CN108853489A - A method of PEDV attenuated vaccine is produced using serum free medium - Google Patents
A method of PEDV attenuated vaccine is produced using serum free medium Download PDFInfo
- Publication number
- CN108853489A CN108853489A CN201810690966.0A CN201810690966A CN108853489A CN 108853489 A CN108853489 A CN 108853489A CN 201810690966 A CN201810690966 A CN 201810690966A CN 108853489 A CN108853489 A CN 108853489A
- Authority
- CN
- China
- Prior art keywords
- content
- pedv
- taurine
- culture
- sodium selenite
- 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.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
-
- 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
- C12N2770/20051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to a kind of methods using serum free medium production PEDV attenuated vaccine; it is characterized in that; the serum free medium is by basal medium, bovine serum albumin(BSA), cortisol; insulin; transferrins, glutathione, trypsase; taurine and sodium selenite composition, wherein the weight ratio of taurine and sodium selenite is 2-3:1.The culture medium does not add animal blood serum, significantly reduces production cost, improves the economic benefit of production, and also avoids serum and use brought problems.
Description
Technical field：
The present invention relates to field of biotechnology, and in particular to a kind of side using serum free medium production PEDV attenuated vaccine
Method.
Background technique：
Pig epidemic diarrhea（PED）It is by Porcine epidemic diarrhea virus（PEDV）Caused acute and highly infectious enteron aisle disease
Viral disease, symptom include diarrhea, vomiting, anorexia, and dehydration and pig weight mitigate.Although the pig of different age group all may
By different degrees of infection and there is symptom, but the state of an illness especially severe of piglet, wherein the death rate is up to 100%, PEDV master
To enter host by the intestinal mucosal surface of piggy, mainly result in enterocyte damage.Although the pig of different age group all may be used
By different degrees of infection and symptom can occur, but the state of an illness especially severe of piglet, wherein the death rate is up to 100%, PEDV
Host is mainly entered by the intestinal mucosal surface of piggy, mainly results in enterocyte damage.For the great outburst of PEDV, we
PEDV can be usually controlled using vaccination, and has been carried out extensively in China with the PEDV vaccine for killing or being attenuated
Vaccine inoculation is carried out, controls the large-scale outbreak of PEDV to a certain extent.But as Chinese PEDV inactivates or is attenuated bigeminy
Vaccine is widely used, and PED persistently spreads in October, 2010 in China, occurs variant at that time, and PEDV infection is anxious
Increase severely and add, seriously endangers pig breeding industry.Although the country is used for the vaccine of epidemic diarrhea（Including dyad inactivated vaccine and bigeminy epidemic disease living
Seedling）Extensive promotion and application have been obtained, and have reduced the disease incidence of pig epidemic diarrhea in a certain range and degree, but
That the disease still fails to be fully controlled, especially in October, 2010 so far, the disease be in duration, explosive growth trend.
In general, PEDV is usedveroCell is cultivated and is proliferated, and then inactivates and PEDV inactivated vaccine is prepared.Mesh
Before, it is based onveroThe production process of cell culture PEDV vaccine mostly uses two stages operating procedure, i.e.,：There is being serum in early period
Make cell Proliferation to required density in culture medium；Later period by washing removal serum composition and metabolic waste, is changed to low serum
Or serum free medium is to support virus to infect and replicate in cell.However, in existing production of vaccine, early periodveroCell
Culture solution cooperates calf or horse serum to be cultivated and produced with commercialized culture medium.The complexity of serum component and not really
Difference between qualitative and batch increases the difficulty of the cell products Production and quality control such as vaccine, while serum easily causes carefully
The pollution of bacterium, fungi, virus and mycoplasma, increases the safety risks of production of vaccine.And serum price itself also compares
Valuableness, these make application of the serum in the production and research of PEDV vaccine, and there are many unfavorable.Have been proven in practice that no blood
Clear culture medium can not only be largely avoided or improve drawbacks described above brought by serum-containing media, can also obtain compared with
Good culture effect.And since it is explicitly at being grouped as, the different batches or unknown component that can avoid serum train cell
Feeding and experimental study influence, to improve the repeatability of result.Therefore, used based on serum in PEDV production of vaccine
Existing problems and production cost are high, and therefore, the dosage for reducing serum to the greatest extent is always to study in production of vaccine
Project, the development and application of low serum and serum free medium is imperative.
Summary of the invention：
In order to solve the above technical problems, and in the prior art problems and production cost present in serum use are high,
The present invention is intended to provide a kind of method using serum free medium production PEDV attenuated vaccine, the culture medium do not add animal blood
Clearly, production cost is significantly reduced, the economic benefit of production is improved, and also avoids serum using brought many
Problem.
In order to solve the above technical problems, the present invention uses following technological means：
A method of PEDV attenuated vaccine being produced using serum free medium, described method includes following steps：
Step（1）, the recovery of Vero cell；
Step（2）, the passage amplification of Vero cell；
Step（3）, PEDV virus stain is inoculated on Vero cell；
Step（4）, infection cell culture supernatant is harvested, virus liquid carries out clarification filtration；
Step（5）, freeze drying protectant, packing freeze-drying is added；
Wherein step（2）In passage amplification and step（3）The breeding of middle virus is all made of free serum culture of the invention
Base.
The serum free medium is by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, paddy Guang
Sweet peptide, trypsase, taurine and sodium selenite composition, wherein the weight ratio of taurine and sodium selenite is 2-3:
1。
Preferably, in the serum free medium：
The basal medium IMDM（Giboc）The mixture of RPMI-1640 or the two；
The content of the bovine serum albumin(BSA) is 8-15mg/mL；
The content of the cortisol is 15-25 μ g/mL；
The content of the insulin is 10-20 μ g/mL；
The content of the transferrins is 10-15 μ g/mL；
The content of the glutathione is 2-3mM；
The content of the trypsase is 3-5 μ g/mL；
The content of the taurine is 20-30 μ g/mL；
The content of the sodium selenite is 10-12 μ g/mL.
Preferably, in the serum free medium：
The basal medium IMDM（Giboc）The mixture of RPMI-1640 or the two；
The content of the bovine serum albumin(BSA) is 10mg/mL；
The content of the cortisol is 20 μ g/mL；
The content of the insulin is 20 μ g/mL；
The content of the transferrins is 15 μ g/mL；
The content of the glutathione is 3mM；
The content of the trypsase is 4 μ g/mL；
The content of the taurine is 20 μ g/mL；
The content of the sodium selenite is 10 μ g/mL.
The production method of serum free medium of suitable large-scale production PEDV vaccine of the present invention is：Step 1,
Basal medium dry powder, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, pancreas egg are weighed by weight
White enzyme, taurine and sodium selenite；Step 2 adds ultrapure water to 1000mL, stirs molten after the weighed above material mixing
Solution adjusts pH value to 7.0-7.2, then uses 0.22 μm of membrane filtration degerming, 4 DEG C of preservations.
Based on above technical scheme, the invention has the advantages that and beneficial effect：
The embodiment of the present invention 1 and the culture medium of embodiment 2 have preferable cultivation effect to the proliferation of PEDV, compared to existing
There is common calf serum to add DMEM culture medium, can more promote the proliferation of PEDV, the virus titer of proliferation is significantly raised, shows
Culture medium of the invention can substitute traditional serum-containing media and be used for the proliferation of PEDV and the preparation of vaccine, so as to avoid
Immune problem caused by the use of serum in vaccine production process；It is compared with the control experiment group that the present invention is arranged, into one
Walk importance of the ratio of the addition for demonstrating glutathione and taurine and sodium selenite for PEDV cell Proliferation, comparison
Glutathione is not added in test example 1, and there is also differences for the ratio of taurine and sodium selenite, are higher than the present invention, then its disease
Malicious cultivation effect difference more of the present invention；The ratio of taurine and sodium selenite is 1 in comparative experimental example 2：1, i.e., taurine is not using
Foot then causes virus multiplication too late expected, although it is proliferated in the 0-12h stage comparatively fast, in the titre in virus multiplication later period
Rise slower；Glutathione is not added in comparative experimental example 3, then virus multiplication is too late expected, and early stage proliferation is relatively slow, after
Phase proliferation also shows slightly insufficient.The above test results show that in culture medium of the invention, the addition of glutathione and taurine with
The ratio of sodium selenite has great importance for PEDV cell Proliferation, and the two is synergistic in virus multiplication culture,
Achieve unexpected culture effect.
Detailed description of the invention：
Fig. 1：The case where PEDV Virus culture.A, control group, normal vero cell；B connects poison after 24 hours, with control group
Normal cell is compared, and more apparent CPE occurs；C connect poison after 48 hours, and cell rounding has part cells float to fall off, out
The now pathological changes such as seine shape.
Fig. 2：IFA testing result：A is normal vero cell controls；B is carried out using 1 culture medium of the embodiment of the present invention
IFA testing result after culture；C is the IFA testing result after being cultivated using 10% FBS DMEM cell culture fluid, and D is
The IFA testing result cultivated using the culture medium that following check experiment example is shown.
Specific embodiment：
Embodiment 1：A kind of serum free medium of suitable large-scale production PEDV vaccine, which is characterized in that the serum-free training
Base is supported by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, trypsase, taurine
It is formed with sodium selenite, wherein the weight ratio of taurine and sodium selenite is 2:1.
The basal medium IMDM（Giboc）；
The content of the bovine serum albumin(BSA) is 10mg/mL.
The content of the cortisol is 20 μ g/mL.
The content of the insulin is 20 μ g/mL.
The content of the transferrins is 15 μ g/mL.
The content of the glutathione is 3mM.
The content of the trypsase is 4 μ g/mL.
The content of the taurine is 20 μ g/mL.
The content of the sodium selenite is 10 μ g/mL.
The production method of serum free medium of suitable large-scale production PEDV vaccine of the present invention is：Step 1,
Basal medium dry powder, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, pancreas egg are weighed by weight
White enzyme, taurine and sodium selenite；Step 2 adds ultrapure water to 1000mL, stirs molten after the weighed above material mixing
Solution adjusts pH value to 7.0-7.2, then uses 0.22 μm of membrane filtration degerming, 4 DEG C of preservations.
Embodiment 2：A kind of serum free medium of suitable large-scale production PEDV vaccine, which is characterized in that the serum-free training
Base is supported by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, trypsase, taurine
It is formed with sodium selenite, wherein the weight ratio of taurine and sodium selenite is 3:1.
The basal medium IMDM（Giboc）；
The content of the bovine serum albumin(BSA) is 10mg/mL.
The content of the cortisol is 20 μ g/mL.
The content of the insulin is 20 μ g/mL.
The content of the transferrins is 15 μ g/mL.
The content of the glutathione is 3mM.
The content of the trypsase is 4 μ g/mL.
The content of the taurine is 30 μ g/mL.
The content of the sodium selenite is 10 μ g/mL.
The production method of serum free medium of suitable large-scale production PEDV vaccine of the present invention is：Step 1,
Basal medium dry powder, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, pancreas egg are weighed by weight
White enzyme, taurine and sodium selenite；Step 2 adds ultrapure water to 1000mL, stirs molten after the weighed above material mixing
Solution adjusts pH value to 7.0-7.2, then uses 0.22 μm of membrane filtration degerming, 4 DEG C of preservations.
Embodiment 3：The small-scale proliferation test of PEDV
Test strain：SX-YL plants of PEDV, it is obtained from Xibei Univ. of Agricultural & Forest Science & Technology, the isolation of strains is from Yangling Shaanxi scale
Farm.
Test cell：Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method：
（One）The recovery of Vero cell strain：
（1）The Vero cell being removed from liquid nitrogen quickly is put into 37 DEG C of water-baths, gently shaking makes its quick-thawing；
（2）300 rpm are centrifuged 10 min；
（3）Supernatant is abandoned, the cell culture fluid of 37 DEG C of pre-temperatures is added（Containing 5% FBS DMEM）, gently dispel Vero cell；
（4）Cell suspension after mixing is added in sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid, is put
It is cultivated in the cell incubator that condition is suitable for；
（5）After passage twice, vero cell suspension is added in 24 orifice plates, culture to its cell in incubator is placed in and grows up to list
Layer, after being rinsed 2 times using the culture medium of the embodiment of the present invention 1, the culture medium for pouring into the embodiment of the present invention 1 is cultivated.
（Two）Inoculation, culture and the IFA detection of virus：
PEDV SX-YL strain virus liquid is diluted using the culture medium of the embodiment of the present invention 1, by Vero cell culture in 24
Well culture plate, after cells grow up to the individual layer are inoculated with PEDV, while setting the normal cell controls for not connecing poison, occur to cell slight
Ethyl alcohol is used after lesion（- 20 DEG C of pre-coolings）20 min are fixed, are then washed 3 times with PBS, each 5min.PEDV N protein list is added
It is anti-（Xibei Univ. of Agricultural & Forest Science & Technology's present）, 37 DEG C of incubation 60min, PBS wash three times, and the sheep anti mouse Ig G of FITC label is added.?
Result is observed under inverted fluorescence microscope.Concrete outcome is referring to attached Fig. 1 and 2.
Wherein attached drawing 1 illustrates the case where carrying out PEDV Virus culture using 1 culture medium of the embodiment of the present invention, and it is small to connect poison 24
There is more apparent CPE compared with the normal cell of control group in Shi Hou, connects poison after 48 hours, and cell rounding has part thin
Born of the same parents' floating falls off, and the pathological changes such as seine shape occurs.
Fig. 2 illustrates IFA detection case, and wherein A is normal vero cell controls；B is trained using the embodiment of the present invention 1
Feeding base cultivated after IFA testing result；C is the IFA detection after being cultivated using 10% FBS DMEM cell culture fluid
As a result, D is the IFA testing result cultivated using the culture medium that following check experiment example is shown.The result above table of comparisons
Bright, after the culture medium of the embodiment of the present invention 1 is cultivated, fluorescence intensity and density are obviously higher than serum in the prior art
The culture medium of culture medium and check experiment example 1, illustrates：On the one hand, the culture medium of the embodiment of the present invention 1 is proliferated in PEDV
It is better than the culture medium containing serum in terms of effect, culture medium of the invention, which is fully able to replace existing serum-containing media, to be used for
The culture of PEDV virus and the preparation of proliferation and vaccine.On the other hand, 1 relative comparison test example 1 of the embodiment of the present invention,
The proportional difference of taurine and sodium selenite and the addition of glutathione are distinguished, the difference of result shows that the present invention is implemented
The addition of the ratio and glutathione of taurine and sodium selenite has important shadow for the proliferation of PEDV virus in example 1
It ringing, the addition of appropriate ratio and glutathione between the two has the function of synergy for the proliferation of PEDV virus,
It demonstrates culture medium of the invention and achieves the unexpected technology effect of those skilled in the art in terms of PEDV virus multiplication
Fruit.
Check experiment example 1 set forth below：A kind of serum free medium, which is characterized in that the serum free medium is trained by basis
Base, bovine serum albumin(BSA), cortisol, insulin, transferrins, trypsase, taurine and sodium selenite composition are supported, wherein ox
The weight ratio of sulfonic acid and sodium selenite is 4:1.
The basal medium IMDM（Giboc）；
The content of the bovine serum albumin(BSA) is 10mg/mL.
The content of the cortisol is 20 μ g/mL.
The content of the insulin is 20 μ g/mL.
The content of the transferrins is 15 μ g/mL.
The content of the trypsase is 4 μ g/mL.
The content of the taurine is 40 μ g/mL.
The content of the sodium selenite is 10 μ g/mL.
The production method of the serum free medium is：Step 1 weighs basal medium dry powder, cow's serum by weight
Albumin, cortisol, insulin, transferrins, trypsase, taurine and sodium selenite；Step 2, by the weighed above object
After matter mixing, add ultrapure water to 1000mL, stirring and dissolving is adjusted pH value to 7.0-7.2, then removed using 0.22 μm of membrane filtration
Bacterium, 4 DEG C of preservations.
Embodiment 4：The large-scale production test of PEDV inactivated vaccine
Test strain：SX-YL plants of PEDV, it is obtained from Xibei Univ. of Agricultural & Forest Science & Technology, the isolation of strains is from Yangling Shaanxi scale
Farm.
Test cell：Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method：
（One）The recovery of Vero cell strain：
（1）The Vero cell being removed from liquid nitrogen quickly is put into 37 DEG C of water-baths, gently shaking makes its quick-thawing；
（2）300 rpm are centrifuged 10 min；
（3）Supernatant is abandoned, the cell culture fluid of 37 DEG C of pre-temperatures is added（Containing 5% FBS DMEM）, gently dispel Vero cell；
（4）Cell suspension after mixing is added in sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid, is put
It is cultivated in the cell incubator that condition is suitable for；
（5）After passage twice, it is placed in culture to its cell in incubator and grows up to single layer, using the culture medium of the embodiment of the present invention 1
After rinsing 2 times, the culture medium replacement culture in glassware liquid after cell is adherent using the embodiment of the present invention 1 continues culture to cell and grows up to
Fine and close single layer, then digests, and cell suspension is made, and it is real that in 4 T125 square vases 3/4 cell suspension is injected the 1 L present invention
It applies and is sufficiently shaken up in the medium bottle of example 1, finally 1 L cell suspension is poured into 10L rolling bottle, bottle stopper is stoppered and is put into Rotary Machine
It is cultivated.
（Two）The culture of Vero cell in the bioreactor
Weigh appropriate I type microcarrier of Cytodex（Purchased from GE）It sets in reactor tank, and 1 L acid-base buffer of addition (
PBS), dissolved oxygen, pH value electrode are calibrated, prepares alkali bottle (7. 5% sodium bicarbonate solutions), tank body is after the installation is completed at 121 DEG C
It sterilizes in pressure cooker, is taken out after 30 mim and carry out sterile test（500 mL culture mediums are added and carry out preculture, mixing speed is
50 r/h, oxyty 50%, pH value are 7. 20, temperature is 37 DEG C, lose pollution condition after 10 h), it is then discharged out tank
Interior PBS, then will digest after the cell suspension to get off mixes and be injected into the culture medium of embodiment 1 of preparation in rolling bottle, utilize pipe
Road is injected into reactor, regulating gas（Compressed air, O2, N2, CO2) and be changed to automatically, alkali bottle is adjusted to add automatically
Add, microcarrier suspension culture can be carried out by regulating revolving speed, every the cell density of monitoring in 15 minutes.
（Three）The inoculation and culture of PEDV
When culture vero cell Proliferation to 60 cells/ball cell density in bioreactor, add into bioreactor
Enter the PEDV SX-YL strain virus liquid being diluted using the culture medium of the embodiment of the present invention 1, the titre of dilution restrovirus liquid is
105.5TCID50, the volume ratio of the cell culture in virus liquid and bioreactor after dilution is 1：50.Dilution virus is added
After liquid, regulates revolving speed and carry out Virus culture, cultivate 24-26 hours, harvest virus-culturing fluid.
（Four）The preparation of PEDV inactivated vaccine
By the virus-culturing fluid of harvest at -80 DEG C, three times, 6000 rpm are centrifuged 20 min to multigelation, collect supernatant, as
Virus liquid；Virus liquid is added 37 DEG C of 0.2% formaldehyde inactivations and obtains PEDV vaccinogen liquid in 24 hours after 20 times of concentrations；It will be obtained
PEDV vaccinogen liquid and 206 adjuvant of adjuvant Montanide ISA press 1.5:1 volume ratio in gnotobasis, is stirred
It is even, obtain pig epidemic diarrhea inactivated vaccine.
（Five）Vaccine test method and result
5.1 characters examine inactivated vaccine appearance pinkiness emulsion state.
5.2 steriling test inactivated vaccines are according to existing《Republic of China Veterinary Pharmacopoeia》Version third portion's annex in 2010 into
Performing check, T.G, G.P pipe and G.A slant medium do not observe bacterium colony.
5.3 mycoplasmas examine inactivated vaccine according to《Republic of China Veterinary Pharmacopoeia》Version third portion's annex in 2010 into
Performing check does not find that significant change occur in bottle and tubule culture color, and the liquid culture of transplanting is on solid medium
Without " fried egg " shape mycoplasma bacterium colony.
5.4 exogenous virus examine inactivated vaccine according to《Republic of China Veterinary Pharmacopoeia》Version third portion annex in 2010
It tests, is passed without swine fever virus, bovine viral diarrhea virus, pig parvoviral, porcine pseudorabies virus, rotavirus, pig
The pollution such as metachromia marcy agent.Prove that seed culture of viruses is pure.
It is negative 3 age in days pig 24 that 5.5 safety verifications, which take pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into
4 groups, every group 6,10 part vaccines of intramuscular injection, clinical observation 14 days, equal 100% strong work had no adverse reaction.
Embodiment 5：The test of PEDV attenuated vaccine large scale preparation
Test strain：CV777 plants of PEDV, obtain the strain of Shaanxi Nowe Li Hua Biotechnology Co., Ltd preservation.
Test cell：Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method：
（One）The recovery of Vero cell strain：
（1）The Vero cell being removed from liquid nitrogen quickly is put into 37 DEG C of water-baths, gently shaking makes its quick-thawing；
（2）300 rpm are centrifuged 10 min；
（3）Supernatant is abandoned, the cell culture fluid of 37 DEG C of pre-temperatures is added（Containing 5% FBS DMEM）, gently dispel Vero cell；
（4）Cell suspension after mixing is added in sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid, is put
It is cultivated in the cell incubator that condition is suitable for；
（5）After passage twice, it is placed in culture to its cell in incubator and grows up to single layer, using the culture medium of the embodiment of the present invention 1
After rinsing 2 times, the culture medium replacement culture in glassware liquid after cell is adherent using the embodiment of the present invention 1 continues culture to cell and grows up to
Fine and close single layer, then digests, and cell suspension is made, and it is real that in 4 T125 square vases 3/4 cell suspension is injected the 1 L present invention
It applies and is sufficiently shaken up in the medium bottle of example 1, finally 1 L cell suspension is poured into 10L rolling bottle, bottle stopper is stoppered and is put into Rotary Machine
It is cultivated.
（Two）The culture of Vero cell in the bioreactor
Weigh appropriate I type microcarrier of Cytodex（Purchased from GE）It sets in reactor tank, and 1 L acid-base buffer of addition (
PBS), dissolved oxygen, pH value electrode are calibrated, prepares alkali bottle (7. 5% sodium bicarbonate solutions), tank body is after the installation is completed at 121 DEG C
It sterilizes in pressure cooker, is taken out after 30 mim and carry out sterile test（500 mL culture mediums are added and carry out preculture, mixing speed is
50 r/h, oxyty 50%, pH value are 7. 20, temperature is 37 DEG C, lose pollution condition after 10 h), it is then discharged out tank
Interior PBS, then will digest after the cell suspension to get off mixes and be injected into the culture medium of embodiment 1 of preparation in rolling bottle, utilize pipe
Road is injected into reactor, regulating gas（Compressed air, O2, N2, CO2) and be changed to automatically, alkali bottle is adjusted to add automatically
Add, microcarrier suspension culture can be carried out by regulating revolving speed, every the cell density of monitoring in 15 minutes.
（Three）The inoculation and culture of PEDV
When culture vero cell Proliferation to 60 cells/ball cell density in bioreactor, add into bioreactor
Enter the PEDV CV777 strain virus liquid being diluted using the culture medium of the embodiment of the present invention 1, the titre of dilution restrovirus liquid is
105.5TCID50, the volume ratio of the cell culture in virus liquid and bioreactor after dilution is 1：50.Dilution virus is added
It after liquid, regulates revolving speed and carries out Virus culture, cultivate 24-26 hour, harvest virus-culturing fluid, the virus liquid of harvest is with 0.45/
1.0 microns of membrane filtration clarification.
（Four）The preparation of PEDV attenuated vaccine
Take filtered virus liquid, addition 2%（W/W）Mannitol, 3%（W/W）Dextran, 2%（W/W）Sodium glutamate, 1%
（W/W）Urea, 0.2%（W/W）R-gene, 0.2%（W/W）Freeze-drying half is made in the protective agents ingredient such as bovine serum albumin(BSA)
Finished product, the semi-finished product do not contain serum.Packing is lyophilized into cillin bottle, and every cillin bottle dispenses 0.5ml, through following procedure into
Row freeze-drying：Pre-freeze：- 45 DEG C, 3h；Drying temperature：- 45 DEG C~30 DEG C；Drying process：- 45 DEG C, 0.01 MPa 3h are maintained, so
Increase 2 DEG C per hour afterwards.
In use, being redissolved using sterilized water for injection.（Six）Vaccine test method and result
6.1 steriling test attenuated vaccines are according to existing《Republic of China Veterinary Pharmacopoeia》Version is tested within 2010, T.G, G.P
Pipe and G.A slant medium do not observe bacterium colony.
6.2 mycoplasmas examine attenuated vaccine according to《Republic of China Veterinary Pharmacopoeia》Version is tested within 2010, is not sent out
There is significant change in existing bottle and tubule culture color, and the liquid culture of transplanting is on solid medium without " fried egg " shape branch
Bovis colony.
6.3 exogenous virus examine attenuated vaccine according to《Republic of China Veterinary Pharmacopoeia》Version is tested within 2010,
Without swine fever virus, bovine viral diarrhea virus, pig parvoviral, porcine pseudorabies virus, rotavirus, transmissible gastroenteritis of swine disease
The pollution such as poison.Prove that seed culture of viruses is pure.
It is negative 3 age in days pig 24 that 6.4 safety verifications, which take pig epidemic diarrhea neutralizing antibody, antigen, is randomly divided into
4 groups, every group 6,10 part vaccines of intramuscular injection, clinical observation 14 days, equal 100% strong work had no adverse reaction.
Embodiment 7：The comparison of different culture medium culture vero cell production PEDV CV777 virus
Test strain：CV777 plants of PEDV, obtain the strain of Shaanxi Nowe Li Hua Biotechnology Co., Ltd preservation.
Test cell：Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method：96 orifice plates use 1-2 of the embodiment of the present invention, 10% newborn bovine serum+DMEM, check experiment example 1,
The culture medium culture vero cell of check experiment example 2 and check experiment example 3, every 50 μ LPEDV CV777 virus of hole inoculation, sealing
Culture plate is placed in 37 DEG C, cultivates in 5% carbon dioxide incubator afterwards, the virus titer in 12 hours measurement cell supernatants.
Comparative experimental example is set：
Comparative experimental example 1：Referring to the comparative experimental example 1 in embodiment 3.
Comparative experimental example 2：A kind of serum free medium, which is characterized in that the serum free medium by basal medium,
Bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, trypsase, taurine and sodium selenite composition,
The weight ratio of middle taurine and sodium selenite is 1:1.
The basal medium IMDM（Giboc）；
The content of the bovine serum albumin(BSA) is 10mg/mL.
The content of the cortisol is 20 μ g/mL.
The content of the insulin is 20 μ g/mL.
The content of the transferrins is 15 μ g/mL.
The content of the glutathione is 3mM.
The content of the trypsase is 4 μ g/mL.
The content of the taurine is 10 μ g/mL.
The content of the sodium selenite is 10 μ g/mL.
The production method of the serum free medium is：Step 1 weighs basal medium dry powder, ox blood by weight
Pure albumen, cortisol, insulin, transferrins, glutathione, trypsase, taurine and sodium selenite；Step 2, will
After the weighed above material mixing, add ultrapure water to 1000mL, stirring and dissolving adjusts pH value to 7.0-7.2, then uses 0.22
μm membrane filtration degerming, 4 DEG C of preservations.
Comparative experimental example 3：A kind of serum free medium, which is characterized in that the serum free medium by basal medium,
Bovine serum albumin(BSA), cortisol, insulin, transferrins, trypsase, taurine and sodium selenite form, wherein taurine
Weight ratio with sodium selenite is 2:1.
The basal medium IMDM（Giboc）；
The content of the bovine serum albumin(BSA) is 10mg/mL.
The content of the cortisol is 20 μ g/mL.
The content of the insulin is 20 μ g/mL.
The content of the transferrins is 15 μ g/mL.
The content of the trypsase is 4 μ g/mL.
The content of the taurine is 20 μ g/mL.
The content of the sodium selenite is 10 μ g/mL.
The production method of the serum free medium is：Step 1 weighs basal medium dry powder, ox blood by weight
Pure albumen, cortisol, insulin, transferrins, trypsase, taurine and sodium selenite；Step 2, more than weighed
After material mixing, add ultrapure water to 1000mL, stirring and dissolving adjusts pH value to 7.0-7.2, then uses 0.22 μm of membrane filtration
Degerming, 4 DEG C of preservations.
Test result：The following table 1 is participated in, as shown in Table 1, PEDV CV777 virus inoculation is in different culture mediums
State, in inoculation 12-48 hours, the virus titer highest for the culture medium being prepared with the embodiment of the present invention 1 and 2 was 6.5-
7.2, the virus titer of the embodiment of the present invention 1 and 2 is above 10% newborn bovine serum+DMEM, check experiment example 1, check experiment example
2 and check experiment example 3 culture medium.
Virus titer of the 1 PEDV CV777 virus inoculation of table after vero cell in different culture medium culture solution supernatant
（log2）
| 0h | 12h | 24h | 36h | 48h | |
| Embodiment 1 | 0 | 4.2 | 5.7 | 6.5 | 7.2 |
| Embodiment 2 | 0 | 3.9 | 5.4 | 6.2 | 6.5 |
| 10% newborn bovine serum+DMEM | 0 | 3.2 | 4.3 | 5.0 | 5.5 |
| Check experiment example 1 | 0 | 3.1 | 3.9 | 4.5 | 5.0 |
| Check experiment example 2 | 0 | 3.5 | 4.2 | 4.8 | 5.2 |
| Check experiment example 3 | 0 | 2.9 | 4.1 | 4.7 | 5.3 |
By the result of the above table 1 it is found that the embodiment of the present invention 1 and the culture medium of embodiment 2 to the proliferation of PEDV have compared with
Good cultivation effect adds DMEM culture medium compared to existing common calf serum, can more promote the proliferation of PEDV, proliferation
Virus titer is significantly raised, show culture medium of the invention can substitute traditional serum-containing media for the proliferation of PEDV and
The preparation of vaccine, immune problem caused by the use so as to avoid serum in vaccine production process；It is arranged with the present invention
Control experiment group compare, further demonstrate the addition of glutathione and the ratio of taurine and sodium selenite for PEDV
The importance of cell Proliferation is not added with glutathione in comparative experimental example 1, and there is also differences for the ratio of taurine and sodium selenite
It is different, it is higher than the present invention, then its virus multiplication effect difference more of the present invention；The ratio of taurine and sodium selenite in comparative experimental example 2
It is 1：1, i.e. taurine then causes virus multiplication too late expected using deficiency, although it is proliferated comparatively fast in the 0-12h stage,
Rise in the titre in virus multiplication later period slower；Glutathione is not added in comparative experimental example 3, then virus multiplication is too late expected,
Its early stage proliferation is relatively slow, and later period proliferation also shows slightly insufficient.The above test results show that in culture medium of the invention, paddy Guang
The ratio of the addition of sweet peptide and taurine and sodium selenite has great importance for PEDV cell Proliferation, and the two is in disease
It is synergistic in malicious Multiplying culture, achieve unexpected culture effect.
It is any in spirit and/or range of the invention due to describing the present invention by the above preferred embodiment
Implement the present invention for replacement/or combination of the invention, will be apparent from for those skilled in the art, and
Among the present invention.
Claims (5)
1. a kind of method using serum free medium production PEDV attenuated vaccine, described method includes following steps：
Step（1）, the recovery of Vero cell；
Step（2）, the passage amplification of Vero cell；
Step（3）, PEDV virus stain is inoculated on Vero cell；
Step（4）, infection cell culture supernatant is harvested, virus liquid carries out clarification filtration；
Step（5）, freeze drying protectant, packing freeze-drying is added；
Wherein step（2）In passage amplification and step（3）The breeding of middle virus is all made of free serum culture of the invention
Base.
2. the method according to claim 1, wherein the serum free medium is by basal medium, cow's serum
Albumin, cortisol, insulin, transferrins, glutathione, trypsase, taurine and sodium selenite form, wherein ox sulphur
The weight ratio of acid and sodium selenite is 2-3:1.
3. method according to claim 1 or 2, which is characterized in that in the serum free medium：
The basal medium IMDM（Giboc）The mixture of RPMI-1640 or the two；
The content of the bovine serum albumin(BSA) is 8-15mg/mL；
The content of the cortisol is 15-25 μ g/mL；
The content of the insulin is 10-20 μ g/mL；
The content of the transferrins is 10-15 μ g/mL；
The content of the glutathione is 2-3mM；
The content of the trypsase is 3-5 μ g/mL；
The content of the taurine is 20-30 μ g/mL；
The content of the sodium selenite is 10-12 μ g/mL.
4. method according to claim 1 or 2, which is characterized in that in the serum free medium：
The basal medium IMDM（Giboc）The mixture of RPMI-1640 or the two；
The content of the bovine serum albumin(BSA) is 10mg/mL；
The content of the cortisol is 20 μ g/mL；
The content of the insulin is 20 μ g/mL；
The content of the transferrins is 15 μ g/mL；
The content of the glutathione is 3mM；
The content of the trypsase is 4 μ g/mL；
The content of the taurine is 20 μ g/mL；
The content of the sodium selenite is 10 μ g/mL.
5. the method according to any one of claim 2-4, it is characterised in that：The production side of the serum free medium
Method is：Step 1 weighs basal medium dry powder, bovine serum albumin(BSA), cortisol, insulin, transferrins, paddy by weight
The sweet peptide of Guang, trypsase, taurine and sodium selenite；Step 2 adds ultrapure water extremely after the weighed above material mixing
1000mL, stirring and dissolving adjust pH value to 7.0-7.2, then use 0.22 μm of membrane filtration degerming, 4 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810690966.0A CN108853489B (en) | 2018-06-29 | 2018-06-29 | A method of PEDV attenuated vaccine is produced using serum free medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810690966.0A CN108853489B (en) | 2018-06-29 | 2018-06-29 | A method of PEDV attenuated vaccine is produced using serum free medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108853489A true CN108853489A (en) | 2018-11-23 |
| CN108853489B CN108853489B (en) | 2019-07-02 |
Family
ID=64296551
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810690966.0A Active CN108853489B (en) | 2018-06-29 | 2018-06-29 | A method of PEDV attenuated vaccine is produced using serum free medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108853489B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747176A (en) * | 2019-11-19 | 2020-02-04 | 畜科生物工程有限公司 | Culture method for improving virus titer of porcine epidemic diarrhea viruses |
| CN114929855A (en) * | 2019-12-24 | 2022-08-19 | 株式会社Lg化学 | Low serum media compositions for culturing VERO cells and uses thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220347A (en) * | 2007-12-28 | 2008-07-16 | 天津百若克医药生物技术有限责任公司 | Human embryo kidney 293 cell amplifying protein-free medium |
| CN103958665A (en) * | 2011-11-11 | 2014-07-30 | 基本药品有限责任公司 | Kit comprising serum replacement and labile factors |
| CN104640974A (en) * | 2012-07-24 | 2015-05-20 | 日产化学工业株式会社 | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN105441378A (en) * | 2015-12-22 | 2016-03-30 | 肇庆大华农生物药品有限公司 | Serum-free medium used for culturing Vero cells, and preparation method thereof |
| CN105832759A (en) * | 2015-01-17 | 2016-08-10 | 广州自远生物科技有限公司 | Pharmaceutical composition for preventing and/or treating diseases caused by coronaviruses and/or rotaviruses |
| CN106062205A (en) * | 2013-12-20 | 2016-10-26 | 基本药品有限责任公司 | Media for cell culture |
-
2018
- 2018-06-29 CN CN201810690966.0A patent/CN108853489B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220347A (en) * | 2007-12-28 | 2008-07-16 | 天津百若克医药生物技术有限责任公司 | Human embryo kidney 293 cell amplifying protein-free medium |
| CN103958665A (en) * | 2011-11-11 | 2014-07-30 | 基本药品有限责任公司 | Kit comprising serum replacement and labile factors |
| CN104640974A (en) * | 2012-07-24 | 2015-05-20 | 日产化学工业株式会社 | Culture medium composition, and method for culturing cell or tissue using said composition |
| CN106062205A (en) * | 2013-12-20 | 2016-10-26 | 基本药品有限责任公司 | Media for cell culture |
| CN105832759A (en) * | 2015-01-17 | 2016-08-10 | 广州自远生物科技有限公司 | Pharmaceutical composition for preventing and/or treating diseases caused by coronaviruses and/or rotaviruses |
| CN105441378A (en) * | 2015-12-22 | 2016-03-30 | 肇庆大华农生物药品有限公司 | Serum-free medium used for culturing Vero cells, and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747176A (en) * | 2019-11-19 | 2020-02-04 | 畜科生物工程有限公司 | Culture method for improving virus titer of porcine epidemic diarrhea viruses |
| CN110747176B (en) * | 2019-11-19 | 2021-08-17 | 畜科生物工程有限公司 | Culture method for improving virus titer of porcine epidemic diarrhea viruses |
| CN114929855A (en) * | 2019-12-24 | 2022-08-19 | 株式会社Lg化学 | Low serum media compositions for culturing VERO cells and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108853489B (en) | 2019-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101934074B (en) | Porcine circovirus II vaccine and production method thereof | |
| CN105311630B (en) | A kind of mammalian cell suspension culture prepares the method and application of vaccine | |
| CN101804203A (en) | Method for mass production of pseudorabies virus vaccine | |
| CN101869702B (en) | Vaccine produced by suspension microcarrier cell culture system and method thereof | |
| CN108853489B (en) | A method of PEDV attenuated vaccine is produced using serum free medium | |
| CN105582533B (en) | Avian influenza virus and avian adenovirus bivalent inactivated vaccine | |
| CN108721615A (en) | A kind of method and its vaccine preparing duck tembusu virus inactivated vaccine | |
| CN114276981B (en) | Vero-E6 suspension cell strain sVero-E6 suitable for porcine epidemic diarrhea virus and application thereof | |
| CN103861097A (en) | Method for preparing porcine epizootic diarrhea inactivated vaccines and product thereof | |
| CN108421037A (en) | A kind of porcine pseudorabies/porcine parvovirus bivalent inactivated vaccine and its culture preparation method that suspends | |
| CN103923885B (en) | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof | |
| CN108815516B (en) | A method of PEDV inactivated vaccine is produced using serum free medium | |
| CN102727877A (en) | Method for preparing highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) by utilizing bioreactor and application thereof | |
| CN102727878A (en) | Preparation method of porcine reproductive and respiratory syndrome inactivated vaccine (NVDC-JXA1 strain) by bioreactor and application thereof | |
| CN102586195B (en) | Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells | |
| CN113384692A (en) | Duck reovirus and duck circovirus bivalent inactivated vaccine and preparation method thereof | |
| CN108841780B (en) | It is suitble to the serum free medium of large-scale production PEDV vaccine | |
| CN105816872A (en) | Preparation method of mink parvoviral enteritis inactivated vaccine and vaccine prepared by using same | |
| CN103861096A (en) | Preparation method of live vaccine for treating porcine reproductive and respiratory syndrome with high pathogenicity and live vaccine product | |
| CN117417900A (en) | Serum-free cell culture method of human diploid cells for rabies vaccine and application | |
| CN117431220A (en) | Method for preparing rabies virus antigen by serum-free culture of human diploid cells and application | |
| CN110747176B (en) | Culture method for improving virus titer of porcine epidemic diarrhea viruses | |
| CN108159412A (en) | It is a kind of to produce cell source newcastle disease, method of bird flu bivalent inactivated vaccine and products thereof | |
| CN106237324A (en) | A kind of method using full suspension technology to produce transmissible gastroenteritis of swine vaccine | |
| CN104740627B (en) | A kind of large-scale method for producing of pseudo- mad dog attenuated live vaccines for animals |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |