CN101926991A - Classical swine fever virus vaccine and production method thereof - Google Patents
Classical swine fever virus vaccine and production method thereof Download PDFInfo
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- CN101926991A CN101926991A CN2010102732241A CN201010273224A CN101926991A CN 101926991 A CN101926991 A CN 101926991A CN 2010102732241 A CN2010102732241 A CN 2010102732241A CN 201010273224 A CN201010273224 A CN 201010273224A CN 101926991 A CN101926991 A CN 101926991A
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Abstract
The invention discloses a method for preparing a classical swine fever (CSF) vaccine by using a cell microcarrier suspension culture system, which comprises the following steps of: (1) inoculating cells for preparing the vaccine to a carrier tank containing culture solution and a microcarrier, and uniformly mixing the cells and the microcarrier to make the cells attached to the microcarrier; (2) when the concentration after cell proliferation is 5 to 40 times of the initial inoculation concentration, inoculating CSF virus (lapinized virus) to the cells according to multiplicity of infection (M.O.I.) of the virus of 0.01-1 and reproducing the virus; and (3) mixing prepared virus liquid, adding an appropriate freeze-drying protective agent, fully and uniformly mixing, quantitatively packaging, and freeze-drying to obtain the CSF vaccine. The CSF vaccine produced by the method has the advantages of high density of cultured cells, continuous culture, high yield of the virus, high immune effect, high safety, complete immune protection on attack of violent CSF, and the like.
Description
Technical field
The invention belongs to biological product technical field, relate to a kind of classical swine fever virus vaccine and production method thereof.
Background technology
Swine fever (Classical Swine fever, abbreviation CSF) is called hog cholera (Hog cholera) or Europe class swine fever (European swine fever) again, it is the viral infectious disease of the only acute or super febris acuta of a boar, feature clinically is to disseminate rapidly, have a fever, and can see typical hemorrhage pathological changes when dissecting check.The pig that swine fever can infect the various ages only, popular throughout the year, M ﹠ M is all high, and is only very harmful to pig.Primary disease is to threaten one of most important infectious disease of pig industry.OIE (OIE) classifies swine fever one of as 16 kinds of Notifiable diseases of category-A, and China then is decided to be a class deadly infectious disease.
Many countries and regions use swine fever malicious vaccine alive to force immunoprophylaxis, also are the best approaches of preventing and treating swine fever at present.
The production method of swine fever (CSF) vaccine has following several at present: (1) tissue Seedling method: promptly with swine fever virus (rabbitization low virulent strain) inoculation rabbit, collect spleen and the lymph node of rabbit then and produce swine fever spleen pouring Seedling; (2) former generation bull testis cell vaccine method: promptly gather nascent calf testis,, cultivate, produce cell vaccine with the rolling bottle culture systems through EDTA-trypsinization cell dispersion; (3) cell line method: with cell line (for example ST) process EDTA-trypsinization cell dispersion, cultivate with the rolling bottle culture systems, obtain hog cholera lapinised virus vaccine, harvesting virus liquid, add suitable stabilizing agent, make the cell line live vaccines of hog cholera through lyophilisation.
Wherein organize Seedling method, not high, the shortcomings such as efficient is low, exogenous virus pollution, the malicious titre of product is not high, differences between batches are big of former generation bull testis cell vaccine method ubiquity output; The cell line method though solved the exogenous virus pollution problem, is produced malicious titre and is also improved a lot, and differences between batches have also reduced, and vaccine output still can not be greatly improved, and technology is loaded down with trivial details, inefficiency, causes very big personnel's waste.
Summary of the invention
Main purpose of the present invention provides a kind of production method of swine Fever Vaccine, comprises the steps:
1) in the microcarrier bioreactor, adds microcarrier, passage cell and cell growth medium, start cell absorption program, after making microcarrier and passage cell fully combining, start the cell culture program, cultivate passage cell.Wherein microcarrier density is 30～60g/L, and passage cell density is 1.6 * 10
7～3.2 * 10
7The cells/g microcarrier;
2) above-mentioned passage cell is cultured to 4 * 10
10Cells/L～3.2 * 10
11Using cell maintenance medium during cells/L instead, is M.O.I.=0.01～1 inoculation swine fever virus according to infection multiplicity, starts viral absorption program, and virus is fully contacted with passage cell, uses the Virus culture program instead, the amplification swine fever virus;
3) Virus culture was gathered in the crops viral liquid after 2～5 days, obtained swine fever virus, 4 ℃ of preservations;
4) Shou Huo viral liquid adds freeze drying protectant behind the purification, makes swine Fever Vaccine.
Preferably, bioreactor of the present invention is the microcarrier suspension culture bioreactor.
Preferably, bioreactor of the present invention is for being tidal type microcarrier suspension culture bioreactor.
Preferably, microcarrier of the present invention is spherical, netted or lamellar
Preferably, the composition of microcarrier of the present invention is polyester, gelatin or polysaccharide.
Preferably, cell growth medium described in the step 1) of the present invention is the DMEM culture medium that contains sheep blood serum or Ox blood serum.
Preferably, cell maintenance medium step 2 of the present invention) is the DMEM culture medium that contains sheep blood serum or Ox blood serum.
Preferably, cell growth medium described in the step 1) of the present invention is the DMEM culture medium that contains 3%～15% sheep blood serum or Ox blood serum.
Preferably, cell maintenance medium step 2 of the present invention) is the DMEM culture medium that contains 1%～5% sheep blood serum or Ox blood serum
Preferably, the condition of culture of microcarrier bioreactor of the present invention is 34 ℃～37 ℃ of temperature, CO
2Concentration is 2.5%～5%, and medium pH value is 7.0～7.6.
Preferably, the cell attaching program described in the step 1) of the present invention is: cell attaching program is: up 2000～3000ml/min; Hold 1min; Down 2000～3000ml/min; Hold 30s; The cell culture program is: up 1000～3000ml/min; Hold 1min; Down 1000～3000ml/min; Hold 1min; Step 2) described viral absorption program is: up 1000～3000ml/min; Hold 1min; Down 1000～3000ml/min; Hold 30s; The Virus culture program parameter is: up 1000～2000ml/min; Hold 1min; Down:1000～2000ml/min; Hold 1min.
Preferably, the program of cell attaching described in the step 1) of the present invention is: up2800ml/min; Hold 1min; Down 2800ml/min; Hold 30s; Set maximum and change liquid measure 9000ml; The cell culture program is: up 2000ml/min; Hold 1min; Down2000ml/min; Hold 1min; Set maximum and change liquid measure 9000ml;
Preferably, viral absorption program is step 2 of the present invention): up2000ml/min; Hold 1min; Down 2000ml/min; Hold 30s; Set maximum and change liquid measure 9000ml; The Virus culture program parameter is: up 1400ml/min; Hold 1min; Down 1400ml/min; Hold 1min; Set maximum and change liquid measure 9000ml.
In the present invention, term " Up " is the rate of climb of culture fluid by microcarrier, " Down " speed that to be culture fluid descend by carrier, and " Hold " is immersed in time in the culture fluid for microcarrier.
Preferably, the volume of swine fever virus liquid described in the step 3) of the present invention is 2.5L～1000L.
Preferably, the virus of the results described in the step 3) of the present invention liquid is the continous way results.
Preferably, the number of times of the results described in the step 3) of the present invention is 3～15 times.
Another aspect of the invention is the swine fever virus of using the inventive method preparation.
Another aspect of the present invention is for using the classical swine fever virus vaccine of the inventive method preparation.
Technique effect
Compared with prior art, the production method of swine fever virus of the present invention has following beneficial effect:
(1) malicious valency height: it only is 10 that the swine fever virus of traditional rolling bottle explained hereafter is tired
4.5TCID
50/ ml, and the present invention adopts new technical parameter, utilization tidal type microcarrier suspension culture technology high density is cultivated the virus titer of producing and can be reached 10
6.5TCID
50/ ml, its malicious valency will exceed 100 times of traditional methods.
(2) big, single batch of output height of production scale: present domestic employing stirring-type suspension culture technology is cultivated zooblast, and separate unit bioreactor maximum-norm is no more than 100L; And the present invention adopts new technical parameter, and utilization tidal type microcarrier suspension culture technology is cultivated zooblast, and separate unit bioreactor scale reaches 500L, and maximum can reach 1000L, and the separate unit scale improves 5-10 doubly.
(3) the closed production of continous way, results virus: method of the present invention can totally-enclosedly be produced, and moves liquid certainly, and harvesting approach is collected viral liquid continuously, has reduced the probability of polluting, the product quality stable homogeneous, and differences between batches are little.The cell inoculation amount of the inventive method preparation is low, and control is easy, and viral infection efficient height, and the antigen of the high titre that obtains can improve the immunocompetence of vaccine greatly.The inventive method has solved traditional handicraft only can control temperature and rotating speed, the problem that the different batches mass discrepancy is big, differences between batches are big.The inventive method, directly linear amplification is used for producing.
(4) the cell inoculation amount of the inventive method preparation is low; control easily; and viral infection efficient height; can improve the immunocompetence of vaccine greatly with the live vaccines of hog cholera of the antigen manufacturing of high titre; show through immune challenge test result; to the swine fever strong virus attack can 100% protection, have good safety and immune effect.Adopt the inventive method, solved not only that spleen drenches Seedling, the ubiquitous production efficiency of former generation bull testis cell vaccine is low, exogenous virus pollutes, produce the not high shortcoming of malicious titre, and single batch of problem such as output is not high, differences between batches big, unstable product quality, labor intensity are big, production cost height when having solved traditional rolling bottle large-scale production.
(5) easy to operate, the working place is little: the carrier that adopts in the inventive method is netted polyester fiber, possess hydrophilic property and biological innocuousness, the 1g carrier can provide 2400cm
2Adherent area, in same space, increased the adherent area of cell greatly, increased the density of cell growth, cell number can reach 1.0 * 10
9More than, its usefulness is tens of times of traditional rolling bottle culture systems, can save many costs and manpower.500L working volume of the present invention only needs 20m
2Operating area, only need 2 people just can finish whole operations, and 2000 big square vases of traditional handicraft needs or rolling bottle, the minimum 600m that needs
2Operating area, minimum needs 100 people just can finish.Adopt the inventive method, single batch of problem such as output is not high, differences between batches big, unstable product quality, labor intensity are big, production cost height when having solved traditional rolling bottle and producing.
(6) technological parameter control accurately: controllable parameter had temperature, pH value, dissolved oxygen amount, gas concentration lwevel, carrier concn when the present invention used tidal type microcarrier suspension culture technology to produce, the parameter that can realize on-line monitoring has glucose, lactic acid and ammonium concentration, batch steady quality, and traditional rolling bottle culture process only can be controlled temperature and rotating speed, and the different batches mass discrepancy is big.The inventive method is utilized bioreactor, solve the problem that antigen concentration is low, production cost is high, labor intensity is big, and can continuous culture, take up an area of shearing force, the generation of no bubble, pair cell little, that production scale big, pair cell forms during no stirring-type suspension culture and injure little.
Description of drawings
Fig. 1 is cell inoculation microphotograph on the microcarrier in the time of 2 days;
Fig. 2 is virus inoculation microphotograph on the microcarrier in the time of 3 days;
Fig. 3 is virus inoculation microphotograph on the microcarrier in the time of 8 days;
Fig. 4 is a tidal type microcarrier suspension culture bioreactor construction sketch map.
The specific embodiment
In the present invention, term " Up " is the rate of climb of culture fluid by microcarrier, " Down " speed that to be culture fluid descend by carrier, and " Hold " is immersed in time in the culture fluid for microcarrier.
Used tidal type microcarrier suspension culture bioreactor in the embodiment of the invention, the microcarrier suspension culture bioreactor of other types, as stirring-type, rotary or filling type microcarrier suspension culture bioreactor, all can use the present invention's method large-scale production swine fever virus or vaccine.Preferably, the present invention uses tidal type microcarrier suspension culture bioreactor, the culture medium in the time of can improving cultivation and the supply of dissolved oxygen, and no bubble and shearing force are little, and the pair cell injury is little.
In the inventive method, bioreactor mainly is made up of carrier tank, fluid reservoir, pH controller, DO monitor, input and output system.Work process is as follows: cell is attached in the carrier tank grows on the carrier, and when the culture fluid of fluid reservoir was pumped to carrier tank, the culture fluid liquid level rose and to supply with nutrient and to promote the removal of cell metabolism product to cell; When the culture fluid of carrier tank pumps into fluid reservoir, cultivate liquid level and descend thereupon, cell is ventilated, promote to breathe, reduce the cell tangential pressure, no O
2The supply restriction, the non-foam worry.This multiple motion makes the cell on the carrier can access enough nutrition and O
2, produced simultaneously metabolic waste is as CO
2Can effectively be discharged from, thus amplifying cells that can be a large amount of and increment virus, and this kind technology is referred to as tidal type microcarrier suspension culture technology.
The bioreactor that adopts in the embodiment of the invention is the tidal type bioreactor.Structural representation as shown in Figure 4.Wherein, each labelling is respectively: constant temperature stirring system 1, the culture medium constant temperature cell body 2 of getting the raw materials ready, present material system 3 automatically, constant incubator 4, carrier bottle 5, microcarrier 6, DO detector and PH controller 7, catcher 8.More preferably, adopted the microcarrier suspension culture bioreactor that designs according to the morning and evening tides principle in the embodiment of the invention, culture systems is divided into two partly; One is carrier bottle 5, and another is that culture medium stirs bag (groove).Cell fixation is at the carrier bottle, and media flow causes intermittent exposure and floods carrier between carrier bottle and agitator tank.Carrier bottle 5 volumes that the present invention has tested reactor are 5L, 10L, 20L, 50L, 100L, all can control automatically temperature, pH value, dissolved oxygen, gas concentration lwevel.Carrier bottle 5 volumes are 20L in the embodiments of the invention.
In the method for the invention, in cell absorption microcarrier stage and cell culture stage, cell absorption program and cell culture program have been started, optimized the control parameter of reactor in the embodiment of the invention, but the inventive method is not limited only to the parameter among the embodiment, those skilled in the art can be according to technology enlightenment provided by the invention, according to different bioreactors, adjust relevant parameters, after reaching microcarrier and cell fully combining, the purpose of a large amount of amplifying cells.
In the method for the invention, in virus absorption microcarrier stage and Virus culture stage, viral absorption program and Virus culture program have been started, optimized the control parameter of reactor in the embodiment of the invention, but the inventive method is not limited only to the parameter among the embodiment, those skilled in the art can be according to technology enlightenment provided by the invention, according to different bioreactors, adjust relevant parameters, after reaching virus and cell and microcarrier fully combining, the purpose of a large amount of amplicon virus.
The microcarrier of the embodiment of the invention is reticular fiber a---polyester fiber, the microcarrier of this area other types---spherical, netted or flaky polyester, gelatin or polysaccharide microcarrier, also can play the effect of fixed cell, also can be used for the present invention's method large-scale production swine fever virus or vaccine.
In the embodiment of the invention, passage cell has used pig testis (ST) cell, and the passage cell that other this areas are commonly used such as PK15, BHK21, Marc 145, MDCK, VERO also can be used for the present invention's method large-scale production swine fever virus or vaccine.
It is 30～60g/L that the present invention has tested microcarrier density, adds 1.6 * 10
7～3.2 * 10
7The passage cell of cells/g microcarrier, more preferably, the microcarrier density of using in the embodiment of the invention is 50g/L, cell density is 3.0 * 10
11Cells/L.
It is M.O.I.=0.01～1 inoculation swine fever virus that the present invention has tested according to infection multiplicity, the swine fever virus poison valency of results is the highest, preferably, has used the M.O.I.=0.2 virus inoculation in the embodiment of the invention, start viral absorption program and Virus culture program, the amplification swine fever virus.
The volume of the swine fever virus liquid of single batch of results of bioreactor of the present invention is 2.5L～1000L, the volume that the embodiment of the invention has been tested the swine fever virus liquid of single batch of results is 50L, those skilled in the art can utilize the present invention's method to carry out linear amplification to the viral liquid of single batch of results is long-pending, can be amplified to 1000L.
The viral liquid of results of the present invention is the continous way results; Preferably, the number of times of results of the present invention is 3～15 times, more preferably, has tested in the embodiment of the invention every 3 days results, gather in the crops 15 times the virus and the vaccine of results virus preparation continuously, reach the requirement of " People's Republic of China's veterinary drug allusion quotation " through checking all.
The preparation method of vaccine is a centrifugal purification in the embodiment of the invention, adds stabilizing agent behind the purification, after the vacuum lyophilization, makes classical swine fever virus vaccine.Other vaccine production methods commonly used of this area also can be used for preparing vaccine of the present invention.The embodiment of the invention has adopted the 50%V/V lactose as stabilizing agent, and the stabilizing agent of other vaccine production commonly used of this area as skim milk or other freeze drying protectant, also can be used for preparing vaccine of the present invention.
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in to limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment 1 tidal type microcarrier suspension bioreactor large-scale production swine fever virus
Employed bioreactor is a tidal type microcarrier bioreactor in the present embodiment, and employed microcarrier is a polyester fiber.Wide 5mm, long 10mm, the 1g carrier provides 2,400cm
2Adherent area, can provide 1.0 * 10
9The cell growth of above quantity.
1. virus and cell strain
Being used to prepare the antigenic Strain of swine fever virus is hog cholera lapinised virus strain (available from China Veterinery Drug Inspection Office)., use and infect and a large amount of breeding swine fever virus as seedling cell line with pig testis (ST) cell line.
2 preparation methoies
1) in tidal type microcarrier bioreactor, adds polyester fiber microcarrier, cell growth medium and pig testis (ST) cell, in 37 ℃, 5%CO
2Under the culture environment, start cell absorption program, microcarrier is fully combined with pig testis (ST) cell, after cell attaches 4h, start the cell culture program, cultivate pig testis (ST) cell.Wherein microcarrier density is 50g/L, and cell growth medium is that the DMEM fluid medium (comprises 10% Ox blood serum, 0.01mol/LNaHCO
3, 0.1mg/ml kanamycin sulfate, 100,000IU penicillin G sodium salt, pH value are 7.2), pig testis (ST) cell initial inoculation density is 3.0 * 10
7The cells/g microcarrier, cell attaching program is: up 2800ml/min; Hold 1min; Down2800ml/min; Hold 30s; Set maximum and change liquid measure 9000ml; The cell culture program is: up 2000ml/min; Hold 1min; Down 2000ml/min; Hold 1min; Set maximum and change liquid measure 9000ml; Up is the rate of climb of culture fluid by microcarrier, and Down is the speed that culture fluid descends by carrier, and Hold is that microcarrier is immersed in the time in the culture fluid.
2) above-mentioned pig testis (ST) cell culture to 3.0 * 10
11Using cell maintenance medium during cells/L instead, is M.O.I.=0.2 inoculation swine fever virus according to infection multiplicity, starts viral absorption program, and virus is fully contacted with pig testis (ST) cell, switches to the Virus culture program behind the 4h, the amplification swine fever virus.Wherein cell maintenance medium is that the DMEM fluid medium (comprises 2% sheep blood serum, 0.01mol/LNaHCO
3, 0.1mg/ml kanamycin sulfate, 100,000IU penicillin G sodium salt, pH value are 7.2); Virus absorption program is: up 2000ml/min; Hold 1min; Down 2000ml/min; Hold 30s; Set maximum and change liquid measure 9000ml; The Virus culture program parameter is: up 1400ml/min; Hold 1min; Down 1400ml/min; Hold 1min; Set maximum and change liquid measure 9000ml.
3) in 37 ℃, 5%CO
2Continue under the culture environment to cultivate; Gather in the crops viral liquid every 3 days, gather in the crops 15 times and place 4 ℃ of preservations continuously.The viral liquid mixing of results is also carried out following check:
(a) pure property check: the relevant regulations by 15,19,20 pages of " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005 is tested, and meets fully, pig safety is had no side effect no antibacterial, mycete, mycoplasma and exogenous virus pollution.
(b) viral level is measured: virus is done 10 times of gradient series dilutions with containing 2% sheep blood serum cell maintenance medium, from 10
-1To 10
-6Each 8 hole of dilution factor inoculation sets up feminine gender not connect the poison contrast simultaneously, puts into 5%CO
2Cultivate 48-72h for 37 ℃ in the incubator, 80% acetone fixed is measured the hole count that each dilution factor contains swine fever virus (CSFV) positive cell (green fluorescence) with indirect immunofluorescence antibody (IFA) method, calculates viral TCID according to the Reed-Muench method
50, every 1.0ml contains virus 〉=10
6.5TCID
50
(c) specificity: measure with indirect immunofluorescence antibody (IFA) method.With virus inoculation in 96 porocyte plate ST cells, each sample 4 hole, every hole 100 μ L set up negative control simultaneously, put into 5%CO
2Cultivate 48～72h for 37 ℃ in the incubator; Discard growth-promoting media,, add 80% acetone soln of 100 μ l pre-coolings then, 4 ℃ of fixing 30min with PBS buffer (pH7.4) washed cell of 0.01mol/L 3 times.With after the PBS washing 3 times, every hole adds 100 μ l PBS1 then: the anti-CSFV serum of pig of 400 dilutions, 37 ℃ of effect 1h; With PBS washing 3 times, behind each 10min; Add and use PBS1: the anti-pig IgG of fluorescently-labeled rabbit (IgG-FITC) of 500 dilutions, every hole 100 μ l, 37 ℃ of effect 1h; With PBS washing 3 times, each 10min observes under fluorescence microscope.Cell control well should not have the specificity yellow-green fluorescence and occurs, and the virus inoculation cell hole should have a large amount of specificity yellow-green fluorescences to occur.
4) viral level of every milliliter through being up to the standards 〉=10
6.5TCID
50Swine fever virus (rabbitization low virulent strain) antigen stock through behind the centrifugal purification, as stabilizing agent, freeze vacuum drying after fully shaking up with 50% (volume ratio) lactose, and quantitatively packing, be stored in 4 ℃ after sealing and obtain finished product.
Make 3 batches of vaccines with quadrat method, numbering is respectively CSF-T01, CSF-T02, CSF-T03.
3 results
Viral level: behind swine fever virus (rabbitization low virulent strain) the inoculation ST cell, gather in the crops viral liquid, totally 3 batches, measure viral level and vaccine character respectively, result of the test sees Table 1.
Three batches of vaccine character of table 1 result of the test
The check of pure property: the relevant regulations by 15,19 pages of " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005 is tested, and meets fully, pig safety is had no side effect no antibacterial, mycete and mycoplasma.
Exogenous virus check: do not have other exogenous virus and pollute, the results are shown in Table 2.
Three batches of vaccine exogenous viruses of table 2 assay
BVDV: bovine viral diarrhoea/bovine diarrhoea virus; PRV: pseudorabies virus; PPV: pig parvoviral
The comparative test of the swine fever that embodiment 2 the present invention make malicious vaccine alive and like product
1. material
(1) 3 batches of the swine fever that vaccine: embodiment 1 makes malicious vaccines alive, lot number: CSF-T01, CSF-T02, CSF-T03.Swine fever malicious vaccine (making) 1 batch alive with the rolling bottle culture systems, lot number: CSF-RB01.
(2) pig is used in experiment: select the feed lot or the supply of fixed point pig farm that meet national laboratory animal standard for use, and the susceptible piglet of weaning for 8 ages in week, body weight is 18～25kg, hog cholera antibody all negative (serum neutralizing antibody); Observed 7 before annotating Seedling, respectively observe morning and afternoon every day 1 time, select body temperature, spirit, the normal person's use of appetite.
(3) counteracting toxic substances virus: swine fever virus (CSFV) crossdrift is a virulent strain.
2. method
(1) character check: perusal vaccine physical behavior.Two groups of vaccines are faint yellow spongy loose agglomerate, and no foreign body is easy to bottle wall and breaks away from, and add dissolving rapidly behind the diluent, concentration homogeneous, free from extraneous odour.
(2) steriling test: test for 15 pages by " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, two groups of vaccines are no bacterial growth.
(3) mycoplasma check: test for 19 pages by " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, two groups of vaccines are no mycoplasma growth.
(4) specificity check: measure with indirect immunofluorescence antibody (IFA) method.With virus inoculation in 96 porocyte plate ST cells, each sample 4 hole, every hole 100 μ l set up negative control simultaneously, put into 5%CO
2Cultivate 48～72h for 37 ℃ in the incubator; Discard growth-promoting media,, add 80% acetone soln of 100 μ l pre-coolings then, 4 ℃ of fixing 30min with PBS buffer (pH7.4) washed cell of 0.01mol/L 3 times.With after the PBS washing 3 times, every hole adds the pig anti-CSFV serum of 100 μ l with PBS dilution in 1: 400 then, 37 ℃ of effect 1h; With PBS washing 3 times, behind each 10min; Add two anti-(IgG-FITC), every hole 100 μ l, 37 ℃ of effect 1h with the anti-pig IgG of fluorescently-labeled rabbit of PBS dilution in 1: 500; With PBS washing 3 times, each 10min observes under fluorescence microscope.Cell control well should not have the specificity yellow-green fluorescence and occurs, and the virus inoculation cell hole should have a large amount of specificity yellow-green fluorescences to occur.
(5) exogenous virus check: test for 20 pages by " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, two groups of vaccines are no exogenous virus and pollute.
(6) residual moisture is measured: test for 31 pages by " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, two groups of vaccines are all up to specification.
(7) vacuum is measured: test for 31 pages by " People's Republic of China's veterinary drug allusion quotation " version appendix in 2005, two groups of vaccines are all up to specification.
(8) safety testing:
A.100 multiple dose test: get 8 of 8 week CSF antiviral antibody feminine gender in age (serum neutralizing antibody≤1: 4) wean susceptible piglets, be divided into 4 groups at random, each group is injected CSF-T01, CSF-T02 respectively, CSF-T03 criticizes and the CSF-RB01 vaccine, every equal intramuscular injection 100 multiple dose vaccines, observed 21 situations such as thermometric, observation simultaneously searched for food, breathing continuously.
B.1/100 multiple dose test: get 8 of 8 week CSF antiviral antibody feminine gender in age (serum neutralizing antibody≤1: 4) wean susceptible piglets, be divided into 4 groups at random, each group is injected CSF-T01, CSF-T02 respectively, CSF-T03 criticizes and the CSF-RB01 vaccine, every equal muscle is annotated 1/100 multiple dose vaccine, observed 21 situations such as thermometric, observation simultaneously searched for food, breathing continuously.
(9) antibody test and counteracting toxic substances protection test: get 39 of 8 week CSF antiviral antibody feminine gender in age (serum neutralizing antibody≤1: 4) wean susceptible piglets, get wherein 36 and be divided into 4 groups at random, every group 9, each group is subdivided into 3 groups again, 3 every group, 1st, distinguish each intramuscular injection CSF-T01, CSF-T02, three batches of vaccines of CSF-T03 for 2,3 groups, the 4th group of intramuscular injection CSF-RB01 vaccine, every group the 1st group injection 1 multiple dose vaccine, the 2nd group of injection 1/100 multiple dose vaccines, the 3rd group of injection 1/500 multiple dose vaccines; Remaining 3 piglets are not injected any vaccine, to contrast as negative; Immunity is blood sampling separation of serum mensuration NAT after 14 days, and every pig is with 10 simultaneously
5The swine fever crossdrift of minimum lethal dose (MLD) is that virulent strain virus is carried out challenge test, observes continuously 16, measures and observe the piglet clinical manifestation.
3. result
(1) safety testing
After the susceptible piglet of weaning for 8 ages in week is distinguished the vaccine (CSF-T01, CSF-T02, CSF-T03) of 100 times of 1/100 multiple dose of injecting immune, CSF-RB01 vaccine, piglet does not have fervescence, search for food, mental status and growth promoter situation be all normal, the test piglet all survives.The result shows, inoculation is safe to the target animals overdose for the three batches of experimental vaccines and CSF-RB01 vaccine.The results are shown in Table 3.
Table 3 vaccine safety result of the test
(2) antibody test and counteracting toxic substances protection test
With the serum neutralizing antibody behind the neutralization test method detection vaccine immunity; the result show no matter be behind CSF-T01, CSF-T02, CSF-T03 vaccine or the CSF-RB01 vaccine immunity antibody all at (except the CSF-RB01 vaccine of 1/500 multiple dose) more than 2; equal 3/3 protection behind the counteracting toxic substances; negative contrast 3/3 morbidity the results are shown in Table 4.
Table 4 vaccine immunity potency test result
-: symptom shapes such as fever do not appear behind the counteracting toxic substances
+: symptoms such as fever appear behind the counteracting toxic substances
*: occur hyperpyrexia symptom and dead behind the counteracting toxic substances
NA: total dead, can't measure
(3) in addition, relatively with the present invention's " cell microcarrier suspension culture system " and rolling bottle culture systems commonly used, cultivate pig testis cell line (ST), with the propagation swine fever virus, the viral correlation ratio that two system cells are cultivated is more as shown in table 5.
The correlation ratio of the different culture systems propagation of table 5 swine fever virus
Remarks: the adherent area 1g=2 of polyester fiber microcarrier, 400cm
2, the amount of 1 tidal type microcarrier suspension culture system adding carrier is pressed 1000g and is calculated.
4. brief summary
Above-mentioned comparative test result as can be known, the vaccine of producing with the swine fever of microactuator suspension carrier cell culture systems production of the present invention malicious vaccine alive and common method all has good safety and immune effect to the susceptible piglet, and, produce the output of swine Fever Vaccine much larger than using the rolling bottle culture systems always with the output that microactuator suspension carrier cell culture systems of the present invention is produced swine fever malicious vaccine alive.
The above only is preferred embodiment of the present invention, and is in order to restriction the present invention, within the spirit and principles in the present invention not all, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (16)
1. the production method of a swine Fever Vaccine is characterized in that, comprises the steps:
1) in the microcarrier bioreactor, adds microcarrier, passage cell and cell growth medium, start cell absorption program, after making microcarrier and passage cell fully combining, start the cell culture program, cultivate passage cell.Wherein microcarrier density is 30～60g/L, and passage cell density is 1.6 * 10
7～3.2 * 10
7The cells/g microcarrier;
2) above-mentioned passage cell is cultured to 4 * 10
10Cells/L～3.2 * 10
11Using cell maintenance medium during cells/L instead, is M.O.I.=0.01～1 inoculation swine fever virus according to infection multiplicity, starts viral absorption program, and virus is fully contacted with passage cell, uses the Virus culture program instead, the amplification swine fever virus;
3) Virus culture was gathered in the crops viral liquid after 2～5 days, obtained swine fever virus, 4 ℃ of preservations;
4) Shou Huo viral liquid adds freeze drying protectant behind the purification, makes swine Fever Vaccine.
2. method according to claim 1 is characterized in that, described bioreactor is the microcarrier suspension culture bioreactor.
3. method according to claim 1 is characterized in that, described bioreactor is for being tidal type microcarrier suspension culture bioreactor.
4. method according to claim 1 is characterized in that, described microcarrier is spherical, netted or lamellar; The composition of described microcarrier is polyester, gelatin or polysaccharide.
5. method according to claim 1 is characterized in that cell growth medium described in the step 1) is the DMEM culture medium that contains sheep blood serum or Ox blood serum.
6. method according to claim 1 is characterized in that step 2) described in cell maintenance medium be the DMEM culture medium that contains sheep blood serum or Ox blood serum.
7. method according to claim 1 is characterized in that, cell growth medium described in the step 1) is the DMEM culture medium that contains 3%～15% sheep blood serum or Ox blood serum.
8. method according to claim 1 is characterized in that step 2) described in cell maintenance medium be the DMEM culture medium that contains 1%～5% sheep blood serum or Ox blood serum.
9. method according to claim 1 is characterized in that, the condition of culture of described microcarrier bioreactor is 34 ℃～37 ℃ of temperature, CO
2Concentration is 2.5%～5%, and medium pH value is 7.0～7.6.
10. method according to claim 1 is characterized in that: the cell attaching program described in the step 1) is: cell attaching program is: up 2000～3000ml/min; Hold 1min; Down 2000～3000ml/min; Hold 30s; The cell culture program is: up 1000～3000ml/min; Hold 1min; Down 1000～3000ml/min; Hold 1min; Step 2) described viral absorption program is: up 1000～3000ml/min; Hold 1min; Down 1000～3000ml/min; Hold 30s; The Virus culture program parameter is: up 1000～2000ml/min; Hold 1min; Down:1000～2000ml/min; Hold 1min.
11. method according to claim 1 is characterized in that: the attaching of cell described in step 1) program is: up 2800ml/min, and hold 1min, Down 2800ml/min, hold30s sets maximum and changes liquid measure 9000ml; The cell culture program is: up 2000ml/min, and hold1min, Down 2000ml/min, hold 1min sets maximum and changes liquid measure 9000ml; Step 2) viral absorption program is described in: up 2000ml/min; Hold 1min; Down2000ml/min, hold 30s sets maximum and changes liquid measure 9000ml; The Virus culture program parameter is: up 1400ml/min, and hold 1min, Down 1400ml/min, hold 1min sets maximum and changes liquid measure 9000ml.
12. method according to claim 1 is characterized in that: the volume of the liquid of swine fever virus described in the step 3) is 2.5L～1000L.
13. method according to claim 1 is characterized in that, the results virus liquid described in the step 3) is the continous way results.
14. method according to claim 1 is characterized in that, the number of times of the results described in the step 3) is 3～15 times.
15. swine fever virus as any method preparation of claim 1-14.
16. classical swine fever virus vaccine as any method preparation of claim 1-14.
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| CN102743748A (en) * | 2011-12-12 | 2012-10-24 | 中牧实业股份有限公司 | Method for preparing swine fever live vaccine by using oscillatory type bioreactor |
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| CN104873978A (en) * | 2015-06-09 | 2015-09-02 | 浙江美保龙生物技术有限公司 | Freeze-drying protective agent for hog cholera live vaccine (spleen and lymph tissue origin) |
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