CN108815516B - A method of PEDV inactivated vaccine is produced using serum free medium - Google Patents

A method of PEDV inactivated vaccine is produced using serum free medium Download PDF

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CN108815516B
CN108815516B CN201810691800.0A CN201810691800A CN108815516B CN 108815516 B CN108815516 B CN 108815516B CN 201810691800 A CN201810691800 A CN 201810691800A CN 108815516 B CN108815516 B CN 108815516B
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pedv
taurine
sodium selenite
free medium
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CN108815516A (en
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杜恩岐
陈瑞
刘项羽
左文峰
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Shaanxi Lihua Norwich Biotechnology Co Ltd
Northwest A&F University
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Shaanxi Lihua Norwich Biotechnology Co Ltd
Northwest A&F University
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Abstract

The present invention relates to a kind of methods using serum free medium production PEDV inactivated vaccine; 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.This method 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

A method of PEDV inactivated vaccine is produced using serum free medium
Technical field:
The present invention relates to field of biotechnology, and in particular to a kind of to produce PEDV inactivated vaccine using serum free medium Method.
Background technique:
Pig epidemic diarrhea (PED) is acute and highly infectious intestines as caused by Porcine epidemic diarrhea virus (PEDV) Road viral disease, symptom include diarrhea, vomiting, anorexia, and dehydration and pig weight mitigate.Although the pig of different age group is all By different degrees of infection and symptom may occur, but the state of an illness especially severe of piglet, wherein the death rate is up to 100%, The intestinal mucosal surface that PEDV mainly passes through piggy enters host, mainly results in enterocyte damage.Although different age group Pig all by different degrees of infection and symptom may occur, but the state of an illness especially severe of piglet, and wherein the death rate is up to The intestinal mucosal surface that 100%, PEDV mainly pass through piggy enters host, mainly results in enterocyte damage.For PEDV's Great outburst, we can usually control PEDV using vaccination, and carried out with kill extensively or subtracted in China The PEDV vaccine of poison carries out vaccine inoculation, controls the large-scale outbreak of PEDV to a certain extent.But as Chinese PEDV goes out Living or attenuation bigeminy vaccine to be widely used, PED persistently spreads in October, 2010 in China, makes a variation at that time Strain, PEDV infection sharply increase, and seriously endanger pig breeding industry.Although the country for epidemic diarrhea vaccine (including bigeminy inactivation Seedling and bigeminal live vaccine) extensive promotion and application have been obtained, and reduce pig epidemic in a certain range and degree The disease incidence rushed down, but the disease still fails to be fully controlled, especially in October, 2010 so far, which is in duration, quick-fried Hair property 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, it may be assumed that 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 high production cost present in serum use High, the present invention is intended to provide a kind of method using serum free medium production PEDV inactivated vaccine, which can not add Object serum, significantly reduces production cost, improves the economic benefit of production, and also avoids brought by serum use Problems.
In order to solve the above technical problems, the present invention uses following technological means:
A method of PEDV inactivated 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) is inoculated with PEDV virus stain on Vero cell；
Step (4), harvests infection cell culture supernatant, and virus liquid carries out clarification filtration；
Step (5), inactivation, that is, be prepared PEDV inactivated vaccine；
Wherein breeding viral in the passage amplification and step (3) in step (2) is all made of serum-free of the invention Culture medium.
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。
In the serum free medium:
The basal medium IMDM(Giboc) RPMI-1640 or the two mixture；
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) RPMI-1640 or the two mixture；
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 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.
The invention further relates to the method using serum free medium culture PEDV virus of the present invention, the method packets Include following steps:
Step (1), the recovery of Vero cell；
Step (2), the passage amplification of Vero cell；
Step (3) is inoculated with PEDV virus stain on Vero cell；
Step (4), harvests infection cell culture supernatant, and virus liquid carries out clarification filtration to get sick to the PEDV Venom；
Wherein breeding viral in the passage amplification and step (3) in step (2) is all made of serum-free of the invention Culture medium.
Based on above technical scheme, the invention has the advantages that and the utility model has the advantages that
The embodiment of the present invention 1 and the culture medium of embodiment 2 have preferable cultivation effect, phase to the proliferation of PEDV Adding DMEM culture medium than existing common calf serum, can more promote the proliferation of PEDV, the virus titer of proliferation is significantly raised, Show that culture medium of the invention can substitute traditional serum-containing media for the proliferation of PEDV and the preparation of vaccine, to keep away Immune problem caused by the use of serum in vaccine production process is exempted from；Its compared with the control experiment group that is arranged of the present invention, Further demonstrate glutathione addition and taurine and sodium selenite ratio for PEDV cell Proliferation importance, Glutathione is not added in comparative experimental example 1, and there is also differences for the ratio of taurine and sodium selenite, are higher than the present invention, then Its virus multiplication effect difference more of the present invention；The ratio of taurine and sodium selenite is 1:1 in comparative experimental example 2, i.e. taurine makes With deficiency, then cause virus multiplication too late expected, although it is proliferated in the 0-12h stage comparatively fast, in the virus multiplication later period Titre rises slower；Glutathione is not added in comparative experimental example 3, then virus multiplication is too late expected, and early stage is proliferated relatively Slowly, later period proliferation also shows slightly insufficient.The above test results show that in culture medium of the invention, the addition of glutathione and ox The ratio of sulfonic acid and sodium selenite has great importance for PEDV cell Proliferation, and the two cooperates in virus multiplication culture Synergy achieves unexpected culture effect.
Detailed description of the invention:
The case where Fig. 1: PEDV Virus culture.A, control group, normal vero cell；B connects poison after 24 hours, and compares The normal cell of group is compared, and more apparent CPE occurs；C connect poison after 48 hours, and cell rounding has part cells float de- It falls, the pathological changes such as seine shape occurs.
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:
A kind of embodiment 1: serum free medium of suitable large-scale production PEDV vaccine, which is characterized in that the no blood Clear culture medium is by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, trypsase, ox Sulfonic acid and sodium selenite composition, 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 the 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.
A kind of embodiment 2: serum free medium of suitable large-scale production PEDV vaccine, which is characterized in that the no blood Clear culture medium is by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, glutathione, trypsase, ox Sulfonic acid and sodium selenite composition, 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 the 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.
The small-scale proliferation test of embodiment 3:PEDV
Test strain:, being obtained from Xibei Univ. of Agricultural & Forest Science & Technology by SX-YL plants of PEDV, which advises from Yangling Shaanxi Modelling farm.
Test cell: Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method:
(1) 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 (containing 5% FBS DMEM) of 37 DEG C of pre-temperatures is added, gently dispels Vero cell；
(4) cell suspension after mixing is added to sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid In, it is put in the suitable cell incubator of condition and cultivates；
(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 Single 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.
(2) viral inoculation, culture and IFA detection:
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 plates, after cells grow up to the individual layer is inoculated with PEDV, while setting the normal cell controls for not connecing poison, occurs to cell 20 min are fixed with ethyl alcohol (- 20 DEG C of pre-coolings) after Minimal change, are then washed 3 times with PBS, each 5min.PEDV N egg is added Bai Dankang (Xibei Univ. of Agricultural & Forest Science & Technology's present), 37 DEG C of incubation 60min, PBS are washed three times, and the sheep anti mouse Ig of FITC label is added G.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 by base Basal culture medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, trypsase, taurine and sodium selenite composition, The weight ratio of middle taurine 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.
The large-scale production test of embodiment 4:PEDV inactivated vaccine
Test strain:, being obtained from Xibei Univ. of Agricultural & Forest Science & Technology by SX-YL plants of PEDV, which advises from Yangling Shaanxi Modelling farm.
Test cell: Vero cell strain, the present of Xibei Univ. of Agricultural & Forest Science & Technology animal medicine institute veterinary vaccination laboratory.
Test method:
(1) 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 (containing 5% FBS DMEM) of 37 DEG C of pre-temperatures is added, gently dispels Vero cell；
(4) cell suspension after mixing is added to sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid In, it is put in the suitable cell incubator of condition and cultivates；
(5) it after passage twice, is placed in culture to its cell in incubator and grows up to single layer, using the training of the embodiment of the present invention 1 After supporting base flushing 2 times, culture is continued to cell using the culture medium replacement culture in glassware liquid of the embodiment of the present invention 1 after cell is adherent Grow up to fine and close single layer, then digest, cell suspension is made, by 4 T125 square vases 3/4 1 L this hair of cell suspension injection Sufficiently shake up in the medium bottle of bright embodiment 1, finally pour into 1 L cell suspension in 10L rolling bottle, be stoppered bottle stopper be put into turn Bottle machine is cultivated.
(2) culture of Vero cell in the bioreactor
It weighs appropriate I type microcarrier of Cytodex (purchased from GE) to set in reactor tank, and 1 L acid-base buffer is added (PBS), dissolved oxygen, pH value electrode are calibrated, prepares alkali bottle (7. 5% sodium bicarbonate solutions), tank body is after the installation is completed 121 Sterilizing in DEG C pressure cooker takes out after 30 mim and carries out sterile test and (500 mL culture mediums are added and carry out preculture, mixing speed For 50 r/h, oxyty 50%, pH value be 7. 20, temperature is 37 DEG C, lose pollution condition after 10 h), be then discharged out PBS in tank, 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, it utilizes Pipeline is injected into reactor, and regulating gas (compressed air, O2, N2, CO2) is simultaneously changed to automatically, and alkali bottle is adjusted to automatically Addition, microcarrier suspension culture can be carried out by regulating revolving speed, every the cell density of monitoring in 15 minutes.
(3) inoculation and culture of PEDV
When culture vero cell Proliferation to 60 cells/ball cell density in bioreactor, to bioreactor It is middle that the PEDV SX-YL strain virus liquid being diluted using the culture medium of the embodiment of the present invention 1 is added, dilute the drop of restrovirus liquid Degree is 105.5TCID50, the volume ratio of the cell culture in virus liquid and bioreactor after dilution is 1:50.Dilution is added After virus liquid, regulates revolving speed and carry out Virus culture, cultivate 24-26 hours, harvest virus-culturing fluid.
(4) 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 system PEDV vaccinogen liquid and 206 adjuvant of adjuvant Montanide ISA press the volume ratio of 1.5:1, in gnotobasis, stirring is mixed It closes uniformly, obtains pig epidemic diarrhea inactivated vaccine.
(5) vaccine test method and result
5.1 characters examine inactivated vaccine appearance pinkiness emulsion state.
5.2 steriling test inactivated vaccines 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.
The test of embodiment 5:PEDV attenuated vaccine large scale preparation
It tests strain: CV777 plants of PEDV, obtaining 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:
(1) 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 (containing 5% FBS DMEM) of 37 DEG C of pre-temperatures is added, gently dispels Vero cell；
(4) cell suspension after mixing is added to sterile T25 bottles containing 5m L5% FBS DMEM cell culture fluid In, it is put in the suitable cell incubator of condition and cultivates；
(5) it after passage twice, is placed in culture to its cell in incubator and grows up to single layer, using the training of the embodiment of the present invention 1 After supporting base flushing 2 times, culture is continued to cell using the culture medium replacement culture in glassware liquid of the embodiment of the present invention 1 after cell is adherent Grow up to fine and close single layer, then digest, cell suspension is made, by 4 T125 square vases 3/4 1 L this hair of cell suspension injection Sufficiently shake up in the medium bottle of bright embodiment 1, finally pour into 1 L cell suspension in 10L rolling bottle, be stoppered bottle stopper be put into turn Bottle machine is cultivated.
(2) culture of Vero cell in the bioreactor
It weighs appropriate I type microcarrier of Cytodex (purchased from GE) to set in reactor tank, and 1 L acid-base buffer is added (PBS), dissolved oxygen, pH value electrode are calibrated, prepares alkali bottle (7. 5% sodium bicarbonate solutions), tank body is after the installation is completed 121 Sterilizing in DEG C pressure cooker takes out after 30 mim and carries out sterile test and (500 mL culture mediums are added and carry out preculture, mixing speed For 50 r/h, oxyty 50%, pH value be 7. 20, temperature is 37 DEG C, lose pollution condition after 10 h), be then discharged out PBS in tank, 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, it utilizes Pipeline is injected into reactor, and regulating gas (compressed air, O2, N2, CO2) is simultaneously changed to automatically, and alkali bottle is adjusted to automatically Addition, microcarrier suspension culture can be carried out by regulating revolving speed, every the cell density of monitoring in 15 minutes.
(3) inoculation and culture of PEDV
When culture vero cell Proliferation to 60 cells/ball cell density in bioreactor, to bioreactor It is middle that the PEDV CV777 strain virus liquid being diluted using the culture medium of the embodiment of the present invention 1 is added, dilute the drop of restrovirus liquid Degree is 105.5TCID50, the volume ratio of the cell culture in virus liquid and bioreactor after dilution is 1:50.Dilution is added It after virus liquid, regulates revolving speed and carries out Virus culture, cultivate 24-26 hours, harvest virus-culturing fluid, the virus liquid of harvest is used 0.45/1.0 the membrane filtration clarification of micron.
(4) preparation of PEDV attenuated vaccine
Filtered virus liquid is taken, 2%(W/W is added) mannitol, 3%(W/W) dextran, 2%(W/W) glutamic acid Sodium, 1%(W/W) urea, 0.2%(W/W) R-gene, 0.2%(W/W) the protective agents ingredient such as bovine serum albumin(BSA), it is made Semi-finished product are lyophilized, which does not contain serum.Packing is lyophilized into cillin bottle, and every cillin bottle dispenses 0.5ml, through following Program is lyophilized: pre-freeze: -45 DEG C, 3h；Drying temperature: -45 DEG C~30 DEG C；Drying process: -45 DEG C are maintained, 0.01 MPa Then 3h increases 2 DEG C per hour.
In use, being redissolved using sterilized water for injection.(6) vaccine test method and result
6.1 steriling test attenuated vaccines are tested according to existing " Republic of China Veterinary Pharmacopoeia " version in 2010, T.G, G.P pipe and G.A slant medium do not observe bacterium colony.
6.2 mycoplasmas examine attenuated vaccine to test according to " Republic of China Veterinary Pharmacopoeia " version in 2010, do not send 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 to test according to " Republic of China Veterinary Pharmacopoeia " version in 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
It tests strain: CV777 plants of PEDV, obtaining 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.
A kind of comparative experimental example 2: 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.
A kind of comparative experimental example 3: 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)
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 have the proliferation of PEDV There is preferable cultivation effect, adds DMEM culture medium compared to existing common calf serum, can more promote the proliferation of PEDV, increase The virus titer grown is significantly raised, shows the increasing that culture medium of the invention can substitute traditional serum-containing media for PEDV The preparation with vaccine is grown, immune problem caused by the use so as to avoid serum in vaccine production process；Itself and the present invention The control experiment group of setting is compared, further demonstrate glutathione addition and taurine and sodium selenite ratio for The importance of PEDV cell Proliferation is not added with glutathione in comparative experimental example 1, and the ratio of taurine and sodium selenite is also deposited In difference, it is higher than the present invention, then its virus multiplication effect difference more of the present invention；Taurine and sodium selenite in comparative experimental example 2 Ratio 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, But rise in the titre in virus multiplication later period slower；Glutathione is not added in comparative experimental example 3, then virus multiplication is not as good as pre- Phase, 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 Ratio the having great importance for PEDV cell Proliferation of the addition of the sweet peptide of Guang and taurine and sodium selenite, the two exists It is synergistic in virus multiplication 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 (3)

1. a kind of method using serum free medium production PEDV inactivated vaccine, described method includes following steps:
Step (1), the recovery of Vero cell；
Step (2), the passage amplification of Vero cell；
Step (3) is inoculated with PEDV virus stain on Vero cell；
Step (4), harvests infection cell culture supernatant, and virus liquid carries out clarification filtration；
Step (5), inactivation, that is, be prepared PEDV inactivated vaccine；
Wherein breeding viral in the passage amplification and step (3) in step (2) is all made of the serum free medium；
The serum free medium is by basal medium, bovine serum albumin(BSA), cortisol, insulin, transferrins, gluathione Peptide, trypsase, taurine and sodium selenite composition, wherein the weight ratio of taurine and sodium selenite is 2-3:1；
In the serum free medium:
The mixture of the basal medium IMDM 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.
2. the method according to claim 1, wherein in the serum free medium:
The mixture of the basal medium IMDM 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.
3. -2 described in any item methods according to claim 1, it is characterised in that: the production method of the serum free medium Be: step 1 weighs basal medium dry powder, bovine serum albumin(BSA), cortisol, insulin, transferrins, paddy Guang by weight Sweet peptide, 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.
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