 DEG C keep 2h.
3. preparation method according to claim 2, is characterized in that step 1) in microcarrier is placed in PBS damping fluid and keeps the operation of 2h to repeat 3 times in 37 DEG C, then perform sterilizing again.
4. preparation method according to claim 1, is characterized in that described microcarrier is Cytodex series microcarrier.
5. preparation method according to claim 1, is characterized in that step 2) described in bio-reactor volume be 14L.
6. preparation method according to claim 1, is characterized in that step 2) in nutrient solution pH be 6.8 ~ 7.2.
7. preparation method according to claim 1, is characterized in that step 2) in culture temperature be 37 ~ 38 DEG C.
8. preparation method according to claim 1, is characterized in that step 3) in when discarding in mixed culture medium cell growth medium the accumulative incubation time of ST cell be 72 ~ 120h.
9. preparation method according to claim 1, is characterized in that step 3) described in the pH of viral maintenance medium be 7.0 ~ 7.4.
10. preparation method according to claim 1, is characterized in that step 4) in results nutrient solution time, the ST cell quantity come off mutually with microcarrier reaches more than 70% of ST cell total amount.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106237324A (en) * | 2016-08-30 | 2016-12-21 | 齐鲁动物保健品有限公司 | A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine |
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Application publication date: 20160316 |